SUPPLEMENT 1

生物物理Vol. 52

2012 年 8 月（増刊号）

ISSN 0582-4052 CODEN : SEBUAL
2012 年 8 月（増刊号）

SUPPLEMENT 1
Vol.52

日本生物物理学会第2012

回年会講演予稿集

50

昭和42年8月1日 国鉄大局特別扱承認雑誌第216号
平成24年8月15日
増刊号

2012 年 Vol.52
SUPPLEMENT 1＜資料請求番号 S1-3＞

第 50回年会講演予稿集
2012.9.22 (土) ～ 24 (月)名古屋大学・東山キャンパス

主催

日本生物物理学会

＜資料請求番号 S1-1＞＜資料請求番号 S1-2＞

–(S1)–

–(S2)–

■ 会場案内図
理学Ｅ館

食

1F Ｉ会場
諸会議室

堂

名古屋大学 消費生活協同組合 ダイニング・フォレスト
9/22・23 11:30〜13:30、9/24 11:00〜20:00

理学南館

1F ポスター会場SＡ会場
年会本部
3F 諸会議室

工学研究科9号館

環境総合館
環境学研究科

工学研究科5号館
北部厚生会館

工学研究科1号館

E館

理学Ｃ館

5F Ｈ会場

C館

工学研究科7号館
学生相談総合センター

南館

ES総合館
素粒子宇宙起源研究機構

B館
IB電子情報館
留学生相談室工学研究科3号館

地下鉄 地下鉄
3 番出口 ２番出口

工学研究科2号館

情報文化学部

文学研究科・文学部

豊田講堂

バス停

地下鉄
1 番出口

1F 休憩室2F Ｇ会場
5F Ｆ会場
ランチョン控室

多元棟

理学部A館
理学部A館

バス停

附属図書館
(中央図書館)

理学Ｂ館

総合案内

経済学研究科・経済学部

多元数理科学棟

広報プラザ

1F
2F
3F
4F

Ｅ会場
諸会議室
Ｄ会場
Ｃ会場
ランチョン控室
5F Ｂ会場

豊田講堂

教育発達科学研究科・教育学部
発達心理精神科学教育研究センター

南部厚生会館

懇親会会場

食懇親会会場 法政国際教育協力
留学生センター堂

レストラン
シェジェロー
9/22〜24
11:30〜14:30

研究センター

南部生協 Mei-dining【1F】ポスター会場T
展示会場
S会場
受付

ポスター番号
PT201−PT240

T

ポスター会場
展示会場

WC
（女）

ホワイエ

S 会場

豊田講堂ホール

ポスター番号
PT101−PT192
事務室

WC
（男）

▼ ▼

豊田
講堂

1F ポスター会場T
展示会場
Ｓ会場
受付
ランチョンセミナー
整理券配布所クローク

法学研究科・法学部

入口

EV

WC
（女）

受 付
EV

–(S3)–

ポスター番号
PT001−PT011

WC（男）■ 会場案内図【1F】A会場、
年会本部、ポスター会場S

理学
南館

諸会議室
年会本部
102

S

ポスター会場107【3F】諸会議室
302

WC
（女）

WC
（男）

WC
（女）

ポスター番号
PS001−PS054

▼

入口

A 会場

坂田・平田ホール

EV

EV

WC

WC

WC

WC

EV

B 会場
509

【4F】C会場、
ランチョン控室

C 会場
409【3F】D会場

WC

WC

WC

WC

WC

WC

EV

D 会場
309

【2F】諸会議室
EV

諸会議室
209

【1F】E会場

入口

▼

EV

E 会場
109

–(S4)–入口

ランチョン控室
453

EV

▼

多元数理
科学棟

【5F】B会場

理学B館【5F】F会場、
ランチョン控室
WC

WC

ランチョン控室
504

EV

F 会場
501

【2F】G会場

G会場

WC

WC

212

EV

【1F】休憩室
WC

WC

休憩室
116

EV

▼

入口

理学
C館

【5F】H会場

諸会議室
101

EV
EV

H会場
517

【1F】I会場、
諸会議室

I会場

WC

WC

131

EV

EV

▼

理学
E館

WC
（女）

入口
–(S5)–

WC■ 9月22日（土） 年会1日目
8:00

理学南館
Sci. south
bldg.

坂田・
平田ホール A会場
1F
Sakata Room A
Hirata Hall【Day1】22th Sep.
9:00

10:00

11:00

12:00

1SA

1LA
GEヘルスケア・ジャパン㈱GE Healthcare Japan
Corporation

新学術領域研究 「少数性生物学−個と多数の狭間が織りなす生命現象の探求−」共催
1分子生物学と生化学の狭間に潜むナノシステム動作力学の理解を目指して

Bridging single molecule biology and biochemistry tounderstand operation principles of bio-nanosystems

302

会議室
Meeting
Room

5F

509

B会場
Room B

1SB
身体ｰ細胞ダイナミクス連関：やわらかさの階層と連携
Body-cell dynamic linkage: softness, flexibility,
fluctuation and controllability

4F

409

C会場
Room C

1SC
生物学における数学的手法の最前線
Frontiers in mathematical methods in biology

多元数理
科学棟
3F
Mathematics
bldg.

309

D会場
Room D1SD
核内分子動態から観る遺伝子発現機構
Molecular Dynamics in the Nucleus andGene Regulation

2F

209

会議室
Meeting
Room

1F

109

E会場
Room E

1SE
コンフォメーション動力学：フォールディングから機能まで
Conformational dynamics: Folding to function

1LE㈱菱化システム
Ryoka
Systems Inc.

5F

501

F会場
Room F

1SF
生物の動きと変形への理論的アプローチ
Theoretical Approaches to Biological Motion
and Deformation

1LF
日本エフイー・アイ㈱FEI Company

2F

212

G会場
Room G

理学C館
5F
Sci. bldg.C

517

H会場
Room H1SH
レチナール蛋白質と光遺伝学
Retinal proteins and optgenetics

131

I会場
Room I

1SI
分子モーター複合体の力発生機構を探る：1分子～細胞の架け橋
Exploring force-generating mechanism of molecular motor ensembles;building bridges between single molecules and cells

101

会議室
MeetingRoom

名 古 屋 大 学

3F

東 山 キ ャ ン パ ス

理学B館
Sci. bldg.B

理学Ｅ館
1F
Sci. bldg.E

豊田講堂ホール
S会場
ToyodaAuditorium Room S
Hall
豊田講堂
Toyoda 1F
Auditorium

ホワイエ
Foyer

BIOPHYSICS
編集委員会

107

1LB
浜松ホトニクス㈱Hamamatsu
Photonics K.K

平成24年度
第4回運営委員会

若手奨励賞受賞講演
Early Research in Biophysics Award

ポスター
会場T
PosterSession T

ポスター展示Poster Viewing

企業展示
Exhibition

理学南館
Sci. south 1F
bldg.

13:00

企業展示
Exhibition

ポスター
会場S
PosterSession S

ポスター展示Poster Viewing–(S6)–

BIOPHYSICS
論文賞
受賞講演

公開講座
生命の不思議を解き明かす14:00

15:00

16:00

17:00

18:00

19:00

1A
分子モーター I
Molecular motors I

HYSICS
委員会

若手賞選考委員会

1B
蛋白質-構造機能相関 I
Proteins: Structure & Function I
1C
蛋白質-計測、解析、エンジニアリング
Proteins: Measurement, Analysis, Engineering
1D
発生・分化、神経
Development & Differentiation, Neuroscience

16:10

1E
蛋白質-物性 I
Proteins: Property I

新旧合同委員会

1F
光生物-視覚、光受容Photobiology: Vision & Photoreception I

16:10

1G
細胞生物的課題 I
Cell biology I
1H
バイオイメージング
Bioimaging
1I
生命情報科学、
バイオエンジニアリング
Bioinformatics & Bioengineering

開講座
の不思議
き明かす
ポスター発表ポスター発表Poster Presentation Poster Presentation 撤去
奇数／Odd number 偶数／Even number Removal
16:30～17:30
17:30～18:30

ポスター展示Poster Viewing

企業展示
Exhibition

ポスター展示Poster Viewing

ポスター発表ポスター発表Poster Presentation Poster Presentation 撤去
奇数／Odd number 偶数／Even number Removal
16:30～17:30
17:30～18:30–(S7)–

20:00

21:00

■ 9月23日（日） 年会2日目
8:00

1F
理学南館
Sci. south
bldg.

坂田・
平田ホール A会場
Sakata Room A
Hirata Hall【Day2】23th Sep.
9:00

10:00

11:00

2SA
海外招聘シンポジウム：生物物理学最前線
Invited symposium from abroad: Forefront ofbiophysics

302

会議室
Meeting
Room

5F

509

B会場
Room B

2SB
若手オーガナイズシンポジウム：生命科学の新しい地平を切り開く若手研究者たち
Symposium organized by younger generation:
Young scientists pioneering future life sciences

4F

409

C会場
Room C2SC
非平衡リビングマター：DNAから細胞骨格、細胞へ
Living matter far from equilibrium: from DNA
to cytoskeletons and cells

多元数理
科学棟
3F
Mathematics
bldg.

309

D会場
Room D2SD
スーパーコンピューティング：分子ネットワークと細胞内ダイナミクス
Supercomputing in Molecular Network to
Cellular Dynamics

2F

209

会議室
Meeting
Room

1F

109

E会場
Room E

5F

501

2F

名 古 屋 大 学

3F

若手の会会議男女共同参画委員会
2SE

東 山 キ ャ ン パ ス

F会場
Room F

2SF
生命システムのばらつきと情報処理
Inherent variability and information coding in
biological systems

2LF
Protein Data
Bank Japan

212

G会場
Room G

2SG
染色体場のダイナミクス
Dynamics of chromatin as thephysicochemical field for genetic activities

517

H会場
Room H

2SH
先端顕微計測が照らす生命の輝き
Star of life shined by frontier microscopies

131

I会場
Room I

2SI
一分子レベルで生体分子機械のエネルギーと機能効率を考える
The art of energetic and functional efficiency inbiomolecular machines on the single molecule level

101

会議室
Meeting
Room

新学術領域研究 「運動超分子マシナリーが織りなす調和と多様性」共催
Harmonized supramolecular machinery for
motility and its diversity

豊田講堂ホール
S会場
ToyodaAuditorium Room S
Hall
ポスター
会場T
Poster
ホワイエ Session T

ポスター展示Poster Viewing

Foyer

企業展示
Exhibition

理学南館
Sci. south 1F
bldg.

2LB
オリンパス㈱Olympus
Corporation

男女共同参画・
若手問題シンポジウム
Promoting Gernder
Equality

理学Ｅ館
1F
Sci. bldg.E

豊田講堂
Toyoda 1F
Auditorium

分野別
専門委員会

運動超分子マシナリーが織りなす調和と多様性

理学B館
Sci. bldg.B

理学C館
5F
Sci. bldg.C

12:00

107

企業展示
Exhibition

ポスター
会場S
PosterSession S

ポスター展示Poster Viewing–(S8)–

2LH
スペクトリス㈱ マルバーン事業部
Malvern Japan Division ofSpectris Co., Ltd.

13:00

総会
Assembly

会
mbly

14:00

15:00

16:00

2A
分子モーター II: ダイニン
Molecular motors II: Dynein

17:00

18:00

19:00

20:00

16:10

2B
蛋白質-構造機能相関 II: 理論、凝集
Proteins: Structure & Function II:
Theory, Aggregation
2C
数理生物学
Mathematical biology
16:10

2D
計測
Measurements

2E
蛋白質-物性 II
Protein: Property II
2F
光生物-視覚、光受容 II
Photobiology: Vision & Photoreception II

16:10
懇親会
南部生協
Mei-dining

2G
生体膜・人工膜
Biological & Artifical membrane
2H筋肉
Muscle2I
細胞生物学的課題 II: Prokaryotes
Cell biology II: Prokaryotes

ポスター発表ポスター発表Poster Presentation Poster Presentation 撤去
奇数／Odd number 偶数／Even number Removal
16:30～17:30
17:30～18:30

ポスター展示Poster Viewing

企業展示
Exhibition

ポスター展示Poster Viewing

ポスター発表ポスター発表Poster Presentation Poster Presentation 撤去
奇数／Odd number 偶数／Even number Removal
16:30～17:30
17:30～18:30–(S9)–

21:00

■ 9月24日（月） 年会3日目
8:00

理学南館
Sci. south
bldg.

坂田・
平田ホール A会場
1F
Sakata Room A
Hirata Hall

【Day3】24th Sep.
9:00

10:00

11:00

12:00

11:10
3A
分子モーター III: F1-ATPase、マイコプラズマ
Molecular motors III: F1-ATPase,
Mycoplasma

302

会議室
Meeting
Room

5F

509

B会場
Room B

3B
蛋白質-構造機能相関 III:動態、生体リズム
Proteins: Structure & Function III:
Dynamics, Circadian rhythm

4F

409

C会場
Room C

3C
核酸
Nucleic acids

多元数理
科学棟
3F
Mathematics
bldg.

309

D会場
Room D

2F

209

会議室
Meeting
Room

1F

109

E会場
Room E

5F

501

F会場
Room F

2F

212

G会場
Room G

3G
生体膜・人工膜-構造・物性
Biological & Artifical membranes:
Structure & Property

理学C館
5F
Sci. bldg.C

517

H会場
Room H

3H
細胞生物的課題 III:細胞骨格、細胞運動
Cell biology III:Cytoskeleton & Motility

131

I会場
Room I

101

会議室
MeetingRoom

名 古 屋 大 学

3F

東 山 キ ャ ン パ ス

理学B館
Sci. bldg.B

理学Ｅ館
1F
Sci. bldg.E

3LA㈱ニコンインステック
Nikon Instech
Co.,Ltd.

11:10

3D
光生物-光合成
Photobiology: Photosynthesis

3E
蛋白質-構造Proteins: Structure

科研費説明会
11:10

3F
膜蛋白質
Membrane proteins

3I
ヘム蛋白質
Heme proteins

13:00

11:10

平成24年度
第5回運営委員会

豊田講堂ホール
S会場
ToyodaAuditorium Room S
Hall
豊田講堂
Toyoda 1F
Auditorium

ポスター
会場T
Poster
ホワイエ Session T

ポスター展示Poster Viewing

ポス
Poster
奇数／
13:1

Foyer

企業展示
Exhibition

理学南館
Sci. south 1F
bldg.

107

企業展示
Exhibition

ポスター
会場S
PosterSession S

ポスター展示Poster Viewing–(S10)–

ポス
Poster奇数／
13:1

14:00

15:00

16:00

17:00

ポスター発表ポスター発表Poster Presentation Poster Presentation 撤去
奇数／Odd number 偶数／Even number Removal
13:15～14:15
14:15～15:15
企業展示
Exhibition
ポスター発表ポスター発表Poster Presentation Poster Presentation 撤去
奇数／Odd number 偶数／Even number Removal
13:15～14:15
14:15～15:15–(S11)–

18:00

19:00

20:00

21:00

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◇ 参加証（名札）
参加証は会場内では必ずご着用ください。参加証のない方のご入場は固くお断りいたします。
事前送付された参加証は必ず会場にご持参ください。（ネームホルダーは会場内で配布します）◇ 領収書の発行
参加証とともに領収書をお渡しいたしますが、別の形式の領収書が必要な場合、お渡しした領収書と引き換えに発行いたします。

◇ 講演予稿集
年会予稿集は日本生物物理学会会員に事前送付されます。残部がある場合に限り年会受付にて当日販
売（3,500 円）を行います。プログラム（タイトル、発表者、所属）は予稿集発行日以後に、年会ホームペー
ジにて公開します。また年会終了後は、半年ほど経て、日本生物物理学会ホームページの年会の記録（http://www.biophys.jp/ann/ann02.html）から CiNii（国立情報学研究所の論文情報ナビゲータ）にリンクが張られ、CiNii の生物物理のページ（http://ci.nii.ac.jp/organ/journal/INT1000001547_jp.html）で予稿本文が公開されます。

◇ 年度会費の支払いと入会の手続き
日本生物物理学会の年度会費が未納の場合は、年会受付の年度会費未納者・新規入会受付デスクでお支払いください。また、日本生物物理学会への新規入会も受け付けます。

２． 会場内のサービス・施設◇ クローク
場

所:

利用時間:

豊田講堂入口
9 月 22 日（土）、23 日（日） 8:00-18:45
9 月 24 日（月） 8:30-15:30※貴重品や傘、またコンピュータなどについては、破損、紛失などの責任は負いかねますので、各自でお持ちください。
※懇親会へ移動される際は荷物をお引き取りの上、懇親会会場に持参ください。

◇ 昼食
ランチョンセミナーならびに男女共同参画・若手問題シンポジウムでは、お弁当とお茶が無料で提供されます。積極的にご参加ください。また会期中以下の食堂をご利用できます。
◇名古屋大学消費生活協同組合 ダイニング・フォレスト
http://www.nucoop.jp/shop/list-sci.html#03
9 月 22 日（土）、23 日（日） 11:30-13:30
9 月 24 日（月）11:00-20:00◇レストラン シェジロー
http://chezjiroud.jp/
9 月 22 日（土）、23 日（日）、24 日（月）11:30-14:30–(S13)–

◇呼び出し㻌
会場内での呼び出しは、緊急の場合を除いて一切行いません。参加者間の連絡用として、年会受付に㻌
伝言板を設置しますので、ご利用ください。㻌
㻌◇駐車場㻌
会場には参加者用駐車場はございません。会場へは公共交通機関をご利用ください。㻌
㻌◇宿泊案内㻌
宿泊に関しては年会ホームページ「宿泊について」をご参照ください。㻌
㻌◇インターネットならびにドリンクコーナーのご案内㻌・インターネット㻦豊田講堂において無線㻸㻭㻺がご利用になれます。㻌
パスワード等については受付にてご確認ください。㻌
・ドリンクコーナー㻦展示会場（豊田講堂）をご利用ください。㻌
㻌◇託児所㻌
年会期間中は、託児所を設置いたします。詳しくは年会ホームページをご覧ください。㻌
㻌
㻌
㻌

３．

年会行事・プログラム㻌

㻌

◇総会㻌
日本生物物理学会第 㻡㻝 回定例総会を、年会 㻞 日目、㻥 月 㻞㻟 日（日）㻝㻞㻦㻡㻜㻙㻝㻟㻦㻡㻜 に 㻭 会場（理学南館 㻝階坂田・平田ホール）で開催しますのでご出席ください。詳しくは開催通知をご覧ください。㻌
㻌◇若手招待講演㻌
日本生物物理学会若手奨励賞の選考会である講演会を、年会 㻝 日目㻌 㻥 月 㻞㻞 日（土）㻥㻦㻜㻜㻙㻝㻝㻦㻟㻜 に、㻌㻿 会場（豊田講堂ホール）で開催します。㻌㻌◇㻮㻵㻻㻼㻴㼅㻿㻵㻯㻿 論文賞受賞講演㻌㻮㻵㻻㻼㻴㼅㻿㻵㻯㻿 論文賞受賞の講演会を、年会 㻝 日目㻌 㻥 月 㻞㻞 日（土）㻝㻝㻦㻟㻜㻙㻝㻝㻦㻡㻡 に、㻌㻿 会場（豊田講堂ホール）で開催します。㻌
㻌◇懇親会㻌
日時㻦㻌 㻞㻜㻝㻞 年 㻥 月 㻞㻟 日（日）㻝㻥㻦㻜㻜㻌㻙㻌㻞㻝㻦㻜㻜㻌
会場㻦㻌 名古屋大学㻌 南部生協 㻝㻲㻌㻹㼑㼕㻙㼐㼕㼚㼕㼚㼓㻌〒㻠㻢㻠㻙㻜㻤㻝㻠㻌 名古屋市千種区不老町 㻝㻌㻌 㻌 㻌 㻌 㻌 㻌 㻌 㻌 㻌 㻌 㻌 㻌 㼀㼑㼘㻦㻜㻡㻞㻙㻣㻟㻠㻙㻢㻝㻜㻜㻌 㻌㻌 㻌 㻌 㻌 㻌 㻌 㻌 㻌 㻌 㻌 㻌 㻌 㼔㼠㼠㼜㻦㻛㻛㼣㼣㼣㻚㼚㼡㼏㼛㼛㼜㻚㼖㼜㻛㼟㼔㼛㼜㻛㼘㼕㼟㼠㻙㼟㼛㼡㼠㼔㻚㼔㼠㼙㼘㻏㻜㻠㻌㻌
懇親会の当日参加も受け付けます（会場前でも受け付けする予定です）。㻌
㻌㻌㻌–(S14)–

㻌◇男女共同参画・若手問題シンポジウム㻌日時㻦㻌 㻞㻜㻝㻞 年 㻥 月 㻞㻟 日（日）㻝㻞㻦㻜㻜㻌㻙㻌㻝㻟㻦㻜㻜㻌
会場㻦㻌 㻱 会場（多元数理科学棟㻙㻝㻜㻥）㻌
昼食㻦㻌 お弁当とお茶が無料で提供されます。㻌
㻌◇科研費説明会㻌
日時㻦㻌 㻞㻜㻝㻞 年 㻥 月 㻞㻠 日（月）㻝㻞㻦㻜㻜㻌㻙㻌㻝㻟㻦㻜㻜㻌
会場㻦㻌 㻱 会場（多元数理科学棟㻙㻝㻜㻥）㻌
昼食㻦㻌 お弁当とお茶が無料で提供されます。㻌

◇ランチョンセミナー㻌
昼食（お弁当とお茶、無料）をとりながらの協力企業によるセミナーに、ご参加ください。なお、お弁当の数に限りがあるため当日午前中に整理券を配布いたします。セミナー開始前に、会場入り口で整理券と引き換えにお弁当を受け取り、ご入場ください。㻌
㻌◆整理券の発券について㻌
ランチョンセミナー整理券は配付デスクにて配付いたします㻌
時間㻦会期中毎日㻌 㻤㻦㻜㻜㻙㻝㻝㻦㻜㻜（当日開催されるセミナー分のみ発券）㻌
場所㻦豊田講堂㻌 入口㻌
※整理券はランチョンセミナー共催の企業、団体よりご提供頂く昼食の引換券になります。㻌
昼食数はセミナーごとに異なりますので、発券は数が無くなり次第終了となります。㻌
㻌◆整理券の注意事項㻌
整理券は各日㻝㻞㻦㻜㻜を過ぎると無効になります。㻌
午前のプログラム終了後、ランチョンセミナー開始時間までにご来場ください。㻌
上記有効時間までにご来場されない場合、整理券は無効となり、お弁当は整理券をお持ちでない㻌
セミナー参加者にご利用いただきますことをご了承ください。㻌
㻌◇機器・試薬・書籍等展示会㻌
機器、試薬、ソフトウエア、書籍などの展示会を豊田講堂で行います。㻌
㻌◇公開講座㻌テーマ㻦㻌 生命の不思議を解き明かす㻌 㻌㻌 㻌 㻌 日時㻦㻌 㻌 㻌 㻥 月 㻞㻞 日（土）㻌 㻝㻟㻦㻜㻜㻙㻝㻠㻦㻜㻜㻌㻌 㻌 㻌 会場㻦㻌 㻌 㻌 名古屋大学㻌 豊田講堂ホール㻌
㻌㻌 㻌 㻌 お問い合わせ㻦㻌 年会事務局までお願いします。㻌㻌 㻌 㻌 㻌 㻌 㻌 㻌 㻌 㻌 㻌 㻌 㻌 㻌 㻌 㻱㻙㼙㼍㼕㼘㻦 㼍㼙㼎㼟㼖㻡㻜㻬㼎㼡㼚㼟㼔㼕㻠㻚㼎㼕㼛㻚㼚㼍㼓㼛㼥㼍㻙㼡㻚㼍㼏㻚㼖㼜㻌㻌㻌㻌㻌

–(S15)–

㻌

４．

禁止事項㻌

㻌

◇撮影・録音㻌
会場内でのカメラ、ビデオ、携帯電話などによる撮影や講演音声の録音などを禁止します。㻌
㻌◇喫煙・飲食㻌
学内は終日禁煙です。講演会場内での飲食はランチョンセミナー、男女共同参画・若手問題シンポジウム、各種委員会など食事が提供される場合を除いて禁止します。㻌
㻌◇携帯電話㻌
シンポジウム、口頭発表、ポスター発表等の講演会場内での携帯電話による通話を禁止します。㻌
講演会場内では電源をオフにするかマナーモードに設定し、呼び出し音が鳴らないようご注意ください。㻌
㻌
㻌

５．

年会についての問い合わせ㻌㻌◇会期中㻌
年会本部㻌 （㻼㼔㼛㼚㼑㻌㼚㼡㼙㼎㼑㼞㻌㼞㼑㼍㼏㼔㼍㼎㼘㼑㻌㼐㼡㼞㼕㼚㼓㻌㼠㼔㼑㻌㼙㼑㼑㼠㼕㼚㼓）㻌㻌 㻌 㻌 㻌 㻌 㻌 㻌 㻌 㻌 㻌 㼀㼑㼘㻦㻌㻜㻤㻜㻙㻝㻝㻡㻤㻙㻤㻞㻥㻡㻌㻌◇会期外㻌
年会事務局㻌
㻱㻙㼙㼍㼕㼘㻦㼍㼙㼎㼟㼖㻡㻜㻬㼎㼡㼚㼟㼔㼕㻠㻚㼎㼕㼛㻚㼚㼍㼓㼛㼥㼍㻙㼡㻚㼍㼏㻚㼖㼜㻌㻌
参加登録・演題登録㻌 システムサポートデスク㻌〒㻝㻝㻟㻙㻜㻜㻟㻟㻌 東京都文京区本郷㻡㻙㻞㻥㻙㻝㻞㻙㻝㻟㻜㻠㻌
中西印刷株式会社㻌 東京事務所㻌 内㻌㼀㻱㻸㻦㻌㻜㻟㻙㻟㻤㻝㻢㻙㻜㻣㻟㻤㻌 㻲㻭㼄㻦㻌㻜㻟㻙㻟㻤㻝㻢㻙㻜㻣㻢㻢㻌㻱㻙㼙㼍㼕㼘㻦 㼎㼟㼖㻬㼚㼍㼏㼛㼟㻚㼏㼛㼙㻌㻌
年会実行委員会サポート・展示・広告問い合わせ先㻌〒㻝㻜㻝㻙㻜㻜㻡㻝㻌 東京都千代田区神田神保町 㻟㻙㻞㻙㻤㻌 昭文館ビル 㻟Ｆ㻌株式会社エー・イー企画㻌 展示会事業部㻌

㼀㼑㼘㻦㻌㻜㻟㻙㻟㻞㻟㻜㻙㻞㻣㻠㻠（代表）㻌 㻲㼍㼤㻦㻌㻜㻟㻙㻟㻞㻟㻜㻙㻞㻠㻣㻥㻌実行委員会サポート関連㻦㻌㼖㼎㼜㻞㻜㻝㻞㻬㼍㼑㼜㼘㼍㼚㻚㼏㼛㻚㼖㼜㻌
広告関連 㻱㻙㼙㼍㼕㼘㻦㻌㼍㼐㼕㼚㼒㼛㻬㼍㼑㼜㼘㼍㼚㻚㼏㼛㻚㼖㼜㻌
展示関連 㻱㻙㼙㼍㼕㼘㻦㻌㼑㼋㼖㼎㼜㻡㻜㻬㼍㼑㼜㼘㼍㼚㻚㼏㼛㻚㼖㼜㻌㻌㻌㻌
–(S16)–

㻌
㻢．㻌

発表者へのご案内㻌㻌◇使用言語㻌
シンポジウムおよび一般の口頭発表は、原則として、英語をお使いください。㻌
㻌◇映写機器㻌
発表に使用できる映写機器は、液晶プロジェクターのみです。音声出力には対応しません。㻌
会場にはコンピュータを用意しませんので、ご自身のノートパソコンを必ずご持参ください。㻌
㻌注意 㻝）㻌各自会場係員の指示に従い、コンピュータを会場に備え付けられた切り替え装置㻌
㻌（㼂㼕㼐㼑㼛㻌㻿㼣㼕㼠㼏㼔㼑㼞）に接続してください。㻌
㻌注意 㻞）㻌切り替え装置に繋がるコンピュータの映像出力端子は、㻌㻌「ミニ 㻰㻙㼟㼡㼎㻝㻡 ピン端子（メス）」㻖（㻟 列あるもの）のみです。㻌
㻌
端子の形状が異なる場合（㻹㼍㼏㼕㼚㼠㼛㼟㼔 等）、変換アダプターをご持参ください。㻌
㻖
読み㻦みに 㻰 さぶ 㻝㻡 ピンたんしメス㻌㻌㻌注意 㻟）㻌発表に使用するパワーポイントファイルが入った 㼁㻿㻮 メモリーを念のためにお持ちください。㻌
㻌注意 㻠）㻌バッテリー切れに備え、必ず電源アダプターをご持参ください。㻌
㻌㻌◇シンポジウム、若手招待講演の座長の方へ㻌
受付㻦㻌 座長の方はシンポジウム開始 㻝㻡 分前までに各会場の「座長席」までおいでください。㻌
㻌
進行㻦㻌 シンポジウムの進行と時間管理は座長に一任いたしますが、終了予定時間を越えないよう㻌
ご注意ください。㻌
㻌◇シンポジウム、若手招待講演の講演者の方へ㻌受付㻦㻌 講演者の方は、シンポジウム開始の 㻝㻡 分前までに各会場においでください。㻌
ご持参いただいたノートパソコンを使用してスライドが正しく映写されることを確認ください。㻌
スライド映写の確認は、あらかじめ理学 㻮 館 㻝㻝㻢（試写室㻌 兼㻌 休憩室）で行うことも可能です。㻌
㻌※モニターと接続し外部出力の確認のみです。機材オペレーターは常駐しておりません。㻌
㻌
講演時間㻦㻌 時間配分は座長に一任します。㻌㻌㻌 㻌 㻌 若手招待講演の講演時間は、発表 㻝㻜 分、討論 㻟 分、コンピュータの交換に 㻞 分です。㻌
㻌◇口頭発表の座長の方へ㻌㻌受付㻦㻌 口頭発表の座長の方は、担当されるセッション開始の 㻝㻜 分前までに各会場の「座長席」まで㻌
お越しください。会場には時間を計測するスタッフを置いています。㻌㻌
進行㻦㻌 㻌 多くの講演者の発表を滞りなく進めるために、時間厳守をお願いいたします。㻌
㻌–(S17)–

㻌◇口頭発表の講演者の方へ㻌
講演者は、各自の発表が含まれるセッションが始まる 㻝㻜 分前までには会場においでください。㻌
口頭発表の講演時間は、発表 㻥 分、質疑応答 㻟 分（交代時間含む）です。㻌
㻌
発表者は各自コンピュータを持参し、会場に備え付けの液晶プロジェクターにより、図等をスクリーンに㻌映写して発表します。使用ソフトはパワーポイント（米国マイクロソフト社）を標準とします。㻌
㻌
画面の解像度は 㻝㻜㻞㻠×㻣㻢㻤 ピクセル（㼄㻳㻭）です。この環境下で発表データを作成ください。㻌
これより大きい画面サイズでデータを作成すると、スクリーン映写時に画面をはみ出す等の不具合が㻌起こる可能性がある旨ご理解ください。㻌
㻌
またパソコンを切替機（セレクター）に繋ぎ、発表していただきますので、以下の時間に会場前の㻌接続デスクにお越しください。㻌
㻌◇休憩時間以前（初日、㻞 日目㻦㻝㻡 時以前、㻟 日目㻦㻝㻜 時以前）に発表がある方㻌
セッション開始 㻝㻡 分前に会場前の接続デスクにお越しください。㻌
㻌◇休憩時間以降（初日、㻞 日目㻦㻝㻡 時 㻝㻜 分以降、㻟 日目㻦㻝㻜 時 㻝㻜 分以降）に発表がある方㻌
休憩時間中に会場前の接続デスクにお越しください。㻌

㻌◇ポスター発表の方へ㻌
ポスターの貼付、展示、説明・討論、撤去㻦㻌㻌㻥 月 㻞㻞 日（土）㻌
㻥 月 㻞㻟 日（日）㻌
㻥 月 㻞㻠 日（月）㻌
展示㻌㻤㻦㻟㻜㻙㻝㻢㻦㻟㻜㻌㻤㻦㻟㻜㻙㻝㻢㻦㻟㻜㻌㻤㻦㻟㻜㻙㻝㻟㻦㻝㻡㻌
奇数番号㻌㻝㻢㻦㻟㻜㻙㻝㻣㻦㻟㻜㻌㻝㻢㻦㻟㻜㻙㻝㻣㻦㻟㻜㻌㻝㻟㻦㻝㻡㻙㻝㻠㻦㻝㻡㻌
説明・討論㻌
偶数番号㻌㻝㻣㻦㻟㻜㻙㻝㻤㻦㻟㻜㻌㻝㻣㻦㻟㻜㻙㻝㻤㻦㻟㻜㻌㻝㻠㻦㻝㻡㻙㻝㻡㻦㻝㻡㻌撤去㻌
㻝㻤：㻠㻡 までに撤去㻌 㻝㻤：㻠㻡 までに撤去㻌 㻝㻡：㻟㻜 までに撤去㻌㻌㻌 㻝．㻌 ポスターは日替わりで貼り替えてください。㻌
㻌 㻞．㻌 ポスターボードの大きさは、幅 㻥㻜㻌㼏㼙、高さ 㻞㻝㻜㻌㼏㼙。貼付に必要な押しピンは会場に用意します。㻌
㻟．㻌 発表代表者の氏名には左肩に小さな○印を付けてください。㻌
㻌 㻠．㻌 撤去時間を過ぎて残ったポスターは年会事務局にて破棄しますので、ご了承ください。㻌
㻌◇ポスター発表要項㻌
ポスターは英語で作成してください。ただし、タイトル、所属、発表者名は、可能であれば日本語の併記もお願いいたします。㻌
㻌◇発表形式と演題番号（各抄録左上の番号）の見方㻌
発表には、一般口頭発表（㻿㼔㼛㼞㼠㻌 㼀㼍㼘㼗）、シンポジウム発表（㻿㼥㼙㼜㼛㼟㼕㼡㼙㻌 㼀㼍㼘㼗）、若手招待講演（㻱㼍㼞㼘㼥㻌㻾㼑㼟㼑㼍㼞㼏㼔㻌㼕㼚㻌㻮㼕㼛㼜㼔㼥㼟㼕㼏㼟㻌㻭㼣㼍㼞㼐㻌㻯㼍㼚㼐㼕㼐㼍㼠㼑㻌㻼㼞㼑㼟㼑㼚㼠㼍㼠㼕㼛㼚㼟）、ポスター発表（㻼㼛㼟㼠㼑㼞㻌㻼㼞㼑㼟㼑㼚㼠㼍㼠㼕㼛㼚）があります。㻌
㻌
一般口頭発表㻦（例）演題番号 㻞㻰㻝㻠㻞㻠㻌㻝 文字目は発表日（㻌 㻝㻦㻌㻥 月 㻞㻞 日、㻞㻌㻦㻥 月 㻞㻟 日、㻟㻦㻌㻥 月 㻞㻠 日）、続くアルファベットは会場名（㻭〜㻵 の合
計 㻥 会場）、最後の 㻠 桁の数字は発表開始時刻を表します。上の例の発表は、㻥 月 㻞㻟 日に 㻰 会場で、㻌
㻝㻠 時 㻞㻠 分に始まる発表です。㻌
㻌–(S18)–

シンポジウム発表:（例）1SA03
1 文字目は発表日（1:9 月 22 日、2:9 月 23 日）、次の S はシンポジウム発表であることを表します。
続くアルファベットは会場名、最後の 2 桁の数字は発表順を示します。
若手招待講演:（例）1YS1045
1 文字目は発表日（1:9 月 22 日）、2 文字目は Young （Scientists）、3 文字目は会場名（S）、最後の 4 桁の数字は講演開始時刻です。
ポスター発表:（例 1）1PT001、（例 2）1PS001
1 文字目は発表日（1:9 月 22 日）、2 文字目は Poster、3 文字目は会場名（T:豊田講堂、S:南館）、
最後の 3 桁の数字はパネル番号を示します。

◇ Language
English◇ Presentation equipments
Please bring your own laptop and connect it to our projector for your presentation.
We will not provide a sound output.
Attention1）

Please connect the computer to a video switcher.

Attention2）

The video output connector of presenter’s laptop should be “miniD-sub15pin （female）”.
If the connector is not this type （for example, that of Macintosh computer）, please bringan adaptor.

Attention3）

Speaker is advised to bring his/her PowerPoint file in a USB memory.

◇ To Symposium Speakers
Please come to the symposium room 15 min before the start of the symposium, and check fitness of theconnection between your PC and our projector. You can also check the connection in advance in the Sci.
bldg. B 1F 116.
◇ Instructions for Short Talks
1． Speaker is requested to tell his/her arrival to the session room attendant.
2． Speaker will make a 12 min-presentation using his/her own computer.
All session rooms will be equipped with a data projector and a lavaliere microphone.
3． PowerPoint® （Microsoft） is supposed to be used as a software.
4． Speaker will make a presentation through your computer, which must be connected to a video switcher.
Please bring your computer with you to connecting desk in front of the presentation room by following
times;◇Those who are scheduled to make presentations before the break or before 15:00 for Day1 and Day2,
before 10:00 for Day3, please bring your computer 15 minutes prior to your presentation.
◇Those who are scheduled to make presentations after the break or after 15:10 for Day1 and Day2,
after 10:10 for Day3, please bring your computer during the break time.

–(S19)–

㻌◇㼀㼛㻌㻼㼛㼟㼠㼑㼞㻌㻼㼞㼑㼟㼑㼚㼠㼑㼞㼟㻌 㻌㻌㻰㼍㼥㻌㻝㻘㻌 㻌 㻿㼑㼜㻚㻌㻞㻞㻌㻰㼍㼥㻌㻞㻘㻌 㻌 㻿㼑㼜㻚㻌㻞㻟㻌㻰㼍㼥㻌㻟㻘㻌 㻌 㻿㼑㼜㻚㻌㻞㻠㻌㻤㻦㻟㻜㻙㻝㻢㻦㻟㻜㻌㻤㻦㻟㻜㻙㻝㻢㻦㻟㻜㻌㻤㻦㻟㻜㻙㻝㻟㻦㻝㻡㻌㼛㼐㼐㻌㼚㼡㼙㼎㼑㼞㻌㻝㻢㻦㻟㻜㻙㻝㻣㻦㻟㻜㻌㻝㻢㻦㻟㻜㻙㻝㻣㻦㻟㻜㻌㻝㻟㻦㻝㻡㻙㻝㻠㻦㻝㻡㻌㼑㼢㼑㼚㻌㼚㼡㼙㼎㼑㼞㻌㻝㻣㻦㻟㻜㻙㻝㻤㻦㻟㻜㻌㻝㻣㻦㻟㻜㻙㻝㻤㻦㻟㻜㻌㻝㻠㻦㻝㻡㻙㻝㻡㻦㻝㻡㻌㻼㼛㼟㼠㼑㼞㻌㻰㼕㼟㼜㼘㼍㼥㻌㻼㼛㼟㼠㼑㼞㻌
㻼㼞㼑㼟㼑㼚㼠㼍㼠㼕㼛㼚㻌㻵㼒㻌㼥㼛㼡㻌㼔㼍㼢㼑㻌㼍㼚㼥㻌㼝㼡㼑㼟㼠㼕㼛㼚㼟㻘㻌㼜㼘㼑㼍㼟㼑㻌㼏㼛㼚㼠㼍㼏㼠㻌㼡㼟㻌㼎㼥㻌㼑㼙㼍㼕㼘㻌 （㼍㼙㼎㼟㼖㻡㻜㻬㼎㼡㼚㼟㼔㼕㻠㻚㼎㼕㼛㻚㼚㼍㼓㼛㼥㼍㻙㼡㻚㼍㼏㻚㼖㼜）㻚㻌
㻌◇㻵㼚㼟㼠㼞㼡㼏㼠㼕㼛㼚㼟㻌㼒㼛㼞㻌㻼㼛㼟㼠㼑㼞㻌㻼㼞㼑㼟㼑㼚㼠㼍㼠㼕㼛㼚㼟㻌
Posters will be mounted at the poster room day by day.
Poster board size is 90 cm in width and 210 cm in height.

◇㻼㼞㼑㼟㼑㼚㼠㼍㼠㼕㼛㼚㻌㼀㼥㼜㼑㼟㻌㼍㼚㼐㻌㻰㼑㼏㼛㼐㼕㼚㼓㻌㻼㼞㼑㼟㼑㼚㼠㼍㼠㼕㼛㼚㻌㻺㼡㼙㼎㼑㼞㼟㻌㻼㼞㼑㼟㼑㼚㼠㼍㼠㼕㼛㼚㻌㼠㼥㼜㼑㼟㻌㼍㼞㼑㻌㻿㼔㼛㼞㼠㻌㼀㼍㼘㼗㻘㻌㻿㼥㼙㼜㼛㼟㼕㼡㼙㻌㼀㼍㼘㼗㻘㻌㼅㼛㼡㼚㼓㻌㻿㼏㼕㼑㼚㼠㼕㼟㼠㼟㻌㻵㼚㼢㼕㼠㼑㼐㻌㼀㼍㼘㼗㻌㼍㼚㼐㻌㻼㼛㼟㼠㼑㼞㻚㻌㻌㻿㼔㼛㼞㼠㻌㼀㼍㼘㼗㼟㻦㻌 （㻱㼤㻚）㻌 㼍㼎㼟㼠㼞㼍㼏㼠㻌㼚㼡㼙㼎㼑㼞㻘㻌㻞㻰㻝㻠㻞㻠㻌㼀㼔㼑㻌㻝㼟㼠㻌㼘㼑㼠㼠㼑㼞㻌㼟㼠㼍㼚㼐㼟㻌㼒㼛㼞㻌㼠㼔㼑㻌㼜㼞㼑㼟㼑㼚㼠㼍㼠㼕㼛㼚㻌㼐㼍㼥㻌 （㻝㻘㻌㻞㻌㼍㼚㼐㻌㻟㻌㼒㼛㼞㻌㻿㼍㼠㻘㻌㻿㼡㼚㻌㼍㼚㼐㻌㻹㼛㼚）㻘㻌㼠㼔㼑㻌㻞㼚㼐㻌㼘㼑㼠㼠㼑㼞㻌㼒㼛㼞㻌㼠㼔㼑㻌㼚㼍㼙㼑㻌㼛㼒㻌㼟㼑㼟㼟㼕㼛㼚㻌㼞㼛㼛㼙㻌㼍㼚㼐㻌㼠㼔㼑㻌㼒㼛㼘㼘㼛㼣㼕㼚㼓㻌㻠㻌㼐㼕㼓㼕㼠㼟㻌㼒㼛㼞㻌㼠㼔㼑㻌㼟㼠㼍㼞㼠㼕㼚㼓㻌㼠㼕㼙㼑㻌㼛㼒㻌㼠㼔㼑㻌㼜㼞㼑㼟㼑㼚㼠㼍㼠㼕㼛㼚㻚㻌 㻌㻌㻿㼥㼙㼜㼛㼟㼕㼡㼙㻌㼀㼍㼘㼗㻦㻌 （㻱㼤㻚）㻌 㻝㻿㻽㻜㻟㻌㼀㼔㼑㻌㻝㼟㼠㻌㼘㼑㼠㼠㼑㼞㻌㼟㼠㼍㼚㼐㼟㻌㼒㼛㼞㻌㼠㼔㼑㻌㼜㼞㼑㼟㼑㼚㼠㼍㼠㼕㼛㼚㻌㼐㼍㼥㻌 （㻝㻌㼍㼚㼐㻌㻞㻌㼒㼛㼞㻌㻿㼍㼠㻌㼍㼚㼐㻌㻿㼡㼚）, the next ‘S’ for Symposium Talk,
㻟㼞㼐㻌㼘㼑㼠㼠㼑㼞㻌㼒㼛㼞㻌㼠㼔㼑㻌㼚㼍㼙㼑㻌㼛㼒㻌㼟㼑㼟㼟㼕㼛㼚㻌㼞㼛㼛㼙㻚㻌㻸㼍㼟㼠㻌㻞㻌㼐㼕㼓㼕㼠㼟㻌㼞㼑㼜㼞㼑㼟㼑㼚㼠㻌㼠㼔㼑㻌㼛㼞㼐㼑㼞㻌㼛㼒㻌㼠㼔㼑㻌㼠㼍㼘㼗㻌㼕㼚㻌㼑㼍㼏㼔㻌㼟㼑㼟㼟㼕㼛㼚㻚㻌 㻌㻌㻱㼍㼞㼘㼥㻌㻾㼑㼟㼑㼍㼞㼏㼔㻌㼕㼚㻌㻮㼕㼛㼜㼔㼥㼟㼕㼏㼟㻌㻭㼣㼍㼞㼐㻌㻯㼍㼚㼐㼕㼐㼍㼠㼑㻌㻼㼞㼑㼟㼑㼚㼠㼍㼠㼕㼛㼚㼟㻦㻌 （㻱㼤㻚）㻌 㻝㼅㻿㻝㻜㻠㻡㻌㼀㼔㼑㻌㻝㼟㼠㻌㼘㼑㼠㼠㼑㼞㻌㼟㼠㼍㼚㼐㼟㻌㼒㼛㼞㻌㼠㼔㼑㻌㼜㼞㼑㼟㼑㼚㼠㼍㼠㼕㼛㼚㻌㼐㼍㼥㻌 （㻿㼍㼠）㻘㻌㼠㼔㼑㻌㻞㼚㼐㻌㼘㼑㼠㼠㼑㼞㻌㼅㻌㼒㼛㼞㻌‘Young （㻿㼏㼕㼑㼚㼠㼕㼟㼠㼟）’ and the‘㻿’ for session room 㻿㻚㻌㼀㼔㼑㻌㼘㼍㼟㼠㻌㻠㻌㼐㼕㼓㼕㼠㼟㻌㼟㼠㼍㼚㼐㻌㼒㼛㼞㻌㼠㼔㼑㻌㼟㼠㼍㼞㼠㼕㼚㼓㻌㼠㼕㼙㼑㻌㼛㼒㻌㼠㼔㼑㻌㼠㼍㼘㼗㻚㻌㻌㻼㼛㼟㼠㼑㼞㻌㻼㼞㼑㼟㼑㼚㼠㼍㼠㼕㼛㼚㼟㻦㻌㻔㻱㼤㻚㻕㻌㻝㻼㼀㻜㻜㻝㻘㻌㻝㻼㻿㻜㻜㻝㻌㼀㼔㼑㻌㼒㼕㼞㼟㼠㻌㼘㼑㼠㼠㼑㼞㻌㼟㼠㼍㼚㼐㼟㻌㼒㼛㼞㻌㼠㼔㼑㻌㼜㼞㼑㼟㼑㼚㼠㼍㼠㼕㼛㼚㻌㼐㼍㼥㻌㻔㻝㻦㻌㻭㼡㼓㼡㼟㼠㻌㻞㻞㼚㼐㻕㻘㻌㼠㼔㼑㻌㼟㼑㼏㼛㼚㼐㻌㼘㼑㼠㼠㼑㼞㻌㻼㻌㼟㼠㼍㼚㼐㼟㻌㼒㼛㼞㻌㻼㼛㼟㼠㼑㼞㻌㻼㼞㼑㼟㼑㼚㼠㼍㼠㼕㼛㼚㻌㼍㼚㼐㻌㼠㼔㼑㻌㼠㼔㼕㼞㼐㻌㼘㼑㼠㼠㼑㼞㻌㼟㼠㼍㼚㼐㼟㻌㼒㼛㼞㻌㼜㼞㼑㼟㼑㼚㼠㼍㼠㼕㼛㼚㻌㼞㼛㼛㼙㼟㻌㻔㼀㻦 㼀㼛㼥㼛㼐㼍㻌㻭㼡㼐㼕㼠㼛㼞㼕㼡㼙㻘㻌㻿㻦 㻿㼏㼕㻚㻌㼟㼛㼡㼠㼔㻌㼎㼘㼐㼓㻚㻕㻚㻌 㻌㼀㼔㼑㻌㼘㼍㼟㼠㻌㻟㻌㼐㼕㼓㼕㼠㼟㻌㼚㼡㼙㼎㼑㼞㼟㻌㼟㼠㼍㼚㼐㻌㼒㼛㼞㻌㼠㼔㼑㻌㼜㼍㼚㼑㼘㻌㼚㼡㼙㼎㼑㼞㼟㻚㻌㻌

–(S20)–

㻌

㻣．㻌

日本生物物理学会第 㻡㻝 回定例総会開催通知㻌

㻌
日時㻦㻌 㻞㻜㻝㻞 年 㻥 月 㻞㻟 日（日）㻝㻞㻦㻡㻜〜㻝㻟㻦㻡㻜㻌場所㻦㻌 名古屋大学東山キャンパス㻌 理学南館㻌 㻝 階㻌 坂田・平田ホール（㻭 会場）㻌日本生物物理学会第 㻡㻝 回定例総会を開催いたします。主な議題は下記の通りです。是非ご出席ください。
都合で出席できない方は委任状（葉書、切手貼付、本誌とじこみ）を総会開始前にご送付ください。㻌
また、委任状は年会受付にも用意します。総会開始前に年会受付にご提出ください。㻌
㻌
議長㻌 年会実行委員長㻌 本間道夫㻌
㻌㻌㻌
総会議題㻌
（㻝） 報告、承認事項㻌
（会長㻌 難波啓一）㻌
平成 㻞㻟 年度決算報告ならびに監査結果報告 㻌
平成 㻞㻠 年度会計ならびに事業の中間報告、平成 㻞㻠 年度の今後の計画㻌㻌 㻌 㻌 㻌 㻌 㻌
平成 㻞㻡 年度次期会長および平成 㻞㻡・㻞㻢 年度役員選挙結果の報告㻌㻌 㻌 㻌 㻌 㻌 㻌
平成 㻞㻡 年度予算案および事業計画 㻌 㻌 㻌㻌（㻞） その他㻌
㻌
㻌
㻌

㻤．㻌
㻌

運営委員会、各種委員会の案内㻌

生物物理編集委員会㻌

㻥 月 㻞㻝 日（金）㻝㻡㻦㻜㻜〜㻝㻤㻦㻜㻜㻌

理学 㻱 館㻌 㻝㻲㻌 㻱㻝㻜㻝㻌

ホームページ編集委員会㻌

㻥 月 㻞㻝 日（金）㻝㻢㻦㻜㻜〜㻝㻤㻦㻜㻜㻌

理学南館㻌 㻟㻲㻌 㻿㻿㻟㻜㻞㻌

平成 㻞㻠 年度第 㻠 回運営委員会㻌

㻥 月 㻞㻞 日（土）㻝㻞㻦㻜㻜〜㻝㻟㻦㻜㻜㻌

理学 㻱 館㻌 㻝㻲㻌 㻱㻝㻜㻝㻌㻮㻵㻻㻼㻴㼅㻿㻵㻯㻿 編集委員会㻌

㻥 月 㻞㻞 日（土）㻝㻟㻦㻜㻜〜㻝㻠㻦㻜㻜㻌

理学南館㻌 㻟㻲㻌 㻿㻿㻟㻜㻞㻌

若手賞選考委員会㻌

㻥 月 㻞㻞 日（土）㻝㻢㻦㻟㻜〜㻝㻣㻦㻟㻜㻌

理学南館㻌 㻟㻲㻌 㻿㻿㻟㻜㻞㻌

新旧合同委員会㻌

㻥 月 㻞㻞 日（土）㻝㻤㻦㻜㻜〜㻝㻥㻦㻜㻜㻌

多元数理科学棟㻌 㻝㻲㻌㻝㻜㻥（㻱 会場）㻌男女共同参画委員会㻌

㻥 月 㻞㻟 日（日）㻝㻜㻦㻟㻜〜㻝㻞㻦㻜㻜㻌

多元数理科学棟㻌 㻞㻲㻌㻞㻜㻥㻌

若手の会会議㻌㻥 月 㻞㻟 日（日）㻝㻝㻦㻠㻡〜㻝㻞㻦㻠㻡㻌

理学南館㻌 㻟㻲㻌 㻿㻿㻟㻜㻞㻌

分野別専門委員会㻌

㻥 月 㻞㻟 日（日）㻝㻞㻦㻜㻜〜㻝㻞㻦㻠㻡㻌

理学南館㻌 㻝 階㻌 坂田・平田ホール（㻭 会場）㻌平成 㻞㻠 年度第 㻡 回運営委員会㻌

㻥 月 㻞㻠 日（月）㻝㻞㻦㻜㻜〜㻝㻟㻦㻜㻜㻌

理学 㻱 館㻌 㻝㻲㻌 㻱㻝㻜㻝㻌–(S21)–

謝 辞
本年会の開催・運営に当たり、以下の団体よりご援助いただきました。
関係者一同より御礼申し上げます。
新学術領域研究 「少数性生物学－個と多数の狭間が織りなす生命現象の探求－」
新学術領域研究 「運動超分子マシナリーが織りなす調和と多様性」
名古屋大学大学院 理学研究科
日本生物物理学会第 50 回年会
実行委員長 本間 道夫–(S22)–

㻌㻌「男女共同参画・若手問題シンポジウム」㻌
㻌「博士号を取得して多様なキャリアパスを手に入れる」㻌
㻭㼘㼠㼑㼞㼚㼍㼠㼕㼢㼑㻌㻯㼍㼞㼑㼑㼞㼟㻌㼍㼒㼠㼑㼞㻌㻼㼔㻰㻌㻯㼛㼡㼞㼟㼑㻌㻌
オーガナイザー：日本生物物理学会㻌 男女共同参画・若手問題検討委員会㻌
㻻㼞㼓㼍㼚㼕㼦㼑㼞㼟㻦㻌㻯㼛㼙㼙㼕㼠㼠㼑㼑㻌㼒㼛㼞㻌㻼㼞㼛㼙㼛㼠㼕㼚㼓㻌㻱㼝㼡㼍㼘㻌㻼㼍㼞㼠㼕㼏㼕㼜㼍㼠㼕㼛㼚㻌㼛㼒㻌㻹㼑㼚㻌㼍㼚㼐㻌㼃㼛㼙㼑㼚㻌㼍㼚㼐㻌㼒㼛㼞㻌㻱㼚㼏㼛㼡㼞㼍㼓㼕㼚㼓㻌㼅㼛㼡㼚㼓㻌㻾㼑㼟㼑㼍㼞㼏㼔㼑㼞㼟㻌㼕㼚㻌㼠㼔㼑㻌㻮㼕㼛㼜㼔㼥㼟㼕㼏㼍㼘㻌㻿㼛㼏㼕㼑㼠㼥㻌㼛㼒㻌㻶㼍㼜㼍㼚㻌㻌
日時：㻌 㻥 月 㻞㻟 日（日）㻝㻞㻦㻜㻜〜㻝㻞㻦㻡㻜（ランチョンセミナーの時間帯）㻌
場所：㻌 Ｅ会場㻌 （多元数理科学棟㻌 㻝 階㻌 㻝㻜㻥）㻌
昼食：㻌 お弁当とお茶が無料で提供されます。ただし、数に限りがあります。㻌
形式：講演会㻌㻌
講演者：当日発表。３名程度を予定㻌
㻌㻌
経済の悪化にともない、大学、研究機関および企業における新規雇用が極端に減り、若者が社会で活躍する場が非常に少なくなっています。大学で博士号を取得して研究者としてがんばっていこうと考えていたのに、思うように道を拓くことができないという話も、残念ながらよく聞くようになりました。このことで、若い方々が博士号を取得する道に進まなくなってきています。博士号取得者の減少は、長い目で見ると日本の科学技
術力の低下につながり、悪循環に陥ってしまいます。㻌
㻌 この状況を打開するために、日本社会における博士号取得者の評価を高めることも重要ですが、大学で博士号を取得したら、次は大学や研究所で研究をするものであるという固定観念を見直す必要もありそうです。
博士号を取得した後に企業で活躍されている方はかなりおられます。また、政府や大学も、博士号取得後のキャリアに関する様々な取り組みを行ってきています。㻌
㻌 そこで、生物物理学を含む理工系の分野において、博士号取得後にどのようなキャリアの可能性があるのかを、人材派遣会社から講師をお招きして紹介してもらうこととしました。その上で、博士号取得後にさまざまな経歴を歩んでこられた数名の方に、それぞれのご経歴について話していただくことを予定しております。これらのお話を聞きながら、フロアからのご意見や質問を受け、時間が許す限りディスカッションをしていきたいと考えております。特に博士前期および後期課程学生のなまの声が聞こえてくることを期待しています。㻌
㻌 老若男女を問わず、ご関心のある方々の参加をお待ちしております。㻌
㻌–(S23)–

日本生物物理学会第 50 回年会 公開講座

「生物物理学最前線」―生命の不思議を解き明かすー
生命の不思議な営みは、生体分子が集まって個性のある働きをすることによって生まれます。そうした生体分子のなかでも、とりわけタンパク質は素晴らしい働きをしており、その仕組みを明らかにすることは、生物物理学の大きな目標のひとつです。この公開講座では、タンパク質の働きを理解するための最前線の研究をわかりやすく紹介します。
タンパク質の働きを理解するための重要なステップは、タンパク質の立体的な形を知ることですが、その新しい方法として、スーパーコンピューターや電子顕微鏡、電子線を用いた方法が注目されています、本公開講座では、この新しい分
野で独創的な方法を考案し、世界をリードしてきた 2 人の研究者が講演をします。タンパク質の形を解き明かす技術の見事さとともに、タンパク質の形からわかる生命現象、生理現象の論理の見事さは、私たちを惹きつける魅力にあふれています。
日時： 2012 年（平成 24 年） 9 月 22 日（土） 13：00～14：00
場所： 名古屋大学 東山キャンパス 豊田講堂ホール（S 会場）
地下鉄名城線「名古屋大学」駅 2 番出口 徒歩すぐ
参加費 ： 無料（どなたでも自由に参加できます）。
主催：日本生物物理学会第 50 回年会 実行委員会
共催：名古屋大学理学研究科

プログラム
13:00

開会
司会：笹井 理生 教授（名古屋大学 大学院工学研究科）
講演 「スーパーコンピューターでタンパク質の形と働きを探る」
岡本 祐幸 教授（名古屋大学 大学院理学研究科）
準備中

講演 「膜タンパク質の形と働きを観る」
藤吉 好則 教授（名古屋大学 大学院創薬科学研究科）
我々が見て、考えて、行動する場合に、神経細胞などの脂質膜の内にある膜タンパク質が重要な役割を果たしている。それらの働きを深く理解するために、電子顕微鏡を用いて、立体的な形＝構造を観たいと思う。しかし、強い電子線を照射すると生物試料は一瞬で黒焦げになってしまう。しかも、電子の通り道は高い真空状態に保たなければならない。それゆえ、室温の状態で生物試料を観察しようとすると、水分が飛んで干からびてしまい、電子線による損傷も受けてしまう。これらの問題を解決するために、液体ヘリウムでマイナス 270゜C 近くまで冷却した状態で高い分解能の像を撮影できる電子顕微鏡が作られた。この様な装置を用いて、膜タンパク質の立体構造が解析されることで、それらの機能の詳細が理解できるようになった。「膜タンパク質の形と働きを観る」ことによって、ヒトの身体の機能を分子レベルから理解しようとする試みについて紹介したい。

おわりに
美宅 成樹 教授（名古屋大学 大学院工学研究科）
14:00

閉会

–(S24)–

若手招待講演

Early Research in Biophysics Award）／ Day 1 (Sep. 22, Sat.)
第 1 日目（9 月 22 日（土）9:00〜11:30
1YS

S 会場：豊田講堂ホール／ Room S:

日本生物物理学会若手奨励賞選考会
Early Research in Biophysics Award Candidate Presentations

オーガナイザー：男女共同参画・若手問題検討委員会
Organizer: The Committee of Promoting Gender Equality and Young Scientists
In 2005, the Biophysical Society of Japan has founded Early Research in Biophysics Award to recognize distinguished research work by young members of the
BSJ. In this eighth year, we received 41 highly qualified applications. After extremely competitive first round of screening based on written application forms,
the following ten applicants were selected as the "young guest speakers." For the second round of the nomination, each young speaker will be asked to make a
10-minute presentation followed by 3-minute Q&A discussion. At the end of these rounds, up to five award winners will be selected. The award winners will
be announced at the banquet in the evening of Sunday 23rd September, and the winners will deliver a short talk. We welcome all the BSJ members to attend theoral presentations on Saturday 22nd September at the Early Research in Biophysics Award Candidate Presentations and would like the members to foresee thefuture of biophysics in Japan through these speakers and their researches.

9:00

1YS0900

岩城

光宏

○岩城

光宏

1PS049生体分子モーターのストレインセンサー機構の発見とエネルギー変換への寄与の定量化
Discovery of strain-sensor mechanism in motor protein and the quantification for the energy conversion
1,2,3

2

，藤田

恵介 ，岩根2

2

敦子 ，マルクッチ ロレンツォ ，柳田

敏雄

1,2

1

2

3

( 理研・生命システム， 阪大・生命機能， ハーバー

ド・医)Mitsuhiro Iwaki1,2,3, Keisuke Fujita2, Atsuko Iwane2, Lorenzo Marcucci2, Toshio Yanagida1,2 (1QBiC, RIKEN, 2Grad. Sch. Biosci., Osaka
Univ., 3Harvard Med. Sch.)

9:15

1YS0915岡部弘基

3PS010○岡部

弘基 ，内山生細胞内における温度分布のイメージング
Imaging of temperature distribution in a living cell11

聖一 ，稲田2のりこ ，原田3

慶恵 ，船津1123高志 ( 東京大・院薬， 奈良先端大・バイオ， 京都大・iCeMs)

Kohki Okabe1, Seiichi Uchiyama1, Noriko Inada2, Yoshie Harada3, Takashi Funatsu1 (1Grad. Sch. Pharm. Sci., Univ. Tokyo,
2NAIST, 3iCeMs, Kyoto Univ.)

9:30

1YS0930

加藤英明

1PT128○加藤

英明 ，Zhang Feng ，Yizhar Ofer ，Ramakrishnan Charu ，西澤

光駆動性陽イオンチャネルであるチャネルロドプシンの結晶構造解析
Crystal Structure of a light-gated cation channel, channelrhodopsin
塚崎

2

1

1

2

智也 ，林

2

5

2

6

4

1

知宏 ，平田1

3

邦生 ，伊藤2

4

4

順平 ，相田
1

雄亮 ，

1

重彦 ，Hegemann Peter ，Maturana Andres D. ，石谷 隆一郎 ，Deisseroth Karl ，濡木 理 ( 東大・院理・生化，
3

4

5

6

スタンフォード大・生命工学， 理研・播磨， 長岡技大・生命工学， 京大・院理・化学， フンボルト大・実験物理・生物)

Hideaki E. Kato1, Feng Zhang2, Ofer Yizhar2, Charu Ramakrishnan2, Tomohiro Nishizawa1, Kunio Hirata3, Jumpei Ito4, Yusuke Aita4,
Tomoya Tsukazaki1, Shigehiko Hayashi5, Peter Hegemann6, Andres D. Maturana4, Ryuichiro Ishitani1, Karl Deisseroth2, Osamu Nureki1(1Dept. of Biophys. and Biochem., Grad. Sch. of Sci., Univ. of Tokyo, 2Dept. of Bioengineering, Stanford Univ., 3Harima Inst., Riken,
4Bioengineering Dept., Nagaoka Univ, of Tech., 5Dept. of Chem., Grad. Sch. of Sci., Kyoto Univ., 6Inst. of Biol., Experimental Biophys.,
Humboldt-Univ.)

9:45

1YS0945

島袋

勝弥

○島袋

勝弥

2PS020Ascaris 精子をもちいたアメーバ運動装置の in vitro 再構成
Simultaneous reconstitution of MSP-based protrusion and retraction in the amoeboid sperm of Ascaris
6

1,2

，野田

3

直紀 ，吉田

2,4

賢右

5

6

1

2

3

4

5

，Stewart Murray ，Roberts Thomas M. ( 宇部高専， 科技機構， MBL， 京産大， MRC，

フロリダ州立大学)

Katsuya Shimabukuro1,2, Naoki Noda3, Masasuke Yoshida2,4, Murray Stewart5, Thomas M. Roberts6 (1Ube Nat. Colg. Tech., 2ICORP, JST,
3MBL, 4Kyoto Sangyo Univ., 5MRC, 6Florida St. Univ.)

10:00

1YS1000

中村照也

1PT129○中村

照也 ，Zhao Ye

DNA ポリメラーゼηによるホスホジエステル結合形成を観察する
Watching DNA polymerase η make a phosphodiester bond
1

2,3

，山縣

1

3

2

1

2

3

ゆり子 ，Hua Yue-jin ，Yang Wei ( 熊本大・院薬， NIDDK，NIH， INAS，Zhejiang Univ.)

Teruya Nakamura1, Ye Zhao2,3, Yuriko Yamagata1, Yue-jin Hua3, Wei Yang2 (1Grad. Sch. of Pharmaceut. Sci., Kumamoto Univ., 2NIDDK,−(S25)−

NIH, 3INAS, Zhejiang Univ.)

10:15

1YS1015

永田崇

2PT190○永田

崇 ，小柳

クモにおけるピンぼけ像を用いた新規距離知覚メカニズム
A novel depth perception mechanism based on image defocus in a spider
寺北

1
2

2

光正 ，塚本1

2

2

3

寿夫 ，佐伯真二郎 ，磯野3

3

邦夫 ，七田4

4

芳則 ，徳永55

史夫 ，木下1

充代 ，蟻川

1

謙太郎 ，明久 ( 総研大・先導研， 大阪市大・院理， 東北大・院情報科学， 京都大・院理， 大阪大・院理)Takashi Nagata1, Mitsumasa Koyanagi2, Hisao Tsukamoto2, Shinjiro Saeki3, Kunio Isono3, Yoshinori Shichida4, Fumio Tokunaga5,
Michiyo Kinoshita1, Kentaro Arikawa1, Akihisa Terakita2 (1Dept Evol Stud Biol Sys, Sokendai-Hayama, 2Grad. Sch. Sci., Osaka City
Univ., 3Grad. Sch. Information Sci., Tohoku Univ., 4Grad. Sch. Sci., Kyoto Univ., 5Grad. Sch. Sci., Osaka Univ.)

10:30

1YS1030

服部素之

1PT103○服部

素之 ，Gouaux Eric

P2X 受容体の ATP 認識機構およびチャネル活性化機構
Mechanism of ATP binding and channel activation in P2X receptors
1

1,2

1

2

( オレゴン健康科学大学ボラム研究所， ハワード・ヒューズ医学研究所)

Motoyuki Hattori1, Eric Gouaux1,2 (1Vollum Institute, Oregon Health & Science University, 2Howard Hughes Medical Institute)

10:45

1YS1045

濱田勉

3PT153○濱田勉，森田

脂質膜へテロ界面はナノ物質をサイズ依存的に識別する
Size-dependent selective localization of nano-particles on heterogeneous membrane surfaces
雅宗，宮川真紀代，高木昌宏 (北陸先端科学技術大学院大学マテリアルサイエンス研究科)

Tsutomu Hamada, Masamune Morita, Makiyo Miyakawa, Masahiro Takagi (Japan Adv. Inst. of Sci. and Tech.)

11:00

1YS1100

原田

隆平

○原田

隆平 ，杉田1PT201

細胞環境における蛋白質の構造安定性とダイナミクス
Protein stability and dynamics under cellular environments
1

有治

1,2,3

3,4

，Michael Feig

1

2

3

州立大)

Ryuhei Harada1, Yuji Sugita1,2,3, Michael Feig3,4 (1RIKEN AICS, 2RIKEN ASI, 3RIKEN QBiC, 4Michgan state university)

11:15

1YS1115

渡邉

力也

○渡邉

力也，田端

1PS033

FoF1-ATP 合成酵素のプロトン駆動力による回転運動の直接観察
Direct observation of H+-driven rotation of FoF1-ATP synthase
和仁，飯野

亮太，野地

4

( 理研 計算科学研究機構, 理研 基幹研究所， 理研 生命システム研究センター， ミシガン

博行 (東京大学大学院工学系研究科応用化学専攻)

Rikiya Watanabe, Kazuhito V. Tabata, Ryota Iino, Hiroyuki Noji (School of Engineering, The University of Tokyo)（アイウエオ順）−(S26)−

㩷㩷䇸ᣣᧄ↢‛‛ℂቇળ㩷 ╙ 㪈 ࿁ 㪙㪠㪦㪧㪟㪰㪪㪠㪚㪪 ⺰ᢥ⾨ฃ⾨⻠Ṷ䇹㩷㪫㪿㪼㩷㪈㫊㫋㩷㪘㫎㪸㫉㪻㩷㪪㪼㫄㫀㫅㪸㫉㩷㪽㫆㫉㩷㫆㫌㫋㫊㫋㪸㫅㪻㫀㫅㪾㩷㪙㪠㪦㪧㪟㪰㪪㪠㪚㪪㩷㫇㪸㫇㪼㫉㩷㩷䉥䊷䉧䊅䉟䉱䊷䋺ᣣᧄ↢‛‛ℂቇળ 㪙㪠㪦㪧㪟㪰㪪㪠㪚㪪 ⺰ᢥ⾨ㆬ⠨ᆔຬળ㩷㪦㫉㪾㪸㫅㫀㫑㪼㫉㫊㪑㩷㪘㫎㪸㫉㪻㩷㪺㫆㫄㫄㫀㫋㫋㪼㪼㩷㪽㫆㫉㩷㫆㫌㫋㫊㫋㪸㫅㪻㫀㫅㪾㩷㪙㪠㪦㪧㪟㪰㪪㪠㪚㪪㩷㫇㪸㫇㪼㫉㩷㩷ᣣᤨ䋺䋹᦬䋲䋲ᣣ䋨࿯䋩㪈㪈䋺㪊㪇ޯ㪈㪈䋺㪌㪌㩷 㩷႐ᚲ䋺㪪 ળ႐䋨⼾↰⻠ၴ䊖䊷䊦䋩㩷 㩷ᒻᑼ䋺⻠Ṷળ㩷㩷㩷╙ 㪈 ࿁ 㪙㪠㪦㪧㪟㪰㪪㪠㪚㪪 ⺰ᢥ⾨ฃ⾨⠪㩷ฎỈ㩷 ജ㩷 㪚㪿㫀㫂㪸㫉㪸㩷㪝㫌㫉㫌㫊㪸㫎㪸㩷
䋨ℂൻቇ⎇ⓥᚲ䊶↢๮䉲䉴䊁䊛⎇ⓥ䉶䊮䉺䊷㩷 㪨㫌㪸㫅㫋㫀㫋㪸㫋㫀㫍㪼㩷㪙㫀㫆㫃㫆㪾㫐㩷㪚㪼㫅㫋㪼㫉㪃㩷㪩㪠㪢㪜㪥䋩㩷㩷⥄Ꮖⶄ⵾䈜䉎⚦⢩ౝ䉻䉟䊅䊚䉪䉴䈮䈍䈔䉎᥉ㆉ⛔⸘ೣ㩷㪬㫅㫀㫍㪼㫉㫊㪸㫃㩷㫊㫋㪸㫋㫀㫊㫋㫀㪺㪸㫃㩷㫃㪸㫎㫊㩷㫀㫅㩷㫉㪼㫇㫃㫀㪺㪸㫋㫀㫅㪾㩷㪺㪼㫃㫃㫌㫃㪸㫉㩷㪻㫐㫅㪸㫄㫀㪺㫊㩷㩷㩷㪠㫅㩷㪸㩷㪺㪼㫃㫃㪃㩷㪸㫅㩷㪿㫌㪾㪼㩷㫅㫌㫄㪹㪼㫉㩷㫆㪽㩷㫆㫉㪾㪸㫅㫀㫑㪼㪻㩷㪺㪿㪼㫄㫀㪺㪸㫃㩷㫉㪼㪸㪺㫋㫀㫆㫅㫊㩷㪸㫉㪼㩷㫉㪼㫈㫌㫀㫉㪼㪻㩷㫋㫆㩷㫄㪸㫀㫅㫋㪸㫀㫅㩷㫀㫋㫊㩷㫃㫀㫍㫀㫅㪾㩷㫊㫋㪸㫋㪼㪅㩷㪘㫃㫋㪿㫆㫌㪾㪿㩷㪸㫅㪸㫃㫐㫊㫀㫊㩷㫆㪽㩷㪻㪼㫋㪸㫀㫃㪼㪻㩷㪺㪼㫃㫃㫌㫃㪸㫉㩷㫇㫉㫆㪺㪼㫊㫊㪼㫊㩷㫀㫊㩷㫀㫄㫇㫆㫉㫋㪸㫅㫋㩷㪽㫆㫉㩷㪸㩷㪺㫆㫄㫇㫃㪼㫋㪼㩷㪻㪼㫊㪺㫉㫀㫇㫋㫀㫆㫅㩷㫆㪽㩷㪺㪼㫃㫃㫌㫃㪸㫉㩷㪹㪼㪿㪸㫍㫀㫆㫉㪃㩷㫀㫋㩷㫀㫊㩷㪸㫃㫊㫆㩷㫅㪼㪺㪼㫊㫊㪸㫉㫐㩷㫋㫆㩷㫊㪼㪸㫉㪺㪿㩷㪽㫆㫉㩷㫌㫅㫀㫍㪼㫉㫊㪸㫃㩷㫃㪸㫎㫊㩷㫎㫀㫋㪿㩷㫉㪼㪾㪸㫉㪻㩷㫋㫆㩷㫋㪿㪼㩷㫀㫅㫋㫉㪸㪺㪼㫃㫃㫌㫃㪸㫉㩷㫉㪼㪸㪺㫋㫀㫆㫅㫊㩷㪺㫆㫄㫄㫆㫅㩷㫋㫆㩷㪸㫃㫃㩷㫃㫀㫍㫀㫅㪾㩷㫊㫐㫊㫋㪼㫄㫊㩷㪸㫅㪻㩷㫋㪿㪼㫅㩷㫋㫆㩷㫌㫅㫉㪸㫍㪼㫃㩷㫋㪿㪼㩷㫃㫆㪾㫀㪺㩷㫆㪽㩷㫃㫀㪽㪼㩷㫃㪼㪸㪻㫀㫅㪾㩷㫋㫆㩷㫊㫌㪺㪿㩷㫌㫅㫀㫍㪼㫉㫊㪸㫃㩷㪽㪼㪸㫋㫌㫉㪼㫊㪅㩷㪠㫅㩷㫋㪿㫀㫊㩷㫊㫋㫌㪻㫐㪃㩷㫎㪼㩷㪽㫆㫌㫅㪻㩷㫋㫎㫆㩷㪽㫌㫅㪻㪸㫄㪼㫅㫋㪸㫃㩷㫃㪸㫎㫊㩷㪺㫆㫅㪺㪼㫉㫅㫀㫅㪾㩷㪽㫃㫌㪺㫋㫌㪸㫋㫀㫅㪾㩷㪺㪼㫃㫃㫌㫃㪸㫉㩷㪻㫐㫅㪸㫄㫀㪺㫊㩷㫎㫀㫋㪿㩷㫉㪼㪺㫌㫉㫊㫀㫍㪼㩷㪾㫉㫆㫎㫋㪿㩷㪲㪈㪴㪅㩷㪝㫀㫉㫊㫋㫃㫐㪃㩷㫋㪿㪼㩷㪺㪿㪼㫄㫀㪺㪸㫃㩷㪸㪹㫌㫅㪻㪸㫅㪺㪼㫊㩷㫄㪼㪸㫊㫌㫉㪼㪻㩷㫆㫍㪼㫉㩷㫄㪸㫅㫐㩷㪺㪼㫃㫃㫊㩷㫎㪼㫉㪼㩷㪽㫆㫌㫅㪻㩷㫋㫆㩷㫆㪹㪼㫐㩷㪸㩷㫃㫆㪾㪄㫅㫆㫉㫄㪸㫃㩷㪻㫀㫊㫋㫉㫀㪹㫌㫋㫀㫆㫅㪅㩷㪪㪼㪺㫆㫅㪻㫃㫐㪃㩷㫋㪿㪼㩷㫉㪼㫃㪸㫋㫀㫆㫅㫊㪿㫀㫇㩷㪹㪼㫋㫎㪼㪼㫅㩷㫋㪿㪼㩷㪸㫍㪼㫉㪸㪾㪼㩷㪸㫅㪻㩷㫊㫋㪸㫅㪻㪸㫉㪻㩷㪻㪼㫍㫀㪸㫋㫀㫆㫅㩷㫆㪽㩷㫋㪿㪼㩷㪸㪹㫌㫅㪻㪸㫅㪺㪼㫊㩷㫎㪸㫊㩷㪽㫆㫌㫅㪻㩷㫋㫆㩷㪹㪼㩷㫃㫀㫅㪼㪸㫉㪅㩷㪫㪿㪼㩷㫌㪹㫀㫈㫌㫀㫋㫐㩷㫆㪽㩷㫋㪿㪼㫊㪼㩷㫃㪸㫎㫊㩷㫎㪸㫊㩷㪼㫏㫇㫃㫆㫉㪼㪻㩷㪹㫆㫋㪿㩷㫋㪿㪼㫆㫉㪼㫋㫀㪺㪸㫃㫃㫐㩷㪸㫅㪻㩷㪼㫏㫇㪼㫉㫀㫄㪼㫅㫋㪸㫃㫃㫐㪅㩷㪬㫊㫀㫅㪾㩷㪸㫅㩷㪸㪹㫊㫋㫉㪸㪺㫋㩷㫄㫆㪻㪼㫃㩷㫎㫀㫋㪿㩷㪸㩷㪺㪸㫋㪸㫃㫐㫋㫀㪺㩷 㫉㪼㪸㪺㫋㫀㫆㫅㩷 㫅㪼㫋㫎㫆㫉㫂㪃㩷 㫋㪿㪼㩷 㫃㪸㫎㫊㩷 㫎㪼㫉㪼㩷 㫊㪿㫆㫎㫅㩷 㫋㫆㩷 㪼㫏㫀㫊㫋㩷 㫅㪼㪸㫉㩷 㪸㩷 㪺㫉㫀㫋㫀㪺㪸㫃㩷 㫊㫋㪸㫋㪼㩷 㫎㫀㫋㪿㩷 㪼㪽㪽㫀㪺㫀㪼㫅㫋㩷 㫊㪼㫃㪽㪄㫉㪼㫇㫉㫆㪻㫌㪺㫋㫀㫆㫅㪅㩷㪘㪻㪻㫀㫋㫀㫆㫅㪸㫃㫃㫐㪃㩷㪹㫐㩷㫄㪼㪸㫊㫌㫉㫀㫅㪾㩷㪻㫀㫊㫋㫉㫀㪹㫌㫋㫀㫆㫅㫊㩷㫆㪽㩷㪽㫃㫌㫆㫉㪼㫊㪺㪼㫅㫋㩷㫇㫉㫆㫋㪼㫀㫅㫊㩷㫀㫅㩷㪹㪸㪺㫋㪼㫉㫀㪸㩷㪺㪼㫃㫃㫊㪃㩷㫋㪿㪼㩷㫌㪹㫀㫈㫌㫀㫋㫐㩷㫆㪽㩷㫃㫆㪾㪄㫅㫆㫉㫄㪸㫃㩷㪻㫀㫊㫋㫉㫀㪹㫌㫋㫀㫆㫅㩷㫆㪽㩷㫇㫉㫆㫋㪼㫀㫅㩷㪸㪹㫌㫅㪻㪸㫅㪺㪼㫊㩷㫎㪸㫊㩷㪺㫆㫅㪽㫀㫉㫄㪼㪻㪅㩷㪪㪼㫍㪼㫉㪸㫃㩷㫊㫋㫌㪻㫀㪼㫊㩷㪽㫆㫃㫃㫆㫎㫀㫅㪾㩷㫋㪿㪼㩷㪸㪹㫆㫍㪼㩷㪽㫀㫅㪻㫀㫅㪾㫊㪃㩷㫀㫅㪺㫃㫌㪻㫀㫅㪾㩷㪸㫅㪸㫃㫐㫊㫀㫊㩷㫆㪽㩷㫋㪿㪼㩷㪼㪽㪽㪼㪺㫋㩷㫆㪽㩷㫊㫌㪺㪿㩷㪽㫃㫌㪺㫋㫌㪸㫋㫀㫅㪾㩷㪼㫏㫇㫉㪼㫊㫊㫀㫆㫅㩷㪻㫐㫅㪸㫄㫀㪺㫊㩷㫆㫅㩷㪸㪻㪸㫇㫋㪸㫋㫀㫆㫅㩷㪲㪉㪴㩷㪸㫅㪻㩷㪼㫍㫆㫃㫌㫋㫀㫆㫅㩷㪲㪊㪴㪃㩷㪸㫅㪻㩷㫋㪿㪼㩷㫊㪼㫃㪽㪄㫆㫉㪾㪸㫅㫀㫑㫀㫅㪾㩷㫄㪼㪺㪿㪸㫅㫀㫊㫄㩷㫆㪽㩷㫋㪿㪼㩷㪺㫉㫀㫋㫀㪺㪸㫃㩷㫊㫋㪸㫋㪼㩷㫎㫀㫋㪿㩷㪸㩷㫃㫆㪾㪄㫅㫆㫉㫄㪸㫃㩷㪻㫀㫊㫋㫉㫀㪹㫌㫋㫀㫆㫅㩷㫆㪽㩷㪺㪿㪼㫄㫀㪺㪸㫃㩷㪸㪹㫌㫅㪻㪸㫅㪺㪼㩷㪲㪋㪴㩷㫎㫀㫃㫃㩷㪸㫃㫊㫆㩷㪹㪼㩷㫇㫉㪼㫊㪼㫅㫋㪼㪻㪅㩷㩷㪲㪈㪴㩷㪝㫌㫉㫌㫊㪸㫎㪸㩷㪼㫋㩷㪸㫃㪅㪃㩷㪙㫀㫆㫇㪿㫐㫊㫀㪺㫊㩷㪈㪃㩷㪉㪌–㪊㪈㩷㩿㪉㪇㪇㪌㪀㩷㪲㪉㪴㩷㪝㫌㫉㫌㫊㪸㫎㪸㩷㪸㫅㪻㩷㪢㪸㫅㪼㫂㫆㪃㩷㪧㪣㫆㪪㩷㪚㫆㫄㫇㪅㩷㪙㫀㫆㫃㪅㩷㪋㩿㪈㪀㪃㩷㪼㪊㩷㩿㪉㪇㪇㪏㪀㩷㪲㪊㪴㩷㪢㪸㫅㪼㫂㫆㩷㪸㫅㪻㩷㪝㫌㫉㫌㫊㪸㫎㪸㪃㩷㪡㫆㫌㫉㪅㩷㪫㪿㪼㫆㫉㪅㩷㪙㫀㫆㫃㪅㩷㪉㪋㪇㩿㪈㪀㪃㩷㪎㪏㪄㪏㪍㩷㩿㪉㪇㪇㪍㪀㩷㪲㪋㪴㩷㪝㫌㫉㫌㫊㪸㫎㪸㩷㪸㫅㪻㩷㪢㪸㫅㪼㫂㫆㪃㩷㪧㪿㫐㫊㪅㩷㪩㪼㫍㪅㩷㪣㪼㫋㫋㪅㩷㪈㪇㪏㪃㩷㪉㪇㪏㪈㪇㪊㩷㩿㪉㪇㪈㪉㪀㩷

㩷

(S27)

シンポジウムSymposium

第 1 日目（9/22（土））／ Day1(Sep. 22, Sat.)
9:00~11:30

A 会場：理学南館 1 階 坂田・平田ホール／ Room A: Sci. south bldg. 1F Sakata Hirata Hall

新学術領域研究「少数性生物学−個と多数の狭間が織りなす生命現象の探求−」共催

1SA

1 分子生物学と生化学の狭間に潜むナノシステム動作力学の理解を目指して
Bridging single molecule biology and biochemistry to understand operation principles
of bio-nanosystems

オーガナイザー：石島 秋彦 (東北大), 永井 健治 (阪大)Organizer: Akihiko Ishijima (Tohoku Univ.), Takeharu Nagai (Osaka Univ.)
Cooperative function/behavior in biological nanosystems consisting of small number of molecular elements is one of the most important aspects in biologicalphenomena. Although so many studies in terms of biochemistry and single molecule biology, which deal with Avogadro’s number of molecules and single
molecule, respectively, have been reported, there is almost no report showing elementary process of cooperative function/behavior among small number ofmolecules in living cells. In the symposium, we will focus on the methodology and biological phenomena to approach this issue and will discuss the prospect of
biological nanosystems research.

はじめに永井 健治 (阪大・産研)

Takeharu Nagai (ISIR, Osaka Univ.)

1SA-01

細胞内反応ネットワークと「少数性」問題〜理論と計算によるアプローチ
Cracking Reaction Networks Involving "Minorities" in the Cell: Theoretical and Computational Approaches
冨樫 祐一 (神戸大・院システム)

Yuichi Togashi (Grad. Sch. Sys. Informat., Kobe Univ.)

1SA-02

1 分子デジタル ELISA
Single-Molecule Digital ELISA
野地 博行 (東大工・応用化学)

Hiroyuki Noji (Applied Chem. U-Tokyo)

1SA-03遺伝子にコードされた発光型プローブ
Genetically encoded luminescent probes
永井 健治 (阪大・産研, JST・さきがけ)

Takeharu Nagai1,2 (1ISIR, Osaka Univ., 2PRESTO, JST)

1SA-04

Coordinated reversal of flagellar motors on a single Escherichia coli cell
石島 秋彦 (東北大学多元物質科学研究所)

Akihiko Ishijima (Tohoku University)

1SA-05ヒトゲノム DNA の収納とそのダイナミクス
Human genome organization and dynamics

前島 一博 (国立遺伝学研究所構造遺伝学研究センター生体高分子研究室)

Kazuhiro Maeshima (Structural Biology Center, National Institute of Genetics)

1SA-06哺乳類概日時計のシステム生物学・合成生物学
Systems and Synthetic Biology of Biological Timings
上田 泰己 (理化学研究所

発生・再生科学総合研究センター, 理化学研究所

生命システム研究センター)

Hiroki Ueda1,2 (1RIKEN, CDB, 2RIKEN, QBiC)

1SA-07

細菌べん毛形成の分子機構とその制御
Molecular mechanism of bacterial flagellar construction and its regulation
今田 勝巳 (阪大・院理・高分子)

Katsumi Imada1, Tohru Minamino2 (1Dept. Macromol. Sci., Grad. Sch. Sci., Osaka Univ., 2Grad. Sch. Frontier BioSci.,
Osaka Univ.)−(S28)−

9:00~11:30
1SB

B 会場：多元数理科学棟 5 階 509 ／ Room B: Mathematics bldg. 5F 509

身体ー細胞ダイナミクス連関：やわらかさの階層と連携
Body-cell dynamic linkage: softness, flexibility, fluctuation and controllability

オーガナイザー：跡見 順子 (東大), 竹森 重 (東京慈恵医大)
Organizer: Yoriko Atomi (Univ. of Tokyo), Shigeru Takemori (Jikei Univ.)
After 3.11, we all need to acts with sense of responsibility and re-think the science. The aim of this symposium is to explore an innovative and sustainablebiophysical research area based on the platform of human body and mind flexibility and free will inspired by Fumio-Osawa’s finding that biological macro
molecules such as F-actin in muscle is more flexible when its in action. Unique ideas to linking with flexibility such as exercise science, protein and DNA
structural dynamis, extracellular matrix and also control of soft but unstable body under the gravity will be presented.

身体―細胞ダイナミクス連関：やわらかさの階層と連携
跡見 順子 (東京大学アイソトープ総合センター)

Yoriko Atomi (Radioisotope Center The University of Tokyo)

1SB-01

DNA の硬さと柔らかさ：高次構造転移が生み出す時空間秩序
Synergy between Stiffness and Softness on DNA: Spatiotemporal Order Organized through the Higher-Order
Structural Transition of DNA
武仲 能子 (産総研・ナノシステム)

Yoshiko Takenaka (Nanosystem Research Inst., AIST)

1SB-02

Proteins as Mechano-Chemical Transducer
赤坂 一之 (近畿大・先端研・ 高圧センター)

Kazuyuki Akasaka (Inst. Adv. Tech, Kinki UNIV.)

1SB-03

身体―細胞能動 / 受動メカノケミカル連携：身体に生きる細胞のニッチとしての柔らかな細胞外基質環境が支配するやわらかさの階層と連携
Body-cell dynamic mechano-mechanical linkage: softness, flexibility, fluctuation and controllability derived from
ECM environment as “niche for cells in our body”
跡見 順子 (東京大学アイソトープ総合センター)

Yoriko Atomi1, Miho Shimizu2, Eri Fujita2, Tomoaki Atomi3, Noboru Hirose3, Katsuya Hasegawa4 (1Radioisotope Center
The University of Tokyo, 2Dept. of Mechano-Informatics, Univ. of Tokyo, 3Teikyo University of Science, 4JAXA)

1SB-04

多分節立位を可能にした身体の冗長性：柔らかさの制御と破綻
Flexibility of human body with multi-segmental structure enabling bipedal standing
跡見 友章 (帝京科学大学, 首都大学東京大学院)

Tomoaki Atomi1,2, Noboru Hirose1, Miho Shimizu3, Yoriko Atomi3 (1Teikyo Univ. of Sci., 2Grad. Sch., Univ. Tokyo
Metropolitan, 3Univ. of Tokyo)

1SB-05

人工制御した細胞基盤の柔らかさと細胞応答
Softness of the artificial cell niche and its active interplay of forces
清水 美穂 (東京大学大学院情報理工・知能機械)

Miho Shimizu1, Yuki Katsurada2, Toshiyuki Watanabe2, Eri Fujita1, Tomoaki Atomi3, Noboru Hirose3, Katsuya Hasegawa4,
Yoriko Atomi5 (1Dept. of Mechano-Informatics, Univ. of Tokyo, 2Tokyo Univ. of Agri. & Tech., 3Teikyo University of
Science, 4JAXA, 5Radioisotope Center The University of Tokyo)

1SB-06

培養基盤の硬さとの整合性が心筋拍動パターンを最適化する
Rigidity Matching between Cells and the Extracellular Matrix Leads to the Stabilization of Cardiac Conduction
Marcel Hoerning (神戸研究所 発生・再生科学総合研究センター フィジカルバイオロジー研究ユニット)

Marcel Hoerning (RIKEN, Center for Developmental Biology, Physical Biology Unit)

1SB-07

アクチン繊維のゆらぎを基盤とした生理機能
Physiological functions of an actin filament based on its fluctuation
本多 元 (長岡技術科学大学・生物系)

Hajime Honda (Dep. Bioeng. Nagaoka Uni. Tech.)

1SB-08

生命の神秘に迫る多次元アプローチ
Multidimensional Approaches to the Secrets of Life
竹森 重 (慈恵医大・分子生理)

Shigeru Takemori (Jikei Univ. Sch. Med.)−(S29)−

9:00~11:30
1SC

C 会場：多元数理科学棟 4 階 409 ／ Room C: Mathematics bldg. 4F 409

生物学における数学的手法の最前線
Frontiers in mathematical methods in biology

オーガナイザー：宇沢 達 (名大), 大平 徹 (名大)Organizer: Tohru Uzawa (Nagoya Univ.), Toru Ohira (Nagoya Univ.)
In these lectures we explore frontiers of mathematical methods in biology. Phenomenon of varied time/space-scale will be examined, ranging from variations inenzyme concentrations, to bird-flocking.

1SC-01特異極限解析：生物システムに現れるパターンを捉える
Singular limit analysis: Understanding of biological pattern formation
三村 泰昌 (明治大学大学院先端数理科学研究科)

Yasumasa Mimura (Graduate School of Advanced Mathematical Sciences, Meiji University)

1SC-02鳥の群れの集団動力学
Collective dynamics of flocking

早川 美徳 (東北大学教育情報基盤センター)

Yoshinori Hayakawa (Center for Information Technology in Education)

1SC-03バクテリアのコロニー形成―細胞の集団運動―
Colony Formation in Bacteria -Collective Movement of Cells松下 貢 (明治大学先端数理科学インスティテュート)

Mitsugu Matsushita (Meiji Institute for Advanced Study of Mathematical Sciences (MIMS), Meiji University)

1SC-04

生命ネットワークにおける動的ロバスト性の数理的解析
Mathematical analysis of dynamical robustness in biological networks
田中 剛平 (東京大学 生産技術研究所, 東京大学 情報理工学系研究科)

Gouhei Tanaka1,2, Kai Morino2, Kazuyuki Aihara1,2 (1Institute of Industrial Science, The University of Tokyo, 2Graduate
School of Information Science and Technology, The University of Tokyo)

1SC-05

自律的酵素量制御による時間スケール調整；ホメオスタシスと記憶
Homeostasis and memory by autonomous regulation of time-scales through enzyme abundances
金子 邦彦 (東大総合文化)

Kunihiko Kaneko (University of Tokyo, Center for Complex-Systems Biology)
9:00~11:30
1SD

D 会場：多元数理科学棟 3 階 309 ／ Room D: Mathematics bldg. 3F 309

核内分子動態から観る遺伝子発現機構
Molecular Dynamics in the Nucleus and Gene Regulation

オーガナイザー：河野 秀俊 (日本原子力機構), 寺川 剛 (京大)
Organizer: Hidetoshi Kono (JAEA), Tsuyoshi Terakawa (Kyoto Univ.)
Advances in single molecule measurements and molecular imaging techniques enable us to directly observe molecular dynamics in the nucleus. The recent
increase of computational power, which is demonstrated by the K super computer, now allows us to simulate behaviors of single molecules as well as complexes
at different spatial and temporal resolutions. In this symposium, we will discuss how such molecular phenomena observed by experiment and simulation areassociated with gene regulation.

1SD-01

Characterization of Protein-DNA complexes dynamics related to Chromatin structure regulation using SingleMolecule Techniques
韓 龍雲 (京都大学物質̶細胞統合システム拠点)

Yong-Woon Han, Yoshie Harada (iCeMS, Kyoto University)

1SD-02The mechanism of nuclear protein searching on DNA: Coarse-Grained simulation study
寺川 剛 (京都大学大学院)

Tsuyoshi Terakawa (Grad. Sch. Sci., Univ. Kyoto)

1SD-03Differences in dissociation free-energy profiles between cognite and non-cognite protein-DNA complexes
米谷 佳晃 (日本原子力研究開発機構)−(S30)−

Yoshiteru Yonetani, Hidetoshi Kono (Japan Atomic Energy Agency)

1SD-04線虫初期胚における染色体ダイナミクスの定量的解析
Quantitative analyses of chromosome dynamics in C. elegans early embryos
菅原 武志 (国立遺伝学研究所)

Takeshi Sugawara, Ritsuko Arai, Akatsuki Kimura (National Institute of Genetics)

1SD-05蛍光相関分光法を用いた細胞内グルココルチコイド受容体の動態解析
Inside view of molecular dynamics of Glucocorticoid Receptor by using Fluorescence Cross Correlation Spectroscopy
in living cell
金城 政孝 (北大 先端生命)

Masataka Kinjo (Faculty of Advanced Life Science, Hokkaido University)
9:00~11:30
1SE

E 会場：多元数理科学棟 1 階 109 ／ Room E: Mathematics bldg. 1F 109

コンフォメーション動力学：フォールディングから機能まで
Conformational dynamics: Folding to function

オーガナイザー：高田 彰二 (京大), 笹井 理生 (名大)Organizer: Shoji Takada (Kyoto Univ.), Masaki Sasai (Nagoya Univ.)
Proteins, and more generally biomolecules, are intrinsically flexible and change their conformations in multiscales both in time and space. In this symposium, we
explore various recent studies on flexible conformational dynamics both in experiments and theories. We discuss recent studies on protein folding, molecular
recognition, allosteric conformational change upon some reactions, and conformation-dependent aggregation.

1SE-01

蛋白質構造の整合性原理とその拡張
Consistency principle of protein conformation and its extension
笹井 理生 (名古屋大学, 岡崎統合バイオサイエンスセンター, KIAS)

Masaki Sasai1,2,3 (1Nagoya University, 2Okazaki Institute for Integrative Bioscience, 3Korea Institute for Advanced Study)

1SE-02

Dynamic mechanism for the transcription when responses to activators

Wei Wang (National Lab. Solid State Microstructure and Dept. Phys., Nanjing Univ.)

Yaolai Wang, Feng Liu, Wei Wang (National Lab. Solid State Microstructure and Dept. Phys., Nanjing Univ.)

1SE-03

保存アミノ酸に着目したジンクフィンガーの構造と分子認識
New Functions of Zinc Fingers Revealed by Substitution of Conserved Residues
今西 未来 (京大・化研)

Miki Imanishi (ICR, Kyoto Univ.)

1SE-04

ドメインスワッピングによるシトクロム c の多量化
Oligomerization of cytochrome c by domain swapping
廣田 俊 (奈良先端科学技術大学院大学物質創成科学研究科)

Shun Hirota (Grad. Sch. Mat. Sci., Nara Inst. Sci. Tech.)

1SE-05

マルチドメインタンパク質のフォールディングとコンフォメーション動力学
Folding and conformational dynamics of multi-domain proteins
高田 彰二 (京大理生物物理)

Shoji Takada (Dept. Biophys, Kyoto Univ.)
9:00~11:30
1SF

F 会場：理学 B 館 5 階 501 ／ Room F: Sci. bldg. B 5F 501

生物の動きと変形への理論的アプローチ
Theoretical Approaches to Biological Motion and Deformation

オーガナイザー：石原 秀至 (東大), 澤井 哲 (東大)Organizer: Shuji Ishihara (Univ. of Tokyo), Satoshi Sawai (Univ. of Tokyo)
Motion and deformation observed in living cells emerge from their intrinsic active nature and interaction with the environment. Are there physical principles that
could integrate and unify these rich and intriguing phenomena? One of key physical approaches is the framework of active matters that addresses cooperativity of
motions at various length and timescales. The symposium will highlight recent progress in this direction with an emphasis on physical and theoretical approaches.

−(S31)−

1SF-01細胞の変形と運動：理論と実験の挑戦
Deformation and motion of cell: theoretical and experimental challenges
佐野 雅己 (東大院理)Masaki Sano (Grad. Sch. Sci. U. Tokyo)

1SF-02

マウスの左右決定の過程に見られるノード繊毛の回転運動の同調
Synchronization of rotaional movement in mouse node cilia during left-right determination
高松 敦子 (早大・電気・情報生命)

Atsuko Takamatsu1, Kyosuke Shinohara2, Takuji Ishikawa3, Hiroshi Hamada2 (1Dept. Elec. Eng. and Biosci., Waseda
Univ., 2Grad. Sch. Frontier Biosci., Osaka Univ., 3Dept. Bioeng. and Robotics, Tohoku Univ.)

1SF-03鞭毛や繊毛の流体力学相互作用による同期：ミニマルアプローチ
Synchronization of flagella and cilia by hydrodynamic interactions: minimal approach
内田 就也 (東北大・理物理)

Nariya Uchida1, Ramin Golestanian2 (1Dept. Phys., Tohoku Univ., 2Cent. Theor. Phys., Univ. of Oxford)

1SF-04興奮性化学進行波に駆動される遊走細胞の形態ダイナミクスの数理モデル
Modeling morphological dynamics of migrating cells governed by self-organized excitable waves
石原 秀至 (東大・院総合文化)

Shuji Ishihara1, Daisuke Taniguchi1, Satoshi Sawai1,2 (1Grad. Sch. Arts & Sciences, Univ. Tokyo, 2JST PRESTO)

1SF-05

細胞の力学と、人工システムにおける自発運動
Mechanics of gels and spontaneous motion of droplets as biologically-motivated systems
義永 那津人 (東北大 WPI-AIMR)

Natsuhiko Yoshinaga (WPI-AIMR, Tohoku Univ.)

1SF-06這行による移動運動の理解に向けてTowards understanding the locomotion of animals by limbless crawling
小林 亮 (広島大学, 科学技術振興機構, CREST)

Ryo Kobayashi1,4, Toshiya Kazama1,4, Kentaro Ito1,4, Yoshimi Tanaka2, Toshiyuki Nakagaki3,4 (1Hiroshima University,
2Yokohama National University, 3Future University Hakodate, 4JST, CREST)
9:00~11:30
1SH

H 会場：理学 C 館 5 階 517 ／ Room H: Sci. bldg. C 5F 517

レチナール蛋白質と光遺伝学
Retinal proteins and optgenetics

オーガナイザー：神山 勉 (名大), 村上 緑 (名大)Organizer: Tsutomu Kouyama (Nagoya Univ.), Midori Murakami (Nagoya Univ.)
Optogenetics, a new research field in which retinal proteins are utilized for functional analyses of the nervous systems, has recently attracted lots of attention.
New technologies have been explored to express light-gated ion channels (channelrhodopsin) and light-driven ion pumps (halorhodopsin) in a specific region of
the nervous system, making it possible to control nerve action potentials by light illumination at various wavelengths, or to regulate the intracellularconcentrations of second messengers by light stimuli. In this symposium, current progresses in optogenetics will be reported.

1SH-01

ハロロドプシンの構造と分子機能
Structure and Molecular Function of Halorhodopsin
出村 誠 (北大・院先端生命)

Makoto Demura (Fac. Adv. Life Sci., Hokkaido Univ.)

1SH-02

Structural basis for light-gated cation conductance by channelrhodopsin
石谷 隆一郎 (東京大学大学院理学系研究科)

Osamu Nureki, Ryuichiro Ishitani (Graduate School of Science, The University of Tokyo)

1SH-03

チャネルロドプシンによる神経細胞ネットワーク素子の制御
Control of neural network devices by channelrhodopsin

宇理須 恒雄 (名古屋大学革新ナノバイオデバイス研究センター, 科学技術振興機構,CREST)

Tsuneo Urisu1,2 (1Nagoya University, FIRST Research Center for Innovative Nanobiodevices, 2Japan Science andTechnology Agency, CREST)

1SH-04

オプトジェネティクスを用いた睡眠覚醒調節に関わる神経回路の動作原理解明
Optogenetics reveals function of neural network involved in the regulation of sleep/wakefulness−(S32)−

山中 章弘 (名古屋大学 環境医学研究所)

Akihiro Yamanaka (Research Institute of Environmental Medicine, Nagoya University)

1SH-05

レチナールタンパク質の進化と機能多様性
Evolution and functional diversity of animal opsin-based pigments
寺北 明久 (大阪市大・理)Akihisa Terakita (Grad. Sch. Sci., Osaka City Univ.)

1SH-06

イカロドプシンの光活性化機構
Photo-activation mechanism of squid rhodopsin
村上 緑 (名大・院理・物理)

Midori Murakami, Tsutomu Kouyama (Grad. Sch. Sci., Nagoya Univ.)

1SH-07GPCR の細胞内シグナル伝達を制御する構造特徴
Structural elements which control the signal transduction pathway of GPCRs
諏訪 牧子 (青学大 ・理工, 産総研 生命情報工)

Makiko Suwa1,2, Minoru Sugihara2 (1Colledge, Sci. Eng. AGU., 2CBRC, AIST)
9:00~11:30
1SI

I 会場：理学 E 館 1 階 131 ／ Room I: Sci. bldg. E 1F 131

分子モーター複合体の力発生機構を探る：1 分子〜細胞の架け橋
Exploring force-generating mechanism of molecular motor ensembles; building bridges between single
molecules and cells

オーガナイザー：茅 元司 (東大), 矢島 潤一郎 (東大)Organizer: Motoshi Kaya (Univ. of Tokyo), Junichiro Yajima (Univ. of Tokyo)
Single molecule studies have been extensively developed for the last 20 years and revealed the force-generating mechanism of molecular motors, forming theirensembles to contribute to the force generation in various cellular activities. In this symposium, young frontier scientists, studying in two contrasting
perspectives, molecular basis and cell basis approaches, will exchange their unique interpretations of the force-generating mechanism of various molecularmotor (dynein, kinesin and myosin) ensembles in cells.

はじめに

茅 元司 (東大)Motoshi Kaya (Univ. of Tokyo)

1SI-01分子複合体フィラメントにおける骨格筋ミオシン 1 分子の力発生メカニズム
Force generation mechanism of single skeletal myosin molecules in myofilaments
茅 元司 (東京大学・院理物理, 科学技術振興機構・さきがけ)

Motoshi Kaya1,2, Hideo Higuchi1 (1Grad. Sch. Sci, Univ. Tokyo, 2JST, PRESTO)

1SI-02

ダイニンモーター運動発生機構の構造的基盤
Structural basis of dynein motility
昆 隆英 (阪大・蛋白研)

Takahide Kon (Institute for Protein Research, Osaka Univ.)

1SI-03

Microtubule corkscrewing motion driven by multiple non-processive motors
矢島 潤一郎 (東京大学大学院総合文化研究科広域科学専攻生命環境科学系)

Junichiro Yajima (Department of Life Sciences, Graduate School of Arts and Sciences, The University of Tokyo)

1SI-04微小管系分子モーターの複数分子による機能を計測する
Measuring ensemble functions of microtubule-based molecular motors
古田 健也 (情報通研, バイオ ICT)

Ken'ya Furuta (Bio ICT Lab, NICT)

1SI-05A cellular funicular: one active force generation drives two directional organelle movements
木村 暁 (国立遺伝学研究所・細胞建築研究室, 総研大・遺伝学専攻)

Akatsuki Kimura1,2 (1Cell Architecture Laboratory, National Institute of Genetics, 2Dept. Genetics, SOKENDAI)

1SI-06

ミオシンの力生成・応答特性による上皮パターン形成
The emergence of epithelial pattern through force-generating and force-responding properties of myosin−(S33)−

杉村 薫 (京大・iCeMS)

Kaoru Sugimura1, Shuji Ishihara2 (1Kyoto Univ., 2The Univ. of Tokyo)

第 2 日目（9/23（日））／ Day2(Sep. 23, Sun.)
9:00~11:30
2SA

A 会場：理学南館 1 階 坂田・平田ホール／ Room A: Sci. south bldg. 1F Sakata Hirata Hall

海外招聘シンポジウム：生物物理学最前線
Invited symposium from abroad: Forefront of biophysics

オーガナイザー：小嶋 誠司 (名大), 千見寺 浄慈 (名大)Organizer: Seiji Kojima (Nagoya Univ.), George Chikenji (Nagoya Univ.)
The Biophysical Society of Japan will celebrate the 50th annual meeting this year. This symposium is planned as one of the anniversary events organized by theannual meeting organizing committee. In this symposium, we invite four speakers from abroad who have been recognized as their distinguished studies in thefield of biophysics. We do not focus on the particular subject of biophysics, but instead we would like to include different topics with various techniques(structural analysis of proteins, (EM, X-ray), systems biology, single molecule biophysics). The aim of this symposium is to overview the forefront of thebiophysics in the world, and to enjoy their outstanding studies by the omnibus style.

はじめに

小嶋 誠司 (名大)Seiji Kojima (Nagoya Univ.)

2SA-01The Mechanism of Cytoplasmic Dynein Motility

Ahmet Yildiz (University of California Berkeley)

Ahmet Yildiz (University of California Berkeley)

2SA-02

Architecture of the Flagellar Switch Complex
Brian R. Crane (Cornell University)

Brian R. Crane1, R. Sircar1, G. Gonzalez-Bonet1, Koushik Paul2, D. Blair2 (1Cornell University, 2University of Utah)

2SA-03

Imaging Water at the Nanoscale and Protein in Water by TEM

Paul T. Matsudaira (Department of Biological Science, National University of Singapore)

Paul T. Matsudaira (Department of Biological Science, National University of Singapore)

2SA-04

Function dictates topology in biochemical networks

Chao Tang (Peking University, University of California, San Francisco)

Chao Tang (Peking University, University of California, San Francisco)9:00~11:30
2SB

B 会場：多元数理科学棟 5 階 509 ／ Room B: Mathematics bldg. 5F 509

若手オーガナイズシンポジウム：生命科学の新しい地平を切り開く若手研究者たち
Symposium organized by younger generation: Young scientists pioneering future life sciences

オーガナイザー：藤井 高志 (理研), 小山 昌子 (名大)Organizer: Takashi Fujii (RIKEN), Masako Koyama (Nagoya Univ.)
In this symposium, young speakers actively working in diverse research fields introduce recent topics in their own research areas and talk about their progress
and potential in addressing important biological questions.

2SB-01

遺伝子発現ノイズの起源
Examining origins of noise in gene expression

谷口 雄一 (理化学研究所生命システム研究センター)

Yuichi Taniguchi, Yutaka Ogawa, Masae Johmura (Quantitative Biology Center, RIKEN)

2SB-02細胞環境下におけるタンパク質の NMR 計測
Protein NMR spectroscopy in the cellular environment

猪股 晃介 (独立行政法人理化学研究所生命システム研究センター)

Kohsuke Inomata1, Shuhei Murayama2, Shiro Futaki3, Hidekazu Hiroaki4, Yutaka Ito5, Hidehito Tochio2, Masahiro
Shirakawa2 (1Quantitative Biology Center, RIKEN, 2Graduate School of Engineering, Kyoto University, 3Institute for
Chemical Research, Kyoto University, 4Graduate School of Pharmaceutical Sciences, Nagoya University, 5Graduate School−(S34)−

of Science and Engineering, Tokyo Metropolitan University)

2SB-03高速原子間力顕微鏡による生体分子の構造ダイナミクスの直接観察
Direct observation of structural dynamics of biological molecules by high-speed atomic force microscopy
古寺 哲幸 (金沢大・理工・バイオ AFM 先端研究センター)

Noriyuki Kodera1, Takayuki Uchihashi1,2, Toshio Ando1,2 (1Bio-AFM Frontier Research Center, Inst. Sci. & Eng.,
Kanazawa Univ., 2Sch. Math. & Phys., Inst. Sci. & Eng., Kanazawa Univ.)

2SB-04

中心小体 9 回対称構造の謎を解く
Cracking the mystery of nine-ness: mechanisms of centriole formation
北川 大樹 (遺伝研・新分野創造センター)

Daiju Kitagawa (Nat. Inst. of Genetics, Cent. for Front. Res.)

2SB-05低温電子顕微鏡法によるらせん複合体の高分解能構造解析
Recent advances in CryoEM high-resolution structural analysis for helical filaments
藤井 高志 (理化学研究所 生命システム研究センター)

Takashi Fujii (Qbic Riken)

2SB-06

Kinetic and X-ray crystallographic studies of assembly/disassembly of the CRM1 nuclear export complex
小山 昌子 (名古屋大・理・生命理学)

Masako Koyama1, Natsuki Shirai1, Yoshiyuki Matsuura1,2 (1Grad. Sch. Sci., Nagoya Univ., 2Strl. Biol. R. Ctr., Nagoya
Univ.)
9:00~11:30
2SC

C 会場：多元数理科学棟 4 階 409 ／ Room C: Mathematics bldg. 4F 409

非平衡リビングマター：DNA から細胞骨格、細胞へ
Living matter far from equilibrium: from DNA to cytoskeletons and cells

オーガナイザー：島本 勇太 (ロックフェラー大), 前多 裕介 (京大)
Organizer: Yuta Shimamoto (The Rockefeller Univ.), Yusuke Maeda (Kyoto Univ.)
Essential understanding of complex biological systems requires revealing the underlying molecular processes and verifying how they follow the laws of physics.
In this symposium we spotlight young researchers who see biological systems as living “matter” and are tackling fundamental problems in biology, using new
tools and methodologies that allow for measuring quantitative physical parameters. Topics focus on the single and collective behavior of dynamic polymer
systems, from genetic materials to cytoskeletal networks in cells. We believe that such collaborative interactions would facilitate deeper understanding of living
systems as well as the development of physics for systems that are out of thermodynamic equilibrium.

2SC-01

有糸分裂紡錘体の構造形成と機能の物理的理解に向けてToward a physical understanding of spindle assembly and function
島本 勇太 (ロックフェラー大学)

Yuta Shimamoto (Rockefeller Univ.)

2SC-02

ダイニンに駆動されたマイクロチューブによる巨大渦の格子形成
Large-scale vortex lattice of microtubules driven by dyneins
永井 健 (東京大)Ken Nagai1, Yutaka Sumino2, Kazuhiro Oiwa3,4 (1Univ. Tokyo, 2Aichi Univ. Edu., 3NICT, 4Univ. Hyogo)

2SC-03

Non-Gaussian athermal fluctuations in active cytoskeletons
水野 大介 (九州大学理学研究院)

Daisuke Mizuno (Kyushu University)

2SC-04植物器官にみられるねじれた成長の弾性力学的および幾何学的な起原について
Elastic and geometric origin of the twisted growth of plant organs
和田 浩史 (立命館・物理)

Hirofumi Wada (Dep. Phys. Ritsumeikan Univ.)

2SC-05Visualizing Chromosome Structure with Superresolution MicroscopyPeter Carlton (京都大学 物質ー細胞統合システム拠点 (iCeMS))Peter Carlton (Kyoto University, iCeMS Institute)

2SC-06

DNA・RNA の輸送と選択：生命の起源への接近
Transport and selection for DNA and RNA: An approach to the origin of life−(S35)−

前多 裕介 (京大・白眉センター, 京大・理・物理, 科学技術振興機構さきがけ, ロックフェラー大)

Yusuke Maeda1,2,3,4, Albert Libchaber4 (1The Hakubi Center for Advanced Research, Kyoto Univ., 2Dept. Phys., Grad. Sch.
Sci., Kyoto Univ., 3PRESTO, JST, 4The Rockefeller Univ.)
9:00~11:30
2SD

D 会場：多元数理科学棟 3 階 309 ／ Room D: Mathematics bldg. 3F 309

スーパーコンピューティング：分子ネットワークと細胞内ダイナミクス
Supercomputing in Molecular Network to Cellular Dynamics

オーガナイザー：木寺 詔紀 (横市大), 杉田 有治 (理研)
Organizer: Akinori Kidera (Yokohama City Univ.), Yuji Sugita (RIKEN)
K computer (the ten Peta FLOPS supercomputer installed in Kobe) provides an enormous enhancement of the capability in computational life science. Thissymposium intends to bring forth the possibility of supercomputing in life science fully utilizing K computer. Particularly, it focuses on the multi-scale
descriptions of molecular networks and cellular dynamics by integrating the molecular and the cellular simulation methods.

2SD-01

Dynamics and stability of proteins in cellular environments
Michael Feig (ミシガン州立大学)

Michael Feig (Michigan State University)

2SD-02

スーパーコンピュータは細胞生物学をどう変えるか
How supercomputing can help change cell biology

高橋 恒一 (理化学研究所 QBiC, 慶大・先端生命研, 阪大・生命機能)

Koichi Takahashi1,2,3 (1RIKEN QBiC, 2Keio Inst. Adv. Biosci., 3Osaka Univ. Grad. Sch. Front. Biosci.)

2SD-03細胞内の場を考慮したシミュレータ RICS の開発
Development of the cell simulator in consideration of space

横田 秀夫 (理研 基幹研, 理研 次世代生命体統合シミュレーション)

Hideo Yokota1,2, Yasuhiro Sunaga2, Sigeho Noda3 (1ASI, Riken, 2iSLIM, Riken, 3ACCC, Riken)

2SD-04「京」による大規模遺伝子ネットワーク推定
Large scale gene network estimation with K computer
玉田 嘉紀 (東京大・院情報理工)

Yoshinori Tamada1, Teppei Shimamura2, Rui Yamaguchi2, Atsushi Niida2, Ayumu Saito2, Yuto Kataoka1,2, Seiya Imoto2,
Masao Nagasaki3, Satoru Miyano1,4 (1Grad. Sch. Info. Sci. Tech., Univ. Tokyo, 2Inst. Med. Sci., Univ. Tokyo, 3Tohoku Med.
Megabank, Tohoku Univ., 4CSRP, RIKEN)

2SD-05

データ同化法にもとづくデータ統合・生体シミュレーション技術
Biological system modeling and data assimilation
吉田 亮 (情報・システム研究機構 統計数理研究所)

Ryo Yoshida (The Institute of Statistical Mathematics, Research Organization of Information and Systems)
9:00~11:30

E 会場：多元数理科学棟 1 階 109 ／ Room E: Mathematics bldg. 1F 109

新学術領域研究「運動超分子マシナリーが織りなす調和と多様性」共催

2SE

運動超分子マシナリーが織りなす調和と多様性
Harmonized supramolecular machinery for motility and its diversity

オーガナイザー：宮田 真人 (阪市大), 南野 徹 (阪大)Organizer: Makoto Miyata (Osaka City Univ.), Tohru Minamino (Osaka Univ.)"Motility" observed inside and outside of cells has long been an important biophysical research subject. In the last decade, we have learned a lot about many
motile systems, including conventional motor proteins and ATP synthetase and so on. However, the mechanisms of many motility systems are still unclear. In
this symposium, we discuss the possibilities for new research subjects through six presentations.

はじめに

南野 徹 (阪大)Tohru Minamino (Osaka Univ.)

2SE-01

細菌べん毛モーターの回転方向変換制御機構の解明
Elucidation of the directional switching mechanism of the bacterial flagellar motor−(S36)−

宮田 知子 (大阪大学大学院生命機能研究科)

Tomoko Miyata1, Takayuki Kato1, Takasi Fujii1,2, Shuichi Nakamura4, Yusuke Morimoto1,2,3, Tohru Minamino1, Hideyuki
Matsunami5, Keiichi Namba1,2 (1Grad. Sch. Frontier Biosci., Osaka Univ., 2QBiC, RIKEN, 3Grad. Sch. Sci., Osaka Univ.
Department of Applied Physics, 4Tohoku Univ. School of Engineering, 5OIST, Initial Research Project)

2SE-02磁性細菌の超分子複合体ーマグネトソーム，細胞骨格そしてベン毛ーの構造的組織化
Structural Organization of Macromolecular assemblies -Magnetosome, Cytoskeleton and Flagella- in Magnetotactic
Bacteria
福森 義宏 (金沢大学理工研究域)

Yoshihiro Fukumori, Azuma Taoka (College Sci. Eng., Kanazawa Univ.)

2SE-03アクチンフィラメントの構造多型と機能分化
Structural polymorphism and functional differentiation of actin filaments
上田 太郎 (産総研・バイオメディカル)

Taro Q.P. Uyeda1, Nobuhisa Umeki1, Saku Kijima2, Yusuke Nishikawa3, Kiyotaka Tokuraku3, Akira Nagasaki1, Taro Q.P.
Noguchi2 (1Biomedical Res. Inst., AIST, 2Miyakonojo Natl. Col. of Tech., 3Muroran Inst. of Tech.)

2SE-04

マイコプラズマ滑走運動の謎にせまる
New foci to clarify Mycoplasma gliding
宮田 真人 (大阪市大・院理・生物地球)

Makoto Miyata (Grad. Sch. Sci., Osaka City Univ.)

2SE-05

バクテロイデスにおける分泌と滑走
Secretion and Gliding in Bacteroides
佐藤 啓子 (長崎大学医歯薬総合研究科)

Keiko Sato1, Daisuke Nakane1, Hirohumi Wada2, Katsumi Imada3, Mark J. McBride4, Koji Nakayama1 (1Graduate School of
Biomedical Sciences, Nagasaki University, 2Department of Physics Sciences, Ritsumeikan University, 3Graduate School of
Science, Osaka University, 4Department of Biological Sciences, University of Wisconsin-Milwaukee)

2SE-06

Molecular motor driven self-organization in Myxococcus xanthus

Joshua W. Shaevitz (Princeton University, Departments of Physics and Genomics)

Joshua W. Shaevitz (Princeton University, Departments of Physics and Genomics)
総合討論

宮田 真人 (大阪市大・院理・生物地球)

Makoto Miyata (Grad. Sch. Sci., Osaka City Univ.)
9:00~11:30
2SF

F 会場：理学 B 館 5 階 501 ／ Room F: Sci. bldg. B 5F 501

生命システムのばらつきと情報処理
Inherent variability and information coding in biological systems

オーガナイザー：黒田 真也 (東大), 柴田 達夫 (理研)
Organizer: Shinya Kuroda (Univ. of Tokyo), Tatsuo Shibata (RIKEN)
Biological systems can process information and exert functions in the presence of inherent variability in expression levels and activity of molecules. Suchvariability generates diversity of biological functions including early embryonic development and wiring of neural circuits. In this symposium, inherent
variability and information coding in biological systems will be discussed by having speakers from experimental and theoretical sides.

2SF-01

Inconsistency between population and single cell growth rates revealed by dynamics cytometer
若本 祐一 (東大・複雑系生命システム研究センター，JST さきがけ)

Yuichi Wakamoto1 (1Research Center for Complex Systems Biology, Univ of Tokyo, 2JST PRESTO)

2SF-02

Information Processing via Noisy Biochemical Channels

小林 徹也 (東京大学 生産技術研究所, 科学技術振興機構 さきがけ)

Tetsuya J. Kobayashi1,2, Atsushi Kamimura3,4 (1Institute of Industrial Science, the University of Tokyo, 2JST PRESTO,
3Graduate School of Arts and Sciences, The University of Tokyo, 4JSPS)

2SF-03

ERK 経路の情報コード
Information coding of ERK signaling networks
黒田 真也 (東大・理・生物化学)

Shinya Kuroda, Shinsuke Uda, Takeshi Saito (Dept. Biophys. Biochem., Univ. Tokyo)−(S37)−

2SF-04

Fluctuating signals in cellular signal transduction systems
柴田 達夫 (理化学研究所 発生再生科学総合研究センター)

Tatsuo Shibata (RIKEN Center for Developmental BiologyE)

2SF-05

Symmetry breaking in mouse development
柊 卓志 (EMBL, iCeMS Kyoto University)

Takashi Hiiragi1,2 (1EMBL, 2iCeMS Kyoto University)

2SF-06

Stochastic expression of cell adhesion molecules in individual neurons
八木 健 (大阪大学, 科技振興機構)

Takeshi Yagi1,2 (1Osaka University, 2CREST-JST)
9:00~11:30
2SG

G 会場：理学 B 館 2 階 212 ／ Room G: Sci. bldg. B 2F 212

染色体場のダイナミクス
Dynamics of chromatin as the physicochemical field for genetic activities

オーガナイザー：徳永 万喜洋 (東工大), 木村 宏 (阪大)Organizer: Makio Tokunaga (Tokyo Inst. of Tech.), Hiroshi Kimura (Osaka Univ.)
Spatial organization of DNA molecules within the nucleus contains potentially important, and yet mystifying genetic information. Such information includes
physical properties, shapes, and spatio-temporal positioning of DNA and regulatory molecules, comprehensively providing the "physicochemical field" that
ensures arranged storage, timely expression and faithful transmission of genetic materials. We will discuss the structure and function of DNA and protein
complexes that are formed transiently and locally. Understanding of the physicochemical field will provide a tool to control and reconstitute various cellularfunctions.

2SG-01

生細胞における RNA ポリメラーゼ II による転写活性化の動態
Kinetics of RNA polymerase II transcription in living cells
木村 宏 (大阪大学生命機能研究科)

Hiroshi Kimura, Timothy J. Stasevich (Grad. Sch. Frontier Biosci., Osaka. Univ.)

2SG-02染色体場の 1 分子イメージング定量解析
Quantitative analysis of single molecule imaging of the physicochemical field for genetic activities
十川 久美子 (東工大・院生命理工, 理研・免疫セ)

Kumiko Sakata-Sogawa1,2, Makio Tokunaga1,2 (1Grad. Sch. Biosci. Biotech., Tokyo Inst. Tech., 2RCAI, RIKEN)

2SG-03

ヒストンバリアントによるヌクレオソームの構造多様性とダイナミクス
Structural versatility, dynamics, and functions of human nucleosomes containing histone variants
胡桃坂 仁志 (早稲田大学 理工学術院)

Hitoshi Kurumizaka (Waseda University, Faculty of Science and Engineering)

2SG-04細胞のパターンをうみだす遺伝子回路の作製
Construction of genetic circuits underlying cell pattern formation
戎家 美紀 (京都大学生命科学系キャリアパス形成ユニット)

Miki Ebisuya (Career-Path Promotion Unit for Young Life Scientists, Kyoto Univ.)

2SG-05細胞分裂と染色体分配のメカノバイオロジー
Mechanobiology of Cell Division and Chromosome Segregation
石渡 信一 (早大・理工, 早大・WABIOS)

Shin'ichi Ishiwata1,2 (1Fac. Sci. Engn., Waseda Univ., 2WABIOS, Waseda Univ.)

2SG-06

減数分裂特異的な非コード RNA が相同染色体の対合を促進する
Meiosis-specific non-coding RNA mediates robust pairing of homologous chromosomes in meiosis
平岡 泰 (大阪大学大学院生命機能研究科, 情報通信研究機構

未来 ICT 研究所)

Yasushi Hiraoka1,2, Da-Qiao Ding2, Tokuko Haraguchi1,2 (1Graduate School of Frontier Biosciences, Osaka University,
2Advanced ICT Research Institute Kobe, NICT)−(S38)−

9:00~11:30
2SH

H 会場：理学 C 館 5 階 517 ／ Room H: Sci. bldg. C 5F 517

先端顕微計測が照らす生命の輝き
Star of life shined by frontier microscopies

オーガナイザー：井上 圭一 (名工大), 今村 博臣 (京大)
Organizer: Keiichi Inoue (Nagoya Inst. of Tech.), Hiromi Imamura (Kyoto Univ.)
Microscopic techniques have continued to increase their presence in biological field as outstanding research methods to directly observe the spatiotemporal
behaviors of various biological materials and phenomena in recent years. In this symposium, seven leading researchers will introduce their cutting-edge
microscopic techniques and provide topics for discussing what will be possible with microscopic techniques in future biological science.

はじめに

今村 博臣 (京大)

Hiromi Imamura (Kyoto Univ.)

2SH-01

KcsA カリウムイオンチャネルの溶液条件変化応答 1 分子開閉ダイナミクスの解析
Analysis of Single Molecular Gating Dynamics of the KcsA Potassium Channels Responding to Rapid Changes of
Solution Conditions
清水 啓史 (福井大・医・分子生理)

Hirofumi Shimizu1, Masayuki Iwamoto1, Antoine Royant2,5, Stetten David von5, Laurent Guerin3, Yoshimitsu Aoki4,
Michael Wulff5, Shigetoshi Oiki1 (1Mol. Physiol. & Biophys., Univ. Fukui. Fac. Med. Sci., 2CEA-CNRS-Universite Joseph
Fourier, 3Universite de Rennes 1, 4Keio. Univ. Sci. & Tech. Elec., 5ESRF)

2SH-02中赤外波長領域の観察が可能な赤外超解像顕微鏡の開発と生体試料への応用
Development of a mid-IR super-resolution microscope and its application to biological samples
酒井 誠 (東工大・資源研)

Makoto Sakai (Tokyo Tech.)

2SH-03

白色レーザーを用いた CARS 分光イメージングによる生細胞の動態追跡
Tracing dynamical behavior of a single cell by CARS microspectroscopy using a white-light laser source
加納 英明 (筑波大 数理物質科学研究科)

Hideaki Kano1, Masanari Okuno2, Hiroki Segawa2, Hiro-o Hamaguchi3 (1Tsukuba Univ., 2The Univ. of Tokyo, 3National
Chiao Tung Univ.)

2SH-04

イメージングマススペクトロメトリーによって初めて明らかになった、細胞内の脂質分布の分子種特異的な極性について
Imaging mass spectrometry revealed the polarized intracellular distribution of specific lipid molecular species
瀬藤 光利 (浜松医科大学解剖学講座)

Mitsutoshi Setou (Hamamatsu University School of Medicine Department of Cell Biology and Anatomy)

2SH-05

随意運動中のマウス運動野 2 光子イメージング
Two-photon imaging of the mouse motor cortex during voluntary skilled movement
松崎 政紀 (基礎生物学研究所, CREST, 総研大)Riichiro Hira1,2,3, Fuki Ohkubo1,3,4, Katsuya Ozawa2,3, Yoshikazu Isomura3,5, Kazuo Kitamura2,3, Masanobu Kano2, Haruo
Kasai2, Masanori Matsuzaki1,3,4 (1National Institute for Basic Biology, 2Graduate School of Medicine, University ofTokyo, 3CREST, 4SOKENDAI, 5Brain Science Institute, Tamagawa University)

2SH-06

Chemiluminescence imaging of flow-induced ATP release at caveolae in vascular endothelial cells
山本 希美子 (東京大学 大学院医学系研究科 医用生体工学講座 システム生理学)

Kimiko Yamamoto1, Joji Ando2 (1Laboratory of System Physiology, Department of Biomedical Engineering, Graduate
School of Medicine, University of Tokyo, 2Laboratory of Biomedical Engineering, School of Medicine, Dokkyo Medical
University)

2SH-07

バイオイメージング技術
New Fluorescent Probes and New Perspectives in Bioscience
宮脇 敦史 (理化学研究所

脳科学総合研究センター)

Atsushi Miyawaki (Brain Science Institute, RIKEN)
おわりに

井上 圭一 (名工大)Keiichi Inoue (Nagoya Inst. of Tech.)−(S39)−

9:00~11:30
2SI

I 会場：理学 E 館 1 階 131 ／ Room I: Sci. bldg. E 1F 131

一分子レベルで生体分子機械のエネルギーと機能効率を考える
The art of energetic and functional efficiency in biomolecular machines on the single molecule level

オーガナイザー：鎌形 清人 (東北大), 藤芳 暁 (東工大), Chun Biu Li (北大)Organizer: Kiyoto Kamagata (Tohoku Univ.), Satoru Fujiyoshi (Tokyo Inst. of Tech.), Chun Biu Li (Hokkaido Univ.)
How single or a few biomolecules perform functions with high energetic efficiency in a fluctuating environment is an intriguing question that concerns theunderstanding of the general designing and operational principles of biological nano-machineries. By bringing together both experimental and theoreticalexperts from interdisciplinary fields, this symposium aims to explore: 1) Novel experimental techniques to probe the energetic and functional efficiency on the
single molecule level; 2) New theoretical frameworks and analysis methods in modeling and explaining the biophysical mechanisms to achieve energetic andfunctional efficiency; 3) Applications of these biological inspirations in building efficient nano-devices.

2SI-01

生物には効率が大事なのだろうか？
Is high efficiency necessary for organisms?
工藤 成史 (東北大・院応用物理)

Seishi Kudo (Dept. Applied Physics, Grad. Sch. Eng., Tohoku Univ.)

2SI-02

F1-ATPase モーターの一分子エネルギー論
Single molecule energetics of F1-ATPase motor
宗行 英朗 (中央大学理工学部物理学科)

Shoichi Toyabe2, Takahiro Watanabe-Nakayama3, Hiroshi Ueno1, Tetsuaki Okamoto4, Hiroshi Taketani1, Seishi Kudo5, Eiro
Muneyuki1 (1Dept. Phys., Faculty of Science and Engineering, Chuo Univ., 2Faculty of Physics, LMU Munich,
3Interdisciplinary Graduate School of Science and Engineering, Tokyo Inst. Tech., 4Environment Preservation Center,
Kanazawa Univ, 5Dept. Appl. Phys., Sch. Eng., Tohoku Univ.)

2SI-03Observation of vibrational absorption of single proteins at few Kelvins
藤芳 暁 (東工大 物理)

Satoru Fujiyoshi (Tokyo Tech.)

2SI-04

揺らぎの定理によるミトコンドリア輸送の駆動力測定
Estimation of driving force acting on a mitochondrion transported in a living cell: Application of the fluctuation
theorem
林 久美子 (東北大工)

Kumiko Hayashi1, Masaaki Sato2, Kazunari Mouri3, Chang-gi Pack3, Kazunari Kaizu4, Kouichi Takahashi4, Yasushi
Okada4 (1Sch. Eng., Tohoku Univ., 2IMRAM, Tohoku Univ., 3ASI, RIKEN, 4QBiC, RIKEN)

2SI-05

物質界面における構造と電荷移動を可視化する走査型プローブ顕微鏡法の開発
Development of scanning probe methods for investigating structures and charge transfers at material interfaces
木村 建次郎 (神戸大学 大学院理学研究科)

Kenjiro Kimura (Grad. Sch. of Sci., Kobe Univ.)

2SI-06

DNA 折り紙フレームを用いた一分子観察
Direct Observation of Single Molecular Event in DNA Origami Frame杉山 弘 (京大院理, 京大物質ー細胞)

Hiroshi Sugiyama1,2 (1Grad. Sch. Sci. Kyoto Univ., 2iCeMS)−(S40)−

口頭発表

Oral Presentation

第 1 日目（9 月 22 日（土））／ Day1(Sep. 22, Sat.)
14:00〜16:22

A 会場

理学南館 1 階 坂田・平田ホール／ Room A: Sci. south bldg. 1F Sakata Hirata Hall

分子モーター I
Molecular Motors I
1A1400コイルドコイル ミオシンの脆弱部位と屈曲性の保存

The test on the power stroke model of Ncd
Masahiko Yamagishi, Yoko Toyoshima, Junichiro Yajima

Prediction of fragile points and conservation of bending
ability of coiled coil myosin(Dept. Life Sciences, Grad. Sch. Arts and Sciences, Univ. Tokyo)

Mieko Taniguchi1, Hideki Tanizawa2, Sigeki Mitaku1 (1Grad.
Sch. Sci., Univ. Nagoya, 2Inst. Wister, USA)
1A1412

1A1522メカニズム

カーボンナノチューブ上におけるミオシン運動の温度応答

Thermal response of myosin motors on single carbon
nanotubes
Mitsunori Nagata1, Hiroshi Matsutaka1, Takeru Okada3,
Akihiko Ishijima2, Yuichi Inoue2 (1Grad. Sch. Life Sci., Tohoku

Selective permeation mechanism through the channel of thestator complex MotA/B in the flagellar motor
1A1534

Univ., 2IMRAM, Tohoku Univ., 3Institute of Fluid Science,
Tohoku University)
1A1424

1A1436

1A1546Engineered disulfide crosslink in the the periplasmic regionof PomB impaired function of the Na+-driven flagellar stator
complex
Shiwei Zhu, Na Li, Seiji Kojima, Michio Homma (Division of
Biology Science, Graduate School of Science, Nagoya
University)

1A1558高圧力が引き起こすべん毛繊維のダイナミック構造変化

高時間分解能暗視野顕微鏡を用いたキネシン頭部の前方へのステッ
プの観察

Direct observation of the forward stepping motion of kinesin-1
using dark-field microscopy with 50-μs temporal resolution

Dynamic conformational changes of flagellar filament
observed by high-pressure microscopy
Masayoshi Nishiyama1, Yoshiyuki Sowa2 (1The Hakubi Center,
Kyoto Univ., 2Hosei Univ.)

Hiroshi Isojima1, Ryota Iino2, Hiroyuki Noji2, Michio
Tomishige1 (1Dept. Appl. Phys., Sch. Eng., Univ. Tokyo, 2Dept.
Appl. Chem., Sch. Eng., Univ. Tokyo)
1A1448Front-head gating mechanism of kinesin-1 as studied bysingle molecule FRET observation of ATP binding
Yamato Niitani, Michio Tomishige (Department of Applied
Physics, School of Engineering, The University of Tokyo)

1A1610c-di-GMP 結合タンパク質 YcgR のホモログ PlzD の大量発現による Vibrio alginolyticus でのべん毛運動阻害
Flagellar motility inhibition by overexpression of PlzD,a YcgR homolog of c-di-GMP binding protein, in Vibrio
alginolyticus
Takuro Yoneda, Wakako Morimoto, Seiji Kojima, Michio
Homma (Grad. Sci., Univ. Nagoya)

休憩 15:00-15:10
1A1510

Yasutaka Nishihara, Akio Kitao (IMCB, Univ. of Tokyo)
Sodium Dynamics of the Bacterial Flagellar Motor
Chien-Jung Lo1, Yoshiyuki Sowa2, Teuta Pilizota2, Richard
Berry2 (1Department of Physics, Natioinal Central University,
Taiwan, 2Clarendon Laboratory, Department of Physics,
University of Oxford)

生体分子モーターのためのステップ解析アルゴリズム

Powerful Algorithm for analyzing Stepping Motion of
Biological Melecular Motors
Takeshi Nakagawa, Kazuo Sasaki (Dept. of Applied Physics,
Tohoku Univ.)

細菌べん毛モーター固定子複合体 MotA/B チャネルの選択的透過

キネシン様モータータンパク質 Ncd の Power-stroke モデルの検証

14:00〜16:22

B 会場

多元数理科学棟 5 階 509 ／ Room B: Mathematics bldg. 5F 509

蛋白質-構造機能相関 I
Proteins: Structure & Function I
1B1400

1B1412“空隙”ー機能構造創出のための自然の戦略

1B1424溶液 NMR を用いた Na+駆動型べん毛モーターの固定子タンパク

Cavity−a Nature’s strategy for functional sub states in

質 FliG の C 末端領域構造解析

proteins
Kazuyuki Akasaka (Kinki UNIV., Inst. Adv. Tech.)HD-exchange motion of free heptameric GroES studied by
the use of TROSY and DMSO quenching followed by 2D
NMR
Mahesh Chandak1, Takashi Nakamura1, Koki Makabe2, Toshio
Takenaka1, Jin Chen1, Koichi Kato1, Kunihiro Kuwajima1(1Okazaki Institute of Integrative Bioscience, National Institutes

Solution NMR analysis of FliG C-terminal domain derived
from Na+-driven motor of Vibrio
Mizuki Gohara1, Rei Abe-Yoshizumi1,2, Shiori Kobayashi1,
Yohei Miyanoiri3, Yoshikazu Hattori4, Chojiro Kojima4,
Masatsune Kainosho3,5, Michio Homma1 (1Div. Bio. Sci., Grad.
Sch. Sci., Nagoya Univ., 2Department of Frontier Materials,
Nagoya Institute of Technology, 3Structural Biology ResearchCenter, Grad. Sch. Sci., Nagoya Univ., 4Institute for Protein
Research, Osaka Univ., 5Center for Priority Areas, Tokyo

of Natural Sciences, The Graduate University for Advanced
Studies (Sokendai), 2Graduate School of Science andEngineering,Yamagata University)

Metropolitan Univ.)
1B1436

酸化還元状態に依存してプロテインジスルフィドイソメラーゼの基
質結合部位ドメインの空間配置が変化する仕組み−(S41)−

1B1534

Molecular mechanism of redox-dependent domain
rearrangement of the substrate-binding region of protein
disulfide isomerase

ル伝達過程

The possible advantage of structural disorder of intrinsicallydisordered pr oteins in the new type of signaling mechanism
Nobu C. Shirai1,2, Macoto Kikuchi1,2,3 (1Graduate School ofKouya Inagaki1,2, Yoshinori Uekusa1,2, Yukiko Kamiya1,2,
Tadashi Satoh1, Koichi Kato1,2 (1Grad. Sch. Phar. Sci., Nagoya
City Univ., 2Natl. Inst. Nat. Sci.)
1B1448

天然変性タンパク質の構造ゆらぎを生かした密度変化誘起型シグナ

Science, Osaka University, 2Cybermedia Center, Osaka
University, 3Graduate School of Frontier Biosciences, Osaka
University)

Transferred cross-saturation 法を用いた EB1 の CH ドメイ
ンの微小管との結合界面の特定

Microtubule-binding sites of EB1 CH domain revealed bytransferred cross-saturation experiments
Teppei Kanaba1, Ryoko Maesaki2, Tomoyuki Mori2, Yutaka
Ito1, Toshio Hakoshima2, Masaki Mishima1 (1Grad. Sch. of Sci.

1B1546

分子動力学法による capping protein（CP）の動的構造の解析

Structural fluctuations of capping proteins analyzed bymolecular dynamics simulations
Ryotaro Koike1, Shuichi Takeda2, Yuichiro Maeda2, Motonori Ota1
(1Grad. Sch. Info. Sci., Nagoya Univ., 2Struct. Biol. Res. Center, Grad.

2

and Tech., TMU, Grad. Sch.l of Biol. Sci., NAIST)

Sch. of Sci., Nagoya Univ.)

休憩 15:00-15:10

1B1558

シトクローム c 酸化酵素のプロトンポンプ機構に関する分子動力学
シミュレーションによる研究

1B1510

再構築型生体外タンパク質合成系を用いた分子シャペロンによる凝

A Molecular Dynamics Study on Proton Pump Function of
Cytochrome c Oxidase

集抑制効果の大規模解析

Global analysis of aggregation-inhibotion effects of molecularchaperones using a reconstituted cell-free translation system
Tatsuya Niwa1, Takuya Ueda2, Hideki Taguchi1 (1Grad. Sch. of
Biosci&Bioteh, Tokyo Institute of Technology, 2Grad. Sch. of
Frontier Sciences, The university of Tokyo)
1B1522

1B1610DNA 界面での蛋白質の局所的構造変化

Masaru Tateno1 (1Grad. Sch. Sci., Univ. Hyogo, 2Grad. Sch.
Pure and Appl. Sci., Univ. Tsukuba)

Local conformational changes of proteins in DNA interfaces
Tomoko Sunami, Hidetoshi Kono (JAEA)

14:00〜16:22

C 会場

Takefumi Yamashita (Research Center for Advanced Scienceand Technology, University of Tokyo)
Substrate transport mechanisms in GatCAB: the smallest
unidirectional valve in subnano scale
Jiyoung Kang2, Shigehide Kuroyanagi2, Yohsuke Hagiwara2,

多元数理科学棟 4 階 409 ／ Room C: Mathematics bldg. 4F 409

蛋白質-計測，解析，エンジニアリング
Proteins: Measurement, Analysis, Engineering
1C1400

抗菌ペプチドを利用した病原体検出サンドイッチ発色検出技術の開発

New colorimetric sandwich assay for detection of pathogens
by using antimicrobial peptides as detection probes
Chihiro Sakai1, Eri Hojo2, Taichi Nakazumi1, Satoshi
Tomisawa1, Takashi Kikukawa1, Yasuhiro Kumaki1, Masakatsu
Kamiya1, Makoto Demura1, Keiichi Kawano1, Ryuji Ohtsuki2,
Taro Yonekita2, Naoki Morishita2, Takashi Matsumoto2, Fumiki

1C1412

Sci., Grad Sch. Arts and Life Sci., The Univ. Tokyo, 2Dept. Chem.,
Grad Sch. Sci., The Univ. Tokyo)
1C1448低分子抗体の多機能化を可能とするピンポイント化学接合デザイン

Morimatsu2, Tomoyasu Aizawa1 (1Grad. Sch. Life Sci.,
Hokkaido Univ., 2R&D Center, Nippon Meat Packers Inc.)

Protein engineering for site-specific bioconjugation
chemistry: Construction of multiple functional lowmolecular antibodies
Mitsuo Umetsu, Asami Ueda, Takeshi Nakanishi, Kentaro
Hashikami, Ryutaro Asano, Izumi Kumagai (Department of
Biomolecular Engineering, Graduate School of Engineering,

VanX の溶菌活性を用いたルシフェラーゼのスクリーニングの簡

Tohoku University)

略化

休憩 15:00-15:10

Toward a simplified screening method using a novel VanX
cell lysis activity
Nan Wu1, Tetsuya Kamioka1, Shihori Sohya1, Tomoki
Matsuda2, Takahisa Ikegami3, Haruki Nakamura3, Yutaka

1C1510Hybrid nano-cellulosome: functional analysis of various
cellulose binding module as component of artificial cellulosome
Hikaru Nakazawa1, Do-myoung Kim1, Takashi Matuyama2,
Nobuhiro Ishida2, Akinori Ikeuchi2, Izumi Kumagai1, Mitsuo
Umetsu1 (1Tohoku univ. tech., 2Toyota Central R&D lab)

Kuroda1 (1Biotechnology and Department Life Science,
Graduate School of Engineering, Tokyo University of Agriculture
and Technology, 2Research Institute for Electronic Science,
Hokkaido University, 3Institute for Protein Research, Osaka
1C1424

1C1522

University)
Fluorescent Probe for the Direct Detection of Histone
Deacetylase Activity

エンド-1,3-βグルカナーゼの糖結合モジュールα-リピートに存在する Trp273 のラミナリン結合への寄与

Contribution of Trp273 in the α-repeat of the carbohydratebinding module of endo-1,3-β-glucanase to laminarin binding
Tomonari Tamashiro1, Yoichi Tanabe1, Kenji Kanaori2,

Koushik Dhara1, Yuichiro Hori1, Reisuke Baba1, Kazuya
Kikuchi1,2 (1Graduate School of Engineering, Osaka
University, 2Immunology Frontier Research Center, Osaka
University)
1C1436ハイブリッドナノセルロームの構成要素としての CBM の機能解析

Teikichi Ikura3, Nobutoshi Ito3, Masayuki Oda1 (1Grad. Sch. ofLife and Environ. Sci., Kyoto Pref. Univ., 2Grad. Sch. of Sci. andTechnol., Kyoto Inst. of Technol., 3Med. Res. Inst., Tokyo Med.
and Dent. Univ.)

RaPID システムによる Akt2 選択的環状ペプチド阻害剤の探索とその阻害ペプチドの生化学的活性の評価

Exploring cyclic peptide inhibitors against the Akt2 kinase by the
RaPID system and evaluation of their biochemical properties

1C1534

Yuuki Hayashi1, Jumpei Morimoto2, Hiroaki Suga2 (1Dept. Life−(S42)−

Construction of an in vitro gene screening system for
membrane proteins
Haruka Soga1, Satoshi Fujii3, Tetsuya Yomo2,3, Hajime

Watanabe1, Tomoaki Matsuura1,3 (1Dept. Biotech., Grad. Sch.
Eng., Osaka Univ., 2Dept. Bioinfo. Eng., Grad. Sch. IST, Osaka

Development of novel rapid buffer-exchange system andmicrofluidic mixer for time-resolved spectroscopic study of
low-yield biomolecules

Univ., 3ERATO, JST.)
1C1546 Orientation-controlled immobilization of pharaonis
halorhodopsin onto gold revealed by SEIRAS

Tetsunari Kimura1,2,3, Yuji Furutani1,2,4 (1Inst. Mol. Sci., 2Dept.
Struct. Mol. Sci., Sokendai, 3CREST/JST, 4PRESTO/JST)

Hao Guo1,2, Tetsunari Kimura1,2,3, Yuji Furutani1,2,4(1Department of Structural Molecular Science, the Graduate
University for Advanced Studies, 2Department of Life andCoordination-Complex Molecular Science, InstituteMolecular Science, 3JST CREST, 4JST PRESTO)
1C1558

1C1610溶液中の金属タンパク質の共鳴 X 線散乱法の開発

Development of resonant X-ray scattering method of
metalloproteins in solutions
Mitsuhiro Hirai1, Kazuki Takeuchi1, Ryota Kimura1, Noboru

forOhta2 (1Grad. Sch. Eng., Gunma Univ., 2SPring-8)

低収量生体分子の時間分解分光計測を目指した新規溶液交換・混合装置の開発

14:00〜16:22

D 会場

多元数理科学棟 3 階 309 ／ Room D: Mathematics bldg. 3F 309

発生，分化，神経Development, Differentiation, Neuroscience
1D1400マウス ES 細胞分化の単 1 細胞解析

1D1510Single-cell-based analysis of differentiation of mouse ES cells
Atsushi Maruyama1, Yuichi Wakamoto2,3, Shogo Nakamura1,

A model of cell-division stop for the limb regeneration
Hiroshi Yoshida (Faculty of Math. Kyushu Univ.)

1D1522ラット培養神経回路網の長期発達過程における同期活動に関わる機能的分子の探索

Tatsuo Michiue2, Shin-ichi Sakai4, Bayar Hexig4, Toshihiro
Akaike4, Kiyoshi Ohnuma1 (1TRI, Nagaoka Univ of Tech, 2Grad.
Sch. Arts. & Sci., Univ. Tokyo, 3Sakigake, JST, 4Grad. Sch. Biosci.

Analysis of functional molecules underlying synchronous
activity during long-term development of rat cultured
neuronal networks
Daisuke Ito1, Keiko Yokoyama2, Kazutoshi Gohara2 (1Fac. Adv.

& Biotech., Tokyo Inst of Tech)
1D1412

顕微ラマン分光法による分化因子刺激後の単一細胞ダイナミクスの計測Life Sci., Hokkaido Univ., 2Fac. Eng., Hokkaido Univ.)

Measurements of Single Cell Dynamics upon Stimulation
with a Differentiation Factor Using Raman MicroSpectroscopy
Sota Takanezawa1,2, Shin-ichi Morita1, Yukihiro Ozaki2,
Yasushi Sako1 (1Cellular informatics Lab., RIKEN, 2Grad. Sch.

1D1534Insulin and glucose for memory in a snail
Etsuro Ito1, Ryuichi Okada1, Mika Morikawa1, Satoshi
Takigami2, Akiko Okuta3, Manabu Sakakibara2 (1Kagawa Schoolof Pharmaceutical Sciences, Tokushima Bunri University, 2School

Sci. and Tech., Kwansei Gakuin Univ.)
1D1424of High-Technology for Human Welfare, Tokai University,
Graduate School of Science, Kyoto University)

1 分子定量計測技術を用いた C. elegans 受精卵における細胞極性
のモデル化

3

1D1546

Quantitative measurement and mathematical modeling ofpolarity-protein PAR-2 in C. elegans embryos
Yukinobu Arata1, Tetsuya Kobayashi2, Michio Hiroshima1,
Chan-gi Pack1, Masashi Tachikawa3, Kenichi Nakazato3, Tatsuo
Shibata4, Yasushi Sako1 (1Cell. Info., ASI, Riken, 2Inst. Ind. Sci.,
Univ. Tokyo, 3Theo. Biol., ASI, Riken, 4CDB, Riken)
1D1436

光捕捉された神経細胞内シナプス小胞群の集合ダイナミクス

Assembling dynamics of optically trapped synaptic vesicles inneuronal cell
Yusuke Ueda1,2, Suguru N. Kudoh2, Takahisa Taguchi1, Chie
Hosokawa1,2 (1Health Res. Inst., AIST, 2Grad. Sci. Eng.,
Kwansei Gakuin Univ.)
1D1558

膜電位感受性色素・カルシウムイメージングに使える新しい超高速形態形成中の組織の力学場を推定する

共焦点顕微鏡の開発

Estimating forces in the growing epithelial tissues
Shuji Ishihara1,2, Kaoru Sugimura3,4 (1Grad. Sch. Arts &A new class of confocal microscope for a fast voltagesensitive dye (VSD) and Ca2+ imaging

Sciences, Univ. Tokyo, 2JST PRESTO, 3iCeMS, Kyoto Univ.,

Takashi Tominaga, Yoko Tominaga (Dept. Neurophysiol.,Kagawa Sch Pharm Sci, Tokushima Buniri Univ)

4

1D1448

モノアラガイの長期記憶におけるインスリンとグルコースの役割

RIKEN BSI)

ショウジョウバエ形態形成におけるアポトーシスの能動的力学的寄与

1D1610De novo アセンブルによる軟体動物脳の全トランスクリプトーム

Active Mechanical Role of Apoptosis in Tissue Dynamics

解析

during Drosophila Morphogenesis
Yusuke Toyama1,2,3 (1Dep. of Biol. Sci., National University ofSingapore, 2Mechanobiology Inst., National University ofSingapore, 3Temasek Life Sciences Lab.)De novo sequencing and transcriptome analysis of themolluscan brain by deep RNA sequencing

休憩 15:00-15:10

14:00〜16:10

E 会場

Hisayo Sadamoto1, Hironobu Takahashi2, Taketo Okada1,
Yoshinori
Asakawa2(1Facul.
Hiromichi
Kenmoku2,
Pharmaceut. Sci. Kagawa Camp, Univ. Tokushima Bunri, 2Inst.
Pharmacognosy, Univ. Tokushima Bunri)

多元数理科学棟 1 階 109 ／ Room E: Mathematics bldg. 1F 109

蛋白質-物性 I
Proteins: Property I
1E1400分子動力学シミュレーションによる Hsp90 と ADP の結合自由エ
ネルギープロファイル

Free energy profile for binding of ADP to Hsp90 with−(S43)−

molecular dynamics simulation
Kazutomo Kawaguchi, Hiroyuki Takagi, Masashi Iwayama,
Megumi Nishimura, Hiroaki Saito, Hidemi Nagao (Inst. Sci.

Eng., Kanazawa Univ.)
1E1412制限

Three-Dimensional Imaging of the Protein Transfer Free
Energy into the Molecular Crowding Condition with TMAO
Isseki Yu1, Kyoko Nakada1, Masataka Nagaoka2 (1Dep. of Cem.

The conformational restriction of denatured protein
encapsulated in the chaperonin cageand Biol. Sci. Aoyama Gakuin Univ., 2Grad. School of Info. Sci.,
Nagoya Univ.)

シャペロニン空洞内の変性タンパク質が受けるコンフォメーション

Fumihiro Motojima, Yuko Motojima-Miyazaki, Masasuke
Yoshida (Kyoto Sangyo Univ.)
1E1424

1E1522Free-energy analysis of urea effect on cytochrome c
Yasuhito Karino, Nobuyuki Matubayasi (Inst. Chem. Res.,
Kyoto Univ.)

小角 X 線散乱によるアミロイド線維形成の初期会合プロセスの解析

Investigation of initial assembling process of fibrillation bysmall angle X-ray scattering
Eri Chatani1, Rintaro Inoue2, Koji Nishida2, Toshiji Kanaya2,
Masahide Yamamoto3 (1Dept. Chem., Grad. Sch. Sci., Kobe

1E1534

Distinguishing crystal-like amyloid fibrils and glass-like
amorphous aggregates from their kinetics of formation
Yuichi Yoshimura1, Yuxi Lin1, Hisashi Yagi1, Young-Ho Lee1,
Hiroki Kitayama1, Kazumasa Sakurai1, Masatomo So1, Hirotsugu

クスとの動的カップリング

Hiroshi Nakagawa1, Mikio Kataoka1,2 (1Japan Atomic Energy
Agency, Quantum Beam Science Directorate, 2Nara Institute of
Science and Technology, Graduate School of Materials Science)
1E1546

Temperature and hydration effects of low-frequency spectraof native and denatured proteins studied by terahertz time-

細胞濃度に近い無添加細胞抽出液の調製

A method to prepare cell extract near intracellularconcentration without additional salts and buffers
Kei Fujiwara, Shin-ichiro M. Nomura (Dept. Biorobot., Tohoku
Univ.)

domain spectroscopy
Naoki Yamamoto1, Atsuo Tamura2, Keisuke Tominaga1,2(1Molecular
Photoscience
ResearchCenter,Kobe
University, 2Graduate School of Science, Kobe University)
1E1558

誘電緩和分光法によるアルキルカルボン酸の水和特性

Hydration Properties of Alkyl Carboxylate Series byDielectric Relaxation Spectroscopy
Yangtian Wang, George Mogami, Tetsuichi Wazawa, Nobuyuki
Morimoto, Makoto Suzuki (Grad. Sch. Eng., Tohoku. Univ)

休憩 15:00-15:10
1E1510

テラヘルツ時間領域分光を用いた天然および変性状態タンパク質における低振動スペクトルの水和および温度依存性の観測

Ogi2, Hironobu Naiki3, Yuji Goto1 (1Inst. Protein Res., Osaka
Univ., 2Grad. Sch. Eng. Sci., Osaka Univ., 3Fac. Med. Sci., Univ.
Fukui)
1E1448タンパク質の水和水の水素結合ダイナミクスとタンパク質ダイナミ

Kinetics of hydrogen-bonding of protein hydration water andits dynamical coupling with protein dynamics

Univ., 2Inst. Chem. Res., Kyoto Univ., 3Kyoto Univ.)
1E1436シトクロム c に対する尿素効果の自由エネルギー解析

TMAO による分子クラウディング環境への蛋白質移相自由エネ
ルギーの三次元描像

14:00〜16:10

F 会場

理学 B 館 5 階 501 ／ Room F: Sci. Bldg. B 5F 501

光生物-視覚，光受容 I
Photobiology: Vision & Photoreception I
1F1400サル緑感受性視物質の全反射赤外分光解析

Univ. Tokushima Grad.Sch.)ATR-FTIR study of monkey green-sensitive visual pigment

1F1448

Kota Katayama1, Yuji Furutani1,2, Hiroo Imai3, Hideki Kandori1(1Department of Frontier Materials, Nagoya Institute ofTechnology, 2Department of Life and Coordination-Complex

Spectroscopic study of photoreaction dynamics ofGroeobacter rhodopsin
Kumiko Nagata, Keiichi Inoue, Hideki Kandori (Nagoya Inst.

Molecular Science, Institute for Molecular Science, 3Primate

Of Technol.)Research Institute, Kyoto University)
1F1412

休憩 15:00-15:10

精製したタコロドプシンの SAXS 測定

SAXS measurements of purified octopus rhodopsin
Shingo Watanabe1, Mitsuhiro Hirai2, Tatsuo Iwasa1,3 (1CEDAR,
Muroran IT, 2Grad. Sch. Eng., Gunma Univ., 3Grad. Sch. Eng.,

1F1510

ナノディスク試料を用いたロドプシンと錐体視物質によるトランス
デューシン活性化効率の比較解析

Comparative analysis of transducin activation by rhodopsin
and cone pigments in nanodiscs
Keiichi Kojima, Ryo Maeda, Yasushi Imamoto, Takahiro
Yamashita, Yoshinori Shichida (Dept. Biophys., Grad. Sch. Sci.,

1F1522Large deformation of helix F upon formation of the M

脊椎動物非視覚オプシン Opn5 とその類似光受容タンパク質の分intermediate of the azide-bound purple form of pharaonis
halorhodopsin
Taichi Nakanishi1, Soun Kanada1, Midori Murakami1, Kunio
Ihara2, Tsutomu Kouyama1 (1Grad. Sch, Sci., Univ.

子的性質の解析

Nagoya, 2Center for Gene Research., Univ. Nagoya)

Kyoto Univ.)
1F1436

ASR の Pro206 はレチナール構造と光反応を制御する
Pro206 of Anabaena sensory rhodopsin is responsible for theisomeric composition of retinal and photochromism
Yoshitaka Kato1, Akira Kawanabe2, Kwang-Hwan Jung3, Hideki
Kandori1 (1Grad. Sch. Eng., Nagoya Inst. Tech., 2Grad. Sch.
Med., Osaka Univ., 3Sogang Univ. Korea)

Muroran IT)
1F1424

グロイオバクターロドプシンの光反応ダイナミクスの分光研究

Analysis of the molecular properties of vertebrate non-visual
opsins, Opn5 and Opn5-like protein
Takahiro Yamashita1, Hideyo Ohuchi2, Sayuri Tomonari3, Sari
Fujita-Yanagibayashi1, Kazumi Sakai1, Sumihare Noji3,
Yoshinori Shichida1 (1Grad. Sch. Sci., Kyoto Univ., 2Grad. Sch.
Med. Dent. Pharm. Sci., Okayama Univ., 3Inst. Technol. Sci.,

1F1534−(S44)−

センサリーロドプシン II-トランスデューサー複合体の比較シミュ
レーションにより明らかにされた基底状態と M 中間体の差異

Differences between the ground state and the Mintermediate of Sensory Rhodopsin II-Transducer complex
revealed by comparative simulations
Koro Nishikata1,3, Mitsunori Ikeguchi1, Akinori Kidera1,2

1F1546

1F1558(1Grad. Sch. of Nanobioscience, Yokohama City Univ., 2Research
Program for Computational Science, RIKEN, 3Bioinformatics And

Light activated states of photoreceptor membrane proteins as
revealed by in-situ photo-irradiated solid-state NMR

Systems Engineering division (BASE), RIKEN)
Effect of potential on the photo reaction of Sensory rhodopsin
II monolayer studied by Surface Enhanced IR absorption

Akira Naito1, Yuya Tomonaga1, Tetsurou Hidaka1, Yusuke
Shibafuji1, Yoshiteru Makino1, Izuru Kawamura1, Yuki Sudo2,
Akimori Wada3, Takashi Okitsu3, Naoki Kamo4 (1Graduate

Spectroscopy
Kenichi Ataka, Joachim Heberle (Freie Universitaet Berlin,
Fachbereich Physik)

School of Engineering, Yokohama National University, 2Graduate
School of Science, Nagoya University, 3Kobe Pharmaceutical
University, 4Matsuyama University)

In-situ 光照射固体 NMR による光受容膜蛋白質の光活性状態の解明

14:00〜16:22

G 会場

理学 B 館 2 階 212 ／ Room G: Sci. bldg. B 2F 212

細胞生物的課題 I
Cell Biology I
1G1400Larger Intercellular Adhesion Strength Generates More
Coherent and Directional Multicellular Movement

Mechanisms of signal transduction activated by heavy-ion
microbeam induced bystander responses- Measurement and Simulation
Tsuyoshi Hirashima1, Takanori Iino2, Yoichiroh Hosokawa2,
Masaharu Nagayama3,4 (1Anatomy and Developmental Biology,

Masanori Tomita1, Hideki Matsumoto2, Tomoo Funayama3,
Yuichiro Yokota3, Kensuke Otsuka1, Munetoshi Maeda1,4, Yasuhiko
Kobayashi3 (1Radiat. Safety Res. Cent., CRIEPI, 2Biomed. Imaging

Faculty of Medicine, Kyoto University, 2Graduate School ofMaterials Science, Nara Institute of Science and Technology,

Res. Cent., Univ. Fukui, 3QuBS, JAEA, 4R&D, WERC)
1G1522

3

Research Institute for Electronic Science, Hokkaido University,
4
CREST, Japan Science and Technology Agency)
1G1412

Effects of conformational fluctuation of chromosomes inbudding yeast on the pattern of gene expression
Naoko Tokuda, Masaki Sasai (Grad. Sch. Eng., Univ. Nagoya)

GPI アンカー型タンパク質の短寿命ホモダイマーはラフト組織
化・機能のための最小単位である

1G1534

Transient GPI-anchored protein homodimers are units forraft organization and function
Kenichi G. N. Suzuki1, Rinshi S. Kasai1, Koichiro M.
Hirosawa1, Yuri L. Nemoto1, Munenori Ishibashi1, Yoshihiro
Miwa2, Takahiro K. Fujiwara1, Akihiro Kusumi1 (1iCeMS, Kyoto

2

Graduate School of Information Science and Technology, Osaka
University, 3Graduate School of Frontier Bioscience, Osaka
University)

1G1448容体の核移行解析

引き起こす

Nuclear translocation analysis of glucocorticoid receptor by
spatiotemporal analysis with multipoint fluorescencecorrelation spectroscopy
Johtaro Yamamoto, Masataka Kinjo (Fuc. Adv. Life Sci.,
Hokkaido Univ.)

弾性膜モデルを用いたゴルジ再集合過程シミュレーション

1G1558細胞内 FRET イメージングによる情報処理タンパク質 RAF の構

Simulation study on Golgi reassembly process based on

造と EGF 応答の相関解析

elastic membrane model
Masashi Tachikawa, Atsushi Mochizuki (Theoretical Biology
Laboratory, ASI, RIKEN)

Correlation between the molecular conformation and EGF
response of RAF by FRET imaging in individual cells
Kayo Hibino1, Masahiro Ueda1, Yasushi Sako2 (1RIKEN,

Analysis of cell behavior near silicon based micro-scale pore
Yo Otsuka1, Shinya Murakami1, Manabu Sugimoto1, Toshiyuki
Mitsui2 (1Grad. Sch. Sci., Aogaku Univ, 2Assoc. Sci., Aogaku

QBiC, 2RIKEN, ASI)
1G16101 分子キネティクス解析により示された、ヘレギュリンと ErbB
タンパク質のダイナミックに変化する相互作用

Interaction between heregulin and ErbB proteins varies

Univ)dynamically shown by single-molecule kinetic analysis in living
cells
Michio Hiroshima1,2, Yuko Saeki3, Mariko Okada-Hatakeyama3,
Yasushi Sako1 (1RIKEN ASI, 2RIKEN QBiC, 3RIKEN RCAI)

休憩 15:00-15:10
1G1510多点蛍光相関分光を用いた時空間解析によるグルココルチコイド受

ナノ秒パルス高電界は新規細胞ストレスとして作用して翻訳抑制を

Nanosecond pulsed electric fields act as a novel cellular stressthat induces translational suppression
Ken-ichi Yano, Keiko Morotomi-Yano (Bioelectrics Res. Center,
Kumamoto Univ.)
1G1436

人工自己複製システムのダーウィン進化

Darwinian Evolution of artificial self-replication system
Norikazu Ichihashi1,2, Tetsuya Yomo1,2,3 (1JST ERATO,

1G1546

Univ., 2Dept. Pharmacol., Univ. Tsukuba)
1G1424出芽酵母の遺伝子発現パターンの変化における染色体揺らぎの影響

重イオンマイクロビーム照射によるバイスタンダー応答で誘導されるシグナル伝達機構の解明

14:00〜16:22

H 会場

理学 C 館 5 階 517 ／ Room H: Sci. bldg. C 5F 517

バイオイメージング
Bioimaging
1H1400

ラスター画像相互相関分光法（ccRICS）を用いた外来 DNA に対Akira Sasaki, Masataka Kinjo (Fac. Adv. Life Sci., Hokkaido

する細胞内防御機構の可視化

Visualization of intracellular defense system against
exogenous DNAs by using cross-correlation raster image

1H1412

correlation spectroscopy

Univ.)
Non-invasive in vivo imaging of vesicle movement inneutrophils in mouse ear
Kenji Kikushima, Sayaka Kita, Hideo Higuchi (Dept of Phys,−(S45)−

1H1534

School of Science, The Univ. of Tokyo)
1H1424

1H1436

脂質二重膜を用いた T 細胞活性化の 1 分子イメージング解析

Single molecule analysis of T cell activation with stimulatory
lipid bilayers by live cell imaging
Yuma Ito1,2, Hirofumi Oyama1,2, Kumiko Sakata-Sogawa1,2,

Anti-charging Phase Plates for Phase Contrast Electron
Microscopy
Kuniaki Nagayama1, Toshiyuki Itoh2, Yoko Kayama2, Zenpei
Saitoh2, Yukinori Nagatani1, Yoshihiro Arai2, Masahiro Ohara1,

Makio Tokunaga1,2 (1Grad. Sch. Biosci. Biotech., Tokyo Inst.
Tech., 2RCAI, RIKEN)

Noriko Kai2, Kazuyoshi Murata1 (1National Institute forPhysiological Sciences, 2Terabase Inc.)GTP 交換促進因子 Sos の生細胞一分子計測

1H1546

Single molecule imaging of guanine nucleotide exchange
factor Sos in living cells
Yuki Nakamura1,2 (1RIKEN sako celluar informatics
laboratory, 2Osaka university Yanagida lab)
1H1448(1National Inst. Physiological Sci., 2Terabase Inc., 3Tokyo Inst.
Tech., 4RTS)
1H1558

光学顕微鏡とカソードルミネッセンス顕微鏡を用いたマルチモーダ

観察による測定

ル細胞イメージング

Single-molecule imaging revealed acceleration of trimeric
G-protein turnover by transient dimerization of liganded

Multimodal cellular imaging with light microscopy andcathodoluminescence microscopy

β2adrenergic receptor
Rinshi Kasai1, Takahiro Fujiwara2, Akihiro Kusumi1,2 (1Inst.
For Frontier Med. Sci., Kyoto Univ., 2WPI-iCeMS, Kyoto Univ.)

Hirohiko Niioka1, Taichi Furukawa2, Masayoshi Ichimiya3,
Tomohiro Nagata4, Masaaki Ashida2, Tsutomu Araki2, Mamoru
Hashimoto2 (1Inst. for NanoSci. Design, Osaka Univ., 2Eng. Sci.,
Osaka Univ., 3Osaka Dental Univ., 4Tsukuba Institute for Super
Materials, ULVAC Inc.)

休憩 15:00-15:10
1H1610光学顕微鏡と同時観察可能な高速 AFM

Naoyuli Miyazaki1,2, Mineo Iseki3, Koji Hasegawa4, Akihiro
Narita5, Shinichi Adachi6, Masakatsu Watanabe7, Kenji Iwasaki1(1Institute for Protein Research, Osaka University, 2National
Institute for Physiological Sciences, 3Pharmaceutical sciences,High-speed AFM observation of intrinsically disordered
proteins (NTAIL-GFP, PNT-GFP and SIC1-GFP)
Sujit Kumar Dora1, Kodera Noriyuki1, David Bloquel2, Johnny
Habchi2, Antoine Gruet2, Sonia Longhi2, Toshio Ando1 (1Bio
AFM frontier research center, Institute of physics andmathematics, Kanazawa University, 2AFMB Laboratory,
University of Aix-Marseille)

14:00〜16:22

I 会場

精製タンパク質の構造から in situ の構造まで：電顕を使用してFrom purified protein structures to in situ structures using
EM

High-speed AFM combined with optical microscopy
Shingo Fukuda, Takayuki Uchihashi, Toshio Ando (Depertmentof Mathmatics and Physics, Grad School of Natural Science andTechnology Kanazawa Univ)
1H1522500kV LinacTEM の開発
Development of the 500kV Linac TEM
Yukinori Nagatani1, Yoshihiro Arai2, Takumi Sannomiya3, Tadao
Shirai4, Ryuzo Aihara2, Giichi Iijima2, Kuniaki Nagayama1

リガンド結合したβ 2 アドレナリン受容体の短寿命ダイマーは三量体 G タンパク質のターンオーバーを加速する：2 色同時 1 分子

1H1510位相差電子顕微鏡用の帯電防止位相板

Toho University, 4AdvanceSoft Corp., 5Structural Biology ResearchCenter, Nagoya University, 6Institute of Materials Structure
Science, High Energy Accelerator Research Organization(KEK), 7The Graduate School for the Creation of New Photonics
Industries)

理学 E 館 1 階 131 ／ Room I: Sci. bldg. E 1F 131

生命情報科学，バイオエンジニアリング
Bioinformatics & Bioengineering
1I1400

Divya Shaji1, Takayuki Amemiya1, Satoshi Fukuchi2, Motonori

Analyzing the conservation of the folding nucleuses among
the Ferredoxin-like fold in their amino acids sequence

Ota1 (1Graduate school of information science, Nagoya
University, Japan, 2Faculty of Engineering, Maebashi Institute ofTechnology, Japan)

Masanari Matsuoka, Takeshi Kikuchi (Dept. Bioinf., Coll. Life
Sci., Ritsumeikan Univ.)
1I1412RNA とタンパク質の相互作用におけるタンパク質構造変化のデー

1I1448

タベース解析

Database analysis of the degree of protein conformationalchanges in RNA-protein interactions
Chihiro Kubota1, Kei Yura1,2,3 (1Graduate School of Humanities
and Sciences, Ochanomizu University, 2Center for Informational

休憩 15:00-15:10

Biology, Ochanomizu University, 3Center of Simulation Sciences,
Ochanomizu University)
1I1424

天然変性タンパク質のデータベース IDEAL の構築

1I1510Construction of IDEAL database for the investigation of
intrinsically disordered proteins
Takayuki Amemiya1, Shigetaka Sakamoto2, Yukiko Nobe1,
Seiko D. Murakami1, Kazuo Hosoda3, Ryotaro Koike1, Hidekazu
Hiroaki4, Motonori Ota1, Satoshi Fukuchi3 (1Grad. Schl of Info.
Sci., 2HOLONICS

1I1436

Co., Ltd., 3Fac. Engr., Maebashi
4Tech., Grad. Schl of Pharm. Sci., Nagoya Univ.)
Computational analysis of protean segments (ProSs) inintrinsically disordered proteins (IDPs)

MODIC: a novel ab inito identification system oftranscription factor binding motifs in genome DNA sequences
Masaru Tateno1, Ryo Nakaki2, Jiyoung Kang2 (1Grad. Sch.
Sci., Univ. Hyogo, 2Grad. Sch. Pure and Appl. Sci., Univ.
Tsukuba)

Interaction of DNA molecules with nanopore studied byfluorescence microscopy
Genki Ando1, Kazuteru Yamada2, Toshiyuki Mitsui1,2 (1Grad.
Mat.Sci., Univ. Aoyama, 2Phys., Univ. Aoyama)

1I1522

診断用 DNA 計算に向けた核酸応答型 DNA 合成システムの構築

Construction of a nucleic-acid-responsive DNA synthesis
system for diagnostic DNA-based computing
Ken Komiya, Asako Kobayashi, Masayuki Yamamura (Interdis.
Grad. Sch. of Sci. and Engi., Tokyo Tech.)Ins.

1I1534−(S46)−

DNA scaffold logic: logic operation on molecular inputs using

FRET cascades
Takahiro Nishimura1, Yusuke Ogura1, Hirotsugu Yamamoto2,

1I1558

Electrochemical regulation of bacterial respiratory activity of
Shewanella, a dissimilatory metal reducing bacteria
Shoichi Matsuda1, Huan Liu3, Shuji Nakanishi2, Kazuhito
Hashimoto1,2,3 (1Grad. Sch. Engr., Univ. Tokyo, 2RCAST, Univ.

Kenji Yamada3, Jun Tanida3 (1Osaka University, 2The Universityof Tokushima, 3Osaka University)
1I1546

寄生虫による接着性宿主細胞への侵入過程観察のためのマイクロ加
工デバイスの作製

Observation of parasite invasion with the micro-fabricateddeviceTetsuhiko Teshima1, Hiroaki Onoe1, Kaori Kuribayashi-

金属還元菌 Shewanella における微生物呼吸代謝の電気化学的制御

1I1610

Shigetomi1, Hiroka Aonuma2, Hirotaka Kanuka2, Shoji
Takeuchi1 (1The Institue of Industrial Science, 2Jikei University
School of Medicine)−(S47)−

Tokyo, 3ERATO/JST)
Spatiotemporal control of cell motility in nano- to microscale topographical environment
Hiromi Miyoshi1, Hiroyoshi Aoki1, Jungmyoung Ju1, Sang Min
Lee2, Jong Soo Ko2, Yutaka Yamagata1 (1ASI, RIKEN, 2Grad.
Sch. Mech. Eng., Pusan National Univ.)

第 2 日目（9 月 23 日（日））／ Day2(Sep. 23, Sun.)
14:00〜16:10

A 会場

理学南館 1 階 坂田・平田ホール／ Room A: Sci. south bldg. 1F Sakata Hirata Hall

分子モーター II：ダイニン
Molecular Motors II: Dynein
2A1400クライオ電子線トモグラフィーを用いた細胞中骨格タンパク質およ

Shibata, Yuta Watanabe, Tomonori Hata, Yoko Toyoshima (Grad.
Sch. of Arts & Sci., Univ. of Tokyo)

びモータータンパク質の非侵襲的構造解析

Observation of intracellular cytoskeleton and motor proteinin the non-invasive cells with electron cryo-tomography
Shinji Aramaki1, Kazuhiro Aoyama2,3, Kota Mayanagi4, Takuo
Yasunaga1 (1Dept. of Bioscience and Bioinformatics, Grad.

2A14122A1510A direct interaction between dynein two heads is notnecessary for processive movements of dynein
Keitaro Shibata, Yui Utsumi, Takayuki Torisawa, Yoko

Bioregulation, Kyushu Univ.)

Toyoshima (Dept. Life Sci., Grad. Sch. Arts and Sci., Univ.
Tokyo)

クライオ電子線トモグラフィーを用いたクラミドモナス軸糸外腕ダ
2A1522The mechanochemical cycle of human cytoplasmic dynein

axonemal ODA by electron cryo-tomography
Mingyue Jin1,4, Haru-aki Yanagisawa2, Kotaro Koyasako1,4,
Ritsu Kamiya2, Kota Mayanagi3,4, Takuo Yasunaga1,4 (1Dep.

under external force
Taketoshi Kambara1, Yoshiaki Tani1, Motoshi Kaya1,
Tomohiro Shima2, Hideo Higuchi1 (1Grad. Sch. Sci., Univ.
2A1534

クライオ電子顕微鏡法とメタロチオネインラベルを用いた軸糸構造の解明

Elucidation of Axoneme architecture by cryo-electron
microscopy and metallothionein labeling
Reiko Chijimatsu1, Mingyue Jin1,2, Takuo Yasunaga1,2(1Kyushu Institute of Technology, 2JST)

2A1546

電子線トモグラフィー法により明らかになった、LIS1 による細胞

Regulation of cytoplasmic dynein-motility on microtubules by
LIS1 revealed by electron-computed tomography
Shiori Toba1, Kotaro Koyasako2,3, Shinji Hirotsune1, Takuo
Yasunaga2,3,4 (1Dept. Genetic Disease Research, Osaka City Univ.
Grad. Sch. of Medicine, 2Dept. Bioscience & Bioinformatics,
Faculty of Computer Science and Systems Engineering, Kyushu
Inst. Tech., 3JST-SENTAN, 4JST-CREST)Tokyo, 2QBiC, Riken)
Rotation of microtubules driven by Tetrahymena 22S outerarm dynein and its sub-particles
Shin Yamaguchi, Yoko Toyoshima, Junichiro Yajima (Dept Life
Sciences, Graduate School of Arts and Sciences, Univ. of Tokyo.)
哺乳類細胞質ダイニンが一方向性を獲得する機構について

The mechanism of the transition from diffusion to directedmovement in mammalian cytoplasmic dynein
Takayuki Torisawa1, Furuta Ken'ya2, Muneyoshi Ichikawa1,
Yoko Toyoshima1 (1Dept Life Sciences, Graduate School of Arts
and Sciences, the Univ of Tokyo, 2Bio ICT lab, NICT)

質ダイニンの運動活性制御メカニズム

2A1448外部負荷存在下でのヒト細胞質ダイニンのメカノケミカルサイクル

Detection of metallothionein-tagged LC2 on chlamydomonas

Biosci. Bioinf., Inst. Tech. Kyushu, 2Dep. Biol. Sci., Grad. Sch.
Sci., Univ. Tokyo, 3Div. Struct. Biol., Med. Inst. Bioreg., Univ.
Kyushu, 4JST)

2A1436

頭部間の直接的相互作用は細胞質ダイニンの連続的な運動に必要ない

School of Computer Sci. and Sys. Eng., Kyushu Inst. of
Tech., 2FEI Company Japan Ltd., Application Lab., 3Grad.
School of Frontier Biosciences, Osaka Univ., 4Medical Inst. of

イニンにおけるメタロチオネイン標識 LC2 の検出

2A1424

休憩 15:00-15:10

2A1558

骨格筋形成過程における細胞質ダイニンの分布変化

The dynamic change of the distribution of cytoplasmic dynein
during skeletal muscle differentiation process
Takuya Kobayashi, Motoshi Kaya, Hideo Higuchi (Departmentof physics, Graduate school of science, The university of Tokyo)
2A1610ダイニンアダプター Bicaudal-D2 の細胞周期依存的核膜局在の分子機構

ヌクレオチド状態に依存したダイニン分子の形態変化

Molecular mechanism of cell-cycle dependent nuclear

Nucleotide-dependent morphological change of cytoplasmic
dyneinenvelope localization of Bicaudal-D2
Takashi Murayama, Hiroki Ota, Takashi Sakurai (Dept.

Muneyoshi Ichikawa, Kei Saito, Takayuki Torisawa, Keitaro

Pharmacol., Juntendo Univ. Sch. Med.)

14:00〜16:22

B 会場

多元数理科学棟 5 階 509 ／ Room B: Mathematics bldg. 5F 509

蛋白質-構造機能相関 II：理論，凝集Proteins: Structure & Function II: Theory, Aggregation
2B1400

単一アミノ酸ポテンシャル力場の改良と短鎖ペプチドの構造予測へ

2B1424の応用

Improvement of the Single Amino Acid Potential Force Field
and the Application to Structure Prediction of Short Peptides
Michio Iwaoka, Kenichi Dedachi, Taku Shimosato, Toshiya

Relaxation mode analysis for simulations of protein systems
Ayori Mitsutake, Toshiki Nagai, Hiroshi Takano (Keio Univ.)
2B1436

オンサーガー・マハラップ作用を用いたペプチド系のパスサンプリ
2B1448ング

A multi-body potential for normal mode analysis of protein
structure
Bhaskar Dasgupta, Narutoshi Kamiya, Haruki Nakamura, Akira
Kinjo (Institute for Protein Research, Osaka University)

Minezaki (Tokai Univ)
2B1412

蛋白質系のシミュレーションの緩和モード解析

Path sampling for small peptide systems using the OnsagerMachlup action method
Hiroshi Fujisaki1,2, Yasuhiro Matsunaga2, Akinori Kidera2,3(1Nippon Medical School, 2RIKEN, 3Yokohama City University)−(S48)−

アクチン線維へのコフィリンの協同的結合の統計力学モデルの作成

A theoretical model of cooperative binding of cofilin to actin
filaments
Shotaro Sakakibara2, Kimihide Hayakawa1, Hitoshi Tatsumi2,Masahiro Sokabe1,2 (1FIRST Research Center for InnovativeNanobiodevice, Univ. Nagoya, 2Grad. Sch. Med., Univ. Nagoya)

2B1546

A copper chaperone-independent mechanism for activationof Cu, Zn-superoxide dismutase

休憩 15:00-15:10
2B1510Yasuyuki Sakurai, Yoshiaki Furukawa (Dept. of Chem, Keio
Univ.)

リゾチーム分子中のアミロド性ぺプチド

Amyloidogenic Peptides from Hen Lysozyme
Hideki Tachibana1,3, Masao Fujisawa1,3, Ryohei Kono2,3, Minoru
Kato4 (1Sch Biol-Oriented Sci Tech, Kinki Univ, 2Wakayama Med

2B15582B1534タンパク質凝集体の形態を制御する「凝集後修飾」のメカニズム

A mechanism controlling the morphologies of protein
aggregates by “post-aggregation oxidation”
Yasushi Mitomi1, Takao Nomura1, Masaru Kurosawa2,

Univ, 3High Press Prot Res Center, Kinki Univ, 4Coll Pharm Sci,
2B1522

銅シャペロンに依存しない SOD1 酵素の活性化制御メカニズム

Nobuyuki Nukina2, Yoshiaki Furukawa1 (1Dept. of Chem, Keio
Univ., 2RIKEN Brain Science Institute)

Ritsumeikan Univ)
Effects of raft components on the membrane-mediatedaggregation of IAPP

2B1610ジスルフィド結合の組換えによる不溶性 SOD1 オリゴマーの新た

Kenji Sasahara (Kobe University)

な形成メカニズム−筋萎縮性側索硬化症における分子病理変化

タンパク質が生体内レドックス環境に依存して異なる凝集体を形成

Destabilization of SOD1 facilitates abnormal scrambling of
its disulfide bond in the familial form of amyotrophic lateralsclerosis

するメカニズムとその病理学的意義

Redox environment is an intracellular factor to operatedistinct pathways for protein aggregation

Keisuke Toichi, Yoshiaki Furukawa (Dept. of Chem, Keio Univ.)

Yoshiaki Furukawa (Dept. of Chemistry, Keio Univ.)

14:00〜16:22

C 会場

多元数理科学棟 4 階 409 ／ Room C: Mathematics bldg. 4F 409

数理生物学
Mathematical Biology
2C1400A Brownian particle undergoing reversible binding in asurface: exact theoretical results and an application in single
molecule biophysics
Ziya Kalay (Institute for Integrated Cell-Material Sciences,
Kyoto University)
2C1412 Single-particle-level simulation reveals effects of molecular
crowding on biochemical signaling response
Kazunari Kaizu, Koichi Takahashi (Laboratory for Biochemical
Simulation, RIKEN Quantitative Biology Center (QBiC))
2C14242 つのヒートショック反応系における反応機構の比較
Conflicted dissociation constant in two reaction mechanisms
for heat shock response
Masayo Inoue1, Ala Trusina2, Namiko Mitarai2, Kim Sneppen2(1CMC, Osaka Univ., 2CMOL, NBI)

2C1436

Mathematical analysis for vascular and spot patterns by
auxin and PIN dynamics in plant development
Yoshinori Hayakawa1,2, Atsushi Mochizuki1,2 (1Tokyo InstituteJun-ichi Ishihara1,2, Masashi Tachikawa2, Hideo Iwasaki1,
Atsushi Mochizuki2 (1Grad.Sci.Eng., Waseda Univ., 2Wako Inst.,
RIKEN)
2C1522Modeling the mammalian circadian clock
Craig Jolley, Hiroki Ueda (RIKEN Center for Developmental
Biology, Laboratory for Systems Biology)
2C1534 Determining the ratio of longitudinal to transverse
conductivity in a cardiac tissue culture through applicationof FFP
Marcel Hoerning1, Seiji Tagaki2, Kenichi Yoshikawa3,4(1RIKEN, Center for Developmental Biology, Physical Biology
Unit, 2Hokkaido University, Research Institute for Electronic
Science, 3Doshisha University, Life and Medical Sciences,
Kyoto University, Department of Physics)

4

2C1546

Mathematical model of Cl- secretion in airway epithelial cells
Kouhei Sasamoto1, Naomi Niisato2,3, Yoshinori Marunaka2,3of Technology, 2Wako Inst., Riken)
2C1448Grad. Sch. Of Med., 4Dept. of Phys., Chiba Univ., Grad. Sch. ofSci.)
休憩 15:00-15:10
2C1510(1Undergraduate
Student,
Kyoto
Prefectural
Univ.
Med., 2Molecular Cell Physiology, Kyoto Prefectural Univ.
Med., 3Japan Institute for Food Education and Health, HeianModeling wave propagation dynamics in MDCK woundhealing assay
Yusuke Sawabu1, Masaharu Nagayama2, Takashi Miura3,
Hiroyuki Kitahata4 (1Inst. of Nat. Sci. and Tech., Kanazawa
Univ., 2Res. Inst. for Elect. Sci. Hokkaido Univ., 3Kyoto Univ.

Jogakuin (St. Agnes') Univ.)
2C1558る研究

Quasispecies Population through the Error Catastrophe
Kouji Harada (Toyohashi Univ. of Tech. Dept. of Comp. Sci.
and Eng.)
2C1610 Characterization of Death and Division Process in Synthetic

スの解析

Analysis of the internal dynamics for pattern formation in
multi cellular cyanobacteria by CA model

D 会場

エラーカタストロフィーによる HIV-1 擬種集団の自壊過程に関す

A Study on a Self-Destruction Process of a HIV-1

CA モデルによる多細胞性シアノバクテリアの形態形成ダイナミク

14:12〜16:10

気道上皮細胞における Cl-分泌の数理モデル

Bacterial Population with Antibiotic Treatment
Takashi Nozoe1, Reiko Okura1, Yuichi Wakamoto2,3 (1Grad.
Sch. Arts and Sci., Univ. Tokyo, 2Research Center for Complex
Systems Biology, Univ of Tokyo, 3JST PRESTO)

多元数理科学棟 3 階 309 ／ Room D: Mathematics bldg. 3F 309

計測
Measurements
2D1412クライオ電子線トモグラフィーに対する超分解能技術

Ryuzo Azuma1,2, Takuo Yasunaga1,2 (1Grad. Sch. of Comp. Sc.
& Eng., Dept. of Biosc. & Bioinfo., Kyushu Inst. of Tech., 2JST)

Super-resolution cryo-electron tomography−(S49)−

2D1424

定量位相顕微鏡による生細胞におけるナノスケールの形状変化の非Yasuda (Institute of Biomaterials and Bioengineering, Tokyo
Medical and Dental University)

染色ビデオレートイメージング
2D1522

Label-free and video-rate imaging of live cell membranemotion in the nanometer-scale by quantitative phase

Cryogenic Coherent X-ray Diffraction Imaging of
non-crystalline particles at SACLA

microscope
Toyohiko Yamauchi1, Takashi Sakurai2, Hidenao Iwai1, Yutaka
Yamashita1 (1Hamamatsu Photonics, 2EIIRIS, Toyohashi Univ.
of Tech.)
2D1436

Masayoshi Nakasako1,2, Yuki Takayama1,2, Tomotaka Oroguchi1,2,
Yuki Sekiguchi1,2, Masaki Yamamoto2, Koji Yonekura2, Takaaki
Hikima2, Saori Maki-Yonekura2, Yukio Takahashi3, Akihiro Suzuki3,

ラマン分光を用いた超音波照射による細胞への影響の評価

Sachihiro Matsunaga4, Shoichi Kato4, Takahiko Hoshi5(1Department of Physics, Keio University, 2RIKEN SPring-8 Center,
RIKEN Harima Institute, 3Department of Precision Science andTechnology,
Graduate
SchoolofEngineering,
Osaka

Evaluation of Ultrasound Irradiation effects on Cells by using
Raman Spectroscopy
Manabu Sugimoto1, Yo Otsuka1, Shinya Murakami1, Toshiyuki
Mitsui2 (1Grad. Sci Eng., Aogaku Univ, 2Assoc Prof, Aogaku
Univ)
2D1448脂肪酸と 7-ヒドロキシ-4-メチルクマリンを用いた質量と蛍光測定によるアンモニアの二次元検出

Two dimensional ammonia sensing by measurement of massand fluorescence using 7-hydroxy-4-methylcoumarin film andfatty acidYuta Ando, Masahiro Terashita, Ryohei Matsueda, Yutaka

University, 4Department of Applied Biological Science, Faculty of
Science and Technology, Tokyo University of Science, 5Kohzu
Precision Co.,Ltd.)
2D1534 A protocol for structure analysis of non-crystalline particles
with X-ray free electron laser
Tomotaka Oroguchi, Masayoshi Nakasako (Department of
Physics, Faculty of Science and Technology, Keio University)
2D1546

休憩 15:00-15:10

Okajima1 (1Grad. Sch. Inform. Sci. Tech.,
Univ., 2Grad. Sch. Life Sci., Hokkaido Univ.)

インビトロ心毒性予測のための心筋細胞ネットワークの時間的細胞

2D1558

外電位ゆらぎと空間的伝達速度ゆらぎの評価

Evaluation of temporal fluctuation and spatial fluctuation on
cardiomyocyte network for in vitro predictive cardiotoxicity
measurement
Tomoyo Hamada, Fumimasa Nomura, Tomoyuki Kaneko, Kenji

14:00〜16:22

E 会場

イオンコンダクタンス顕微鏡による細胞膜揺らぎの測定

Fluctuation of cell membrane investigated by ion
conductance microscopy
Yusuke Mizutani2, Satoshi Ichikawa1, Zen Ishikura1, Takaharu

Tsujiuchi (Department of Material Science and Engineering,
Akita University)

2D1510

SACLA における非結晶粒子の低温 X 線回折イメージング実験

Hokkaido

Relationship between Intercellular Adhesion Strength andCommunication Detected by Femtosecond Laser-induced
Impulsive Force
Takanori Iino1, Man Hagiyama2, Tadahide Furuno3, Akihiko
Ito2, Yoichiroh Hosokawa1 (1Nara Institute of Science andTechnology, 2Kinki University, 3Aichi Gakuin University)

多元数理科学棟 1 階 109 ／ Room E: Mathematics bldg. 1F 109

蛋白質-物性 II
Proteins: Property II
2E1400

AFM を用いたホロミオグロビンの一分子力学計測によるアン

休憩 15:00-15:10

フォールディング経路の探索

Mechanical unfolding pathways of holo-myoglobin exploredby AFM-based single molecule force spectroscopy
Aya Yoshida, Masaru Kawakami (Materials Sci, JAIST)
2E1412

2E1510The effect of flexibility on the stability of Human Leucocyte
Antigen
Saeko Yanaka1, Kenjji Sugase2, Takamasa Ueno3, Kouhei

走査型プローブ顕微鏡を用いた癌抑制因子 p53 の一分子構造解析

Single-molecule structural analysis of tumor suppressor p53

Tsumoto1 (1Med. Genome Sci., Grad. Sch. Frontier Sci., the Univ.

using scanning probe microscopes
Seiya Takahashi1, Yasuyuki Sainoo1, Risa Kashima2, Takashi
3

1

2E1424of Tokyo, 2Suntory Institue for Bioorganic research, 3Center for
AIDS Research, Kumamoto Univ.)1

Tokino , Tadahiro Komeda , Satoshi Takahashi , Kiyoto
Kamagata1 (1IMRAM, Tohoku Univ., 2Univ. California, 3Res.
Inst. Front. Med., Sapporo Med. Univ. Sch. Med.)

2E1522β-Galactoside-binding activity of human galectin-1 at basic pH
Hirotsugu Hiramatsu, Tomohide Nishino, Hideo Takeuchi

2E1436

2E1448Cavity-dependent dynamics of c-Myb R2R3 revealed by highpressure fluorescence and NMR spectroscopy
Satomi Inaba1, Akihiro Maeno2, Hisayuki Morii3, Harumi
Fukada4, Kazuyuki Akasaka2, Masayuki Oda1 (1Grad. Sch. ofLife and Environ. Sci., Kyoto Pref. Univ., 2High Pressure Protein
Res. Center, Kinki Univ., 3Biomed. Res. Inst., Natl. Inst. Adv. Ind.
Sci. Technol., 4Grad. Sch. of Life and Environ. Sci., Osaka Pref.

塩基性条件下でのヒトガレクチン 1 のβガラクトシド結合能力(Graduate School of Pharmaceutical Sciences, Tohoku
University)
A Molecular Mechanism of Induced-fit of U1A Protein
Ikuo Kurisaki1,2, Masayoshi Takayanagi1,2, Masataka

ヒト主要組織適合複合体の安定化に置ける揺らぎの重要性について

Univ.)
2E1534

単純化 BPTI に融合したペプチド系タグ配列を用いたアミノ酸溶

Nagaoka1,2 (1Grad. Sch. Info. Sci. Univ. Nagoya, 2CREST, JST)

解性の測定

Single mutation on a loop alters the key dynamics of the core,
but not the average structure
Akihiro Maeno1, Sunilkumar P.N.1, Yuji Wada2, Eiji Ohmae2,
Shin-ichi Tate2, Kazuyuki Akasaka1 (1HPPRC, Kinki
Univ., 2Grad. Sch. Sci., Univ. Hiroshima)

Analysis of amino acid contributions to protein solubility
using short peptide tags fused to a simplified BPTI variant
Yutaka Kuroda1, Alam, M. Khan1, Mohammad, M. Islam1,2(1Dept of Biotech and Life Sci, TUAT, 2Dept of Mol Biol andBiochem, Chittagon University)
2E1546−(S50)−

Protein Aggregation Kinetics Using Short Amino acid

Peptide Tags
Monsur A. Khan1, Mohammad M. Islam1,2, Yutaka Kuroda1

2E1558Le Chang, Shoji Takada (Grad. Sch. Sci., Kyoto Univ.)
2E1610(1Tokyo University of Agriculture and Technology, 2University of
Chittagong, Bangladesh)Protein specific partial charges by Ab-initio fragment

Jumpei Torii, Shoji Yamashita, Etsuko Nishimoto (Institute ofBiophysics, Faculty of Agriculture, Graduate school of Kyushu

molecular orbital method for more accurate protein
simulations

14:00〜16:10

F 会場

Regulatory Mechanism of the fluorescence emission fromGreen Fluorescent Protein Chromophore

University)

理学 B 館 5 階 501 ／ Room F: Sci. bldg. B 5F 501

光生物-視覚，光受容 II
Photobiology: Vision & Photoreception II
2F1400全原子量子化学計算によるレチナールタンパク質の吸収波長制御機

Time-resolved study on photoreaction dynamics of full-構の解析

length phototropin from Chlamydomonas reinhardtii
Yusuke Nakasone1, Koji Okajima2, Yusuke Aihara3, Akira
Nagatani3, Satoru Tokutomi2, Masahide Terazima1 (1Grad.
Chem. Sci, Kyoto Univ., 2Grad. Sci, Osaka Prefecture

Full-Quantum Chemical Calculations of the Absorption
Maxima of Retinal Proteins
Tomohiko Hayashi1, Azuma Matsuura2, Hiroyuki Sato2, Minoru
Sakurai1 (1Center for Biological Resources and Informatics,
Tokyo Institute of Technology, 2Fujitsu Laboratories, Ltd.)
2F1412

Univ., 3Grad. Biol. Sci, Kyoto Univ.)
2F1522

トリプトファン残基の位置に影響しない

的波長制御メカニズム

N-Terminal Truncation Does Not Affect the Location of aConserved Tryptophan in the BLUF Domain of AppA from
Rhodobacter sphaeroide

Mechanism of color change of proteorhodopsin by mutations
at distant cytoplasmic loop
Yuya Ozaki1, Rei Abe-Yoshizumi1, Susumu Yoshizawa2,2

1

Masashi Unno1, Yuuki Tsukiji1, Kenshuke Kubota1, Shinji
Masuda2,3 (1Depart. Chem. Appl. Chem., Saga University, 2Center
for BioRes. & Inform., Tokyo Inst. Tech., 3PRSTO, JST)

1

Kazuhiro Kogure , Hideki Kandori ( Grad. Sch. Eng., Nagoya
Inst. Tech, 2AORI, Univ. Tokyo)
2F1424

キ メ ラ タ ン パ ク 質 を 用 い た Rhodobacter capsulatus 由 来

2F1534

Photoactive Yellow Protein の相互作用部位の解明

2F1436

Analysis of interaction sites on the Photoactive Yellow
Protein of Rhodobacter capsulatus with chimeric proteins
Mayu Shimad, Yoichi Yamazaki, Hironari Kamikubo, Mariko
Yamaguchi, Mikio Kataoka (Grad. Sch. Mat. Sci., NAIST)FTIR study of Aureochrome that possesses LOV domain at
the C-terminus
Shota Ito1, You Zhang1, Tatsuya Iwata2, Osamu Hisatomi3,
Fumio Takahashi4, Hironao Kataoka5, Hideki Kandori1 (1Nagoya
Inst. Tech, 2Ctr. Fost. Yng. Innov., Nagoya Inst. Tech, 3Grad.
Sch. Sci., Osaka Univ, 4PRESTO, JST, 5Botanical Gardens,

2F1546

Getzoff4, Hideki Kandori1 (1Nagoya Inst. Tech., 2Ctr. Fost. Yng.
Innov. Res., Nagoya Inst. Tech., 3Grad. Sch. Eng. Sci., Osaka
Univ., 4The Scripps Res. Inst. USA)
2F1558The study of the dissociation and recovery reaction kinetics
for photo-sensor protein UVR8

休憩 15:00-15:10

Takaaki Miyamori1, Yusuke Nakasone1, Kenichi Hitomi2, John
M. Christie3, Elizabeth D. Getzoff2, Masahide Terazima1 (1Grad.
Sci. Chem., Univ. Kyoto, 2Scripps Research Inst., 3Univ.
Glasgow)

クラミドモナス由来の全長フォトトロピンが示す光反応ダイナミク
スの時間分解検出

14:00〜16:22

G 会場

光センサータンパク質 UVR8 の解離・回復反応カイネティックスの測定

Of Technol., 2Ctr. Fost. Yng. Innov. Res., Nagoya Inst. Tech.,)2F1510

アニオンラジカル型の(6-4)光回復酵素による DNA 修復

Repair of damaged DNA by the anion radical form of (6-4)
photolyase
Daichi Yamada1, Yu Zhang1, Tatsuya Iwata2, Junpei
Yamamoto3, Kenichi Hitomi4, Shigenori Iwai3, Elizabeth

LOV ドメインを鋳型としたレドックス感受性蛍光タンパク質の開発
Construction of LOV-based redox-sensitive fluorescentproteins
Yukiko Ono1, Tatsuya Iwata2, Hideki Kandori1 (1Nagoya Inst.

Substrate-Dependent Measurement Revealed Different
Protein Conformations on Photoactivation and DNA Repairof E. coli CPD Photolyase
I Made Mahaputra Wijaya1, Yu Zhang1, Junpei Yamamoto2,
Kenichi Hitomi3, Shigenori Iwai2, Elizabeth D. Getzoff3, Hideki
Kandori1 (1Nagoya Institute of Technology, 2Osaka University,
3
The Scripps Research Institute, La Jolla, USA)

Tohoku Univ)
2F1448

N 末アミノ酸残基の有無は AppA BLUF ドメインの保存されたプロテオロドプシンにおける細胞質側ループの変異がもたらす遠隔理学 B 館 2 階 212 ／ Room G: Sci. bldg. B 2F 212

生体膜・人工膜
Biological & Artificial Membranes
2G1400

膜融合におけるストーク構造からの変形：分子動力学シミュレー

electrophysiological measurement of membrane proteins
Taishi Tonooka1, Ryuji Kawano2, Koji Sato1, Toshihisa Osaki2,

ション

Shoji Takeuchi1 (1Institute of Industrial Science, The Universityof Tokyo, 2Kanagawa Academy of Science and Technology)

Morphology change of fusion stalk studied by molecular
dynamics simulation
Shuhei Kawamoto, Wataru Shinoda (Nano. AIST)
2G1412

2G1424

蛋白質毒素ライセニンがスフィンゴミエリンを含む脂質膜中に誘起蛍光検出と電気計測による膜タンパク質計測に向けた脂質膜チャンするポア形成： 単一 GUV 法による研究

バアレイ

Single Giant Unilamellar Vesicle Method Reveals LyseninInduced Pore Formation in Lipid Membranes Containing

Lipid bilayer chamber array toward fluorescent and−(S51)−

Masumoto2, Takashi Goto3, Yutaka Tsujiuchi1 (1Department of

Sphingomyelin
Jahangir Md.

1

2

Alam ,

Material Science and Engineering, Akita University, 2Center for

Toshihide Kobayashi , Masahito
Yamazaki ( Int. Biosci. Sec., Grad. Sch. Sci. Tech., Shizuoka
University, 2Lipid Bio. Lab., RIKEN)
1

2G14361

人工細胞ベシクルにおける脂質ドメインへの化合物局在を介したドInterdisciplinary Research, Tohoku University, 3Institute forMaterials Research, Tohoku University)
2G1534

メインサイズと膜崩壊率の制御

Single molecular observation of CFTR channels by highspeed AFM
Hayato Yamashita1, Kazuhiro Mio2, Muneyo Mio2, Takayuki

Localization of bulky-molecules in raft-like domains onphase-separated liposomes: Control of domain size andbursting rate of liposome
1

2

Uchihashi3, Masato Yasui1, Toshio Ando3, Yoshiro Sohma1,4(1Pharmacol., Keio Univ. Med. Sch., 2AIST, 3Dept of Physics,
Kanazawa Univ., 4Dalton Cardiovas. Res. Cen., Univ. MissouriColumbia)

3

Miho Yanagisawa , Damien Baigl , Kenichi Yoshikawa(1Grad. Sch. Sci., Kyushu Univ., 2Depart. Chem., Ecole Normale
Superieure, 3Depart. Biomed. Info., Doshisya Univ.)
2G1448

PI4P と脂質膜曲率による Kes1 ステロール輸送活性の制御

2G1546

Regulation of Kes1 sterol transport activity by PI4P and lipid
membrane curvature
Hirokazu Yokoyama1, Masaki Wakabayashi1, Yasushi
Ishihama1,
MinoruNakano2(1Graduate
Schoolof2

Pharmaceutical Sciences, Kyoto University, Graduate School of
Medicine and Pharmaceutical Sciences, University of Toyama)(1GPI, 2RIKEN)
2G1558でのゲート開閉構造の直接観察

KcsA in Lipid Bilayer by Atomic Force Microscopy
Ayumi Sumino1, Takashi Sumikama2, Masayuki Iwamoto2,
Takahisa Dewa1,3, Shigetoshi Oiki2 (1Grad. Sch. Eng., Nagoya

リン脂質輸送タンパク質 Sec14 の脂質輸送メカニズムの解明

Inst. Tech., 2Deprt. of Mol. Physiol. and Biophys., Univ. of Fukui
Fac. Med. Sci., 3JST-PRESTO)
2G1610

Yuji Furutani1,2, Hirofumi Shimizu3, Yusuke Asai1, Tetsuya
Fukuda1, Shigetoshi Oiki3, Hideki Kandori1 (1Grad. Sch. Tech.,
Nagoya Inst. Tech., 2Inst. Mol. Sci., 3Facul. Med. Sci., Univ.

ン伝導整流素子

Photo-controlled ion conductive rectification element usinggel, amino acids and hydrogenated amorphous silicon film
Takaaki Ichikawa1, Hiroki Suzuki1, Ryohei Matsueda1, Hiroshi

H 会場

全反射赤外分光法による KcsA のイオン選択フィルターの振動解析

The Vibrational Analysis of the Selectivity Filter of KcsA byusing ATR-FTIR Spectroscopy

ゲル、アミノ酸、水素化アモルファスシリコンを用いた光制御イオ

14:00〜16:22

原子間力顕微鏡によるカリウムイオンチャネル KcsA の脂質膜中

Direct Observation of Ion Entryway of Potassium Channel

Elucidation of lipid transfer mechanism of phospholipid
transfer protein Sec14
Chisato Takahashi1, Makiko Yamada1, Masaki Wakabayashi1,
Yasushi Ishihama1, Minoru Nakano2 (1Grad. Sch. Pharm., Kyoto
Univ., 2Grad.Sch.Pharm., Toyama Univ.)
2G1522

KcsA チャネルの細胞内ドメインは不活性化に影響を与える
The KcsA channel cytoplasmic domain effects on theinactivation gating
Minako Hirano1,2, Yukiko Onishi2, Daichi Okuno2, Toru Ide1,2

休憩 15:00-15:10
2G1510高速 AFM による CFTR チャネルの動態観察

Fukui)

理学 C 館 5 階 517 ／ Room H: Sci. bldg. C 5F 517

筋肉
Muscle2H14002H1412

中性子散乱による心筋症関連トロポニン変異体のダイナミクス測定

2H14483 次元トラッキングによる in vitro アクチンフィラメントモーティ

Dynamics of cardiomyopathy-causing mutant of troponin

リティーアッセイ

observed by neutron scattering
Tatsuhito Matsuo1, Francesca Natali3, Giuseppe Zaccai2,3,
Satoru Fujiwara1 (1QuBS, JAEA, 2CNRS, 3ILL)

Three-dimensional tracking of gliding actin filament in an invitro motility assay
Tatsuya Naganawa, Togo Shimozawa, Tomoko Masaike,
Takayuki Nishizaka (Gakushuin Univ.)

スピンラベル ESR 距離測定による細いフィラメントのアクチン、
トロポミオシン、トロポニンの動的構造解析

Structural dynamics of actin, tropomyosin, and troponin in
the thin filament as studied by distance measurements using
spin-labeling ESR
Keisuke Ueda1,2, Chenchao Zhao1, Akie Yamamoto1, Takayasu
Somiya1, Tomoki Aihara1,3, Shoji Ueki4, Masao Miki5, Toshiaki

休憩 15:00-15:10
2H1510Ca2+-independent on-off regulation of a cardiomyocyte bymicroscopic heat pulses
Kotaro Oyama1, Akari Mizuno1, Seine Shintani1, Hideki Itoh1,
Takahiro Serizawa1, Norio Fukuda2, Madoka Suzuki3,4, Shin’ichi
Ishiwata1,3,4 (1Sch. Adv. Sci. Eng., Waseda Univ., 2Dept. Cell

Arata1 (1Grad. Sch. Sci., Osaka Univ., 2Inst. Prot. Res., Osaka
Univ., 3Harima Inst., Riken, 4Tokushima-Bunri Univ., 5Univ.
Fukui)
2H1424

アクチン周りのハイパーモバイル水の形成とその熱力学的考察

Physiol., Jikei Univ. Sch. Med., 3Org. Univ. Res. Initiatives,
Waseda Univ., 4WABIOS, Waseda Univ.)

Formation of hyper-mobile water around actin and its
thermodunamic considerations
Makoto Suzuki, George Mogami, Tetsuichi Wazawa, Asato
Imao, Noriyoshi Ishida (Tohoku Univ. Grad. Sch. Eng.)
2H1436

2H1522

ミオシン分子の非線形弾性とフィラメントの弾性変形を考慮した筋収縮のシミュレーション

The Effects of Nonlinear Elasticity of Myosin Molecules on

アクチン様トレッドミリングモータが満たすべき最小条件

Muscle Contraction Studied by Numerical Simulation
Keita Miyamoto, Kazuo Sasaki (Dept. Appl. Phys., Sch. Eng.,
Tohoku Univ.)

Minimum requirements for the actin-like treadmilling motor
system
Akihiro Narita (Grad. Sch. Sci., Nagoya Univ.)

Ca2+に依存しない熱パルスによる心筋細胞の On-Off 制御

2H1534−(S52)−

高塩濃度溶液中における熱変性したミオシン分子のミオシンフィラメントへの付着

2H1558Adhesion of heat-denatured myosin molecules to myosin
filament at high salt concentration
Masato Shimada1, Eisuke Takai1, Daisuke Ejima2, Kentaro Shiraki1(1Facul. of Pure and Appl. Sci., Univ. of Tsukuba., 2Institute for

Stability of Myofilament Lattice in Striated Muscle Sarcomere
Shigeru Takemori1, Masako Kimura2, Maki Yamaguchi1,
Tetsuo Ohno1, Naoya Nakahara1, Shunnya Yokomizo3 (1Jikei
Univ. Sch. Med., 2Kagawa Nutrition Univ., 3Tokai Univ.

Innovation, Ajinomoto Co. Inc.)
2H1546

Graduate Sch. Physical Education)

ラット幼若心筋細胞内サルコメア集団の自励振動(SPOC)特性

2H1610I 会場

羽ばたくマルハナバチ中で拮抗する 2 種の飛翔筋の超高速 X 線回折像同時記録Auto-oscillation (SPOC) properties of sarcomeres in rat
neonetal cardiomyocytes
Seine Shintani1, Kotaro Oyama1,2, Norio Fukuda2, Shin'ichi
Ishiwata1,3 (1Sch. Adv. Sci. Eng., Waseda Univ., 2Dept. Cell Physiol.,
Jikei Univ. Sch. Med., 3WABIOS, Waseda Univ.)

14:00〜16:22

横紋筋サルコメアのフィラメント格子の安定性

Simultaneous ultrafast X-ray recordings of actions of two
antagonistic flight muscles during wingbeat of live bumblebee
Hiroyuki Iwamoto, Naoto Yagi (SPring-8, JASRI)

理学 E 館 1 階 131 ／ Room I: Sci. bldg. E 1F 131

細胞生物的課題 II：Prokaryotes
Cell Biology II: Prokaryotes
2I1400海洋性ビブリオ菌の周毛性べん毛形成の抑制に関与する DnaJ モ

driven stator complex from Vibrio alginolyticus

チーフを持った新規遺伝子の解析

Tetsuya Oba, Seiji Kojima, Michio Homma (Division of
Biological Science, Graduate school of science, Nagoya

A novel gene of dnaJ family plays a role in the suppression offlagellation in Vibrio alginolyticus
Takehiko Nishigaki1, Maya Kitaoka1, Kunio Ihara2, Noriko
Nishioka1, Seiji Kojima1, Michio Honma1 (1Grad. Sch. Sci., Univ.

University)
2I1522ク質間の相互作用解析

Nagoya, 2Gene., Univ. Nagoya)
2I1412

2I1424

2I1436

Analysis of interactions between rotor proteins of flagellar
motor using Fluorescence Correlation Spectroscopy
Takaaki Kishi, Seiji Kojima, Michio Homma (Division of
Biological Science, Graduate School of Science, Nagoya
University, Chikusa-ku, Nagoya, Japan.)

バクテリアⅣ型線毛の粘着脱離動力学

Unbinding Dynamics of Type IV Pili
Tomonari Sumi1,2, Rahul Marathe2, Stefan Klumpp2 (1Dept.
Comp. Sci. Eng., Toyohashi Univ. Tech., 2Dept. Theo. & Biosyst., Max Planck Inst. Colloids & Interfaces)

2I1534

異による協調作用の解析

Role of the C-terminal α-helix of FliT chaperone in the exportof its cognate substrate FliD
Tohru Minamino1, Miki Kinoshita1,2, Noritaka Hara1, Katsumi
Imada2, Keiichi Namba1,3 (1Grad. Sch. Frontier Biosci., Osaka
Univ., 2Grad. Sch. Sci., Osaka Univ., 3QBiC RIKEN)

Synergetic effects of the mutations of charged residues between
the rotor and the stator in the Na+-driven flagellar motor
Norihiro Takekawa, Seiji Kojima, Michio Homma (Div. of Biol.
Sci., Grad. Sch. of Sci., Nagoya Univ.)
2I1546

べん毛輸送装置近傍の局所 pH に対する FliI ATPase の効果

Na+駆動型べん毛モーター固定子タンパク質 PomA の細胞質ルーUniv.)
2I1558プ領域の示差走査熱量測定を用いた性質検討

CheZ 極局在の有無における大腸菌細胞内シグナル伝達の計測
Propagation of intracellular signaling molecule in the

Characterization of cytoplasmic loop of PomA, Na+-driven
flagellar stator protein, using differential scanningcalorimetry
Shiori Kobayashi1, Rei Abe-Yoshizumi1,2, Mizuki Gohara1,
Seiji Kojima1, Michio Homma1 (1Division of Biological Science,
Graduate School of Science, Nagoya University, 2Department ofFrontier Materials, Nagoya Institute of Technology)

光分解されたセリンへの大腸菌の応答計測

Measurement of cellular response of single E. coli to thephotoreleased serine
Takashi Sagawa1, Hajime Fukuoka2, Yuichi Inoue2, Hiroto
Takahashi2, Takahiro Muraoka2, Kazushi Kinbara2, AkihikoIsjijima2 (1Grad. Sch. Life Sci., Tohoku Univ., 2IMRAM, Tohoku

Biosci., Osaka Univ., 2QBiC, RIKEN)

presence and absence of polar localization of CheZ in a single
E. coli cell
Hajime Fukuoka1, Takashi Sagawa2, Yuichi Inoue1, Hiroto
Takahashi1, Akihiko Ishijima1 (1IMRAM, Tohoku Univ., 2Grad.
Sch. life Sci., Tohoku Univ.)
2I1610

休憩 15:00-15:10
2I1510

Na+駆動型べん毛モーターの固定子と回転子間における荷電残基変

細菌べん毛特異的シャペロン FliT の C 末αヘリックの役割

Effect of FliI ATPase on local pH around the bacterial
flagellar protein export apparatus
Yusuke V. Morimoto1,2, Nobunori Kami-ike1, Tomoko Miyata1,
Keiichi Namba1,2, Tohru Minamino1 (1Grad. Sch. Frontier
2I1448蛍光相関分光法（FCS）を用いたべん毛モーターを構成するタンパ

プラグを欠失した Na+駆動型べん毛モーター固定子複合体の精製・再構成系の構築

Purification and reconstitution of the plug-deleted Na+-−(S53)−

大腸菌の遊泳速度に及ぼすγ線照射の効果

Effect of gamma ray irradiation on the swimming speed ofEscherichia coli
Eriko Fujimoto1, Masakazu Furuta2, Tatsuo Atsumi3, Mikio
Kato2 (1Osaka Prefecture University School of Science, 2Osaka
Prefecture University Graduate School of Science, 3Gifu
University of Medical Science)

第 3 日目（9 月 24 日（月））／ Day3(Sep. 24, Mon.)
9:12〜11:10

A 会場

理学南館 坂田・平田ホール／ Room A: Sci. south bldg. 1F Sakata Hirata Hall

分子モーター III：F1-ATPase, マイコプラズマ
Molecular Motors III: F1 ATPase and Mycoplasma
3A0912

F1-ATPase と繊毛軸糸の機能に関わる構造変化

Engineering, The University of Tokyo, 2Graduate School of

Motions in F1-ATPase and ciliary axonemes that drive
functions
Tomoko Masaike1, Koji Ikegami2, Rinako Nakayama1, Mitsutoshi
Setou2, Takayuki Nishizaka1 (1Department of Physics, Gakushuin

Frontier Biosciences, Osaka University)
3A1022

残基はない

None of the rotor residues of F1-ATPase are essential for
torque generation

University, 2Department of Cell Biology and Anatomy, Hamamatsu

Ryohei Chiwata1, Tomoya Kawakami1, Ayako Kohori1, Shou
Furuike2, Katsuyuki Shiroguchi3, Kazuo Sutoh1, Masasuke
Yoshida4, Kazuhiko Jr. Kinosita1 (1Dept. Physics, Fac. Sci. Eng.,
Waseda Univ., 2Fac. Physics, Osaka Med. Col., 3Dept. Chem.

University School of Medicine)
3A0924高速暗視野照明による金ナノロッドの方向検出システムを用いた

F1-ATPase の回転の検出
Detection of rotation of F1-ATPase using high-speed
orientational detection of gold nanorod
Sawako Enoki1,2, Ryota Iino1,2, Hiroyuki Noji1,2 (1Grad. Sch.
Engineering., Univ. Tokyo, 2CREST)
3A0936and Chem. Biol., Harvard Univ., 4Dept. of Mol. Biosci., Kyoto
Sangyo Univ.)
3A1034

UTP を基質とした時の F1-ATPase の回転運動

Single Molecule Analysis of Inhibitory Pausing States of V1ATPase
Naciye Esma Tirtom1, Yoshihiro Nishikawa2, Daichi Okuno3,
Masahiro Nakano4, Ken Yokoyama5, Hiroyuki Noji1

3A1046

リポソーム膜中に再構成した好熱菌由来 Fo F1 - ATP 合成酵素による ATP 駆動 H+輸送の定量

Quantification of ATP-driven H+ transport by thermophilic
Bacillus PS3 FoF1-ATP synthase reconstituted in a liposomal
membrane(1Department of Applied Chemistry, School of Engineering,
University of Tokyo, 2Dept. of Biotechnology, Osaka
University, 3RIKEN, Osaka, Japan, 4The Institute of Scientific

Yuzo Kasuya1, Naoki Soga1, Toshiharu Suzuki2, Masasuke
Yoshida2, Kazuhiko Kinosita1 (1Dept. Phys., Facl. Sci. Eng.,and Industrial Research, 5Dept. of Biolmolecular Sciences,
Kyoto Sangyo University)

Waseda Univ., 2Dept. Mol. Bio., Facl. Life Sci., Kyoto Sangyo
Univ.)
3A1058

休憩 10:00-10:10
3A1010F1-ATPase のシリンダーから中心軸を引き抜く力の測定
UNBINDING FORCE MEASUREMENTS OF THE SHAFTFROM THE CYLINDER OF F1-ATPase
Tatsuya Naito, Kaoru Okada, Tomoko Masaike, Takayuki
Nishizaka (Dept Phys, Gakushuin Univ.)

Characterization of UTP driven rotation of F1-ATPase
Hidenobu Arai, Rikiya Watanabe, Hiroyuki Noji (Grad.
applchem., Univ. Tokyo)
3A0948

F1-ATPase の回転子γサブユニットに有限のトルク発生に必要なDetection of steps of Mycoplasma mobile gliding ghost
Yoshiaki Kinosita1, Daisuke Nakane2,3, Kana Mizutani1,
Makoto Miyata2, Takayuki Nishizaka1 (1Dept.phys., Gakushuin
Univ, 2Dept. Biol., Osaka City Univ, 3Present address: Mol.

F1-ATPase のトルク伝達における DELSEED ループの役割
The role of DELSEED loop in torque-transmission of F1ATPase
Kazuma Koyasu1, Rikiya Watanabe1, Mizue Tanigawara2,
Hiroyuki Noji1 (1Department of Applied Chemistry, School of

9:00〜11:22

B 会場

マイコプラズマモービレゴーストのステップ検出

Microbiol. and Immunol., Grad. Sch. Biomed. Sci., Nagasaki
Univ)

多元数理科学棟 5 階 509 ／ Room B: Mathematics bldg. 5F 509

蛋白質-構造機能相関 III：動態，生体リズム
Proteins: Structure & Function III: Dynamics and Circadian Rhythm
3B0900

原核生物由来ナトリウムチャネルにおける C 末端 4 ヘリックスバ

Kyoto Pref. Univ., 2Jpn. Syn. Rad. Res. Inst., 3Grad. Sch. of Fron.

ンドルを用いたゲーティング制御

and Sci., Univ. of Tokyo., 4Res. Ins. for Biol. Sci., Tokyo Univ. ofSci.)

The cytosolic C-terminal four-helix bundle regulates thegating of prokaryotic sodium channel

3B0912

3B0936

Mechanical control of light-induced protein conformational

Univ. Nagoya)change of photoactive yellow protein
Yasushi Imamoto, Take Matsuyama, Yoshinori Shichida (Grad.
Sch. Sci., Kyoto Univ.)[NiFe]ヒドロゲナーゼ成熟化因子 HypE，HypF の X 線結晶構
造解析
3B0948

X-ray structural analysis of HypE and HypF, maturation

3B0924

PYP の光構造変化の機械的な制御

Katsumasa Irie, Takushi Shimomura, Yoshinori Fujiyoshi (CeSPI.,

αカテニンによるアクトミオシン収縮の阻害

factors for [NiFe]-hydrogenases
Yasuhito Shomura1,2, Yoshiki Higuchi1,2 (1Grad. Sch. Life Sci.,
Univ. Hyogo, 2RIKEN/SPring-8 Center)

Inhibition of actomyosin contractility by α-catenin,
a component of adherens junctions
Shuya Ishii1, Takashi Ohki1, Hiroaki Kubota1,

Dynamic structural and antigen binding analyses of antibody
single-chain Fvs
Yusuke Tanaka1, Hiroshi Sekiguchi2, Yuji C. Sasaki3, Takachika
Azuma4, Masayuki Oda1 (1Grad. Sch. of Life and Environ. Sci.,

Ishiwata1,2,3 (1Dept. of Phys., Faculty of Sci. and Eng., Waseda−(S54)−

Shin'ichi

Univ., 2Adv. Res. Inst. for Scie. and Eng., Waseda Univ., 3WABIOS,
Waseda Univ.)

3B1046

休憩 10:00-10:10
3B1010Interactions between cyanobacterial clock proteins KaiA and
KaiC revealed by ESR analysis
Kentaro Ishii1, Toshiaki Arata2, Masahiro Ishiura1 (1Center forgene research Nagoya Univ., 2Grad. Sch. of Science, Osaka

ダイニンモータードメインの溶液中の分子動力学シミュレーション

Molecular dynamics simulations of dynein motor domain in
explicit water
Narutoshi Kamiya1, Tadaaki Mashimo2, Yu Takano1, Takahide
Kon1, Genji Kurisu1, Haruki Nakamura1 (1Institute for Protein
3B1022

Univ.)
3B1058Autodephosphorylation of cyanobacterial circadian clock
protein KaiC occurs via formation of ATP as an intermediate
Taeko Ohkawa-Nishiwaki, Takao Kondo (Div.Biol.Sci., Grad.
Schl. Sci., Nagoya Univ.)
3B1110

シアノバクテリアの時計タンパク質 KaiC の ATPase に備わる分
子内フィードバック制御(1RIKEN Research Center for Allergy and Immunology, 2RIKEN
Advanced Science Institute, 3RIKEN Quantitative Biology
Center)
3B1034

ATP を中間体とするシアノバクテリア概日時計蛋白質 KaiC の新規脱リン酸化機構

Research, Osaka Univ., 2BIRC, AIST)
A computational Investigation into the MHC-I Recognition
Mechanism of MIR2 from Kaposi's Sarcoma-Associated
Herpesvirus
Pai-Chi Li1,2, Naoyuki Miyashita3, Satoshi Ishido1, Yuji Sugita2

時計タンパク質 KaiA-KaiC 相互作用の ESR 解析

タンパク質構造変化経路予測法の新規開発

Intramolecular feedback regulation of cyanobacterial KaiC
ATPaseAtsushi Mukaiyama1,2, Yasuhiro Onoue1,2, Masato Osako1,2,

A Novel Method to simulate protein conformational change
upon ligand binding

Takao Kondo1,2, Shuji Akiyama3 (1Grad. Sch. of Sci., Nagoya
Univ., 2CREST/JST, 3IMS, SOKENDAI)

Koichi Tamura, Shigehiko Hayashi (Grad. Sch. Sci., Univ.
Kyoto)

9:00〜11:22

C 会場

多元数理科学棟 4 階 409 ／ Room C: Mathematics bldg. 4F 409

核酸
Nucleic Acids
3C0900

DNA/RNA 結合タンパク質 TDP-43 が特異的な塩基配列を認識

Naoto Hori, Shoji Takada (Grad. Sch. Sci., Kyoto Univ.)

するメカニズム

休憩 10:00-10:10

Tandem-like fusion of two heterologous RNA-recognition
motifs in TDP-43 enhances the specificity to its target
nucleotide sequence
Yo Suzuki, Yoshiaki Furukawa (Dept. of Chem, Keio Univ.)
3C0912

3C0924

3C1010

A structural basis for the antibiotic resistance conferred byan A1408G mutation in 16S rRNA
Jiro Kondo (Fac. Sci. Tech., Sophia University)

Hybridization Rates of DNA Strands with Unstable Selffolded Secondary Structures
Hiroaki Hata1, Akira Suyama1,2 (1Grad. Sch. Sci., Univ.
Tokyo, 2Grad. Sch. Arts and Sci., Univ. Tokyo)

3C1022Q β replicase を構成する翻訳因子の RNA 合成における役割

長鎖 DNA の 1 分子内折り畳みはカチオン性ポリマー凝縮剤の長3C1034

不安定な二次構造をつくる DNA のハイブリダイゼーション速度

Non-canonical functions of translational factors as RNA
replication cofactors
Daijiro Takeshita, Kozo Tomita (Biomed. Res. Inst., AIST)
反応機構の解明

Folding of a Single Giant Duplex DNA Chain Expresses

Elucidation of the location-dependent deamination reaction

Contrastive Behaviors depending on the Length of Cationic
Polymer
Tatsuo Akitaya1, Norio Hazemoto2, Toshio Kanbe3, Makoto

mechanism of an anti-HIV factor, APOBEC3G
Ayako Furukawa1,2, Kenji Sugase2, Ryo Morishita3, Taashi
Nagata1, Akifumi Takaori4, Akihide Ryo5, Masato Katahira1

Demura4,(1Inst. of Adv. Energy, Kyoto Univ., 2Bioorg. Res. Inst., Suntory
Found. Life Sci., 3CellFree Sci., 4Grad. Sch. Med., Kyoto
Univ., 5Grad. Sch. Med., Yokohama City Univ.)

Hideaki Yamaguchi1, Koji Kubo5, Anatoly
Zinchenko , Shizuaki Murata5, Kenichi Yoshikawa6 (1Fac.
Pharm., Meijo Univ., 2Grad. Sch. Pharm. Sci., Nagoya City
5

Univ., 3Sch. Med., Nagoya Univ., 4Grad. Sch. Life Sci., Hokkaido
Univ., 5Grad. Sch. Env. Study, Nagoya Univ., 6Grad. Sch. Life
Med. Sci., Doshisha Univ.)
3C0936

3C1046

き戻す

インテリジェントリボザイムの創製
3C1058リボソーム-SecM 翻訳アレスト配列間相互作用の 1 分子顕微解析

Single-molecule force measurement for the interaction
between ribosome and SecM arrest sequence
Zhuohao Yang, Ryo Iizuka, Takashi Funatsu (Grad. Sch. of

Yudai Yamaoki1,2, Tsukasa Mashima1,2, Yu Sakurai3, Yukari
Hara3, Takashi Nagata1,2, Masato Katahira1,2 (1Inst. Adv. Energy,
Kyoto Univ., 2Grad. Sch. Energy Sci., Kyoto Univ., 3Grad. Sch.
3C1110Nanobio., Yokohama City Univ.)

大腸菌非六量体型 DNA ヘリカーゼ UvrD は多量体で DNA を巻

Single-molecule visualization of a non-hexameric helicase
reveals active roles of its oligomeric forms in DNA unwinding
Hiroaki Yokota, Yoshie Harada (iCeMS, Kyoto Univ.)

カリウムイオン依存的な四重鎖構造形成によって活性がオンになる

Development of intelligent ribozyme whose activity switches
on in response to K+ via quadruplex formation

3C0948

抗 HIV 宿主因子 APOBEC3G の位置依存的デアミネーション

さによってモードが変わる

翻訳伸長におけるリボソームと tRNA の挙動−粗視化分子シミュ
レーションによる解析

Dynamical motions of tRNA and ribosome complex duringtranslation elongation studied by coarse-grained molecularsimulations−(S55)−

Pharm. Sci., The Univ. of Tokyo)
The probability of double-strand breaks in genome-sized
DNA decreases markedly as the DNA concentration increases
Shunsuke Shimobayashi1, Takafumi Iwaki2, Toshiaki Mori3,
Kenichi Yoshikawa1,4 (1Grad. Sch. of Sci., kyoto Univ., 2Fukui
Inst., Kyoto Univ., 3Rad. Res. Cent., Osaka Pref. Univ., 4Grad.
Sch. of Life and Med. Sci., Doshisha Univ.)

9:00〜11:10

D 会場

多元数理科学棟 3 階 309 ／ Room D: Mathematics bldg. 3F 309

光生物-光合成
Photobiology: Photosynthesis
3D0900

膜配向した PS II の ENDOR 法による Mn クラスター周辺のプ

布: 時間平均 XRD 構造の照射 X 線量依存性

X-ray dose dependence of time-averaged XRD structure ofmulti-photoreduced Mn4CaO5Hx clusters in photosystem II
Masami Kusunoki (Department of Physics, School of Science
Technology, Meiji University)
3D1022光化学系 II 酸素発生系 S2状態における Mn 間磁気的相互作用の解明

Determination of magnetic couplings of S2 state Mn-cluster in
Photosystem II studied by PELDOR measurement
Mizue Asada1, Hiroki Nagashima1, Faisal Hammad Mekky

BLUF ドメインの光反応における水素結合環境の変化

Nagoya, 2Grad. Sch. Natl. Sci. & Tech., Univ. Okayama)

Koua2, Jian-Ren Shen2, Hiroyuki Mino1 (1Grad. Sch. Sci., Univ.
3D1034

光化学系 II におけるキノン電子受容体の酸化還元電位制御機構

Mechanism of controlling the redox potentials of the quinone

Tatsuya Iwata1, Shota Ito2, Mineo Iseki3, Masakatsu Watanabe4,
Hideki Kandori2 (1Cent. Fost. Young Innov. Res., NITech, 2Dept.
Front. Mater., NITech, 3Faculty Pharm. Sci., Toho Univ., 4Grad.

electron acceptors in photosystem II
Ryota Ashizawa1, Takuya Iwasa1, Miwa Sugiura2, Takumi
Noguchi1 (1Division of Material Science, Graduate School ofSch. Creat. New Photo. Ind.)

Science, Nagoya University, 2Division of Material Science,
Graduate School of Science, Nagoya University)

励起移動を記述する変分マスター方程式の改良および PSII への適用

Improvement of variational master equation describing
excitation energy transfer, and its application to PSII
Yuta Fujihashi, Akihiro Kimura (Department of Physics,
Graduate School of Science, Nagoya University)
3D0948at low temperature

the Schiff bases in bacteriorhodopsin and Anabaena sensory
rhodopsin
Hiroshi Ishikita1,2, Keisuke Saito1, Hideki Kandori3 (1CareerPath, Kyoto U, 2JST PRESTO, 3Nagoya Inst Tech)
Structural changes of hydrogen-bonding environment upon
the photoreaction of the BLUF domains

3D0936低温光化学系 II における多重光還元 Mn4CaO5Hxクラスター分

Determine the location of protons surrounding Mn cluster of
PS II by proton matrix ENDOR using oriented PS II
membrane
Hiroki Nagashima, Hiroyuki Mino (Division of Material
Science, Graduate School of Science, Nagoya University)
3D0912 Factors that differentiate the H-bond strengths of water near

3D0924

3D1010ロトンの位置の決定

3D1046

Electron Transfer Pathway Analysis in Bacterial
Photosynthetic Reaction Center
Hirotaka Kitoh-Nishioka, Koji Ando (Grad. Sch. Sci. Kyoto
Univ.)

光化学系 II コア複合体での非光化学的消光の可能性

Possible non-photochemical quenching mechanism withinpohotosystem II core complex
Yutaka Shibata1, Shunsuke Nishi2, Keisuke Kawakami3, Jian-Ren
Shen4, Thomas Renger5 (1Grad. Sch. Sci., Tohoku Univ., 2Grad. Sch.
Sci., Nagoya Univ., 3Adv. Res. Inst., Osaka City Univ., 4Grad. Sch.

3D1058

化学制御

休憩 10:00-10:10

E 会場

光合成細菌 Rhodopseudomonas の遺伝子発現パターンの電気Electrochemical regulation of gene expression profiles of
Rhodopseudomonas, a photosynthesis bacterium
Yue Lu1, Syoichi Matsuda1, Shuji Nakanishi2, Kazuhito
Hashimoto1 (1The Univesity of Tokyo, School of Engineering,
Department of Applied Chemistry, 2Research Center for
Advanced Science and Technology)

Sci., Okayama Univ., 5JKU Linz)

9:00〜11:22

光合成バクテリア反応中心の電子移動経路解析

多元数理科学棟 1 階 109 ／ Room E: Mathematics bldg. 1F 109

蛋白質-構造Proteins: Structure
3E0900

核膜孔複合体構造変化による核移行制御機構の構造基盤

Structural basis for nuclear import regulated by molecularrearrangements in the nuclear pore complex
Junya Kobayashi1, Yoshiyuki Matsuura1,2 (1Div. Biol. Sci.,
Grad. Sch. Sci., Nagoya Univ., 2Strl. Biol. R. Ctr., Grad., Nagoya
Univ.)
3E0912

inhibitor of fibrillation as revealed by NMR and docking
simulation
Ken Takeuchi1, Hikari Watanabe1, Javkhlantugs Namsrai1,
Hiroshi Hirota2, Akira Naito1 (1Yokohama National University
Graduate School of Engineering, 2Riken)
3E0948

単粒子クライオ電子顕微鏡解析によるサポウイルスキャプシドのNADH シトクロム b5還元酵素超高分解能結晶構造解析による水8-Å 構造素と外殻電子の観察8-Å structure of sapovirus capsid by single particle electron
cryomicroscopy

Visualizing hydrogens and outer-shell electrons of NADH
cytochrome b5 reductase by ultra-high resolution crystallography
Kiyofumi Takaba1, Kazuki Takeda1, Masayuki Kosugi1, Taro

Naoyuki Miyazaki1, David Taylor1, Grant Houseman2, Kosuke
Murakami2, Kuniaki Nagayama1, Kazuhiko Katayama2,

Tamada2, Ryota Kuroki2, Kunio Miki1 (1Grad. Sch. Sci., Univ.

Kazuyoshi Murata1 (1National Institute for Physiological
Sciences, 2National Institute of Infectious Diseases)

2

Kyoto, JAEA)
3E0924最新の各種固体 NMR 法を用いた絹の構造解析

3E0936

Silk Structure studied by Newly-developed Solid-state NMR
Keiko Okushita, Koji Yazawa, Akihiro Aoki, Tetsuo Asakura(Grad. Sch. of Eng., Tokyo Univ. Agr. Tech.)Interaction of human calcitonin with curcumin as an

休憩 10:00-10:10
3E1010−(S56)−

Structural analysis of the 26S proteasome by cryo-electron
microscopy and Single-Particle Analysis

Zhuo Wang1, Yasuo Okuma1, Daisuke Kasuya2, Kaoru
Mitsuoka3, Yasushi Saeki4, Yasunaga Takuo1 (1Department ofAnalyses of folding processes on the Ferredokin-like foldproteins by means of a coarse grained Go model
Masatake Sugita, Takeshi Kikuchi (Dept. Bioinf., Coll. Life Sci.,

Bioscience and Bioinformatics, Faculty of Computer Science andSystems Engineering, Kyushu Institute of Technology,2
Biomedicinal Information Research Center, Japan Biologycal

Ritsumeikan Univ.)
3E1046構造の最適化

Information Consortium (JBIC), 3Biomedicinal Information
Research Center, National Institute of Advanced Industrial
Science and Technology, 4Laboratory of Protein Metabolism,
3E1022

Tokyo Metropolitan Institute of Medical Science)

Optimization of protein model structures according tostatistical potentials with different reference states
Matsuyuki Shirota1,2, Kengo Kinoshita1,2,3 (1GSIS, Tohoku

大気圧電子顕微鏡（ASEM）によるタンパク質微結晶とマイコプラ

Univ., 2ToMMo, Tohoku Univ., 3IDAC, Tohoku Univ.)

ズマの液中観察3E1058

クーロンレプリカ交換法を用いた A βフラグメントに関する研究

Direct electron microscopy of protein crystals andMycoplasma cells in solution using the Atmospheric SEM

Studies on a Aβ fragment by the Coulomb replica-exchange
methodYuusuke Maruyama1, Tatsuhiko Ebihara1, Daisuke Nakane2,
Hidetoshi Nishiyama3, Takayuki Nishizaka4, Miki Senda5,
Kazuhiro Mio1, Mitsuo Suga3, Toshiya Senda1, Makoto Miyata2,
Chikara Sato1 (1AIST, 2Univ. Osaka City, 3JEOL, 4Univ.

Satoru Itoh1,2, Hisashi Okumura1,2 (1Institute for Molecular
Science, 2The Graduate University for Advanced Studies)
3E1110

レプリカ交換インターフェースプログラム(REIN)

Replica-exchange interface program (REIN)
Naoyuki Miyashita1,2,3, Suyong Re4, Yuji Sugita1,2,3,4 (1RIKEN
QBiC, 2RIKEN CSRC, 3RIKEN AICS, 4RIKEN ASI)

Gakushuin, 5JBIC)
3E1034

異なる参照状態を用いた統計ポテンシャルによるタンパク質モデル

粗視化 Go モデルを用いた Ferredoxine-like fold タンパクのフォールディング機構の解析

9:00〜11:10

F 会場

理学 B 館 5 階 501 ／ Room F: Sci. bldg. B 5F 501

膜蛋白質
Membrane Proteins
3F0900

サポーテット膜を用いた膜タンパク質の顕微鏡下でのイオン輸送活

3F1010と圧力特性

Evaluation of ion-pump activity of membrane proteinsreconstituted on supported membrane under optical microscope

Structure and lateral pressure profile of lipid bilayer
containing gramicidin A by molecular dynamics simulation
Hiroaki Saito, Masashi Iwayama, Megumi Nishimura, Hiroyuki
Takagi, Kazutomo Kawaguchi, Hidemi Nagao (Kanazawa
University)

Jyunan Ki, Rikiya Watanabe, Kazuhito Tabata, Hiroyuki Noji
(Department of Applied Chem., Univ. Tokyo)
3F0912

Catalytic activity of MsbA reconstituted in nanodisc particles
is modulated by remote interactions with the bilayer
Takeaki Kawai1, Jose Caaveiro1,2, Toyomasa Katagiri3, Hisashi
Tadakuma1, Takuya Ueda1, Kouhei Tsumoto1,2,4 (1Grad. Sch. of

3F1022がアゴニストをインバースアゴニストに変える

線虫イネキシン 6 ギャップ結合チャネルの単離と機能特性

Isolation and characterization of C. elegans innexin-6 gap
junction channels
Atsunori Oshima1, Tomohiro Matsuzawa2, Kouki Nishikawa1,
Yoshinori Fujiyoshi1 (1CeSPI, Nagoya Univ., 2Grad. Sch. of Sci.,
3F0936

3F1034non-detergent sulfobetaine (NDSB)による G 蛋白質共役型受容体 (GPCR)の熱安定化

Kyoto Univ.)

Thermal stabilization of G protein-coupled receptor by non-ATR-FTIR 法を用いた KcsA 野生型と不活化しない変異体に対

detergent sulfobetaines (NDSBs)
Toshihide Yamai, Layla Takahashi, Masato Nakajima, Naoki

するカチオン誘起構造変化の赤外分光分析

Cation-induced structural changes in the WT and noninactivating mutant KcsA studied by ATR-FTIR
Chikako Muramatsu1, Masayo Iwaki1, Tetsuya Fukuda1,
Yusuke Asai1, Yuji Furutani1,2, Hideki Kandori1 (1Grad. Sch.
Tech., Nagoya Inst. Tech., 2Inst. Mol. Sci.,)
3F0948代謝型グルタミン酸受容体の膜貫通領域における 1 アミノ酸変異

Single amino acid substitution in the transmembrane domainof metabotropic glutamate receptor changes an agonist into
an inverse agonist
Masataka Yanagawa, Takahiro Yamashita, Yoshinori Shichida(Department of Biophysics, Graduate School of Science, Kyoto
University)

Front. Sci., Univ. of Tokyo, 2Inst. Med. Sci., Univ. of Tokyo, 3Inst. Gen.
Res., Univ. of Tokushima, 4Dep. Chem. Bio., Univ. of Tokyo)
3F0924

分子動力学法によるグラミシジン A を含んだ脂質二重層膜の構造性計測

Yamashita, Takeshi Ishii, Shigeki Takeda, Kaori Wakamatsu(Gunma Univ.)
3F1046ドプシンの構造と運動性変化の解析

Thermal and Pressure induced structural and dynamics

黄色ブドウ球菌の 2 成分性膜孔形成毒素の分子機構

changes of bacteriorhodopsin as studied by solid-state NMR
Izuru Kawamura1, Miyako Horigome1, Hirohide Nishikawa1,
Kana Tajima1, Takashi Okitsu2, Akimori Wada2, Satoru Tuzi3,
Tatsuo Iwasa4, Akira Naito1 (1Grad. Sch. Eng., Yokohama Natl.
Univ., 2Kobe Pharm. Univ., 3Univ. Hyogo, 4Muroran Inst. Tech.)

Molecular basis of staphylococcal bi-component pore forming
toxin
Yoshikazu Tanaka1, Daichi Yamashita2, Keitaro Yamashita2,
Yuka Kawai2, Jun Kaneko3, Noriko Tomita4, Makoto Ohta4,
Yoshiyuki Kamio5, Min Yao1, Isao Tanaka1 (1Fac. of Adv. Life

固体 NMR を用いた熱および圧力により誘起されるバクテリオロ

3F1058

Sci., Hokkaido Univ., 2Grad. Schl. of Life Sci., Hokkaido
Univ., 3Grad. Schl. of Agri. Sci., Tohoku Univ., 4Inst. of Fluid
Sci., Tohoku Univ., 5Grad. Schl. of Life Sci., Tohoku Univ.)
休憩 10:00-10:10−(S57)−OBSERVATION OF TRANSMEMBRANE PROTEIN BY
HIGH SPEED ATOMIC FORCE MICROSCOPY:BACTERIORHODOPSIN D85S MUTANT, A CHLORIDE
PUMP
Maxime Ewald1, Mikihiro Shibata2, Takayuki Uchihashi1,3,
Hideki Kandori4, Toshio Ando1,3 (1School of Mathematics &Physics, Institute of Science & Engineering, Kanazawa

University, 2Department of Neurobiology, Duke University
Medical Center, 3Bio-AFM Frontier Research Center, College of

9:00〜11:22

G 会場

Science and Engineering, Kanazawa University, 4Department ofFrontier Materials, Nagoya Institute of Technology)

理学 B 館 2 階 212 ／ Room G: Sci. bldg. B 2F 212

生体膜・人工膜-構造・物性
Biological & Artificial Membranes: Structure & Property
3G0900

好熱性 Meiothermus ruber H328 株が産生放出する高耐性ケラ

3G1010チン分解性プロテアーゼ複合体のフリーズレプリカ電子顕微鏡観察

Collapse of nonuniform phospholipid monolayers
Masahiro Hibino, Ken Hashimoto, Takuya Fujisawa (Div. Appl.

Freeze-replica observation of keratinolytic protease complex
produced and released from Meiothermus ruber H328
Kazunori Kawasaki1, Maachi Kataoka2, Keiji Nomura2,
Yasushi Shigeri1, Kunihiko Watanabe2 (1AIST, 2Grad. Sch.

Sci., Muroran Inst. Technol.)
3G1022

ジャイアントベシクル

間的制御

Microsphere-containing giant vesicles prepared by water-in-

Spatially controlled accumulation of membrane-boundprotein in a micro-patterned model biological membrane
Fumiko Okada1, Kenichi Morigaki1,2 (1Grad. Sch. Agr., Univ.

oil emulsion centrifugation method
Yuno Natsume1, Taro Toyota1,2 (1Grad. Art. Sci., Univ. Tokyo,
2

JST PRESTO)

3G1046

Kobe, Res. Cent. Env. Genomi., Univ. Kobe)
圧力摂動熱量法によるジミリストイルホスファチジルコリン二分子

An attempt to reveal the relaxation behavior ofdimyristoylphosphatidylcholine bilayer by pressure
perturbation calorimetry
Nobutake Tamai, Sayuri Kakibe, Saeko Tanaka, Masaki Goto,
Hitoshi Matsuki (Dept. Life System, Inst. Technol. & Sci., Univ.
of Tokushima)

3G1058Initial Step of Low pH-Induced Lamellar to Bicontinuous Cubic
Phase Transition in Dioleoylphosphatidylserine/Monoolein
Toshihiko Oka1, Tomoki Takahashi1, Taka-aki Tsuboi1,
Masahito Yamazaki2 (1Fac. Sci., Shizuoka Univ., 2Grad. Sch.
Sci. Tech., Shizuoka Univ.)

限界
3G1110

H 会場

X 線及び電子線回折法を用いた皮膚角層の構造解析
Breakthrough for Unresolved Structural Problems in Skin
Function by Combined Use of X-ray and Electron Diffraction
Methods
Hiromitsu Nakazawa1, Ichiro Hatta2, Satoru Kato1 (1Sch. Sci.

休憩 10:00-10:10

9:00〜11:22

低い pH が誘起する DOPS/MO 膜の液晶相からキュービック相への相転移の初期過程

リン脂質二重膜の圧力誘起指組み構造形成：疎水鎖長依存性と形成

Pressure-induced interdigitation of phospholipid bilayer
membranes: dependence of acyl-chain length and limitationof the formation
Hitoshi Matsuki, Masaki Goto, Nobutake Tamai (Institute ofTechnology and Science, The University of Tokushima)

オイルフリー GUV に封入された分子演算システム RTRACS

Molecular computing system RTRACS encapsulated in oilfree giant unilamellar vesicle
Koh-ichiroh Shohda, Tadashi Sugawara, Akira Suyama (Grad.
Sch. Arts Sci., Univ. Tokyo)

膜の緩和挙動の解明への試み

3G0948

油中水滴エマルション遠心沈降法を用いたミクロスフェア内包型

微細パターン化モデル生体膜における膜結合タンパク質の濃縮の空2

3G0936

structures: lateral domains and vesicular shapes
Hiroki Himeno, Tsutomu Hamada, Masahiro Takagi (1Shool ofMaterials Science, Japan Advanced Institute of Science andTechnology)
3G1034

Informatics, Tokyo Inst. Tech.)

脂質分子の電荷が引き起こす膜構造変化：2 次元相分離と 3 次元曲率

Charge-induced transition in membrane mesoscopic

Kyoto Pref. Univ.)
3G0912 Effects of model peptides for late embryogenesis abundant(LEA) proteins on the thermal properties of liposomes
Takao Furuki, Minoru Sakurai (Center for Biol. Resources and3G0924

均一でないリン脂質単分子膜における崩壊現象について

Tech. kwansei Gakuin Univ., 2Nagoya Industrial Science
Research Inst.)

理学 C 館 5 階 517 ／ Room H: Sci. bldg. C 5F 517

細胞生物的課題 III：細胞骨格，細胞運動
Cell Biology III: Cytoskeleton & Motility
3H0900

GTP 結合状態と GDP 結合状態の微小管分子構造における大きな

屈曲運動

構造変化

Resurrection of flagellar bending movements inchlamydomonas paralyzed mutants at high pressure
Toshiki Yagi1, Masayoshi Nishiyama2 (1Grad. Sch. Medicine,Univ. Tokyo, 2The Hakubi Center, Kyoto Univ.)

Large Conformational Changes in Tubulin in the GTP- and GDPStates Microtubules Observed by Cryo Electron Microscopy
Hiroaki Yajima1, Toshihiko Ogura2, Ryo Nitta1, Yasushi
Okada1, Chikara Sato2, Nobutaka Hirokawa1 (1Grad. Sch. Med.,

3H0936

Univ. Tokyo, 2Biomedical Research Inst., AIST)
3H0912

Directed cell migration induced by travelling waves ofchemoattractant

紡錘体は一方の極から他方へ構造変化を伝搬することで対称形状を維持する

Meiotic spindles maintain the symmetrical shape by
propagating structural changes to the opposite side
Kazuya Suzuki1, Jun Takagi1, Takeshi Itabashi1, Shin'ichi
Ishiwata1,2 (1Dept. Phys., Adv Sci. Eng., Waseda Univ.,
2
WABIOS)
3H0924

走化性誘因物質の進行パルス刺激によって誘発された一方向細胞運動

Akihiko Nakajima1, Satoshi Sawai1,2 (1Grad. Sch. Arts & Sci.,
Univ. Tokyo, 2Res. Cent. for Complex Systems Biology, Univ.
Tokyo)
3H0948高圧負荷によって誘導されるクラミドモナス非運動性変異株鞭毛の−(S58)−

極性を持つアメーバ細胞の走化性運動に関する理論

Theory on the chemotaxis of an amoeboid cell with the cell
polarity
Tetsuya Hiraiwa, Tatsuo Shibata (Kobe, Riken)

A distinct role for the dynamics of p130Cas at focal adhesion
via interaction with Src in the regulation of cell migration

休憩 10:00-10:10
3H1010Controlled cell migration with ultrasoundShinya Murakami1, Yo Otsuka1, Manabu Sugimoto1, Toshiyuki
Mitsui2 (1Grad. Sci. Eng., Aogaku Univ., 2Assoc Prof., Aogaku
Univ.)

3H1022

Hiroaki Machiyama1,2, Hiroaki Hirata1, Yasuhiro Sawada1,2(1Mechanobiology Inst., Nat. Univ. Singapore, 2Dept. Biol. Sci.,
Nat. Univ. Singapore)
3H1058筋肉と異なるストレスファイバ−の収縮特性

Gowrishankar4, Satyajit Mayor5, Madan Rao4 (1iCeMS, Kyoto
University, 2Rudolf Peierls Centre for Theoretical Physics,
University of Oxford, UK, 3Raman Research Institute,Bangalore, India, 4NCBS-TIFR, Bangalore, India)

Contractile properties of stress fibers are distinct from thoseof muscles
Shinji Deguchi, Tsubasa Matsui, Daiki Komatsu, Masaaki Sato
(Tohoku University)
3H1034 Smooth muscle differentiation related transcription factor

3H1046

Spatiotemporal regulation of signaling by active cytoskeletal
remodeling
Bhaswati Bhattacharyya1, Abhishek Chaudhuri3, Kripa

3H1110Arp2/3 とアクチンを内包した油中液滴のフィロポディア様変形

CRP2 directly regulates physical properties of actin filaments
Takanori Kihara1, Satoko Shinohara1, Yasunobu Sugimoto2,
Jun Miyake1 (1Grad. Sch. Eng. Sci., Osaka Univ., 2Nagoya Univ.
Synchrotron Radiat. Res. Center)

Filopodia-like protrusions in water-in-oil droplets induced by
Arp2/3 and actin polymerization
Masataka Chiba1, Makito Miyazaki1, Takashi Ohki1, Shin'ichi
Ishiwata1,2 (1Dept. of Phys., Waseda Univ., 2WABIOS, Waseda

細胞接着班における p130Cas と Src の動的相互作用を介した遊Univ.)

走制御機構の解明

9:12〜11:10

I 会場

理学 E 館 1 階 131 ／ Room I: Sci. bldg. E 1F 131

ヘム蛋白質
Heme Proteins
3I0912

3I0924

ヘモグロビンのアロステリック平衡のリアルな描像

Masayuki Hara1, Sachiko Yanagisawa1, Hiroshi Sugimoto2,

A realistic picture of allosteric equilibrium of hemoglobin
Naoya Shibayama1, Sam-Yong Park2 (1Biophysics, Jichi Med.
Univ., 2Protein Design Lab., Yokohama City Univ.)

Yoshitsugu Shiro2, Takashi Ogura1 (1Grad. Sch. Sci., Univ.
3I1022

ヘムをヘム垂線のまわりに 90°回転した時の共鳴ラマンスペクト
ル変化：ヘムオキシゲネース

Effects of Heme Rotation around the Heme Normal by 90°
on Resonance Raman Spectra of Heme Proteins; Observationfor Heme Oxygenase
Sachiko Yanagisawa1, Hiroshi Fujii2, Saburo Neya3, Takashi
Ogura1, Teizo Kitagawa1 (1University of Hyogo, 2Okazaki Inst.

3I1034Time-Resolved IR Analyses of Proteins Using a Novel Pulse
Flow System Synchronized with Pump/Probe Lasers

ヘムオキシゲナーゼにおける CO 光解離後の構造変化および COの放出経路

Minoru Kubo1, Satoru Nakashima1, Masao Mochizuki1, Kyoko
Shinzawa-Itoh2, Shinya Yoshikawa1,2, Takashi Ogura1,2 (1Picobiology
Inst., Grad. Sch. Life Sci., Univ. Hyogo, 2Dept. Life Sci., Grad. Sch.

Protein motions and CO migration following CO photolysis
in heme oxygenase
Masakazu Sugishima1,2, Keith Moffat2,3, Masato Noguchi1 (1Dept.
Med. Biochem., Kurume Univ. Sch. Med., 2Dept. Biochem. & Mol.
Biol., Univ. Chicago, 3BioCARS, Univ. Chicago)
3I0948Life Sci., Univ. Hyogo)
3I1046チトクロム c 酸化酵素のヘム a 側鎖に由来する共鳴ラマン線の帰属

The assignment of the resonance Raman bands of heme a

多様な生物種によるヘムオキシゲナーゼ反応の微調整戦略：オキシ

peripheral substituents in cytochrome c oxidase

ヘムの安定化Miyuki Sakaguchi1, Yukie Katayama2, Hiroshi Fujii3, Hideo
Shimada2, Takashi Ogura1,2 (1Grad. Sch. Life. Sci., Univ.

Strategy of a variety of organisms for the fine-tuning of heme
oxygenase reactions: Stabilization of the oxyheme
intermediate
Sayuri Takada, Taiko Migita (Dep. Biol. Chem., Fac. Agr.,
Yamaguchi Univ.)

Hyogo, 2Picobiol. Inst., Grad. Sch. Life. Sci., Univ. Hyogo, 3OkazakiInst. Integr. Biosci.)
3I1058シアン結合型構造から推察される一酸化窒素還元酵素の反応機構

Functional Implications from Structural Characterization of
CN-bound Nitric Oxide Reductase
Takehiko Tosha, Nozomi Sato, Norihiro Okada, Hiroshi
Sugimoto, Yoshitsugu Shiro (Harima Inst., RIKEN)

休憩 10:00-10:10
3I1010ポンプ・プローブレーザーと同期したパルスフローシステムの開発とそれを用いたタンパク質の時間分解赤外分光解析

Integrative Biosci., 3Chiba University)
3I0936

Hyogo, 2Harima Inst., Riken)
An Intermediate Conformation of Cytochrome Oxidase in the
Ligand-Free State Identified by Time-Resolved ResonanceRaman Spectroscopy
Izumi Ishigami, Takeshi Nishigaki, Kyoko Shinzawa-Itoh,
Shinya Yoshikawa, Satoru Nakashima, Takashi Ogura (Grad.
Schl. Life Sci., U. Hyogo)

Ultraviolet Resonance Raman Study on Indoleamine 2, 3Dioxygenase−(S59)−

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1SA-01

細胞内反応ネットワークと「少数性」問題〜理論と計算によるアプ
ローチ

Cracking Reaction Networks Involving "Minorities" in the
Cell: Theoretical and Computational Approaches
Yuichi Togashi (Grad. Sch. Sys. Informat., Kobe Univ.)
The activities of biological systems are maintained primarily by chemical
reactions, and often modeled as reaction-diffusion systems represented bypartial differential equations for the "concentrations" of chemicals. However,this classical scheme requires some preconditions. First, each reaction event isinstantaneously completed and not affected by previous reactions. Secondly, themolecules diffuse freely and their excluded volume can be neglected. These
conditions are however not always satisfied; many of enzymes are molecularmachines whose reaction cycles and motions are coupled, and cells are highly
crowded with macromolecules. Moreover, recent experiments have shownthat there are a number of "minority" chemicals, existing only one or a
few molecules per cell. For such rare species, the notion of continuous"concentrations" is no longer valid, and the classical scheme depending on it
breaks down. Using simple autocatalytic models, we previously showed that themolecular discreteness in such rare chemicals, either discreteness in numbers(i.e. integerness) or spatial discreteness (i.e. finite space between molecules),may affect the reaction-diffusion behavior. Toward a theoretical framework to
predict phenomena in the complex molecular networks in the cell, we have
expanded the models for a number of chemical species. In some cases, thebehavior strongly depends on the system size, which suggests importance ofbeing moderately small. Relevance to cell behavior and possible experimental
designs will be also discussed.
1SA-02

1 分子デジタル ELISA

Single-Molecule Digital ELISA
Hiroyuki Noji (Applied Chem. U-Tokyo)
I will introduce the single-molecule digital counting assay based on the arraysystem of a million of water-in-oil droplets that we have recently developed(Lab on a chip 2010). Especially, I will focus the digital ELISA assay as one ofapplication methods of the digital counting. The detection principle of thesingle-molecule digital counting is very simple; by encapsulating a single
molecule of enzyme or enzyme-conjugated antibody in a micron-sized reactor,
the catalytic product molecules are highly accumulated in the micron space.
Individual chambers resultantly give detectable signal, fluorescence fromreaction product molecules in general. By counting the number of chamberswith evidently high fluorescent signal under an optical microscope, the numberof target molecule is determined. As a model reaction, we conducted digital
ELISA of the marker molecule of prostate tumor (PSA). The detection limit was
revealed to be 2 aM that is million-times lower than that of conventional ELISA
assay of PSA, demonstrating the extremely high sensitivity of digital counting
assay. In the last part of the talk, I will also discuss about the prospective of thedigital counting assay for single cells assay in addition to the integration to
CMOS imaging sensor for palm-top digital counting devise.
1SA-03遺伝子にコードされた発光型プローブ

Genetically encoded luminescent probes
Takeharu Nagai1,2 (1ISIR, Osaka Univ., 2PRESTO, JST)
Optogenetic tools including ChR2, LOV domain, and CALI (chromophoreassisted light inactivation) allows us to operate biomolecule function upon lightirradiation, by which we are now able to investigate protein of interest in termsof functional difference according to the existing area inside cells andexpression stage with real time resolution. To understand what‘s going on after
the light-switching of optogenetic tools, many researcher may want to visualize
some other molecular dynamics and function by fluorescence imaging.
However, excitation light for the imaging should mis-activate or mis-inactivate
the optogenetic tools so that we cannot apply the fluorescence imaging in
combination with the optogenetic technology. To overcome this problem, we
have been developing genetically-encoded luminescent probes which are basedon hybrid between a mutated chemiluminescent protein and a fluorescentprotein variant. In the symposium, I will introduce our recent achievement suchas development of color variant of bright luminescent protein, and the use ofthem in conjunction with the optogenetic technologies for understanding
operation principles of bio-nanosystems.

1SA-04

Coordinated reversal of flagellar motors on a single
Escherichia coli cell
Akihiko Ishijima (Tohoku University)
An E. coli cell transduces extracellular stimuli sensed by chemoreceptors to theintracellular signal molecule, which regulates the switching of the rotational
direction of the flagellar motors in both direction changes. The switching ishighly coordinated with a sub-second delay between motors in correlation withthe distance of each motor from the chemoreceptor patch localized at a cell pole.
This result suggested that a transient increase and decrease in the concentrationof CheY-P caused by a spontaneous burst of its production by thechemoreceptor patch. The switching delay of the both switching was
individually investigated. In wild-type cell, the switching delay clearlycorrelated with each motor’s relative distance from the receptor patch in bothswitching. However, in a mutant cell lacking CheAs, which is required for thepolar localization of CheZ, in a CW-to-CCW switching, obvious switching
delay was not observed. This result suggested that the polar localization of CheZ
is critical for the directed propagation of the decrease of CheY-P concentration.
Next, we used caged serine and developed a photoreleasing system of serinefrom the caged compounds. The response time was shortened by photorelease ofserine. The measured response time appeared to increase with increment of
distance between receptor patch and flagellar motor. This result suggested thatthe directed intracellular signal was propagated in cytoplasm from receptor
patch to each flagellar motor with sub-seconds time scale.
1SA-05ヒトゲノム DNA の収納とそのダイナミクス

Human genome organization and dynamics
Kazuhiro Maeshima (Structural Biology Center, National Institute of
Genetics)
Human genome DNA of around 2 m in length is organized into a cell nucleus
having a volume of only 1 picoliter. Each genome has only two sets of genes. In
order to activate a certain gene, a protein factor, which is normally considered to
be in a small number, has to search the whole genome and target the specificgene. In this process, genome organization must play an important role. In fact,
how is the human genome DNA is organized into a nucleus? The DNA iswrapped around histones, forming a nucleosome structure. The nucleosome had
been assumed to be folded into a 30-nm chromatin fiber and other helicalfolding structures. However, our recent cryo-microscopy (cryo-EM) andsynchrotron X-ray scattering analyses have shown almost no visible 30-nm
chromatin fibers or other regular structures in interphase nuclei and mitotic
chromosomes. This suggests that chromosomes consist of irregularly folded
nucleosome fibers. Thus, nucleosome fibers may be constantly moving andrearranging at the local level. Recently we observed these local nucleosome
dynamics, which could be crucial for scanning genome information.
1SA-06哺乳類概日時計のシステム生物学・合成生物学

Systems and Synthetic Biology of Biological Timings
Hiroki Ueda1,2 (1RIKEN, CDB, 2RIKEN, QBiC)
The logic of biological networks is difficult to elucidate without (1)comprehensive identification of network structure, (2) prediction and validation
based on quantitative measurement and perturbation of network behavior, and(3) design and implementation of artificial networks of identified structure andobserved dynamics.
Mammalian circadian clock system is such a complex and dynamic system
consisting of complicatedly integrated regulatory loops and displaying thevarious dynamic behaviors including i) endogenous oscillation with about 24hour period, ii) entrainment to the external environmental changes (temperatureand light cycle), and iii) temperature compensation over the wide range oftemperature. In this symposium, I will take a mammalian circadian clock as an
example, and introduce the systems- and synthetic-biological approaches forunderstanding of biological timings.
References
1. Ueda, H.R. et al, Nature 418, 534-539 (2002).
2. Ueda, H.R. et al, Nat. Genet. 37, 187-92 (2005).
3. Sato T. K. et al, Nat Genet. 38, 312-9 (2006).
4. Ukai H. et al, Nat Cell Biol. 9, 1327-34 (2007).
5. Ukai-Tadenuma M. et al, Nat Cell Biol. 10, 1154-63 (2008).
6. Minami Y. et al, PNAS 106, 9890-5 (2009).
7. Isojima Y. et al, PNAS 106, 15744-49 (2009).

− S1 −

8. Masumoto KH. et al, Curr Biol.20(24),2199-206.(2010).
9. Ukai-Tadenuma M et al. Cell 144(2),268-81 (2011).
10. Hogenesch JB, Ueda HR. Nature Rev. Genet. 12(6),407-16 (2011).
1SA-07

細菌べん毛形成の分子機構とその制御

Molecular mechanism of bacterial flagellar construction andits regulation
Katsumi Imada1, Tohru Minamino2 (1Dept. Macromol. Sci., Grad. Sch. Sci.,
Osaka Univ., 2Grad. Sch. Frontier BioSci., Osaka Univ.)
The bacterial flagellum is a filamentous organelle made up of about 30 proteins
with their copy numbers from a few to a few tens of thousands. Since theflagellum extends from the cytoplasm to the cell exterior, most of thecomponent proteins are exported to the distal end of the growing flagellum
through the flagellar type III protein export apparatus. The export apparatus iscomposed of the export gate made of six membrane proteins (FlhA, FlhB, FliO,
FliP, FliQ, FliR) and three soluble proteins (FliH, FliI, FliJ), which assemble on
and disassemble from the gate during the export cycle. Recent studies revealed
that the export process is regulated by a small number of multifunctional
substrate-specific chaperon proteins (FlgN, FliA, FliS, FliT). By forming a 1:1
complex with their substrates, they prevent their substrates from premature
aggregation in the cytoplasm, and facilitate the export of the substrates throughthe direct interaction with the export apparatus. After release of their substrate,
the free chaperons regulate flagellar protein expression. Thus the flagellar gene
expression and flagellar assembly stage are linked by the export chaperones. In
this symposium, we will show the role of chaperones on the flagellar
construction process based on the recent biophysical, biochemical and genetic
works and discuss the molecular mechanisms of the protein export and flagellar
gene expression.
1SB-01

DNA の硬さと柔らかさ：高次構造転移が生み出す時空間秩序

Synergy between Stiffness and Softness on DNA:
Spatiotemporal Order Organized through the Higher-Order
Structural Transition of DNA
Yoshiko Takenaka (Nanosystem Research Inst., AIST)
Genomic DNA is a semi-flexible polymer chain, which has longer persistence
length (~ 50 nm) than the diameter of a chain (~ 2 nm) and much longer total
length (10¹μm to 1 cm) than the persistence length. That is, DNA behaves as aflexible chain over μm scale and a rigid chain in nm scale. Different from shortoligomeric DNA, long DNA exhibits the property to undergo a discrete
structural transition induced by the change in pH, temperature, concentration ofpolycation, ATP, RNA, etc. We call this kind of structural transition as higherorder structural transition of DNA. In the presentation, we propose a novel
hypothesis to interpret the informational cascade from DNA to macroscopic
spatio-temporal order in multi-cellular systems, based on the unique property ofgenome-sized DNA as a semi-flexible polymer.
We will present a hypothetical gene network model, in which the higher-order
structural transition of DNA plays an essential role in regulating stable on-off
switching and/or the oscillation of a large number of genes under thefluctuations in individual living cells. This model can explain the robust andbroad transcriptional response in a genetic assembly against fluctuations.
We will also interpret the mechanism of the spatiotemporal pattern insomitogenesis. We will stress the importance of spatial discreetness, i.e., actual
size of cells, on the reliable theoretical model. With this model, we can produce
stationary patterns, which are similar to those of somitogenesis.
1SB-02

Proteins as Mechano-Chemical Transducer
Kazuyuki Akasaka (Inst. Adv. Tech, Kinki UNIV.)“Human body has mechanisms for transducing mechanical stress (deformationand motion by force) into biochemical reactions, which is considered the basis
for producing the dynamics of the body.” stated Miyasaka et al. in Seikagaku(81, 494-501, 2009). They further stated that the mechanical stress would
include 1) shearing, 2) hydrostatic pressure, 3) pulling, compressing andpushing, and 4) vibration. We have been studying structural, dynamic andthermodynamic properties of proteins on pressure-axis by NMR spectroscopyusing hydrostatic pressure as variable. We have found that proteins are highly
sensitive to pressure perturbation and change their equilibrium populations fromthe basic sub state often to an excited and functional sub state with increasinghydrostatic pressure (Akasaka K., Chemical Reviews 106: 1814-35, 2006).
Experimentally, a uni-axial mechanical stress applied to a protein solution isused to produce a thermodynamically excited state of the protein with a higher
chemical reactivity. Namely, [mechanical stress to a pump (uni-axial) --hydrostatic pressure (isotropic) ---protein conformational transition(thermodynamics) ---activation of reactivity (chemical reaction, function)] Is ssimilar scheme as above found also in living systems? What is the relationshipof the above scheme with the apparently non-uniform stress to human body likepulling, compressing, shearing and vibrating?
1SB-03身体-細胞能動 / 受動メカノケミカル連携：身体に生きる細胞のニッチとしての柔らかな細胞外基質環境が支配するやわらかさの階層と連携

Body-cell dynamic mechano-mechanical linkage: softness,flexibility, fluctuation and controllability derived from ECM
environment as “niche for cells in our body”
Yoriko Atomi1, Miho Shimizu2, Eri Fujita2, Tomoaki Atomi3, Noboru Hirose3,
Katsuya Hasegawa4 (1Radioisotope Center The University of Tokyo, 2Dept. of
Mechano-Informatics, Univ. of Tokyo, 3Teikyo University of Science, 4JAXA)
After 3.11, we all need re-think the science based on the principle of living
human system. Fumio-Osawa has already discerned that biological macro
molecules such as F-actin in muscle is more flexible/fluctuate in action,suggesting that all physical activities may promote flexibility at the molecularlevels. We human beings belong to eukaryote as multi-cellular organism. Sixty
trillions cells in our body are soft and sticky compared with yeast cells, whichhave hard cell wall like plant. In addition, we should think about specificity ofmulticellular organism in our body, which cells can communicate each otherwith mechano-mechnical linkage and also using paracrine and autocrine, and/or
secretion of ECM molecules. Acquiring controllability of the human body isessential to receive a benefit from cellular activity-dependent manner, which is aprinciple of life. Rapid progress of stem cell technology shed light on an
importance of a special microenvironment termed the “niche” (ECM), a relation
between the cells and our own body as a niche at macro-level is mysterious.
Various phenomenological results from exercising/immobilized knee,
unweighting/gravity loading muscle, heat loading muscle cells, and solubilized
eggshell membrane treated skin which give us “Knowledge” of life and exercise
sciences are included in this topic. Enhanced adaptability for wellbeing broughtby soft and flexible fine-tuned extracellular matrix will link body-to-cell andsolve many problems in aged society where people live with unstable bodyunder the gravity.
1SB-04

多分節立位を可能にした身体の冗長性：柔らかさの制御と破綻

Flexibility of human body with multi-segmental structure
enabling bipedal standing
Tomoaki Atomi1,2, Noboru Hirose1, Miho Shimizu3, Yoriko Atomi3 (1Teikyo
Univ. of Sci., 2Grad. Sch., Univ. Tokyo Metropolitan, 3Univ. of Tokyo)
Human body movements are restricted by the shape/structure of the human bodyparts, like bones, muscles, and joints. Structure-function relationship of the bodyis deduced from dynamic protein structures/ interactions at micro cellular level,
and from bone shapes of the joint which affects how body moves at macro level.
In principle, life has been evolved to the species survival and preservation. If welook at the individual humans, a system to control the gravity center of the bodywithin an appropriate range away from a possible danger of falling down isessential for life survival. Gravity center of the body is affected by the shape ofthe segments primary consisting from bones, and soft tissue consisting ofdiarthrodial joint. Intrinsically unstable bipedal human being controls gravity
center of the body at relatively higher position compare to four-legs animals.
Possible compatibility of multi-segmentality and stability shows that close
relation exists between regulation systems of physical movement and physicalproperty of soft tissue, and gravity center of the body. The present study is
focusing on body elasticity (material) and balance regulation (controllability) tothe physical movement which affects the shape alterations of the body figure(eg. standing, crouch down, lay down, etc.).
1SB-05

人工制御した細胞基盤の柔らかさと細胞応答

Softness of the artificial cell niche and its active interplay of forces
Miho Shimizu1, Yuki Katsurada2, Toshiyuki Watanabe2, Eri Fujita1, Tomoaki
Atomi3, Noboru Hirose3, Katsuya Hasegawa4, Yoriko Atomi5 (1Dept. of
Mechano-Informatics, Univ. of Tokyo, 2Tokyo Univ. of Agri. & Tech., 3Teikyo
University of Science, 4JAXA, 5Radioisotope Center The University of Tokyo)− S2 −

We, human beings are living on earth under 1G through interactive
communication back-and-fourth between natural and artificial environment. By
acquiring large adaptability, namely ‘flexibility’ at cellular level, we willsurvive super ageing society and extreme conditions such as natural disasters
and climate changes within controllable range. Looking at micro-level, adherent
cells reside in our body can ‘feel’ their environment such as stiffness of theECM, known to determine the cell fate (Engler et al, 2006), and alter cell
behavior. Therefore exploring the method for give a good ECM as niche of thecells will provide an ultimate strategy for human wellbeing. To do so, it isimportant to have a fine evaluation system at single cell level. In this study, we
are using micro-needle arrays fabricated by two-photon initiated polymerization(TPIP) of polymer acryl amide gels that can detect an active interplay of forces
between cells and their environment. Stiffness of the cell microenvironment canalter with several factors including gel components, hight and diameters, andcoating materials of the micro-needle. Studies using rat L6 skeletal muscle celltested for different protein coating condition and amount of small heat shockprotein will be presented.
1SB-06

培養基盤の硬さとの整合性が心筋拍動パターンを最適化する

Rigidity Matching between Cells and the Extracellular Matrix
Leads to the Stabilization of Cardiac Conduction
Marcel Hoerning (RIKEN, Center for Developmental Biology, Physical
Biology Unit)
We show, though investigations in the spontaneous substrate-induced
modulation of synchronized beating in cultured cardiomyocyte tissue, that when
the rigidity of the cardiac cell culture environment matches that of the cardiac
cells, the tissue functionality and entrainment dynamics enhances and lead to acloser approximation to native cell functionality [1]. Systematically studies on
entrainment dynamics of spontaneous originated waves showed that the main
entrainment-frequency increases with the increase in substrate rigidity, and theentrainment stability of spontaneous originated waves become maximized when
the substrate rigidity and cell rigidity (~4 kiloPascals) matches. Further,
independent studies on the calcium transient alternates (CTA) in highfrequency-paced tissue showed an significant increase in the critical period.
CTA is known to be responsible for life-threatening arrhythmia in a real beatingheart. We explain our findings by the enhancement in the cell development thatalters the cell morphology (actin skeleton, gap-junction distribution, etc.) on the
one hand, and by optimization of force actuation and coupling between the cells
and extracellular matrix. Our studies suggest that mimicking the extracellularenvironment in in vitro cultures can cause the tissue culture to more closely
approximate native cell functionality, and may help improve reengineering hearttissues for regeneration of diseased hearts.
1SB-07

アクチン繊維のゆらぎを基盤とした生理機能

Physiological functions of an actin filament based on its
fluctuation
Fluctuation of a trajectory of moving actin filaments appears to possess close
relations to their velocity of sliding motion in vitro. The spatio-temporalfluctuation of wiggling filament strongly depends on both the chemical and thephysical conditions imposed on it. We have been focusing on the transitions ofmechanical fluctuations at low ATP concentrations where the filament begins tomove from rigor states to a unidirectional manner. During sliding motion, single
monomers embedded in a single filament supposed to be subjected to push-pullforces from neighboring monomers in addition to the driving forces frommyosin molecule(s). Push-pull forces which should associated with the dynamic
linkage between monomers, and the flexibility and the cooperativity along thefilament. Detailed measurement of the wiggling of monomers within a filament
seemed to insinuate that the spatial range of cooperativity spread not only along
the filament but also perpendicular to it.
We have been investigating the mechanical fluctuation of actin filaments fromthe informational aspects; information in order to systematically control thevelocity of both entire single and spatially distributed groups of filaments.
Various types of dynamic behavior of actin filaments will be demonstrated inthis symposium in association with their physiological functions.
生命の神秘に迫る多次元アプローチ

Multidimensional Approaches to the Secrets of Life
Shigeru Takemori (Jikei Univ. Sch. Med.)

1SC-01特異極限解析：生物システムに現れるパターンを捉える

Singular limit analysis: Understanding of biological patternformation
Yasumasa Mimura (Graduate School of Advanced Mathematical Sciences,
Meiji University)
Reaction-diffusion system is a model that describes patterns that arise inbiological systems. Even though it is one of the most simple system of equations
among non-linear partial differential equations, it nevertheless exhibits diverse
temporal-spatial patterns. Singular limit analysis is a method developed tounderstand the occurrence of such patterns. In this lecture, we give an
introduction to this method, illustrated with concrete examples.
1SC-02鳥の群れの集団動力学

Collective dynamics of flocking
Yoshinori Hayakawa (Center for Information Technology in Education)
Flocking of birds exhibits varieties of collective behavior depending on species
and conditions. To understand the physical properties of such collective
dynamics as self-organization processes of self-propelling elements, we
conducted quantitative field study on the structure of flocks and its
spatiotemporal dynamics for two distinctive examples; skeins of white-fronted
geese and swarm of starlings. In appropriate situations, large populations ofgeese exhibit dynamical rearrangements by repeated mergers and splits among
the groups. We described such grouping process in terms of a mean-field model
based on the Smoluchowski equation of coagulation with fragmentation andobservationally plausible kernels. We also performed a spatiotemporal analysis
of wave excitation in a long one-dimensional chain of airborne geese bymeasuring the phase velocity and the dispersion relation, which leads to asimple mathematical model similar to that of single-lane traffic flow. Cluster ofstarlings exhibits very different shapes and dynamics. By using a portable threedimensional image analysis system to estimate the trajectories of eachindividual in the field, we determined the shape and the proportion of flocks ofstarlings and found a simple scaling relation. We also found that mutual
rearrangement of individuals in a flock can be considered as a short-term
diffusion process, which seems to be a stabilizing factor to tame intrinsic
dynamical instabilities of the system.
1SC-03

Hajime Honda (Dep. Bioeng. Nagaoka Uni. Tech.)

1SB-08To reveal the secrets of spirally streaming cream on coffee surface, diverse
approaches at macroscopic to microscopic levels would certainly be necessary.
In building our understanding of the spiral streaming, some findings intuitively
seem significant in constructing a specific streaming pattern. We hope thepresent symposium under a key concept of flexibility in hierarchical dynamics
has provided a good chance to brush up your intuition in your approach to thesecret of life.

バクテリアのコロニー形成―細胞の集団運動―

Colony Formation in Bacteria -Collective Movement of CellsMitsugu Matsushita (Meiji Institute for Advanced Study of Mathematical
Sciences (MIMS), Meiji University)
We present experimental results of colony formation in bacteria. Common
bacterial species Bacillus subtilis is known to exhibit various colony patterns
such as DLA, Eden, highly branched with smooth circular envelope and disk
ones, depending on the environmental conditions, namely, substrate softness
and nutrient concentration. We have extended experiments of colony formation
to various species of bacteria such as Proteus mirabilis, and Eschelichia coli.
The growth behavior really reminds us of the formation of human colonies suchas villages, towns and cities. We have established the morphological diagram ofcolony patterns, and then examined and characterized both macroscopically andmicroscopically how they grow. The most impressive growth is the formation ofconcentric-ring colony formation. Our experimental results suggest thatmacroscopically the most important factor for its growth is the cell population
density, i.e., that there seem to be higher threshold of the cell population density
to start migrating and lower one to stop migrating. Recently we found thatseveral other common bacterial species such as Serratia marcescens and evenEschelichia coli also exhibit repetitive growth of colonies under appropriate
conditions. This suggests that this type of growth is common in the bacterial
world.
1SC-04− S3 −

生命ネットワークにおける動的ロバスト性の数理的解析

Mathematical analysis of dynamical robustness in biologicalnetworks
Gouhei Tanaka1,2, Kai Morino2, Kazuyuki Aihara1,2 (1Institute of Industrial
Science, The University of Tokyo, 2Graduate School of Information Scienceand Technology, The University of Tokyo)
Biological systems are highly tolerant to some types of perturbations to which
the systems are usually exposed. It is thought that this robustness has beenacquired through an adaptation to the environment in the evolutionary process.
However, they can be extremely fragile to other types of perturbations whichrarely happen. Although this 'robust but fragile' property is an essential featureof biological systems, a mathematical theory to understand this property is yet tobe fully established. A potent mathematical approach to examine structuralrobustness of biological networks has been developed in complex network
theory. It has been revealed that heterogeneously connected networks (e. g.
scale-free networks) are highly robust against random removal of nodes but
extremely vulnerable to targeted removal of hubs. However, dynamics is notconsidered in this framework. Here we focus on another framework to deal withthe robustness of dynamic activity in biological networks consisting of elements
having intrinsic dynamics. When some elements are inactivated, the level ofdynamic activity of the whole network is lowered. There is a critical ratio ofinactivated elements, at which the network dynamics vanishes. By analyzing
this phase transition, we show that heterogeneously connected networks arehighly fragile to targeted inactivation of low-degree elements. This is in strong
contrast to the property of structural robustness. Our result implies that theinterplay between dynamics and structure plays an important role in network
robustness.
1SC-05

自律的酵素量制御による時間スケール調整；ホメオスタシスと記憶

Homeostasis and memory by autonomous regulation of timescales through enzyme abundances
Kunihiko Kaneko (University of Tokyo, Center for Complex-Systems Biology)

expression and is essential for silencing of parasitic DNA, genomic imprinting
and embryogenesis. During DNA replication, methylated CpGs are converted
into hemi-methylated CpGs and newly replicated CpGs should be methylated toinherit methylation pattern. DNA methyltransferase 1 (Dnmt1) is the enzyme tomethylate hemi-methylated CpG regions. Np95 is methylated CpG binding
protein and interacts with Dnmt1, followed by recruitment of Dnmt1 to hemimethylated CpG regions. SRA domain of Np95 is responsible for hemimethylated CpG binding activity. We characterize the process of hemimethylated CpG recognition by SRA domain using Single-Molecule technique,and in this symposium, we will show our present data.
1SD-02The mechanism of nuclear protein searching on DNA: CoarseGrained simulation study
Tsuyoshi Terakawa (Grad. Sch. Sci., Univ. Kyoto)
Various nuclear proteins search their specific binding sites on DNA andfunction at proper time and location. This process is important for gene
expression regulation. Previous theoretical studies have revealed thatcombination of one-dimensional diffusion along the DNA chain and threedimensional diffusion in the bulk solution makes it possible for these proteins tosearch its cognate binding site efficiently. Although this theory is based on anassumption that these proteins can quickly diffuse on DNA, the mechanism of
the quick diffusion at molecular level has been elusive. In addition, it has been
controversial whether the same mechanism is valid in nucleosomal environment
in which histone proteins bind to DNA and possibly hinder the one-dimensional
diffusion.
In order to approach such problems, we have developed coarse-grained model
for protein-DNA complex where most of the parameters are derived fromatomic structural information or atomic simulation results. Using this model, we
conducted molecular dynamics simulations of several proteins (e.g. TF IIIA,
p53, and PCNA) with DNA and got insights into the mechanism of onedimensional diffusion on DNA. We also performed simulations of the proteins
with DNA to which histone proteins bind. Then, we will discuss the validity ofthis mechanism in nucleosomal environment.

Biological systems generally consist of a variety of time scales. These
timescales also change in time depending on their internal state, according to thechange in abundances of enzyme that governs the reaction speed. Here we
discuss that homoeostatic response and memory emerge as autonomous
regulation of enzyme concentrations. First it is shown that slow relaxation
process with some plateaus generally emerge in dynamics of catalytic reaction
networks, where the negative correlation between the enzyme and substrateabundances is a key factor for such ‘glassy’ dynamics [1]. Second, we
demonstrate that the enzyme-limited competition leads to a homeostasis in thesystem. To be specific, we study temperature compensation in period of
circadian rhythm [2]: By considering a simple system consisting just of Kai
proteins, the period of the rhythm is found to be kept constant against
temperature change. The origin of this temperature compensation is attributed toenzyme-limited competition, where negative correlation between abundances ofsubstrates and enzymes again plays an important role. Finally, anotherconsequence of long-term dynamics by autonomous regulation of enzyme
abundances is generation of cellular, epigenetic memory. After discussing thispossibility, I will also illustrate relevance of epigenetic memory to adaptation[3] and differentiation, if I have time.
[1] A. Awazu and K. Kaneko “ “Glassy” Relaxation in Catalytic Reaction
Networks” Phys.Rev. E, 80, 041931 (2009)[2] T. S. Hatakeyama and K. Kaneko “Generic temperature compensation of
biological clocks by autonomous regulation of catalyst concentration” PNAS,
109 (21) 8109-8114, 2012
[3] C. Furusawa and K. Kaneko, “Epigenetic feedback regulation accelerates
adaptation and evolution” submitted.

DNA-binding proteins recognize cognate and non-cognate DNA sequences withtwo different binding modes, in which proteins loosely bind to non-cognate
DNA sequences, but to cognate sequences they tightly bind. Experimental
structures of such complexes provides us an atomic view of the binding modes,
however, it is not so simple to dissect which is cognate or non-cognatecomplexes by seeing the structures.
In this study, free-energy profiles for dissociation of a cognate and a noncognate Lac repressor-DNA complexes were calculated to obtain energetic
views by performing atomic-level molecular dynamics simulations. We
implemented an algorithm of adaptive biasing force (ABF) [E. Darve, A.
Pohorille, J. Chem. Phys. 128, 144120 (2008)] into the AMBER software to
calculate free energy changes in dissociation along a dissociation path. The
results showed that the free energy profiles were clearly distinct between the
cognate and non-cognate complexes. We found that this difference can beinterpreted in terms of changes in the protein-DNA contacts and the number ofinterfacial hydration water. The calculated dissociation process here agrees withthat suggested from an H/D exchange experiment. In the talk, we will discuss
how gene regulatory proteins find their target sites on the DNA and what are
determinants for distinguishing cognate and non-cognate targets.

1SD-01

1SD-04Characterization of Protein-DNA complexes dynamics related
to Chromatin structure regulation using Single-Molecule
Techniques
Yong-Woon Han, Yoshie Harada (iCeMS, Kyoto University)

Eukaryotic gene expression is regulated by chromatin structures and/or DNA
modification such as CpG methylation. The basic unit of eukaryotic chromatin
structure is a nucleosome consisting of approximately 150 bp DNA wrapped in
1.7 superhelical turns around a histone octamer. The histone octamer consists of
two copies each of H2A, H2B, H3 and H4. Posttranslational histone
modifications such as acetylation, methylation, phosphorylation andubiquitylation regulate chromatin structure, resulting in activation or repressionof gene expression. On the other hand, CpG methylation represses gene

1SD-03Differences in dissociation free-energy profiles between cognite
and non-cognite protein-DNA complexes
Yoshiteru Yonetani, Hidetoshi Kono (Japan Atomic Energy Agency)

線虫初期胚における染色体ダイナミクスの定量的解析

Quantitative analyses of chromosome dynamics in C. elegans
early embryos
Takeshi Sugawara, Ritsuko Arai, Akatsuki Kimura (National Institute of
Genetics)
During development and differentiation, it has been suggested that chromatin
remodeling occurs and contributes to cell fate determination. However, there arefew studies for chromatin dynamics during the course of development. We
visualized and tracked chromatin loci in C. elegans early embryos. Based on theexperimental data, we statistically analyzed dynamics of the chromatin locus,
and then revealed quantitative features of chromatin dynamics for several
developmental stages. We found several quantitative features that depend on− S4 −

developmental stages and the size of the nuclei at the stages. In order tocharacterize chromatin dynamics further, we are focusing on the anomaly ofchromatin diffusion. The anomalous diffusion should be caused byuncharacterized intra-nuclear structures that disrupt free diffusion of thechromatin locus. Therefore, we may be able to extract biological information
about intra-nuclear structures.
1SD-05蛍光相関分光法を用いた細胞内グルココルチコイド受容体の動態解析

Inside view of molecular dynamics of Glucocorticoid Receptor
by using Fluorescence Cross Correlation Spectroscopy inliving cell
Masataka Kinjo (Faculty of Advanced Life Science, Hokkaido University)
The glucocorticoid receptor (GR) is a ligand-inducible transcription factor
belonging to the nuclear receptor superfamily. Upon ligand binding, GR
translocates from cytoplasm to the nucleus, then associate as a dimer form withglucocorticoid response element (GRE) in a promoter region, or as a monomericcomplex that operate with other transcription factors to induce transcription.
Besides homodimer of GR can act as repressor by association to negative GRE,GR can tether other transcriptional factors, such as NFkB, as monomer. These
complicated regulation can be unraveled by determination of affinity properties
of GR and/or associated molecules in living cell.
Fluorescence correlation spectroscopy (FCS) provide two molecular properties
such the diffusion time and the number of molecules in defined region. Then,molecular interaction is accessible from the changing of the diffusion time. FCS
measurement revealed interaction between GR and DNA in nucleus could be
modified by difference of ligand.
To understand more detailed of GR dynamics in nucleus, the dimerization
process of GR that is occur before binding to GRE was evaluated by using
Fluorescence Cross Correlation Spectroscopy (FCCS) measurement oftransiently expressed mCherry tandem dimer (mChe2) and EGFP fused GR in
living cell.
In our experiment, the positive cross correlation was obtained after addition of
Dex. Then, Kd (dissociation constant) value of dimerization of GR wild typeand mutant were estimated and compare in living cell.
1SE-01

蛋白質構造の整合性原理とその拡張

Consistency principle of protein conformation and its
extension
Masaki Sasai1,2,3 (1Nagoya University, 2Okazaki Institute for Integrative
Bioscience, 3Korea Institute for Advanced Study)
Proteins are evolutionarily designed polymers which have the ability to fold into
unique conformations (either as single molecules or as parts of molecularcomplexes). The design principle of proteins to facilitate such folding wassummarized as "consistency principle" by N. Go in 1983. The validity of thisprinciple was intensively examined in 1990s and 2000s, from the view point of
the closely related "minimal frustration principle", and the power of thoseprinciples in analyses of folding pathways, structure prediction, and protein
design has been proven. In this talk, we revisit the consistency principle anddiscuss its extension to analyze protein functioning. First, the consistency
principle is represented in 2 or 3 dimensional space of order parameters ofconformation change. In such multi-dimensional expression, competition
among multiple folding pathways and coexistence of multiple intermediates canbe analyzed, and the further application of the principle is discussed. Proteins
have evolved, however, not to fold but to function, so that the functional
necessities may lead to inconsistent interactions in proteins. We discuss the
localized inconsistent (frustrated) regions in proteins, which are important inallosteric transformations and catalytic reactions. We show the chameleon
model, which was introduced by T.P. Terada and colleagues, is the efficient tool
to analyze the roles of inconsistency in proteins.
1SE-02

Dynamic mechanism for the transcription when responses toactivators

Yaolai Wang, Feng Liu, Wei Wang (National Lab. Solid State Microstructure
and Dept. Phys., Nanjing Univ.)
The transcription apparatus (TA) is a complex molecular machine, and involves
complicated interactions between the enhancer and various proteins, and thosebetween the related proteins themselves. The TA detects the time-varying
concentrations of transcriptional activators and initiates mRNA transcripts at
appropriate rates. We propose a dynamic model to describe how the TA candynamically orchestrate reliable transcriptional response based on the generalconfigurations of TA. We argue that there exists a relatively stable clamp-likespace between the Mediator complex and the enhancer, and activators cycle
rapidly in and out this space. The entry of activators into this space results inconformational transition in the Mediator complex, which gives rise to afacilitated circumstance where Pol IIs can initiate/re-initiate transcription
rapidly. As a result, the activators’ concentration can be encoded by a temporal
occupancy rate which modulates the transcription. Furthermore, we build astochastic model which reproduces and reconciles several experimental
observations. These indicate that regulated transcription likely shares the same
dynamic principles. [1] Y. L. Wang, F. Liu, and Wei Wang, “Dynamic
mechanism for the transcription apparatus orchestrating reliable responses toactivators”, Scientific reports, 2: 422, DOI: 10.1038/srep00422
1SE-03

保存アミノ酸に着目したジンクフィンガーの構造と分子認識

New Functions of Zinc Fingers Revealed by Substitution ofConserved Residues
Miki Imanishi (ICR, Kyoto Univ.)
The C2H2-type zinc finger motif is among the most major DNA binding motifs
in eukaryotes. Each finger folds into a globular structure by coordination of azinc ion with the conserved two cysteine (C) and two histidine (H) residues in atetrahedral fashion. This fold is stabilized by hydrophobic interactions formedby the conserved hydrophobic amino acid residues. Usually, multiple zinc finger
motifs are connected by conserved linkers. Though the Zn (II) ligands, thehydrophobic amino acid residues, and the linker are highly conserved among theC2H2-type zinc fingers, their contribution to structure and functions is notclearly understood. Here, the highly conserved amino acid residues weremutated and the function was investigated. Mutation of the conservedhydrophobic amino acids clarified the relationship between formation of ahydrophobic core and DNA binding function. On the other hand, ligand
substituted zinc fingers, in which cysteine residue(s) is substituted to histidine
residue(s), showed hydrolytic ability. In addition, the Zn(II)-dependent DNA
binding domain, CDH2-zinc finger, has been successfully created by reducing
the Zn (II) binding affinity via ligand substitution of the second cysteine toaspartic acid. The linker alteration changed the DNA binding selectivity to
discontinuous target sequences. The new functions of zinc fingers revealed bysubstitution of highly conserved residues will be discussed.
1SE-04

ドメインスワッピングによるシトクロム c の多量化

Oligomerization of cytochrome c by domain swapping
Shun Hirota (Grad. Sch. Mat. Sci., Nara Inst. Sci. Tech.)
Cytochrome c (cyt c) is a stable protein which functions in a monomeric state as
an electron donor for cytochrome c oxidase. It is also released to the cytosol at
the early stage of apoptosis. For nearly half a century, it has been known that cyt
c forms polymers, but the polymerization mechanism remains unknown. We
found that cyt c forms polymers by successive domain swapping, where the Cterminal helix is displaced from its original position in the monomer and Metheme coordination is perturbed significantly [1]. In the crystal structures ofdimeric and trimeric cyt c, the C-terminal helices are replaced by thecorresponding domain of other cyt c molecules and Met80 is dissociated fromthe heme. The solution structures of dimeric, trimeric, and tetrameric cyt c werelinear based on small-angle X-ray scattering measurements, where the trimericlinear structure shifted toward the cyclic structure by addition of PEG and(NH4)2HPO4. The absorption and CD spectra of high order oligomers (~40mer)
were similar to those of dimeric and trimeric cyt c but different from thoseof monomeric cyt c. For dimeric, trimeric, and tetrameric cyt c, the Δ H of
the oligomer dissociation to monomers was estimated to be about -20 kcal/mol
per protomer unit, where Met-heme coordination appears to contribute largely
to H. The present results suggest that cyt c polymerization occurs by
successive domain swapping, which may be a common mechanism of protein
polymerization.
1. S. Hirota et al., Proc. Natl. Acad. Sci. USA, 107, 12854-12859 (2010).
1SE-05

マルチドメインタンパク質のフォールディングとコンフォメーショ
ン動力学

Folding and conformational dynamics of multi-domain
proteins
Shoji Takada (Dept. Biophys, Kyoto Univ.)− S5 −

Although folding reactions and fluctuations of small and single domain proteins
have been relatively well understood by now, much less is clear for folding andfunctional dynamics of larger and multi-domain proteins. Here, we present ourrecent computational work on three case studies of large and/or multi-domain
proteins. First, we discuss both folding and conformational dynamics ofadenylate kinase, a model allosteric protein, which shows rather rich behaviors.
Second, we discuss folding-coupled docking in a split GFP. Finally, importanceof co-translational folding is addressed for a three-domain protein suf1 bycomputer simulations.
1SF-01細胞の変形と運動：理論と実験の挑戦

Deformation and motion of cell: theoretical and experimental
challenges
Masaki Sano (Grad. Sch. Sci. U. Tokyo)
Deformation and motion are indispensable features for the understanding ofliving systems. Recent development in theoretical approaches on deformationand motion of living systems will be discussed in this session. After taking abird’s eye view of these concepts, I will discuss on deformation and motion of
single amoeba cell with regard to a mathematical modeling based on recent
experimental findings.
Cell migration is a highly complex process that integrates many spatial andtemporal cellular events. Motile bacteria and most eukaryotic cells can move ina directed manner or spontaneous fashion depending on the presence or absenceof external cues. In general, motile cells are able to migrate spontaneously in aseemingly random manner even in the absence of external stimuli. It is thischaracter that allows the cells to forage and explore their surroundings bybalancing random and directed migrations. To understand how amoeboid cell
crawls on the substrate by deforming its shape, we performed a multipole
expansion analysis of traction force distribution exerted by individually
migrating amoeboid Dictyostelium cells and looked at correlations to theirmotion. Interestingly enough, some new modes in traction force dynamics arefound in this analysis. In addition to a familiar inchworm-like mode, we foundother modes, i.e. squeezing-stretching mode and crawling mode. These modes
are found to be fundamental modes in deformable objects that migrate on thesubstrate. Possible relations to a recent theoretical approach will be discussed.
1SF-02

マウスの左右決定の過程に見られるノード繊毛の回転運動の同調

Synchronization of rotaional movement in mouse node cilia
during left-right determination
Atsuko Takamatsu1, Kyosuke Shinohara2, Takuji Ishikawa3, Hiroshi
Hamada2 (1Dept. Elec. Eng. and Biosci., Waseda Univ., 2Grad. Sch. Frontier
Biosci., Osaka Univ., 3Dept. Bioeng. and Robotics, Tohoku Univ.)
Animals have slight left-right (LR) asymmetry. The LR body axis in a mouse isconsidered to be established by unidirectional flow in a node cavity of mouseembryo. The unidirectional flow is generated by rotation of tilting monocilia. It
has long been believed that rotation of the numerous cilia is necessary to trigger
the LR determination. However, recently we found rotation of only two cilia issufficient [1].
To investigate what the only two cilia can achieve, we focus on the relation ofrotational movement between a pair of isolated two cilia in a mutant mouse. The
cilia intervals are much larger than those for systems with densely arraigned
cilia such as epithelium or swimming protozoan showing metachronal waves.
Thus the establishment of the cooperative movement is considered to bedifficult. However, we found the phase locking is easily observed in the mousesystem.
To verify the existence of phase locking state theoretically, we applied the phasereduction theory combined with computational fluid dynamics under ahypothesis there is hydrodynamic interaction among cilia. Analysis results using
the same parameter as those of the experimental condition such as cilia length,
intervals, tilting angle, and etc. was compared. The results suggest the phaselocking can be established via only hydrodynamic interaction even though thecilia arrangements are sparse. Finally the biological advantage of the phaselocking will be discussed.
[1] K. Shinohara et al. (2012) Nat. Commun. 3, 622.
1SF-03鞭毛や繊毛の流体力学相互作用による同期：ミニマルアプローチ

Synchronization of flagella and cilia by hydrodynamic
interactions: minimal approach

Nariya Uchida1, Ramin Golestanian2 (1Dept. Phys., Tohoku Univ., 2Cent.
Theor. Phys., Univ. of Oxford)
Unicellular organisms such as E. Coli and Paramecium swim in viscousenvironment using flagella or cilia. Coordinated motion of these active
filaments, known as flagellar bundling and ciliary metachronal waves, areimportant for achieving efficient propulsion. The roles of long-range
hydrodynamic interaction in their synchronized motion were addressed as early
as in 1951 by G. I. Taylor, and are recently attracting increasing attention. Whilelarge-scale numerical simulations of the filament dynamics have beendeveloped, the key mechanism of synchronization is still far from wellunderstood. In this talk, we consider a couple of minimal models ofhydrodynamic synchronization, in which the center-of-mass motion of cilia or
flagella is represented by a rigid bead making a fixed trajectory. In the firstmodel, the driving force acting on the bead is a periodic function of its phase,
which generalizes the effective and recovery strokes of ciliary beating. For
arbitrary trajectory shape, we derive sufficient and necessary conditions for twoparallel rotors to synchronize. In the second model, each bead exerts a radialpumping force on the surrounding fluid, and at the same time is driven by aconstant torque. The model exhibits various collective behavior such as defectcoarsening, turbulent spiral waves, traveling waves, and order-disordertransition. We discuss the results from the perspective of coupled oscillatorswith long-range interactions.
1SF-04興奮性化学進行波に駆動される遊走細胞の形態ダイナミクスの数理
モデル

Modeling morphological dynamics of migrating cells governedby self-organized excitable waves
Shuji Ishihara1, Daisuke Taniguchi1, Satoshi Sawai1,2 (1Grad. Sch. Arts &Sciences, Univ. Tokyo, 2JST PRESTO)
Cell migration is one of the fundamental processes that support various
biological functions such as immune response and development. In randomly
moving cells, phosphatidylinositol (3, 4, 5) -trisphosphate (PIP3) and F-actin
propagate as waves at the basal membrane that act to push out the cell border,and such chemical waves are responsible for the observed cyclic yet complex
dynamics of cell migration. Although many of the molecular mechanisms
underlying the process are known, how they give rise to these patterns is notwell understood. Here, by using Dictyostelium discoideum as a model system,
we address how the complex morphological change arises from the underlying
molecular interactions. We present a theoretical description that integrates
molecular processes inside cell and dynamics of cellular morphological change.
In the model, excitability of the system is governed by positive feedback from
PIP3 to PI3K activation that is mediated by actin polymerization. By
incorporating membrane deformation into the model, we demonstrated how
geometries of competing waves explain most of the observed dynamics ofamoeboid morphology. The parameter dependence of the model, especiallyactin-mediated feedback strength, is compared with experimental observations
for cells in which actin is perturbed.
1SF-05

細胞の力学と、人工システムにおける自発運動

Mechanics of gels and spontaneous motion of droplets as
biologically-motivated systems
Natsuhiko Yoshinaga (WPI-AIMR, Tohoku Univ.)
In this talk, I will present two topics which would be hopefully merged in future:
one is about mechanics of biological gels and the other is spontaneous motion ofdroplets. (i) Cross-linked actomyosin bundles retract when severed in vivo bylaser ablation, or when isolated from the cell and micromanipulated in vitro in
the presence of ATP. We identify the time scale for contraction as a viscoelastic
time, where the viscosity is due to (internal) protein friction. The results aresupported by an hydrodynamic model of a retracting bundle as a cylinder ofisotropic, active matter, from which the order of magnitude of the active stress isestimated. (ii) I shall also show that spontaneous motion (also called as selfpropulsion) of droplets driven by a surface tension gradient (Marangoni effect)
coupled with internal and/or external patterns of chemicals. Such systems havebeen attracting attention in last decades for its potential application to biologicalproblems such as cell motility. Recently several model experiments showing
spontaneous motion have been proposed. The systems in these works consist of
relatively simple ingredients, for instance, oil drops in water nevertheless the
motion is as if the drops are alive. I will address why the particle moves without
external force and why it breaks symmetry and chooses one direction.

− S6 −

1SF-06

Tsuneo Urisu1,2 (1Nagoya University, FIRST Research Center for Innovative
Nanobiodevices, 2Japan Science and Technology Agency, CREST)

這行による移動運動の理解に向けてTowards understanding the locomotion of animals by limblesscrawling
Ryo Kobayashi1,4, Toshiya Kazama1,4, Kentaro Ito1,4, Yoshimi Tanaka2,
Toshiyuki Nakagaki3,4 (1Hiroshima University, 2Yokohama National University,
3
Future University Hakodate, 4JST, CREST)
Limbless crawling is a fundamental form of animal locomotion adopted by awide variety of species, including amoeba, earthworm, flatworm and snake.
Limbless crawling can be classified into several types, which are amoeboid
locomotion, one-dimensional peristaltic crawling (single lane and double lane),
two-dimensional crawling and serpentine locomotion (lateral undulation, side
winding, concertina). An interesting question from a biomechanics perspective
is how limbless crawlers control their flexible bodies in order to realize
directional migration.
In this paper, we mainly concentrate on the one-dimensional peristaltic
crawling, which is driven by elongation-contraction waves propagating along
the body axis. We will demonstrate that the model equation describing this typeof locomotion is an one-dimensional diffusion equation with coefficientsdepending on the space and time variables. Simulations and theoretical analysis
show that the proper control of friction with the substrate is essential fortranslating internal motion into directional migration. We also discuss about thetwo-dimensional crawling adopted by polyclad flatworms.
1SH-01

We have newly developed the incubation type planar patch clamp biosensor
with PMMA (polymethyl methacrylate) substrate by using hot embossing,focused ion beam microfabrication and surface modification by extra cellularmatrix. PMMA is an attractive material as the sensor chip of incubation typeplanar patch clamp from the view points of: low cost, easiness in various
microstructure fabrications and surface chemical modifications and low
dielectric constant compared with Si. Light-gated ion-channel protein is not
only useful in the in vivo and in vitro neural network analysis, but also useful inthe performance test of the newly developed ion-channel biosensor because of
its high time and spacial resolutions and easiness of handling. We haveevaluated the performance of the newly developed incubation type planar patch
clamp biosensor with PMMA sensor chip by using the light-gated ion-channel
protein, channelrhodopsin wide receiver, which was expressed on HEK293
cells. Observed laser-induced current profile almost completely agreed to thoseobserved by pipette patch clamp. PMMA sensor chips showed faster response
characteristics than Si sensor chip. Based on these results, we are now
developing new high throughput screening devices with in vitro neuralnetworks.
1SH-04

作原理解明

Optogenetics reveals function of neural network involved in
the regulation of sleep/wakefulness

ハロロドプシンの構造と分子機能

Akihiro Yamanaka (Research Institute of Environmental Medicine, Nagoya
University)

Structure and Molecular Function of Halorhodopsin
Makoto Demura (Fac. Adv. Life Sci., Hokkaido Univ.)
Halorhodopsin acts as an inward-directed chloride pump. Recently, theelectrical function of Natronomonas pharaonis HR (NpHR) has been applied to
optogenetics, the technique of which has the potential to enhance the basic
knowledge in neuroscience research and to inform the mechanisms andtreatment of brain injury and disease. A detailed structural model of trimeric
HsHR was built by Kolbe et al. in 2000 by means of X-ray crystallography.
More recently, the trimeric NpHR-bacterioruberin complex has been isolated
from the Natronomonas pharaonis strain KM-1 cells and its crystal structure has
been also revealed by Kouyama group. The carotenoid bacterioruberin binds tocrevices between adjacent NpHR subunits in the trimeric assembly and thecentral part of the trimer is filled with three lipid molecules. Although the
crystal structures of HsHR and NpHR are very similar to each other, HsHR
monomers in the homo-trimer are tilted relative to the monomers in the NpHRbacterioruberin complex. In other words, there are two types of the trimeric HR
assembly, a tilted homo-trimer and a trimeric complex with bacterioruberin. In
the case of HR expressed in E. coli cell, NpHR forms a robust homo-trimer in a
detergent DDM solution. Our results revealed that Phe150 was essential to formand stabilize the NpHR trimer in the DDM solution. Further analyses for
examining the structural and functional significance of Phe150 mutant monomer
and dimer in dark and light condition will be discussed.

Orexin, also called hypocretin is a neuropeptide recently identified as a natural
ligand for orphan G protein coupled receptor1. Orexin-producing neurons(orexin neurons) are located specifically in the hypothalamus but project theirefferents throughout the brain. Intriguingly, the mice lacking prepro-orexin gene
showed behavioral characteristics similar to human sleep disorder“Narcolepsy”, that is a fragmentation of sleep/wakefulness and sudden muscle
weakness. Human clinical studies also showed that orexin neurons are
specifically ablated in the narcoleptic patient’s brain. These results suggest thatthe orexin neurons play a critical role in the regulation of sleep/wakefulness.
Previous studies using electrophysiology have identified potential neuronal
pathways or networks connecting orexin neurons with other neurons which are
known to be involved in sleep/ wakefulness regulation. However, it will beessential to analyze living animals for the purpose of elucidating how theseneuronal networks regulate sleep/wakefulness, a physiological phenomenon
only observed in living animals. To address this, we incorporated recently
developed technique “optogenetics”, which allows us to control neuron activity
by illuminating light locally in the brain. In this symposium, I will discuss
physiological importance of the activity of orexin neurons in the maintenance ofarousal using optogenetics.
1SH-05

1SH-02

Structural basis for light-gated cation conductance bychannelrhodopsin

Channelrhodopsins (ChRs) are light-gated cation channels derived from algaethat have been found to provide experimental utility in the technology of
optogenetics; for example, neurons expressing ChRs can be optically controlledwith high temporal precision within systems as complex as freely moving
mammals. Although ChRs have been broadly applied to neuroscience research,
little is known about the molecular mechanism by which these remarkable andpowerful proteins operate. Here we present the crystal structure of a ChR (C1C2chimera between ChR1 and ChR2 from Chlamydomonas reinhardtii) at 2.3 Å
resolution. The structure reveals the essential molecular architecture of ChR,
including the retinal-binding pocket and the cation conducting pathway.
Integration of structural and electrophysiological analyses provide insight into
the molecular basis for the unusual operation of ChR, and pave the way for
precise and principled design of ChR variants with novel properties. I will
discuss the recent results of our study of the structure and variants of ChR.
チャネルロドプシンによる神経細胞ネットワーク素子の制御

Control of neural network devices by channelrhodopsin

レチナールタンパク質の進化と機能多様性

Evolution and functional diversity of animal opsin-based
pigments

Osamu Nureki, Ryuichiro Ishitani (Graduate School of Science, The
University of Tokyo)

1SH-03

オプトジェネティクスを用いた睡眠覚醒調節に関わる神経回路の動Akihisa Terakita (Grad. Sch. Sci., Osaka City Univ.)
Thousands of animal opsins have been identified so far and they are
phylogenetically classified into several groups. Most opsin-based pigments
other than vertebrate visual pigments have a nature of a bistable pigment, ofwhich light-activated state is stable and reverts to the original dark state bysubsequent light absorption. We have investigated functional properties ofvarious bistable pigments. The phylogenetic classification of opsins roughly
corresponds to the difference in molecular function of the opsin-based pigments.
We identified two novel opsin-based pigments, which activate Go and Gs inscallop and jellyfish photoreceptor cells, respectively. Several lines of evidence
including our findings revealed that visual pigments of most invertebrate(protostomes) such as insects and cephalopods as well as vertebrate OPN4
homologues (melanopsins) activate Gq. Recently, it was reported that vertebrate
OPN5 (neuropsin) activates Gi (Yamashita et al., 2010; Kojima et al., 2011).
Therefore, accumulated knowledge suggests that diverged bistable pigments
may drive cyclic nucleotide cascade or phosphoinositide cascade. Interestingly,
some of opsin moieties of bistable pigments can bind all-trans retinal, as achromophore, probably related to non-visual function. Molecular characteristics
including G-protein signaling, photoreaction and retinal binding of various
opsin-based pigments will be compared, and potential availability of the− S7 −

resist external loads solely, implying the synchronization of force generations
between several myosin molecules. Such a coupling of force generation
between myosin motors might be achieved by synchronizing biochemical
reactions through strain-dependent kinetics (e. g., ADP release rate). In this
symposium, we will discuss the force generation mechanism of single skeletal
myosins found uniquely in ensembles of motor.

diversified opsins as an optogenetics tool also will be discussed.
1SH-06

イカロドプシンの光活性化機構

Photo-activation mechanism of squid rhodopsin
Midori Murakami, Tsutomu Kouyama (Grad. Sch. Sci., Nagoya Univ.)
Retinal proteins have proven useful in the field of optogenetics. For further
extention of the optogenetics, it is important to elucidate the activation
mechanisms of indicvidual retinal proteins. The visual pigment rhodopsin is aphoto-sensor protein in retina. Upon absorption of light, rhodopsin undergoes
the photocycle and, via several intermediates, forms a final photoproduct which
stimulates the intracellular signaling pathway to excite visual cells.
Photochemical reactions, conjugated G-protein types and activation efficiencies
of G-protein of invertebrate and non-visual rhodopsins are different largely fromthose of vertebrate visual rhodopsin. To reveal the molecular mechanism of
rhodopsin activation, we have performed crystallographic analysis of squidrhodopsin. Squid rhodopsin extracted from microvillar membrane wascrystallized into a P62 crystal. Spectroscopic data of the P62 crystal showedthat a series of intermediates can be trapped by adjusting illumination condition
and temperature regulation. The difference electron density map derived
from diffraction data of unphotolysed and illuminated crystals exhibits
complementary positive and negative densities around retinal in the transition
from dark-adapted state, via bathorhodopsin, to lumirhodopsin. These datasuggest different structural changes during the early and middle part of thephotocycle of squid rhodopsin from those of bovine rhodopsin. I will discuss its
implications in activation mechanism of squid rhodopsin.
1SH-07GPCR の細胞内シグナル伝達を制御する構造特徴
Structural elements which control the signal transduction
pathway of GPCRs
1,2

2 1

ダイニンモーター運動発生機構の構造的基盤

Structural basis of dynein motility
Takahide Kon (Institute for Protein Research, Osaka Univ.)
Dyneins are microtubule-based motor complexes of 1,000-3,000 kDa that powera wide variety of biological processes within eukaryotic cells, including ciliary
beating, cell division, cell migration, and intracellular trafficking. Compared tothe other cytoskeletal motors, the molecular mechanism of dynein is still poorly
understood, in part due to the lack of high-resolution structural information.
Here we report the crystal structure of the 380-kDa motor domain ofDictyostelium cytoplasmic dynein at 2.8-Å resolution with approximately 90%
of its amino acid residues traced. This atomic structure shows details offunctional units constituting the motor domain, such as the ATP-hydrolyzing
ring composed of six AAA+ modules, the long coiled-coil microtubule-binding
stalk, and the force-generating rod-like linker. We visualizes four ADP
molecules bound to the first four AAA+ modules of the ring, among which
three are active ATP hydrolysis sites. Our analysis also uncovers the junctional
structures between the ring and the two mechanical units, which should becritical for dynein’s motor function. The structure, together with the results of
our EM and functional analyses, provides a framework for understanding themechanism of dynein-based motility.
1SI-03

2

Makiko Suwa , Minoru Sugihara ( Colledge, Sci. Eng. AGU., CBRC, AIST)
Recent optogenetic technology intends to control the intracellular signal of Gprotein coupled receptor (GPCR) by using light instead of chemical substances.
This technology made a chimera in which cytoplasmic loops of a rhodopsin are
exchanged to those of other GPCR (Raag, D, Airan et al., Nature, 2009). We
address a question of how specific partial structural elements of GPCRs
contribute to control the signaling pathway (G-protein coupling selectivity) byperforming combined quantum/molecular mechanical (QM/MM), molecular
dynamics (MD) studies of rhodopsin structure and sequence analysis of GPCRs.
We compared the structures of Bovine and Squid rhodopsin which bind to G
proteins of different kinds, as a model case. In the retinal binding pockets of thetwo rhodopsins, the similar environment containing highly conserved aromatic
residues was observed. However, in the inter-helical environment, the stabilityof hydrogen bonding network formed between highly conserved Trp274(Trp265) and Tyr 315 (Tyr 306) in squid (bovine) was different. Moreover, tworhodopsins show the significant difference about the number of salt bridges
formed on cytoplasmic side loops. We also indicate difference of simulated
structure of cytoplasmic side of rhodopsin according to the interaction withdifferent type (Gq, Gt, Gs) of G-protein. Paying attention to these regions
distributions of hydrogen bonding residues and charged residues weresummarized as profiles. And the correlation of profiles and G coupling
selectivity was investigated.
1SI-01

1SI-02

Microtubule corkscrewing motion driven by multiple nonprocessive motors
Junichiro Yajima (Department of Life Sciences, Graduate School of Arts andSciences, The University of Tokyo)
Microtubule-based molecular motors are engines for producing a mitotic bipolar
spindle architecture and a coordinated beating of cilia. They work by converting
scalar chemical energy into mechanical 3D vector motion along microtubules.
Current models for their mechano-chemistry propose that three dimensional
directed mechanical forces originates either in a directionally-biased selection of
microtubule binding sites, or in a directional conformational change that follows
microtubule binding. In this study, we are exploring these competing proposals
using a 3D optical tracking microscope, which permits studies to be made on
mechanical motions of motors that are masked in conventional 2D-tracking
experiments. To do this, we quantified 3D-microtubule motion driven by
ensembles of minimum motile unit of microtubule-based motors such as singleheaded kinesin-1s, 5s, 6s, 10s, 14s and single-headed outer-arm dyneins. We
found that microtubule corkscrewing motion driven by these motors have theirown intrinsic pitches, handedness and directions which can be influenced bymutations or phosphorylations in the motor head, ATP concentrations andgeometry on the glass surface. Our results indicate that motors are capable ofexerting a directed force separately parallel to a longitudinal axis of
microtubules and perpendicular to the axis of microtubules. The
mechanochemical properties of individual motors might be related to conserved
functions of their motor domains based on a directionally-biased selection of
microtubule binding sites.

分子複合体フィラメントにおける骨格筋ミオシン 1 分子の力発生メ
カニズム

Force generation mechanism of single skeletal myosin
molecules in myofilaments
Motoshi Kaya1,2, Hideo Higuchi1 (1Grad. Sch. Sci, Univ. Tokyo, 2JST, PRESTO)
Skeletal myosins are non-processive motors and form ensembles to generate agreat amount of force during rapid muscle contractions. Hence in order tounderstand the force generation mechanism of single skeletal myosins, we haveinvestigated the mechanical properties of myosins embedded in synthetic
myosin-rod cofilaments. We previously found that the elasticity of skeletal
myosins is non-linear, implying that the non-linear elasticity might be acomplementary function to reduce the drag effect when myosins are negatively
strained after the force generation. Recently, we further investigateddisplacements and forces produced by synthetic myosin-rod cofilaments, which
are composed of ~20 myosin interacting molecules. The results show stepwisechanges of displacement and, the individual step sizes continuously decrease asloads increase up to 30 pN. In such high force ranges, single myosins cannot

1SI-04微小管系分子モーターの複数分子による機能を計測する

Measuring ensemble functions of microtubule-based molecularmotorsKen'ya Furuta (Bio ICT Lab, NICT)
Molecular motors usually work together in teams within the cell. However,studying coordination among these motors has posed a challenge. Here we
present an experimental system that enables a bottom-up approach where adefined number of motors can be self-assembled on a DNA scaffold to form alinear array assembly with defined spacing. Using this system, we firstquantified the motion and force production of assemblies composed of either
kinesin-1 or kinesin-14, which are kinesin motors with different processivity.
We found that, regardless of the motor type, the binding time of assemblies on
microtubules depends on the spacing between motors, suggesting thatcooperative microtubule binding is the key to improving multiple-motor basedtransport. Force measurement of these assemblies demonstrated that

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nonprocessive motors can coordinate with each other to exert much larger force
than can single motors, while processive motors are not able to effectively sharean external load. Using numerical simulations, we reproduced the observed
experimental behavior and hypothesized a formerly unexpected stepping
mechanism of nonprocessive kinesins. We also present a minimal tug-of-war
between defined numbers of kinesin and dynein, which resulted in highly
stochastic, bidirectional movement in the absence of external regulators. Thesefindings highlight the impact of physical motor-motor interactions on collective
transport and thus provide a step toward bridging the gap between single
molecules and ensemble function.
1SI-05A cellular funicular: one active force generation drives two
directional organelle movements
Akatsuki Kimura1,2 (1Cell Architecture Laboratory, National Institute of
Genetics, 2Dept. Genetics, SOKENDAI)
Organelles inside cells move to position themselves at the right place at the righttime. Such positioning is driven by active force generators, typically themolecular motors. Previous studies on organelle transport mainly focused on
cases wherein a single active force generating mechanism is responsible for the
movement of an organelle toward one direction. Alternatively, one active force
generation mechanism in a cell may drive organelle movements in two
directions at the same time, like a funicular. Our recent studies using
Caenorhabditis elegans embryos [1, 2] and embryonic mouse brain cells [3]suggest the existence of such funicular-like coupled movements of organelles
inside cells. Hence, we propose the existence of a “cellular funicular,” [4] inwhich one active force generation mechanism drives organelle movements in
two opposite directions; this may apply to many intracellular movements, andtherefore, it potentially comprises a common force generation mechanism inside
the cell.
[1] Kimura K & Kimura A. Proc. Natl. Acad. Sci. USA 108, 137-142 (2011);[2] Kosodo, Suetsugu, Suda, Mimori-Kiyosue, Toida, Baba, Kimura A &Matsuzaki. EMBO J. 30, 1690-1704 (2011); [3] Niwayama, Shinohara &Kimura A. Proc. Natl. Acad. Sci. USA 108, 11900-11905 (2011); [4]Niwayama & Kimura A. Worm 1, 71-75 (2012)

1SI-06

ミオシンの力生成・応答特性による上皮パターン形成

The emergence of epithelial pattern through force-generating
and force-responding properties of myosin
Kaoru Sugimura1, Shuji Ishihara2 (1Kyoto Univ., 2The Univ. of Tokyo)
In the course of animal development, tissues undergo a series of deformations.
These processes are realized by the coordination of spatio-temporal profiles ofactivity of proteins and mechanical force that directly acts on cell shape. It has
been shown that the non-uniform localization of the force-generating molecularmachinery including myosin results in biased cellular forces that drive thecellular rearrangements underlying tissue morphogenesis. Along these lines, it
was suggested that the stress field-dependent myosin redistribution consists of apositive feedback loop that accelerates such irreversible cell shape changes.
The present study determines that force-responsive property of myosin as wellas its force-generating property is critical for morphogenesis. By using a novel
force-estimation method, we characterize the relationship between the force
distribution, myosin localization, and cell elongation in the Drosophila wing.
Our force-estimation analysis and mechanical manipulation of tissues clarified
that the proximal-distally biased intrinsic cortical tension is generated in order toresist the anisotropic extrinsic stretching force. Moreover, the emergent global
balance between the intrinsic and extrinsic forces plays a pivotal role in theepithelial pattern formation: the maximum stress direction of a cell population
provides the directional information to instruct the local alignment of hexagonalcells, leading to the global organization of the hexagonally packed, structurally
stable wing.
2SA-01The Mechanism of Cytoplasmic Dynein Motility
Ahmet Yildiz (University of California Berkeley)
Cytoplasmic dynein is a cytoskeletal motor driving minus-end directed motility
along microtubules (MTs). In contrast to kinesins and myosins, the mechanism
by which the two catalytic head domains interact and move relative to eachother remains unresolved. By tracking the positions of both heads at nanometer
resolution, we found that the heads remain widely separated and moveindependently along the microtubule. By engineering the mechanical andcatalytic properties of the motor domain, we show that a rigid linkage between

monomers and dimerization between N-terminal tail domains are not essential
for processive movement. Instead, dynein processivity minimally requires thelinker domain of one active monomer to be attached to an inert MT tether
retaining only the MT-binding domain. The release of a dynein monomer from
the MT can be mediated either by nucleotide binding or external load, however,force applied to the linker domain inhibits nucleotide dependent release. Forcedependent release is significantly asymmetric, with faster release towards theminus-end. These results lead us to a detailed mechanistic model of dynein
processivity, directionality and force generation.
2SA-02
Architecture of the Flagellar Switch Complex
Brian R. Crane1, R. Sircar1, G. Gonzalez-Bonet1, Koushik Paul2, D. Blair2(1Cornell University, 2University of Utah)

The switch complex of the bacterial flagella motor comprises a large
cytoplasmic rotor assembly that couples with the intramembrane proton motors
to drive rotation and mediate switching of the flagellum. The switch complex of
E. coli is composed of the proteins FliG, FliM and FliN, with FliG most directly
involved in rotation. In Gram-positive bacteria FliN is replaced by a larger
protein, FliY, which can also act as a phosphatase for the switching signal,
phosphorylated CheY. Through a combination of biochemical, genetic,
crystallographic and spin-labeling studies we have investigated the interactions
and arrangement of key components within the switch complex. The
stoichiometric mismatch between FliG (26 subunits) and FliM (34 subunits) iswell explained by two distinct positions for FliM: one where it binds the FliG
central domain and another where it binds the FliG C-terminal domain. Thisarchitecture provides a structural framework for addressing the mechanisms of
motor rotation and direction switching. Recent data on conformational changes
within the switch complex induced by phosphorylated CheY will also be
discussed.
2SA-03
Imaging Water at the Nanoscale and Protein in Water by TEM
Paul T. Matsudaira (Department of Biological Science, National University ofSingapore)

Interfacial water is the first layers of water molecules in immediate contact witha surface, whether solid, liquid, or gas and represent the “bound” water
surrounding proteins and other macromolecules and structures in a cell. Since
the cell is a “crowded” environment, interfacial water plays an important rolein biological function. However, interfacial water does not display thehydrodynamic properties of bulk water. Studying interfacial water has been
experimentally difficult and requires methods with resolution in the nm-scale.
Recently, we have developed new methods for studying water and protein inwater directly by imaging in the TEM. Key is the refinement of a
microfabricated liquid cell, the dimensions of a TEM grid, which can protect
liquids against the high vacuum of a TEM. Samples are viewed through 10 nm
thick silicon nitride windows. In this device, we can study fluidics at thenanoscale and observe stick-slip motions of nm-diameter water droplets as wellas significantly damped Brownian motion. Furthermore, when applied toprotein, we can image macromolecular protein structures in liquid water andobserve the dynamic motion of proteins that are not absorbed to the window of
the liquid cell. We now show that the resolution is limited by radiation damage
and the thickness of the silicon nitride windows. Our studies show that it is now
possible to study directly dynamics of water and protein in water at the nmscale by TEM.
2SA-04
Function dictates topology in biochemical networks
Chao Tang (Peking University, University of California, San Francisco)

A diverse set of biological functions in a diverse set of cells and organisms are
performed by a myriad of biochemical networks. A major challenge in systems
biology is to seek design principles or general rules that link network function
and topology. I will present two case studies in which the network function
limits the choice of the network topology to a very small set. One case concernswith the biochemical adaptation. We show that there are only two core
topologies that can achieve perfect adaptation. The other case is theparasegments boundary formation in fruit fly development. The network
topology for this patterning function has to be one of the few variants of a core
topology. A function-topology map would guide us to comprehend natural
biochemical networks, identifying key or missing nodes and links andsuggesting the network's function. It can also provide us with a manual tosynthesize artificial networks.

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Finally, future challenges of HS-AFM studies will be discussed.
2SB-01

遺伝子発現ノイズの起源

Examining origins of noise in gene expression
Yuichi Taniguchi, Yutaka Ogawa, Masae Johmura (Quantitative Biology
Center, RIKEN)
Protein and messenger RNA (mRNA) copy numbers vary from cell to cell in
isogenic bacterial populations. However, these molecules often exist in low
copy numbers and are difficult to detect in single cells. We carried out
quantitative system-wide analyses of protein and mRNA expression in
individual cells with single-molecule sensitivity using a newly constructed
yellow fluorescent protein fusion library for Escherichia coli. We found that
almost all protein number distributions can be described by the gamma
distribution with two fitting parameters which, at low expression levels, haveclear physical interpretations as the transcription rate and protein burst size. At
high expression levels, the distributions are dominated by extrinsic noise. We
found that a single cell’s protein and mRNA copy numbers for any given geneare uncorrelated. In the symposium, we will present our recent challenges onmRNA dynamics, in addition to the work on single-cell proteome andtranscriptome analyses.
2SB-02細胞環境下におけるタンパク質の NMR 計測

Protein NMR spectroscopy in the cellular environment
Kohsuke Inomata1, Shuhei Murayama2, Shiro Futaki3, Hidekazu Hiroaki4,
Yutaka Ito5, Hidehito Tochio2, Masahiro Shirakawa2 (1Quantitative Biology
Center, RIKEN, 2Graduate School of Engineering, Kyoto University, 3Institutefor Chemical Research, Kyoto University, 4Graduate School of Pharmaceutical
Sciences, Nagoya University, 5Graduate School of Science and Engineering,Tokyo Metropolitan University)
It has been known that protein structure and dynamics in the cellularenvironment can differ from in vitro. In-cell NMR can be a powerful method tocompare this difference directly. In-cell NMR is an isotope-aided multidimensional NMR technique that enables observation of conformation andfunction of protein in living cells. However, application of in-cell NMR has
been limited to E. coli or Xenopus laevis oocytes. To extend the applicability ofin-cell NMR, we have established a method to obtain high-resolution multidimensional heteronuclear NMR spectra of proteins inside living human cells.
Proteins were delivered to the cytosol by the pyrenebutyrate-mediated action ofcell-penetrating peptides (CPPs) which were covalently linked to the proteins.
Proteins were subsequently released from CPPs by endogenous enzymatic
activity or autonomous reductive cleavage. In this presentation, we show anddiscuss our "in human cell NMR” results. In this course, we especially focus onprotein folding stability in cells, because we observed a significant difference
from in vitro. Moreover, we discuss protein folding stability and other forms ofprotein dynamics analyses under conditions that mimic the cellular environment
such as macromolecular crowded solutions.
2SB-03高速原子間力顕微鏡による生体分子の構造ダイナミクスの直接観察

Direct observation of structural dynamics of biologicalmolecules by high-speed atomic force microscopy
Noriyuki Kodera1, Takayuki Uchihashi1,2, Toshio Ando1,2 (1Bio-AFM
Frontier Research Center, Inst. Sci. & Eng., Kanazawa Univ., 2Sch. Math. &Phys., Inst. Sci. & Eng., Kanazawa Univ.)
Biological molecules (protein molecules in particular) function by changing
their structure. Thus, the direct observation of their structure dynamics is astraightforward approach to understanding how they operate to function.
However, this observation has long been infeasible because of a lack oftechniques that can meet all conditions required for it: single-nanometer
resolution, high temporal resolution, aqueous environment, and lowinvasiveness. To make the observation possible, we have been developing highspeed atomic force microscopy (HS-AFM), which has now reached its maturity[T. Ando, Nanotechnology (2012)]. HS-AFM opens up a new opportunity todirectly visualizing the dynamic structure changes and processes of functioning
biological molecules in physiological solutions at sub-second to sub-100 ms
temporal and submolecular spatial resolution. Recently, the powerfulness of
HS-AFM has continuously been demonstrated by the observations of several
proteins. Unlike fluorescence microscopy, dynamic molecular events
unselectively appear in detail in the AFM movies, facilitating our understandingof how they function. In the presentation, we will briefly overview the principle
and performance of HS-AFM and then show molecular movies captured.

2SB-04

中心小体 9 回対称構造の謎を解く

Cracking the mystery of nine-ness: mechanisms of centriole
formation
Daiju Kitagawa (Nat. Inst. of Genetics, Cent. for Front. Res.)
Centrioles are critical for organizing cilia, flagella and centrosomes acrosseukaryotic evolution. Centrioles are barrel-shaped microtubule-containing
structures characterized by a universal nine-fold radial symmetry. Initiation ofcentriole formation and establishment of its universal 9-fold symmetry require a
structure called the cartwheel. In most species, the centriole is organized arounda cartwheel that comprises a central hub from which nine spokes emanate
outwards towards the microtubules. The mechanisms directing centriole
formation are not understood and represent a fundamental open question in
biology. Recently, we demonstrate that the evolutionarily conserved centriolar
protein SAS-6 forms rod-shaped homodimers that self-interact through theirN-terminal domains to form further oligomers. We establish that sucholigomerization is essential for centriole formation in C. elegans and humancells. Moreover, by using X-ray crystallography and biophysical approaches, we
generate a structural model of the related protein Bld12p from C. reinhardtii, in
which nine homodimers assemble into a ring from which nine coiled-coil rod
domains radiate outwards. Furthermore, we demonstrate that recombinant
Bld12p self-assembles into structures akin to the central hub of the cartwheel,
which serves as a scaffold for centriole formation. Overall, our findings
establish a structural basis for the universal nine-fold symmetry of centrioles,
thus contributing to solving a long-standing question in biology.
2SB-05低温電子顕微鏡法によるらせん複合体の高分解能構造解析

Recent advances in CryoEM high-resolution structural
analysis for helical filaments
Takashi Fujii (Qbic Riken)
CryoEM(cryo-electron microscopy) has made rapid advances in this decade andbecame one of core technologies for high-resolution structural analysis forbiological macromolecules. Helical filaments such as F-actin and Microtubule
without or with their associate proteins working as cytoskeleton and a centralhub of macromolecular interactions play important roles in numerous cellular
processes. We have been working on CryoEM three-demensional reconstructionof the helical filaments, including the flagellar hook and rod, F-actin, the TypeIII needle and the parM filament. Here, we report our recent developments in thehigh-resolution and high-throughput structural analysis for helical filaments.We
have found the optimal conditions of ice thickeness and sample temparature indata collection and the removement of inelastic electron by Ω-type energy filter
made drastic improvement on image contrast. We are now focusing on
development of the automatic data collection, the automatic particle picking
method and the accurate classification program to achieve atomic resolution.
2SB-06

Kinetic and X-ray crystallographic studies of assembly/
disassembly of the CRM1 nuclear export complex
Masako Koyama1, Natsuki Shirai1, Yoshiyuki Matsuura1,2 (1Grad. Sch. Sci.,
Nagoya Univ., 2Strl. Biol. R. Ctr., Nagoya Univ.)
CRM1 is the most versatile exportin that mediates nuclear export of a plethoraof proteins and ribonucleoproteins bearing a leucine-rich nuclear export signal(NES). In the nucleus, cargo and RanGTP bind cooperatively to CRM1, forminga ternary nuclear export complex that translocates through nuclear porecomplexes (NPCs) and then dissociates in the cytoplasm. Translocation through
NPCs appears to be a reversible facilitated diffusive process that is mediated byweak interactions between CRM1 and nuclear pore proteins (nucleoporins) that
contain characteristic FG sequence repeats. The assembly/disassembly of cargocarrier complexes in the appropriate compartments not only imparts
directionality, but also allows accumulation of cargo against a concentration
gradient. Our kinetic study demonstrates that a cytoplasmic protein RanBP1
accelerates dissociation of cargo from CRM1 and RanGTP. A high-resolution
structure of CRM1-RanBP1-RanGTP complex and its comparison with thestructures of CRM1-NES-RanGTP complexes reveal that RanBP1 accelerates
cargo release by inducing allosteric conformational changes in CRM1. Our
kinetic study also shows that a nuclear protein Yrb2 accelerates binding of cargo
to CRM1 in the presence of RanGTP. Yrb2 has a multi-domain structure
containing nucleoporin-like FG repeats and a Ran-binding domain. A highresolution structure of CRM1-Yrb2-RanGTP complex suggests that Yrb2

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functions catalytically as a coordinating scaffold to increase the rate of nuclear
export while preventing futile export of cargo-free CRM1.
2SC-01

有糸分裂紡錘体の構造形成と機能の物理的理解に向けてToward a physical understanding of spindle assembly andfunction
Yuta Shimamoto (Rockefeller Univ.)
The faithful segregation of chromosomes during cell division depends on a
dynamic microtubule-based structure, the metaphase spindle. Althoughnumerous molecular components responsible for spindle assembly and function
have been revealed, it is still unclear how this micron-sized, multi-component
cytoskeletal structure generates and responds to forces while maintaining its
overall stability, as we have a poor understanding of its micromechanicalproperties. Here we combine the use of force-calibrated microneedles, highresolution microscopy and biochemical perturbations to examine the vertebrate
metaphase spindle’s timescale- and orientation-dependent viscoelastic
properties. We find that the metaphase spindle is mechanically anisotropic, andcan deform either elastically (i.e., restore shape) or viscously (i.e., remodel andflow) depending on the timescale and orientation of applied force. We also findthat spindle viscosity depends on the dynamics of microtubule crosslinking.
Spindle elasticity can be linked to the rigidity of two subsets of polymers,
kinetochore and non-kinetochore microtubules, which have different
polymerization dynamics and stability. These data suggest a quantitative modelfor the micromechanics of this dynamic cytoskeletal architecture and provide
insight into how structural and functional stability is maintained in the face ofdifferent forces, such as those that control spindle size and position, and canresult from deformations associated with chromosome transport and stretch.
2SC-02

ダイニンに駆動されたマイクロチューブによる巨大渦の格子形成

dipoles which decays as 1/r2 ensures the ultraslow convergence to the central
limit theorem. As a result, distribution is explained with truncated Levystatistics rather than Gauss. We also found that athermal fluctuations are far
from Gauss due to the extended exponential tails which we believe derived fromrandomly occurring motor detachments from actin networks. We extract anestimate of the velocity of motor heads and average run-length along the actin
filaments. Recording the whole distribution rather than the second moment(mean-squared displacement) thus allowed us to differentiate between the effectof individual motors and the collective action of multiple motors.
2SC-04植物器官にみられるねじれた成長の弾性力学的および幾何学的な起原についてElastic and geometric origin of the twisted growth of plant organs
Hirofumi Wada (Dep. Phys. Ritsumeikan Univ.)
The molecular and cellular basis of the left‑right asymmetry in plantmorphogenesis is a fundamental issue in biology. A rapidly elongatingroot/hypocotyl of twisting mutants of Arabidopsis thaliana exhibits helical
growth with its handedness opposite to that of underlying cortical microtubule
arrays in epidermal cells. However, how such a hierarchical helical orderemerges is currently unknown. We propose a model for investigating
macroscopic chiral asymmetry in mutants. Our elastic model suggests that theobserved helical pattern is a direct consequence of the simultaneous presence ofanisotropic growth and tilting of cortical microtubule arrays. We predict the root
helical pitch angle as a function of the microtubule helical angle, which isconsistent with experimental measurements. The proposed model is versatile
and will be potentially important to other biological systems ranging fromprotein fibrous structures to tree trunks.
2SC-05Large-scale vortex lattice of microtubules driven by dyneins
Ken Nagai1, Yutaka Sumino2, Kazuhiro Oiwa3,4 (1Univ. Tokyo, 2Aichi Univ.
Edu., 3NICT, 4Univ. Hyogo)
Swarming behavior is well-known phenomena in living systems with various
scales: e. g., flocks of birds and bioconvection. Many individuals move together
through interaction between the others around itself.
Collective behaviors of microorganisms are often realized through chemicalinteraction between organisms. One example is spiral formation ofdictyostelium cells mediated by cyclic AMP. The analysis using reaction
diffusion equations also succeeded in pattern formation of colonies of bacillus
subtilis on agar plate.
Not only chemical interaction but steric interaction, which is caused bycollision, works between microorganisms. Theoretically, Vicsek et al. reported
that it is possible to induce swarming only through steric directional interaction
when the density of the self-propelled objects is high enough [1]. This type of
nonequilibrium phase transition has been confirmed using granular materials
experimentally [2]. We will report a novel type of swarming behavior induced
by steric interaction using microtubule-dynein motility assay. Using this system,
we found that periodic pattern of a finite size structure can be formed. Using themathematical model based on the experimentally confirmed properties of
microtubules, we found that the key properties for this phenomenon are the longcorrelation time of the motion of microtubules and nematic interaction between
microtubules [3].
[1] T. Vicsek, et al., Phys. Rev. Lett. (1995).
[2] V. Narayan, et al., Science (2007).
[3] Y. Sumino, et al., Nature (2012).
2SC-03

Non-Gaussian athermal fluctuations in active cytoskeletons
Daisuke Mizuno (Kyushu University)
Dynamic networks designed to model the cell cytoskeleton can be reconstituted
from filamentous actin, the motor protein myosin and a permanent cross-linker.
They are driven out of equilibrium when the molecular motors are active. We
have measured the fluctuations and response of probe particles in such “activegel” systems using active/passive simultaneous microrheology, and observed
both a violation of fluctuation-dissipation theorem and significant stiffening of
the networks due to the active tension. Here we further investigate the full
distribution of athermal fluctuations, also known as the van Hove correlation
function. The distribution exhibits a central Gauss-like region which we assume
derives from the processive action of many but “not too many” independent
motor proteins. Localized nature of displacement field surrounding the force

Visualizing Chromosome Structure with Superresolution
MicroscopyPeter Carlton (Kyoto University, iCeMS Institute)
The correct expression and inheritance of the genome is made possible in partby the cell's exquisite control and regulation of chromatin structure. While
microscopy has been used to elucidate many aspects of chromatin structure, thediffraction limit of light has previously limited the amount of detail available toa resolution of ~250 nanometers. However, super-resolution microscopy
techniques now allow the quantitative imaging of cellular structures at muchhigher detail. Three-Dimensional Structured Illumination Microscopy (3DSIM) uses moire interference between the sample and a striped pattern of lightto increase the resolution by a factor of two. We are using 3D-SIM to analyze
the fine structure of meiotic chromosomes in C. elegans, as well as the covalentmodification of histones in pluripotent cell chromatin. By visualizing the twolateral elements of the synaptonemal complex, separated from each other by adistance of ~150 nanometers, we can quantitatively analyze the progression of
meiotic events to a finer extent than previously possible. I will discuss our use of
3D-SIM to analyze the phenotypes of meiotic mutants and to explore thefunction of the synaptonemal complex.
2SC-06

DNA・RNA の輸送と選択：生命の起源への接近

Transport and selection for DNA and RNA: An approach tothe origin of life
Yusuke Maeda1,2,3,4, Albert Libchaber4 (1The Hakubi Center for Advanced
Research, Kyoto Univ., 2Dept. Phys., Grad. Sch. Sci., Kyoto Univ., 3PRESTO,
JST, 4The Rockefeller Univ.)
Atoms and molecules move along a gradient of external fields as seen inelectrophoresis, which is a motion of charged molecules relative to fluid along
an electric field. One unexplored but relevant alternative is thermophoresis, theSoret effect, that makes a solute moves along a temperature gradient. In this talkwe will present that thermophoresis has a great potential in biology, especiallyin the selection of molecules depending on their size and folding.
Thermophoresis depletes a polyethylene (PEG) polymer of large concentrations
from the hot region and builds a concentration gradient. In such a solution,
solutes of small concentration experience thermophoresis and PEG
concentration-dependent restoring forces. Under focused laser heating, DNA
and RNA as solutes localize as a ring-like structure which diametermonotonically decreases with the size of polymer following a behavior
analogous to gel electrophoresis [1]. Moreover, we show that the selection ofsmall RNA depending on its stem-loop structure is also possible in atemperature gradient in a solution of PEG of large volume fraction. Thus

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trapping and selection of molecules could be physically feasible in a simple way
relying on temperature gradient. Selection of DNA and RNA via transport might
be relevant to molecular evolution at the origin of life: Separation of RNA fromthe large library of RNA world might occur at the thermal vent of the deep
ocean where large temperature gradient is present. [1] YT Maeda, A Buguin, A
Libchaber (2011) Phys Rev Lett 107: 038301.
2SD-01

Dynamics and stability of proteins in cellular environments
Michael Feig (Michigan State University)
Biological cellular environments provide a complex biophysical environment
with a high degree of macromolecular crowding and chemical heterogeneity. Its
effect on biomolecular structure and dynamics remains only partially
understood. Past studies of crowding have focused on the entropic volume
exclusion effect but recent experimental data suggest that enthalpic effects thatwere largely neglected in previous work may be equally important. Here, resultsfrom recent computer simulations of protein and peptide systems in the presenceof physicochemically realistic crowder environments are presented. The overallfinding is a tendency towards destabilization of native protein structures and asignificant reduction in diffusive motion. The role of direct protein-protein
interactions vs. indirect modulation of solvent properties is discussed based on
the simulation results.

2SD-02

スーパーコンピュータは細胞生物学をどう変えるか

How supercomputing can help change cell biology
Koichi Takahashi1,2,3 (1RIKEN QBiC, 2Keio Inst. Adv. Biosci., 3Osaka Univ.
Grad. Sch. Front. Biosci.)
Predictive modeling of cellular behaviors bottom up from molecular interactions
is a holy grail in computational systems biology. Although 'rigorous' chemical
master equations exist since the midst of the 20th century, biochemical network
simulations has been suffering from lack of quantitative bases. I argue that partof this lack of predictive power in systems biology is due to negligence of nonidealistic intracellular environments, including molecular crowding and spatial
heterogeneity of proteins including localization and clustering in microdomains.
Such an environment is beyond scopes of classical theories such as EinsteinSmoluchowski theory of molecular diffusion and Michaelis-Menten's enzymatic
reaction kinetics. The advent of advanced laser micro-/spectro-scopy that candirectly observe macromolecules in living cells made us realize the need ofmodeling at the level of individual molecules and their internal states, instead ofelectrical circuit-like reaction networks. To fulfill this computational need, we
have been developing high-performance simulation platform E-Cell System
Version 4 for supercomputing architectures that can potentially handle theenormous amount of computation required to reproduce macroscopic
physiological responses of cellular systems from microscopic molecular events.
We will also introduce our efforts in modeling various types of cellularsignaling pathways including the epidermal growth factor response pathways incellular environments using K-computer.
2SD-03細胞内の場を考慮したシミュレータ RICS の開発

Development of the cell simulator in consideration of space
Hideo Yokota1,2, Yasuhiro Sunaga2, Sigeho Noda3 (1ASI, Riken, 2iSLIM,
Riken, 3ACCC, Riken)
Cells are the smallest life units, and the ultimate goal of the cell scale simulationteam is to recreate intracellular and intercellular phenomena. However,intracellular phenomena have not, for the most part, been elucidated, and theirclarification is a major objective in cell biology. It is currently impossible todevelop a simulator that can recreate all complicated intracellular phenomena.
Hence, the cell scale simulation team’s objective is to simulate intracellular andextracellular phenomena that have been sufficiently elucidated by past research
and for which mathematical models have been established. In addition, using thecomputational power of next-generation 10-petaflop supercomputers that arescheduled to be completed in 2012, we hopes to enable simulations that consider
intracellular environments that heretofore could not have been calculated due tohigh computational costs. More concretely, intracellular spaces are viewed as
grid coordinates with mesh-shaped positional information. In each grid, various
intracellular phenomena are simulated to recreate intracellular environments,
which are uneven environments containing spaces between organelles. Futureadvances in cell biology, mathematical model construction, and simulation
technologies should markedly expand the scope of cell simulation. In the hopeof contributing to the future development of cell simulation, a cell simulation

platform that can analyze coupled phenomena by linking simulators for multiple
phenomena (RICS) was developed.
2SD-04「京」による大規模遺伝子ネットワーク推定

Large scale gene network estimation with K computer
Yoshinori Tamada1, Teppei Shimamura2, Rui Yamaguchi2, Atsushi Niida2,
Ayumu Saito2, Yuto Kataoka1,2, Seiya Imoto2, Masao Nagasaki3, Satoru
Miyano1,4 (1Grad. Sch. Info. Sci. Tech., Univ. Tokyo, 2Inst. Med. Sci., Univ.
Tokyo, 3Tohoku Med. Megabank, Tohoku Univ., 4CSRP, RIKEN)
We present large scale gene network estimation software called SiGN, which iscurrently developed for the Japanese flagship supercomputer "K computer." Agene network is a graphical representation of regulatory dependencies between
genes. There are many algorithms to estimate gene networks from gene
expression data. However, mainly due to their computational speed, the existingalgorithms can be applied to only thousands of genes, which can cover only afew percent of the whole human genome. Using a supercomputer is a possible
solution in order to speed up these algorithms. Without appropriate
parallelization, however, we cannot receive benefits from its speed, due to is
massively parallel architecture. In our group, we parallelized several gene
network algorithms to overcome this problem to realize the human wholegenome scale gene network estimation. SiGN consists of three different
subprograms: SiGN-BN, SiGN-SSM and SiGN-L1. SiGN-BN estimates gene
networks with static and dynamic Bayesian network models. SiGN-SSM
implements a parameter estimation algorithm for the state space model which issuitable for modeling temporal systems of gene expressions. SiGN-L1 estimatesgene networks with sparse learning techniques. These sub-programs can capture
different aspects of gene expression data, which cannot be observed from asingle gene network model. The estimated gene networks can be viewed andanalyzed by Cell Illustrator Online and Systems Biology integrative Pipeline.
The software will be available our web site at http://sign.hgc.jp/.
2SD-05

データ同化法にもとづくデータ統合・生体シミュレーション技術

Biological system modeling and data assimilation
Ryo Yoshida (The Institute of Statistical Mathematics, Research Organizationof Information and Systems)
Modeling biochemical kinetic systems based on ordinary differential equations(ODEs) or algebraic equations has been practiced widely in metabolicengineering and many researches on systems biology. This has promoted indepth understanding of action, design and control of the potentially complex
dynamics. The system consists of a network of interacting biochemical species,
which involves production, degradation, diffusion and binding of many
substances. This study attempts, as the primary focus, to achieve a previouslyunexplored task of the system model construction; robust biochemical networks
are designed automatically according to the principle of Bayesian robustness.
Certain types of biological systems are hypothesized to maintain robustness
with respect to specific instances of systems perturbation and environmental
uncertainty. The present Bayesian method is aimed at exploring a robust ODE
network such that the model outputs fit given data robustly in response to
artificially-created kinetic perturbations, damages, reaction failures andenvironmental uncertainty. The key idea lies in the use of the Dirichlet process
mixture (DPM) as a prior distribution. Once system perturbations or kinetic
parameter variations are modeled by the DPM prior, relevant robust systems can
automatically be constructed while in the stochastic graph search process. We
provide the general framework for the Bayesian robust networking, technicaldetails on the Markov chain Monte Carlo algorithm, and demonstrations onsome benchmark problems.
2SE-01

細菌べん毛モーターの回転方向変換制御機構の解明

Elucidation of the directional switching mechanism of thebacterial flagellar motor
Tomoko Miyata1, Takayuki Kato1, Takasi Fujii1,2, Shuichi Nakamura4,
Yusuke Morimoto1,2,3, Tohru Minamino1, Hideyuki Matsunami5, Keiichi
Namba1,2 (1Grad. Sch. Frontier Biosci., Osaka Univ., 2QBiC, RIKEN, 3Grad.
Sch. Sci., Osaka Univ. Department of Applied Physics, 4Tohoku Univ. Schoolof Engineering, 5OIST, Initial Research Project)
Many bacteria swim by rotating flagella, which function as a helical propeller
driven by a reversible rotary motor that are embedded in the cell membrane. The
flagellum consists of three parts: the filament, the hook, and the basal body. The
basal body houses machinery for rotation and directional switching as well as− S12 −

for flagellar protein export, and its complex structure is composed of about 20
different proteins. The basal body contains at least five parts according to theirfunctions: the rod, LP ring, MS ring, C ring, and type III protein export
apparatus. The C ring is made of the switch complex proteins FliG, FliM, FliN,
which controls counterclockwise-clockwise (CCW/CW) switching of the motor
rotation and sits on the cytoplasmic face of the MS ring. To understand theswitching mechanisms in detail, we analyzed the C ring structures of CCW-bias
and CW-locked mutant motors by electron cryomicroscopy (cryoEM). The
CCW-bias and CW-locked phenotypes arise from single point mutation(V135L) and the deletion of residues 169-171 (ΔPAA) in FliG, respectively. We
purified the hook basal bodies from the both strains with fliC and clpP genedisrupted, collected cryoEM images, and carried out image analysis. The fittingof the crystal structure of the switch proteins into the 3D map allowed us tobuild a partial swich complex model. We will report the structural change
between the CCW and CW forms and the switching mecanism of flagellar
motor rotation.
2SE-02

マイコプラズマ滑走運動の謎にせまる

New foci to clarify Mycoplasma gliding
Makoto Miyata (Grad. Sch. Sci., Osaka City Univ.)
Many species of Mycoplasma, pathogenic bacteria form a membrane protrusion
at a cell pole, and glide on host cell surfaces. Although the gliding is fast andsmooth, the mechanism is not related to the conventional motor proteins or the
known bacterial motility such as flagella or pili. We have studied mainly on thefastest species, Mycoplasma mobile, which glides up to 4.5 micron per s and 27
pN, and proposed a working model, “The movements generated by ATPhydrolysis inside cell transmits to a “leg” protein through a “crank” protein,
resulting in the repeated catching, pulling, and releasing of sialic acids fixed on
the surface (Miyata M. Annu Rev Microbiol. 2010 64: 519-37).” To obtain the
better images of this gliding mechanism, we are now working on (i) fine andwhole structures of gliding machinery, (ii) actual movements of legs in gliding,and (iii) coupling of ATP hydrolysis and leg behaviors.

磁性細菌の超分子複合体ーマグネトソーム，細胞骨格そしてベン毛ーの構造的組織化

Structural Organization of Macromolecular assemblies-Magnetosome, Cytoskeleton and Flagella- in Magnetotactic
Bacteria
Yoshihiro Fukumori, Azuma Taoka (College Sci. Eng., Kanazawa Univ.)
Magnetotactic behavior in bacteria was discovered over 35 years ago by Richard
P. Blakemore (Science, 190, 377-379, 1975). The discovery was based on thefact that certain motile, aquatic bacteria orient and migrate along magnetic fieldlines when subjected to a magnetic field of the order of the geomagnetic field, or
greater. Furthermore, he reported that the magnetotactic bacteria have uniqueintracytoplasmic membrane vesicles named as magnetosome.
Magnetosomes comprise a chain of regular-sized bio-mineralized magnetite
crystals, each of which is surrounded by a lipid bilayer membrane and organic
components. Recently, we found that magnetosomes of Magnetospirillum
magneticum AMB-1 are covered with TPR-protein MamA oligomers outside a
lipid bilayer in near-native environments using atomic force microscopy.
Furthermore, most magnetosomes are arranged intimately along novel
cytoskeletal filaments composed of actin-like protein, MamK. Interestingly, one
extremity of the MamK filaments is located at the cellular pole. Philippe andWu have proposed that an MCP-like protein may interact with the MamK
filaments (J. Mol. Biol., 400, 309-322, 2010). Based on these results, we will
discuss structural organization of macromolecular assemblies, magnetosome,
cytoskeleton and flagella in magnetotactic bacteria.
2SE-03

2SE-04アクチンフィラメントの構造多型と機能分化

Structural polymorphism and functional differentiation ofactin filaments
Taro Q. P. Uyeda1, Nobuhisa Umeki1, Saku Kijima2, Yusuke Nishikawa3,
Kiyotaka Tokuraku3, Akira Nagasaki1, Taro Q.P. Noguchi2 (1Biomedical Res.
Inst., AIST, 2Miyakonojo Natl. Col. of Tech., 3Muroran Inst. of Tech.)Interaction with specific actin binding proteins (ABPs) is believed to define thefunction of the actin filaments. However, the story may not be that simple,
because actin filaments are intrinsically polymorphic. Moreover, ABPs induce
specific conformational changes of the filaments, often in a cooperative mannerso that ABP-induced conformational changes that occurred in the bound actin
subunit are propagated to neighbor subunits, presumably leading to cooperative
binding of that ABP to the filaments. Along this line, we recently showed thatcooperative binding of cofilin and HMM to actin filaments is mutually
exclusive, so that when cofilin, HMM and actin filaments are mixed, somefilaments bound only cofilin while others bound only HMM. Because
cosedimentation assays showed that this does not involve competition for abinding site on actin, HMM and cofilin likely induce specific conformationalchanges in neighbor actin subunits to differentially modulate their affinities forcofilin, HMM and other ABPs. This property of actin filaments might have twobiological implications. First, binding of a small number of a specific ABPmolecules would initiate a domino effect to drive the conformation of thefilament to one structure and consequently one function, amplifying and fixing
the impact of biochemical signal or thermal fluctuation that induced the initialbinding. Second, because actin filaments change the structure by stretching,
they can function as mechanosensor to locally regulate ABPs and the function of
the actin cytoskeleton.

2SE-05

バクテロイデスにおける分泌と滑走

Secretion and Gliding in Bacteroides
Keiko Sato1, Daisuke Nakane1, Hirofumi Wada2, Katsumi Imada3, Mark J.
McBride4, Koji Nakayama1 (1Graduate School of Biomedical Sciences,
Nagasaki University, 2Department of Physics Sciences, Ritsumeikan University,
3
Graduate School of Science, Osaka University, 4Department of Biological
Sciences, University of Wisconsin-Milwaukee)
Flavobacterium johnsoniae is a Gram-negative bacterium in the phylum
Bacteroidetes, which moves rapidly over surfaces at a speed of 1-3 μ m/s.
Bacteroidete gliding motility is thought to be different from any other motility
systems, but the molecular mechanism is unknown. We recently found that
SprB adhesin is responsible for the gliding motility of F. johnsoniae. Optical
microscopic observation using the fluorescent labeling technique indicated that
SprB adhesin molecules move along a “right-handed closed helical loop track”
on the bacterial cell membrane, and this movement causes the cell rotation andtranslation. SprB adhesin is secreted by the Por secretion system (PorSS), whosegene is widely distributed in the phylum Bacteroidetes, even in nonmotile
bacteria such as periodontal pathogen Porphyromonas gingivalis. The secretion
substrates are recognized by their C-terminal signal domains composed of about
80 amino acid residues. Various proteins, such as chitinase and pathogenic
cysteine proteinase, are secreted. Thus disruption of PorSS resulted not only in
loss of secretion but also motility. In this symposium, we show the uniquegliding motility and secretion system in Bacteroides, and discuss their relation.
2SE-06

Molecular motor driven self-organization in Myxococcus
xanthus
Joshua W. Shaevitz (Princeton University, Departments of Physics andGenomics)
From wildebeest herds to bacterial biofilms and every scale in between, howindividuals self-assemble into large, spatially complex groups is a key problem
in understanding collective behavior, multicellularity and development. The
coordinated motion of individuals at the cellular level can drive the formation ofhigher-scale structure is tissues, organisms and even populations. Thesephenomena arise statistically as cells modulate their direction and speed in
response to both local and global cues. A full understanding of how collective
behavior in cell populations arises has been difficult to achieve because wecurrently lack the ability to cross spatial scales and draw connections between
sensory input, motor control and group formation. Experiments examining theeffect of specific mutations on single cell movement and group morphology
have identified many important molecular players but lack the ability to probe
the behavior of individuals within groups or physical aspects of coordination. I
will present some of my group's recent work studying force production, motion
control and coordination on the molecular to the population scales using themodel social bacterium Myxococcus xanthus, a fascinating organism that takes
advantage of multi-cellular, coordinated motility to feed on colonies of bacteria
in large, pack-like groups and to form giant, spore-filled fruiting bodies during
starvation.
2SF-01− S13 −

Inconsistency between population and single cell growth rates
revealed by dynamics cytometerYuichi Wakamoto1 (1Research Center for Complex Systems Biology, Univ ofTokyo, 2JST PRESTO)
Any phenotypic states of single cells fluctuate in time-course, which produces
spectra of phenotypes in cell populations. Although it is usually assumed thatthe mean inherent phenotypic state of single cells agree with the population
average, heterogeneity of any phenotypic variables correlated with growthcauses persistent natural selection within populations and the resultant
inconsistency between single cell and population phenotypic states. Here we
provide the first experimental evidence of inconsistency between growth rates
of cell populations and inherent mean growth rates of single cells in constantenvironments using a newly developed single-cell measurement device,“dynamics cytometer”. The results showed that growth rates of clonal cell
populations of Escherichia coli under constant growth conditions were always
greater than those of single cells. The extent of growth rate gain of cell
populations quantitatively agreed with the prediction of simple growth modelthat assumes probabilistic age-dependent cell divisions. This model also
successfully predicted the population age structures. Based on these results, we
provide several mathematical relations that interconnect growth-associatedparameters of single cells and populations. Our results provide clear evidence ofinconsistency between single-cell and population properties, which indicate thatthe detailed information about single-cell dynamics is required to understandhow population properties are shaped by inherently heterogeneous single cells.
2SF-02

Information Processing via Noisy Biochemical Channels
Tetsuya J. Kobayashi1,2, Atsushi Kamimura3,4 (1Institute of Industrial
Science, the University of Tokyo, 2JST PRESTO, 3Graduate School of Arts andSciences, The University of Tokyo, 4JSPS)
Microscopic biochemical reactions are intrinsically noisy because of small
number of molecules involved in the reactions. Biological systems, however,can behave reliable even though they are composed of such noisy reactions
channels. From the information-theoretical viewpoint, the total amount of
information transmitted via noisy channel bounds the reliability of outcome inresponse to the input to the systems. Maximization of information transfer,
therefore, is crucial for reliable operation of biological systems. In this talk, wedemonstrate that Bayesian computation implemented by biological reactions
can contribute to the maximization of information transfer. The underlying
mechanism for the efficient operation of the Bayesian computation is clarified
as mapping of biologically irrelevant infinite dimensional trajectories onto
biologically relevant intracellular states. The implication of our results will also
be discussed. [1] T.J. Kobayashi, (2010), “Dynamic Bayesian Decision Making
by Intracellular Kinetics,” Phys. Rev. Lett., vol. 104, no. 228104. [2] T. J.
Kobayashi, A. Kamimura, (2011). " Dynamics of Intracellular Information
Decoding". Physical Biology, vol. 8, 055007. [3] T. J. Kobayashi, (2011),“Connection between noise-induced symmetry breaking and an informationdecoding function for intracellular networks,” Phys. Rev. Lett., vol.106, no.
228101.
2SF-03

ERK 経路の情報コード
Information coding of ERK signaling networks

Shinya Kuroda, Shinsuke Uda, Takeshi Saito (Dept. Biophys. Biochem., Univ.
Tokyo)
Cellular signaling network is capable of regulating multiple cellular functions
depending on growth factors and cell lines. For example, in PC12 cells, EGF
and NGF induce transient and sustained ERK activation, leading to cellproliferation and differentiation, respectively. This indicates that the same
signaling network can encode multiplex signal information in temporal patterns.
We made a system identification of temporal decoding of ERK activation by
selective immediate early genes (IEGs) expression. We found that pulsatile
ERK phosphorylation was decoded by selective expression of EGR1 rather thanc-FOS, and conjunctive NGF and PACAP stimulation was decoded bysynergistic JUNB expression through a switch-like response to c-FOS. Cellularsignaling network can be regarded as a communication channel in theframework of Shannon’s information theory. We can measure the distribution ofphosphorylation of ERK and CREB and expression of IEGs products at a cell
population level. We found that information transmission was generally morerobust than averaged signal intensity despite pharmacological perturbations,and information transmission through unperturbed signaling pathways
compensatorily increased in many signaling pathways. We propose that cells
use information entropy as information, so that messages can be robustly
transmitted despite noise and variation in molecular activities between

individual cells. Information coding will be discussed as a general property ofcellular signaling.
2SF-04
Fluctuating signals in cellular signal transduction systems
Tatsuo Shibata (RIKEN Center for Developmental BiologyE)Many cellular processes respond to internal and external changes by using
networks of interacting molecules. Because intracellular processes arestochastic processes, the signal transduction processes are inherently noisy.
Such inherent noise may deteriorate or improve the performance of signaltransduction systems. For example, it has been shown theoretically that theaccuracy of eukaryotic chemotaxis is limited by stochastic fluctuations in thesignaling processes of receptor on the plasma membrane. Large fluctuations arealso observed in intracellular signaling processes. Such intracellular fluctuations
are often produced by intrinsic mechanism mostly independent of extracellular
stimuli. This implies that the fluctuations are necessary for the functions ofsignaling processes. For instance, a large fluctuation may generate an
intracellular signal, which can be biased or carry an extracellular signals. In thispresentation, I will discuss the roles of such fluctuations on intracellular
processes.
2SF-05

Symmetry breaking in mouse development

Takashi Hiiragi1,2 (1EMBL, 2iCeMS Kyoto University)
A fundamental question in biology is the mechanism by which the embryonicasymmetry is established during development. The primary interest of my lablies in the principles underlying the lineage establishment in mouse
development. We have developed live-imaging for pre-implantation embryos,
demonstrating unexpectedly high dynamicity during blastocyst morphogenesis.
Our studies characterized unique principles of mouse embryonic patterning:
localized determinants play no role in generating asymmetry; mechanicalcontext plays a key role; stochastic processes generate dynamic heterogeneity.
Collectively, an attractive hypothesis is that early mammalian embryogenesis
may be a stochastic process in a particular structural context that leads to selforganization. These features suggest that, in order to fully understand theunderlying mechanisms, it will be essential to address how the diverse inputs
acting on every individual cells are integrated in the embryo at the systems
level. Thus we have recently developed necessary tools and multi-disciplinary
strategies: Venus-trap reporter mouse that visualize molecular dynamics; gene
expression profile of every single cell; computer simulation of the blastocyst
morphogenesis. In particular we have successfully established 9 Venus-traplines active specifically in one of the initial lineages. This allows highresolution 4D-imaging and tracking of dynamic lineage segregation, andcharacterizes the emergence and resolution of molecular heterogeneity. Recent
results will be discussed.
2SF-06

Stochastic expression of cell adhesion molecules in individual
neurons
Takeshi Yagi1,2 (1Osaka University, 2CREST-JST)The brain contains an enormous, but finite, number of neurons. The ability ofthis limited number of neurons to produce nearly limitless neural information
over a lifetime is typically explained by combinatorial explosion; that is, by
the exponential amplification of each neuron’s contribution through its
incorporation into “cell assemblies” and neural networks. In development, eachneuron expresses diverse cellular recognition molecules that permit theformation of the appropriate neural cell assemblies to elicit various brain
functions. The mechanism for generating neural networks must involve
molecular codes that give neurons individuality and allow them to recognize one
another and join appropriate networks. The extensive molecular diversity ofcell-surface proteins on neurons is likely to contribute to their individual
identities. The clustered protocadherin (Pcdh) is a large subfamily within thediverse cadherin superfamily. The Pcdh genes are encoded in tandem by three
gene clusters, and are present in all known vertebrate genomes. The set of Pcdh
genes is expressed in a stochastic and combinatorial manner in each neuron.
Here I present neuronal individuality based on the stochastic and combinatorial
expression of Pcdh isoforms in individual neurons. Their molecular andbiological features suggest that the diverse Pcdh molecules provide themolecular code by which neuronal individuality and cell assembly permit the
combinatorial explosion of networks that supports enormous processing
capability and plasticity of the brain.

− S14 −

discuss about their contribution in functional chromatin architecture.
2SG-01

生細胞における RNA ポリメラーゼ II による転写活性化の動態

Kinetics of RNA polymerase II transcription in living cells
Hiroshi Kimura, Timothy J. Stasevich (Grad. Sch. Frontier Biosci., Osaka
Univ.)RNA polymerase II (RNA pol II) is a multisubunit enzyme responsible fortranscription of most genes in eukaryotes. To understand how transcription isregulated in living cells, the kinetics of RNA polymerase II have been measured
by photobleaching-based techniques like FRAP. However, it has been difficult
to accurately differentiate the multiple kinetic steps, such as the preinitiation
complex assembly, the initiation and elongation, from a single FRAP curve. We
now applied the Fab-based live endogenous modification labeling method,which is a recently developed technique for monitoring post-translational
modification, to RNA polymerase II. The largest subunit of RNA pol II harbors
YSPTSPS heptapeptide repeats at the C-terminal domain (CTD). Upon geneactivation, RNA polymerase II is assembled into the preinitiation complex as
unphosphorylated form, and becomes phosphorylated at Ser5 and Ser2 in the
CTD during the initiation and elongation steps, respectively. By timing therecruitment of these different phosphorylation marks to a gene array upon thestimulation of transcription and by fitting the data to a mathematical model,
it was able to differentiate the initiation kinetics from the recruitment andelongation kinetics. Our observations suggested that transcription on the array isquite efficient and that the elongation efficiency is correlated with thepreexisting histone H3 acetylation level.

2SG-04

Miki Ebisuya (Career-Path Promotion Unit for Young Life Scientists, Kyoto
Univ.)
Our objective is to understand the mechanism and dynamics of biologicalphenomena by constructing genetic circuits. We are particularly interested information of cell patterns that are driven by Notch signaling, such as a signal
propagation pattern and a salt-and-pepper pattern. Recently, we haveconstructed a genetic circuit that generates the signal propagation pattern, (inwhich a gene expression signal propagates within a cell population), in
mammalian cultured cells. In our genetic circuit, binding of Delta (the ligand) to
Notch (the receptor) promotes transcription of Delta. A cell that produces Delta
forces its adjacent cells to produce a more amount of Delta. Namely, weconstructed a positive feedback loop between the adjacent cells. To amplify the
responses of Notch to Delta, we added a transcriptional cascade as well as a
Notch modulator, Lunatic fringe (Lfng), to the cell-cell positive feedback loop.
We found that when the genetic circuit satisfied conditions for cell-populationlevel bistability, the expression signal of Delta propagated from one cell to its
neighboring cells. These results demonstrate the mechanistic sufficiency of thecell-cell positive feedback loop to generate signal propagation.
2SG-05

2SG-02染色体場の 1 分子イメージング定量解析

Quantitative analysis of single molecule imaging of thephysicochemical field for genetic activities
Kumiko Sakata-Sogawa1,2, Makio Tokunaga1,2 (1Grad. Sch. Biosci. Biotech.,Tokyo Inst. Tech., 2RCAI, RIKEN)
Single cells in a living eukaryotic organism harbor the identical genetic
information in the chromatin. The expression of each gene is regulated in both
spatial and temporal manner. The dynamic regulation results in specificity anddiversity of the cells and determines the characteristics of the individualorganism. The nucleus accommodates the field not only to store the genetic
information but also to orchestrate the regulation of the genetic activities. The
functional compartmentation of the nucleus plays an important role in theregulation of the genetic activities. However the information about theorganization and the architecture of the compartment of the nucleus is limited.
Actin and actin related protein (Arps) are known to work as components ofchromatin-remodeling complex. Our recent results of single molecule imaging
analysis provide that transcriptional activation causes dissociation of Arp4,
subfamily of Arp, from the target sites, indicating that Arp4 is involved intranscription control. In addition to the function, recent reports demonstrate theirimportant function in the nuclear compartment. Furthermore actin is reported tobind RNA polymerase II and the binding may be required for the active forms of
the enzyme. This talk will describe the recent results in our group regarding to
the physicochemical field for genetic activities.
2SG-03

ヒストンバリアントによるヌクレオソームの構造多様性とダイナミクス

Hitoshi Kurumizaka
Engineering)(WasedaUniversity,

Facultyof

ScienceandIn eukaryotes, genomic DNA is packaged into chromatin, in which nucleosome
is the fundamental repeating unit. Core histones, H2A, H2B, H3, and H4, are theprotein components of nucleosomes, and histones H2A, H2B and H3, but nothistone H4, have nonallelic isoforms called histone variants. The histone
variants may have distinct functions in formation of specific chromosome
regions and/or in regulation of transcription, replication, recombination, andrepair of DNA. However, contributions of histone variants in these processes arepoorly understood. To reveal the structural basis of functional chromatinarchitecture, we have solved crystal structures of human nucleosomes
containing histone H2A, H2B, and H3 variants, including the centromerespecific histone H3 variant, CENP-A. We also performed biochemical andbiophysical analyses of mono- and poly-nucleosomes containing these histone
variants. In this symposium, I will show the current results of our structural andfunctional analyses of human nucleosomes containing histone variants, and

細胞分裂と染色体分配のメカノバイオロジー

Mechanobiology of Cell Division and Chromosome
Segregation
Shin'ichi Ishiwata1,2 (1Fac. Sci. Engn., Waseda Univ., 2WABIOS, Waseda
Univ.)We have been studying the effects of mechanical perturbation on cell divisionand chromosome segregation, by using a microscopic manipulation technique.
Here, I will present our recent results on the effects of mechanical impulse (MI)on the timing of chromosome segregation in HeLa cells (Itabashi, 2012),
showing that the MI, applied perpendicular to the pole-to-pole axis of a spindle,
accelerates the chromosome segregation, whereas the MI applied along thepole-to-pole axis decelerates it. I will discuss how the mechano-chemical
processes regulate the timing of spindle check point. We are interested in the
structural and functional hierarchy of the cell division system. Next, I will
introduce the visco-elastic properties and the response to the externalmechanical perturbation of meiotic spindle self-organized in Xenopus laevis egg
extracts (Itabashi, 2009). The spindle behaves as an elastic body in response tosmall compression, whereas larger compression results in plastic deformation,
but the spindle adapts to this change, establishing a stable architecture atdifferent sizes. Finally, I will report that the microtubule-depolymerizing
activity of MCAK is accompanied by the force generation at both microtubule
ends (Oguchi, 2011). The results provide a simple model for the regulation ofchromosome segregation through a “side-sliding, end-catching” mechanism.
We propose that the force balanced by molecular motors and polymerizationdepolymerization dynamics of microtubules is essential for the dynamic
stability of spindles.
2SG-06Structural versatility, dynamics, and functions of humannucleosomes containing histone variants

細胞のパターンをうみだす遺伝子回路の作製

Construction of genetic circuits underlying cell patternformation

減数分裂特異的な非コード RNA が相同染色体の対合を促進する

Meiosis-specific non-coding RNA mediates robust pairing ofhomologous chromosomes in meiosis
Yasushi Hiraoka1,2, Da-Qiao Ding2, Tokuko Haraguchi1,2 (1Graduate Schoolof Frontier Biosciences, Osaka University, 2Advanced ICT Research InstituteKobe, NICT)
Pairing and recombination of homologous chromosomes are essential for
ensuring reductional segregation in meiosis. However, the mechanisms bywhich chromosomes recognize their homologous partners are poorly
understood. Here we report that the sme2 gene encodes a meiosis-specific noncoding RNA that mediates homologous recognition in the fission yeast
Schizosaccharomyces pombe. The sme2 gene locus shows extraordinarily robust
pairing from early in meiotic prophase. The sme2 RNA transcripts accumulate
at their respective gene loci, and greatly enhances pairing of homologous loci:deletion of the sme2 sequence eliminates this robust pairing while transposition
to other chromosomal sites confers the robust pairing at those sites. Thus, we
propose that RNA transcripts retained on the chromosome play an active role inrecognition of homologous chromosomes for pairing.

− S15 −

2SH-01

KcsA カリウムイオンチャネルの溶液条件変化応答 1 分子開閉ダイ
ナミクスの解析

Analysis of Single Molecular Gating Dynamics of the KcsA
Potassium Channels Responding to Rapid Changes of Solution
Conditions
Hirofumi Shimizu1, Masayuki Iwamoto1, Antoine Royant2,5, Stetten David
von5, Laurent Guerin3, Yoshimitsu Aoki4, Michael Wulff5, Shigetoshi Oiki1(1Mol. Physiol. & Biophys., Univ. Fukui. Fac. Med. Sci., 2CEA-CNRSUniversite Joseph Fourier, 3Universite de Rennes 1, 4Keio. Univ. Sci. & Tech.
Elec., 5ESRF)
Recently, we succeeded in recording single molecular gating transitions of
KcsA potassium channel from closed to open state with the modified diffracted
X-ray tracking (DXT) method. In the conventional method the motions wererecorded at video rate under equilibrium conditions. To reveal a whole pictureof the gating dynamics we introduced a fast measurement system, pH jumpmeasurement system, optimized X-ray for the method, and an efficient spot
tracking program. The channel was fixed on a glass plate at the extracellular partand a gold nanocrystal was attached to the intracellular end of the channelmolecule as a probe. The sample was irradiated with an optimized white-beamfrom a synchrotron and the motions of the Laue diffraction spots from thenanocrystal were tracked on a CMOS detector at the speed of 5000 frames/s.
Solution pH condition was jumped during the measurements from neutral toacidic pH by laser-photolysis of caged proton, which can trigger the gating
motions of pH-gated KcsA channel. The diffraction spots in the sequential
image data were extracted and tracked by a developed data analysis program.
The trajectories of the diffraction spots were translated into movements of thechannel. With this modified DXT method we succeeded in revealing thedifferent dynamic status of the channel depending on the change of pH
condition and the gating transitions starting from the closed state with a larger
spatial range at the speed of 5000 frames/s which is comparable to the speed ofsingle channel current recordings.
2SH-02中赤外波長領域の観察が可能な赤外超解像顕微鏡の開発と生体試料への応用

Development of a mid-IR super-resolution microscope and its
application to biological samples
Makoto Sakai (Tokyo Tech.)
Infrared (IR) microscope is a powerful tool to measure molecular images of
biological samples based on molecular information. However, microscopic
objects such as biological cells cannot be measured by conventional IR
microscope, because of its low spatial resolution about 10 μm. This is a serious
problem for a microscope.
To overcome this problem, we have developed a novel type of IR superresolution microscope based on vibrational sum-frequency generation (VSFG)
detection. VSFG is a non-linear optical process and we irradiate visible and IR
beams on the sample. If IR beam is resonant to vibrational mode of target
molecule, VSFG signal photon will strongly generate. Therefore, we can detect
IR absorption of the molecule by monitoring VSFG. In addition, IR absorption
can be also imaged at the diffraction limit of visible light (several 100 nm)
because VSFG signal has visible wavelength. By using this method, we
succeeded in obtaining IR super-resolution images of biological cells such as
onion root cells and human lung cancer cells in the 3 μm IR region. The spatial
resolution is approximately 1 μm, that is smaller than the diffraction limit of IR
light.
In this study, we improved an observational wavelength range of it up to the 6-9
μm mid-IR region to observe amide bands, and applied this IR microscope tothe biological samples such as sugar crystals, amino acids, and human hairs. In
the presentation, the result of IR super-resolution micro-spectroscopy of humanhairs will be discussed in detail.
2SH-03

白色レーザーを用いた CARS 分光イメージングによる生細胞の動態追跡

Tracing dynamical behavior of a single cell by CARS
microspectroscopy using a white-light laser source
Hideaki Kano1, Masanari Okuno2, Hiroki Segawa2, Hiro-o Hamaguchi3(1Tsukuba Univ., 2The Univ. of Tokyo, 3National Chiao Tung Univ.)
The mechanism of a solubilization process of living organisms with surfactants

has so far been attributed to the surface adsorption and subsequent disruption ofcellular membranes. In this study, we applied quantitative CARS
microspectroscopy to investigate the molecular dynamics of surfactant
molecules and intracellular molecules in living cells after the exposure ofsurfactant with low concentration (0.1 w%). By using an isotope labeledsurfactant with CD bonds, the dynamic behavior of the surfactant in living cells
was clearly observed. The simultaneous CARS imaging of the cell itself andsurfactant molecules can address the details of the solubilization process withthe surfactant. We observed the accumulation of the surfactant molecules inside
a living cell and a subsequent sudden leak of cytosolic components such asproteins. This indicates the surfactant uptake prior to the solubilization of thecells. Our observation revealed that the surfactant molecules are accumulated in
the lipid rich part of the cells probably due to the similarity of the composition.
Quantitative CARS microspectroscopy enables us to estimate the molecularconcentration of the surfactant molecules accumulated in a cell quantitatively.
We also investigated the drag response to the surfactant uptake dynamics. As
the result of the inhibition of tubulin polymerization, the surfactant uptake rate
was significantly lowered. This suggests that intracellular membrane trafficking
contributes to the surfactant uptake mechanism.
2SH-04

イメージングマススペクトロメトリーによって初めて明らかになった、細胞内の脂質分布の分子種特異的な極性について

Imaging mass spectrometry revealed the polarized
intracellular distribution of specific lipid molecular species
Mitsutoshi Setou (Hamamatsu University School of Medicine Department of
Cell Biology and Anatomy)
Fluid mosaic model of cell membrane tells that fluid part phospholipids flow
like liquid. This model predicts the evenly distributed phospholipids in cell
membrane. However, the distribution of phospholipids was not wellinvestigated experimentally at intracellular level because it was very difficult tovisualize the distribution of distinct lipid molecular species regarding its varietyof length of fatty acids. We applied high resolution imaging mass spectrometry
on cultured neuron to visualize the gradient of phospholipids along the axon.
Surprisingly, the distribution is distinct between the molecular species ofphospholipids along the cellular polarity. We focus on the distribution of
Arachidonic acid (AA)-containing PC (AA-PC), which we found to be enrichedwithin the axon and is distributed across a proximal-to-distal gradient. Inhibitorsof actin dynamics (cytochalasin D and phallacidin) disrupted this gradient,while microtubule dynamics inhibitors did not. A possible model of this newly
found phenomena will be presented and discussed.
2SH-05

随意運動中のマウス運動野 2 光子イメージング

Two-photon imaging of the mouse motor cortex during
voluntary skilled movement
Riichiro Hira1,2,3, Fuki Ohkubo1,3,4, Katsuya Ozawa2,3, Yoshikazu Isomura3,5,Kazuo Kitamura2,3, Masanobu Kano2, Haruo Kasai2, Masanori Matsuzaki1,3,4(1National Institute for Basic Biology, 2Graduate School of Medicine, Universityof Tokyo, 3CREST, 4SOKENDAI, 5Brain Science Institute, Tamagawa University)
Motor cortical neurons are activated at multiple stages during voluntary skilled
movement. However, the fine-scale organization of these activities remains
poorly understood. To address this issue, we developed a self-initiated leverpull task using a forelimb for two-photon calcium imaging of the mouse cortex
in vivo. In layer 2/3 of two forelimb motor areas, the rostral forelimb area andcaudal forelimb area, we identified several types of cortical cells according totheir temporal activity patterns. We found that the trial-to-trial variability of theactivity of individual cells is associated with that of other cells. This variability
is not independent among neurons; rather, pairs of neurons covary theiractivities, depending on their spatial distribution and the extent of their firing
rates. Our results suggest that sequential reconfiguration of functional
ensembles with different correlation structures occurs in the motor cortical
microcircuit.
2SH-06

Chemiluminescence imaging of flow-induced ATP release at
caveolae in vascular endothelial cells
Kimiko Yamamoto1, Joji Ando2 (1Laboratory of System Physiology,
Department of Biomedical Engineering, Graduate School of Medicine,
University of Tokyo, 2Laboratory of Biomedical Engineering, School of
Medicine, Dokkyo Medical University)
Endothelial cells (ECs) alter their functions in response to hemodynamic shear− S16 −

stress, and although their responses play important roles in vascular tone
control, angiogenesis, and atherogenesis, how ECs recognize shear stressand transmit signals into the cell interior is poorly understood. Our previous
studies demonstrated that calcium signaling is crucial to shear-stressmechanotransduction. Shear stress evokes a rapid, dose-dependent increase in
intracellular calcium concentration caused by an influx of extracellular calcium
via ATP-operated P2X4 ion channels. P2X4 activation requires the presence ofendogenous ATP released by ECs, but the mechanism of the ATP releaseremains unknown. To analyze the dynamics of ATP release, we visualized ATP
release at the cell surface by using cell-surface attached luciferase and a CCD
camera. When exposed to shear stress, cultured ECs simultaneously released
ATP in both, a highly concentrated, localized manner and a diffuse manner. The
hot spots of ATP release occurred at caveolin-1-rich regions of the cell
membrane and were blocked by caveolin-1 knockdown with siRNA, indicating
involvement of caveolae in the localized ATP release. Immediately after thislocalized ATP release, an increase in intracellular calcium concentration andsubsequent calcium wave occurred at the same site as the localized ATP release,suggesting that the ATP released at caveolae activates nearby P2X4 ionchannels, which triggers shear stress calcium signaling in ECs.
2SH-07

バイオイメージング技術

New Fluorescent Probes and New Perspectives in Bioscience
Atsushi Miyawaki (Brain Science Institute, RIKEN)
The behavior of biochemical molecules moving around in cells makes me thinkof a school of whales wandering in the ocean, captured by the Argus system on
the artificial satellite. When bringing a whale back into the sea ― with atransmitter on its dorsal fin, every staff member hopes that it will return safely toa school of its species. A transmitter is now minute in size, but it was not thisway before. There used to be some concern that a whale fitted with a transmitter
could be given the cold shoulder and thus ostracized by other whales for“wearing something annoying.” How is whale’s wandering related to the tide ora shoal of small fish? What kind of interaction is there among different speciesof whales? We human beings have attempted to fully understand this fellow
creature in the sea both during and since the age of whale fishing.
In a live cell imaging experiment, a fluorescent probe replaces a transmitter. Welabel a fluorescent probe on a specific region of a biological molecule and bring itback into a cell. We can then visualize how the biological molecule behaves in
response to external stimulation. Since fluorescence is a physical phenomenon, we
can extract various kinds of information by making full use of its characteristics.
For example, the excited energy of a fluorescent molecule donor transfers to an
acceptor relative to the distance and orientation between the two fluorophores. Thisphenomenon can be used to identify interaction between biological molecules or
structural change in biological molecules. Besides, we can apply all othercharacteristics of fluorescence, such as polarization, quenching, photobleaching,photoconversion, and photochromism, in experimentation.
Cruising inside cells in a supermicro corps, gliding down in a microtubule like aroller coaster, pushing our ways through a jungle of chromatin while hoisting aflag of nuclear localization signal ― we are reminded to retain a playful andadventurous perspective at all times. What matters is mobilizing all capabilities
of science and giving full play to our imagination. We believe that suchserendipitous findings can arise out of such a sportive mind, a frame of mind
that prevails when enjoying whale-watching.
2SI-01

生物には効率が大事なのだろうか？

Is high efficiency necessary for organisms?
Seishi Kudo (Dept. Applied Physics, Grad. Sch. Eng., Tohoku Univ.)
I have wondered whether organisms have pursued energetic efficiency. A little
while ago, I simultaneously measured swimming speed and flagellar rotation
rate of bacteria. Obtained ratio of swimming speed to flagellar rotation rate,
which indicates how long a cell progresses during one flagellar rotation, was 7%of flagellar helical pitch for singly flagellated Vibrio alginolyticus. That for
peritrichously flagellated Salmonella typhimurium was 11% of the pitch. These
data show that flagellar screw propeller slips almost 90% during swimming.
Energetic efficiency of the screw propeller is only a few percent. Though theefficiency of the flagellar motor has been reported to be nearly 100%, it seems
to have little meaning. Energetic efficiency may not be the most important
assessment function for organisms. In the case of bacteria, they do not seem tohave pursued the most efficient shape of the screw propellers. Instead, theychose minor changes of morphology of the flagellar filaments which they
happened to get during the evolutional history. To better understand thebiological system, it may be necessary to evaluate the total cost which includes

not only short-term cost of energy but also long-term cost of development.
2SI-02

F1-ATPase モーターの一分子エネルギー論
Single molecule energetics of F1-ATPase motor

Shoichi Toyabe2, Takahiro Watanabe-Nakayama3, Hiroshi Ueno1, Tetsuaki
Okamoto4, Hiroshi Taketani1, Seishi Kudo5, Eiro Muneyuki1 (1Dept. Phys.,
Faculty of Science and Engineering, Chuo Univ., 2Faculty of Physics, LMU
Munich, 3Interdisciplinary Graduate School of Science and Engineering,Tokyo Inst. Tech., 4Environment Preservation Center, Kanazawa Univ, 5Dept.
Appl. Phys., Sch. Eng., Tohoku Univ.)
Since the first single molecule observation of the rotation of F1-ATPase (Nojiet al. Nature 1997), the mechanism of the rotation has been intensively
investigated. However, study in the energetics of the rotation has beensomewhat hampered due to the lack of the method to apply external torque.
Using the electrorotation method, we applied precisely controlled torque on therotational probe and examined the stall torque as a function of the free energy of
ATP hydrolysis (Δ GATP) (Toyabe et al. PNAS 2011). The maximum work
calculated from the stall torque coincided well with Δ GATP under wide
conditions, indicating that the coupling of ATP hydrolysis/synthesis and step
rotation was quite tight. Distant from the stall conditions, applying Harada-Sasa
equality, we found that the difference between ΔGATP and work against theexternal torque dissipated through the nonequilibrium fluctuation and steadymotion in the rotational degree of freedom (Toyabe et al. PRL 2010).
Furthermore, we could successfully reconstitute state-specific free energy
potential from single molecule trajectories (Toyabe et al. epl 2012). We could
also estimate the point where the chemical state switches and found that the statetransition occurred where the free energy potentials intersected. This finding
gives a phenomenological interpretation for the high efficiency of F1-ATPase
motor.
2SI-03Observation of vibrational absorption of single proteins at few
Kelvins
Satoru Fujiyoshi (Tokyo Tech.)Single-protein fluorescence spectroscopy at few K was applied to study
individual structures and conformational dynamics of proteins. The method is
attractive as well in the mid-infrared (MIR) wavelength region, information on
the three dimensional structure of the backbone of individual proteins would be
provided if vibrational infrared-absorption spectroscopy was applied to single
proteins. However, detection of infrared absorption of a backbone vibration of asingle protein has not yet been achieved. Here we report that MIR irradiation
modified temporal behavior at 1.5 K of the fluorescence-excitation spectrum inthe near-infrared region of single bacteriochlorophyll a molecules bound as a
B800 pigment to the light harvesting 2 complex. Out of over 1000 vibrationalmodes that absorbed MIR light, measurement of MIR-frequency dependence ofthe modification revealed MIR-absorption spectrum of the vibration thatinduces change of a hydrogen-bond at the pigment-binding site.
2SI-04

揺らぎの定理によるミトコンドリア輸送の駆動力測定

Estimation of driving force acting on a mitochondriontransported in a living cell: Application of the fluctuation
theorem
Kumiko Hayashi1, Masaaki Sato2, Kazunari Mouri3, Chang-gi Pack3,
Kazunari Kaizu4, Kouichi Takahashi4, Yasushi Okada4 (1Sch. Eng., Tohoku
Univ., 2IMRAM, Tohoku Univ., 3ASI, RIKEN, 4QBiC, RIKEN)
Technological development in both the manipulation and observation of nanoand micro- sized objects has led to a new understanding of thermodynamics andstatistical mechanics in small systems such as colloidal particles and proteins,
moving subject to thermal fluctuation. For example, while the second law ofthermodynamics predicts that an entropy production should be increased, it
became an apparent after the discovery of statistical mechanics that it can haveinstantaneously negative values. Probability of measuring negative values ofentropy production is related to that of measuring positive values by thefluctuation theorem (FT). FT has several important consequences such as the
Jarzynski equality and the Crooks fluctuation theorem. They are useful tocompute the free energy difference between two equilibrium states, and wereexperimentally tested in RNA hairpin systems. Recently, another kind of FT has
been suggested to be useful for measuring the driving forces of motor proteins.
This force measurement method using fluctuations was applied to the singlemolecule experiment on F1-ATPase. In this talk, we extended the applicability− S17 −

of FT, as the force measurement method, to estimate driving force acting on anorganelle transported by motor proteins in a living cell. Among organelle, we
experimentally studied mitochondria transported by linear motor proteins suchas kinesin and dynein in PC12 cells. The result was compared with thesimulation developed by the E-Cell system, which is a software platform formodeling cells.
2SI-05

物質界面における構造と電荷移動を可視化する走査型プローブ顕微鏡法の開発

Development of scanning probe methods for investigating
structures and charge transfers at material interfaces
Kenjiro Kimura (Grad. Sch. of Sci., Kobe Univ.)
It is essential for understanding the working mechanism of functional biomaterials in liquid to study their structures including solvation with higher
spatial resolution. Recently, we have developed a molecular-scale solvation
imaging method using frequency-modulation atomic force microscope withatomic resolution in liquid. In this method, the atomic force microscope tip isthree-dimensionally scanned at solid- liquid interface, and distributions ofapplied forces toward the tip, related to the structures of solid surface and its
solvation, are imaged. Using this method, we have revealed solvation structures
around various materials such as minerals, polymer films, organic monolayers
and bio-molecules and relationships with their solid surface structures. Morerecently, we have developed a subsurface magnetic field imaging method forunderstanding the locations of charge transfers inside materials. In this method,
highly-sensitive magnetic field sensor is two-dimensionally scanned above
materials, and a couple of magnetic field images are acquired at different
heights from the material surface. Then, basic equation of electro-magnetic field
is solved using two images as its boundary conditions, and two-dimensional
magnetic field around the locations of charge transfers inside materials isreconstructed with sensor-size spatial resolution.
2SI-06

DNA 折り紙フレームを用いた一分子観察

Direct Observation of Single Molecular Event in DNA Origami
Frame
Hiroshi Sugiyama1,2 (1Grad. Sch. Sci. Kyoto Univ., 2iCeMS)
The DNA origami method developed for the preparation of fully addressable
two-dimensional (2-D) structures has been utilized for the selective positioningof the functional molecules and nanoparticles and for the design of various 3-Darchitectures. Here we report the design of ” DNA frame” using the DNA
origami method to examine enzymatic action. We newly developed the tensioncontrolled dsDNA substrates in the DNA frame and showed the importance ofDNA strand relaxation in allowing double helix bending during enzymatic
reaction. In our DNA frame, tensed and relaxed dsDNA can be created bybridging the defined length of dsDNA in the DNA scaffold. The relaxed strand
can accommodate the enzymes to bind and bend the target sequences. On theother hand, the tensed strand allows binding of these enzymes, while this strand
is a poor substrate for bending, resulting in the lower reaction efficiency. In
addition, the DNA frame is valuable for analyzing the motion of the enzyme
because of the defined coordinated space. The exact location and displacementof the enzyme in the reaction on the dsDNA can be monitored and analyzed.
Therefore, the time-resolved reaction coordination between the enzymes andsubstrate can be estimated at meso-scale spatial resolution. In this presentation
direct observation of DNA modifying enzymes such as DNA methylase (M.
EcoRI), 8-oxoguanine glycosylase (hOgg1), T4 pyrimidine dimer glycosylase(PDG), and recombinase (Cre recombinase) as well as DNA structural change
will be discussed.

− S18 −

1A1400

コイルドコイル ミオシンの脆弱部位と屈曲性の保存

1A1436Prediction of fragile points and conservation of bending abilityof coiled coil myosin
Mieko Taniguchi1, Hideki Tanizawa2, Sigeki Mitaku1 (1Grad. Sch. Sci., Univ.
Nagoya, 2Inst. Wister, USA)
Myosin is a mechano-chemical motor that converts the chemical energy of ATP
hydorolysis into mechanical energy. Myosin has two heads and a long tail.
Recently a prediction system has developed for identification of heptad breakpoints and fragile points due to the hydrophilic core or hydrophobic outfieldregion in the coiled coil. Here we identified the skeletal myosin rod using thisprediction system. The results showed that the rod has 8 heptad break points
and 10 fragile points. To compare their values from prediction, we precisely
analyzed their bending positions of the rods using AFM. As a result, we foundthat typical bending positions with high bending angles in the rod were tightly
correlated to their fragile points and heptad break points.
Finally we would like to report about sequence homology between their heptad
break points in 22 myosins from different sources.
1A1412カーボンナノチューブ上におけるミオシン運動の温度応答

Thermal response of myosin motors on single carbon
nanotubes
Mitsunori Nagata1, Hiroshi Matsutaka1, Takeru Okada3, Akihiko Ishijima2,
Yuichi Inoue2 (1Grad. Sch. Life Sci., Tohoku Univ., 2IMRAM, Tohoku
Univ., 3Institute of Fluid Science, Tohoku University)
We demonstrated that single carbon nanotubes (CNTs) can be used as a newheat conductor to induce temperature change for skeletal muscle myosin(Annual meeting, 2011). However, dynamic range and local gradient of theinduced temperature were not well characterized. Toward an application ofvarious proteins including motor proteins, thermal response of myosin motors
on single CNT was examined.
Multiwall CNTs (~170 nm diameter, ~10 μm length) were coated with skeletal
myosin to observe sliding of fluorescently-labeled actin filaments in the presenceof 2 mM ATP. During the sliding, an endpoint of the CNT was temporarily heatedwith a laser irradiation (645 nm, φ ~2 μm, ~0.2 s) to supply external heat.
By the local heating with laser, the sliding speed linearly increased to ~12 μm/s
with increasing laser power up to 2.7 mW, indicating temperature jump of~15°C at myosin. However, laser irradiation of 3.7 mW reduced the sliding
speed, suggesting the denaturation of myosin at > 40°C.
Our results show that CNTs work as a heat conductor to induce temperature
jump over 20° C for myosin. Distribution of the speed along a CNT andsimulation with finite element method suggest the temperature gradient along
CNT (~10 mu;m) and perpendicular to CNT (sub mu;m). Possibility of the local
temperature and future applications would be discussed in the meeting.
1A1424生体分子モーターのためのステップ解析アルゴリズム

Powerful Algorithm for analyzing Stepping Motion of
Biological Melecular Motors
Takeshi Nakagawa, Kazuo Sasaki (Dept. of Applied Physics, Tohoku Univ.)
Many of biological molecular motors move stochastically along a linear track ina stepwise manner. The algorithms extracting step sizes and dwell times fromthe noisy experimental data proposed by Kerssemakers et al. [Nature 442, 709(2006)] and by Kalafut et al. [Comput. Phys. Comm. 179, 716 (2008)], among
others, seem excellent in that they do not require assumptions on the underlying
mechanism of the stepping motion or user supplied parameters. We evaluatedthe performance of these algorithms using benchmark data under various
conditions, and found that the Kerssemakers algorithm becomes considerably
worse as the noise level increases and that the Kalafut algorithm shows better
performance than the Kerssemakers algorithm under almost all conditions.
We propose a new step finding algorithm, which turned out to work better thanthe Kalafut algorithm for benchmark data with noise level smaller than about
3/8 of the step size and work equally well with the Kalafut algorithm for datawith larger noise level. Our method adopts the first stage of the Kerssemakers
algorithm to fit a step function to the data by the least-square method. Eachcandidate step is accepted or rejected based on the two-sample Student's t-test.
The significance level for the test is automatically determined depending on thenumber of data points in the two dwells adjoining the step. We will explain thedetails of our method and compare its performance with the other algorithms at
the meeting.

高時間分解能暗視野顕微鏡を用いたキネシン頭部の前方へのステッ
プの観察

Direct observation of the forward stepping motion of kinesin-1
using dark-field microscopy with 50-μs temporal resolution
Hiroshi Isojima1, Ryota Iino2, Hiroyuki Noji2, Michio Tomishige1 (1Dept.
Appl. Phys., Sch. Eng., Univ. Tokyo, 2Dept. Appl. Chem., Sch. Eng., Univ.
Tokyo)
Kinesin-1 moves along microtubule by alternately moving two motor domains,in which the trailing head detaches and displaces toward the 16-nm forward
tubulin-binding site upon ATP binding to the partner head. However, thedetailed process and the mechanism of the forward stepping motion are still
unknown, mainly due to the lack of temporal resolution of the single-molecule
measurements. Here we employed dark-field microscopy with perforated
mirror, and observed the movement of one of kinesin heads labeled with 40-nm
gold nano-particle (GNP) with ~4 nm spatial and ~50 μs temporal resolutions.
In the presence of saturating ATP, the GNPs showed ~16 nm discrete steps
toward the microtubule long axis, which is consistent with the previous
observations. However, our measurements also revealed that the GNP displaced~20 nm rightward, perpendicular to the microtubule axis, accompanied with asignificant increase in the fluctuation of GNP. The rightward asymmetry is
consistent with the kinesin’s structure that the proximal end of the neck linker
is located right side of the head. Then GNPs showed rapid left-forward
displacement followed by decrease in the fluctuation, although we could notdetect clear sub-step. The duration of the highly fluctuating period increased as
the ATP concentration was decreased, indicating that the period represents
ATP-waiting state. These results support the biased binding mechanism inwhich the tethered head freely diffuses and is selectively captured at the forward
tubulin-binding site.
1A1448Front-head gating mechanism of kinesin-1 as studied by single
molecule FRET observation of ATP binding
Yamato Niitani, Michio Tomishige (Department of Applied Physics, School ofEngineering, The University of Tokyo)
Kinesin is a dimeric motor protein that moves along microtubule in a hand-overhand manner. To walk in a coordinated fashion, kinesin’s two motor domains(heads) should alternately move and hydrolyze ATP, however the underlying
mechanism is still controversial. At the last meeting, we presented results using
disulfide-crosslinking showing that ATP hydrolysis cycle of momomerickinesin can be differently regulated depending on the direction of the necklinker extension. Here we investigated the effect of neck linker tension on thehead of dimeric kinesin by directly observing ATP binding/dissociation to/fromthe leading head using single molecule FRET. We employed heterodimerickinesin that is composed of a wild-type head and a mutant head that binds
ATP but hydrolyzes it very slowly. This heterodimer has been shown to
predominantly take two-head-bound state where the wild-type head is theleading. Then we introduced donor fluorophore to the wild-type head near the
nucleotide-binding pocket and observed FRET in the presence of Alea-647
labeled ATP. We observed robust binding of ATP to the trailing head (lowFRET state) but rarely observed binding to the leading head (high FRET state).
When we extended the neck linker of the heterodimer by inserting poly-Gly
after the neck linker, we observed frequent, transient binding of ATP to theleading head. These results support the front-head gating mechanism that the
backward tension posed to the neck linker suppresses ATP binding to theleading head.
1A1510キネシン様モータータンパク質 Ncd の Power-stroke モデルの検証

The test on the power stroke model of Ncd
Masahiko Yamagishi, Yoko Toyoshima, Junichiro Yajima (Dept. Life
Sciences, Grad. Sch. Arts and Sciences, Univ. Tokyo)
Drosophila kinesin-14, Ncd, moves toward the minus-end of microtubules. Ncd
has a neck region between the motor domain, the catalytic core of ATPase, andthe stalk domain, dimerizing region. The neck is composed of a rigid coiled-coilstructure. The widely accepted model of its force-producing mechanism is thepower-stroke model in which Ncd strokes its neck region as a lever-arm aroundthe motor domain as a pivot in the direction to the minus-end of microtubules.
Therefore, the power-stroke model needs the rigid coiled-coil structure. Here, totest the power-stroke model, we have engineered Ncd that was fused to gelsolin at
the C-terminus as a tag to fix to the glass surface. These Ncds are able to be linked− S19 −

to the surface via their C-terminus, leaving the N-terminal coiled-coil structure
hanging free. These C-linked Ncds drove microtubule sliding at similar rates to
N-linked Ncds, thereby ruling out models in which the rigid coiled-coil structureis the only element able to exert or transmit force. Instead, our findings indicate
another force-generating mechanism that shifts both the N & C terminus of thehead, so that force can almost equally well be exerted by either the N or C
terminus of the head. It was previously reported that a chimera Ncd whose Cterminus was replaced with kinesin-1 neck was able to move when it was attachedto the glass at its C-terminus, so the kinesin-1 neck was regarded as a critical part.
But our result shows that the kinesin-1 neck is not necessary.
1A1522

細菌べん毛モーター固定子複合体 MotA/B チャネルの選択的透過メカニズム

Selective permeation mechanism through the channel of thestator complex MotA/B in the flagellar motor
Yasutaka Nishihara, Akio Kitao (IMCB, Univ. of Tokyo)
Bacterial flagellar motor is composed of a rotor and stators. Escherichia coli haveproton-driven motor embedded in the membrane and the stator consists of MotA
and MotB proteins. Experimental studies have suggested that the stator is atorque-generating unit and that binding of protons across the membrane to Asp32
of MotB induces changes of the stator structure and the interaction of MotA withthe rotor. However, atomic-level structure of the transmembrane (TM) regions of
the stator has not been determined yet and the detailed mechanism of torquegeneration is still unclear. In this study, we constructed the structure of TM
regions of the stator using molecular modeling and calculated free energy profiles
for ion permeation through the channel of the stator. For the modeling, thedistance restraints derived from disulfide cross-linking experimental data were
employed in simulated annealing and molecular dynamics (MD) calculations. The
free energy profiles for the permeation of water molecule, hydronium ion andsodium ion were calculated with the steered MD calculations. These profiles
show that water molecule should overcome a high free energy barrier (< 7kcal/mol) whereas no high free energy barrier was found in the hydronium ion
permeation. We identified that difference of interaction between the permeatedmolecules and main chain is the origin of the selectivity.
1A1534

Sodium Dynamics of the Bacterial Flagellar Motor
Chien-Jung Lo1, Yoshiyuki Sowa2, Teuta Pilizota2, Richard Berry2(1Department of Physics, Natioinal Central University, Taiwan, 2Clarendon
Laboratory, Department of Physics, University of Oxford)
Many species of bacteria swim by means of flagella, the long helical filaments
connected to rotary molecular motors embedded in the cell membrane. Motors
can run up to several hundred hertz. The motor is powered by thetransmembrane ion-motive force: a combination of electrical voltage (Vm) andchemical potential. To explore the fundamental mechanism of this powerfulmolecular machine, we measured the torque-speed curves of sodium-driven
chimeric flagellar motor in Escherichia coli. We test the motor speed in 25
different sodium-motive-force conditions. Similar to other report, the generalshape of torque-speed cure is concave down with negative slop. The shape isless sensitive to the membrane voltage but very sensitive to the sodium
concentration. The knee and the zero-torque speed depended on sodium
concentration and the stall torque depended on the total sodium-motive- force.
In high load conditions, the motor operates close to thermodynamic equilibriumwith high efficiency. By energy balance, the ion number required to drive themotor in high load in different smf are 37±2 ion/rev. We also build a 4-statekinetic model to fit the data. Using Monte Carlo Optimization, we found themodel parameters to fit the experimental data. We found the ion binding in low
sodium concentration and the ion transition through stators are the rate limiting
steps. The fitted parameters indicate that the motor mechanism is ‘powerstroke’ and ion transit is channel-like rather than carrier-like.
1A1546Engineered disulfide crosslink in the the periplasmic region of
PomB impaired function of the Na+-driven flagellar stator
complex

Shiwei Zhu, Na Li, Seiji Kojima, Michio Homma (Division of Biology
Science, Graduate School of Science, Nagoya University)
In the functioning bacterial flagellar motor, multiple stator units composed of
PomA and PomB must be incorporated into the motor at the appropriate
places around the rotor. When assembled, stator units are anchored at the
peptidoglycan layer through the periplasmic region of the B subunit. Crystal

structures of this region have been recently solved, and it was proposed that thelarge conformational changes in this region are required for activation andassembly of the stator complex. To test this model, here we employed thedisulfide crosslinking approach. If the large conformational changes are induced
during the assembly of stator, then an intramolecular disulfide bridge that isengineered to covalently link between two movable elements may inhibit statorfunction. Based on structural information of the periplasmic region of PomB
(PomBC), cysteine replacements were introduced into the cysteine-less PomB
protein. We found that mutant strains with single cysteine replacement remain
motile, but when we introduced several pairs of cysteine, one in the α1 helix(154-169) and another in the core domain (C-terminal region from residue 170)
of PomBC, pairs of M157C-I186C, L160C-I186C, I164C-L217C and L168CQ213C inhibited the function of the Cysteine-less PomB. However, after
treating with DTT, all of them restored the motility less than 1 hour. Thus, we
speculated that the crosslink due to the disulfide bridge formed by one pair ofcysteine inhibits the conformational change in the periplamic region of PomB.
(1529/1560)
1A1558高圧力が引き起こすべん毛繊維のダイナミック構造変化

Dynamic conformational changes of flagellar filament
observed by high-pressure microscopy
Masayoshi Nishiyama1, Yoshiyuki Sowa2 (1The Hakubi Center, Kyoto
Univ., 2Hosei Univ.)
The bacterial flagellar motor is a molecular machine that rotates a flagellum inboth directions. CCW rotation allows the left-handed helical filaments to form abundle that propels the cell smoothly, whereas CW rotation of a filament leads
to change the shape of filament in right-handed helix and break the bundle, andinhibits smooth swimming of the cell, called a tumble. The switching in thehelical structure is thought to be caused by directional mechanical actions
arising from abrupt change of exerted torque by the motor rotation. Here, weshow that application of pressure can also change the helical structure offlagellar filaments. The flagellar filaments in E. coli cells were fluorescently
labeled, and then the images were acquired by using an improved high-pressure
microscope. We measured the diameter and pitch of the individual filaments andthen classified them into 11 possible waveforms which are predicted fromstructural data. At 0.1MPa (ambient pressure), all flagellar filaments form lefthanded helical structure (normal form). At 40 MPa, we found not only left-, butalso right-handed structures such as coiled-coil and curly I (or II). At 80 MPa,
only the right-handed structures existed. After the pressure was released, mostfilaments returned to the initial left-handed structures. The application ofpressure is thought to enhance the structural fluctuation and/or association ofwater molecules with the exposed regions of flagellin molecules, and results inswitching the helical from left- to right-handed structure.
1A1610c-di-GMP 結合タンパク質 YcgR のホモログ PlzD の大量発現による Vibrio alginolyticus でのべん毛運動阻害

Flagellar motility inhibition by overexpression of PlzD, a YcgR
homolog of c-di-GMP binding protein, in Vibrio alginolyticus
Takuro Yoneda, Wakako Morimoto, Seiji Kojima, Michio Homma (Grad.
Sci., Univ. Nagoya)
Bacterial flagellum is a locomotive apparatus which is needed to swim or toswarm. At the base of each flagellum, there exists a membrane-embedded
motor. The ion flux through the stator couples to the rotor-stator interaction thatgenerates torques. Recently it was found that YcgR inhibits motility in thepresence of cyclic diguanylate (c-di-GMP) in E. coli. Its N-terminal
YcgR/PilZN domain is considered to be required to interact with flagellar basal
protein (s) and C-terminal PilZ domain is a receptor for c-di-GMP. It wasreported that PlzD of Vibrio chorelae, a homolog of YcgR, affected the flagellar
motility under a poor nutrient condition. In this study, we cloned PlzD from
Vibrio alginolyticus. V. alginolyticus PlzD conserves all 5 residues required forbinding to c-di-GMP for PlzD of Vibrio chorelae. PlzD inhibited motility innutrient-poor plate and EGFP-PlzD was localized at cell pole. N-terminallytruncated PlzD shows weaker motility inhibition than wild type PlzD, and
EGFP-PlzDΔ1-35 exhibits no fluorescent localization. Based on these results, we
speculate that N-terminal region of PlzD is important for interact flagellar basal
protein(s) and that PlzD can assemble around flagellar basal body. PlzD as wellas YcgR repress motility significantly in E. coli MS1467 strain (ΔyhjHΔycgR).
This indicates that PlzD can function in E. coli and may suggest a
conformational similarity between N-terminal region of PlzD and YcgR though
the sequence homology is very low.

− S20 −

1B1400

measurements and the on-going sequential signal assignments. Furthermore, we
tried to create PomA fragments containing the cytoplasmic loop region, withwhich FliG is supposed to interact. We found that overexpression of the PomA
fragment impedes the motility. We are currently trying to measure NMR spectraof FliGC in the presence and absence of PomA fragments, and further results
will be discussed at the meeting.

“空隙”ー機能構造創出のための自然の戦略
Cavity−a Nature’s strategy for functional sub states inproteins

Kazuyuki Akasaka (Kinki UNIV., Inst. Adv. Tech.)
How Nature can provide proteins with the dynamics and sub states required fortheir specific needs for function is a fundamental question in protein scienceand evolution. To address the question, here we focus on cavities. Cavity is
synonymous to void in folded proteins, often discernible as atom defect incrystal structures of proteins. Although cavity has long been known as a factor
that affects thermal stability and compressibility of proteins (Gekko andHasegawa, Biochemistry 1986), its specific role in protein dynamics andfunction remains unclear. In recent years, the question is pursued intently onpressure axis using NMR or X-ray crystallography in functional proteins
including hen lysozyme, cMyb protein and OspA among others (1-3). The
studies start revealing crucial roles of cavities in providing functional dynamics
and sub states, and further suggest their roles in evolution.
References
1. Kamatari et al., Biophys. Chem.156, 24-30 (2011).
2. Sunilkumar P N et al., Biophys. J. 102, L8-L10 (2012).
3. Kitahara et al., Biophys. J. 102, 916-926 (2012).
1B1412

HD-exchange motion of free heptameric GroES studied by theuse of TROSY and DMSO quenching followed by 2D NMR
Mahesh Chandak1, Takashi Nakamura1, Koki Makabe2, Toshio Takenaka1,
Jin Chen1, Koichi Kato1, Kunihiro Kuwajima1 (1Okazaki Institute ofIntegrative Bioscience, National Institutes of Natural Sciences, The Graduate
University for Advanced Studies (Sokendai), 2Graduate School of Science andEngineering,Yamagata University)
The GroEL-GroES is a chaperonin complex which assists target proteins to foldinto native state. To understand structural and functional relationship of GroESGroEL complex, we studied the dynamic structural fluctuations of freeheptameric GroES by the use of hydrogen/deuterium (H/D) exchange and
2DNMR techniques. H/D exchange analysis is used to trace the rate at which
protein backbone amides undergo chemical exchange with deuterated solvent.
NMR spectroscopy is used to study proteins having molecular weight up to
50KDa. However, as the molecular weight of the GroES complex is 70KDa, wecannot get well resolved NMR spectrum. So, we need a modified strategy to study
this complex. In this study, we report the dynamic structural fluctuations of freeheptameric GroES by the use of H/D exchange technique followed by:
1) Transverse relaxation optimized spectroscopy (TROSY) and 2) DMSO
quenching with 2DNMR. In our study, we observed rapid exchange rate of
backbone amide residues in a mobile loop region. During the GroES-GroEL
complex formation, this mobile loop region forms contact with GroEL complex.
Therefore, this result indicates that mobile loop is very flexible in free heptameric
GroES.
1B1424溶液 NMR を用いた Na+駆動型べん毛モーターの固定子タンパク質 FliG の C 末端領域構造解析

Solution NMR analysis of FliG C-terminal domain derived
from Na+-driven motor of Vibrio
Mizuki Gohara1, Rei Abe-Yoshizumi1,2, Shiori Kobayashi1, Yohei
Miyanoiri3, Yoshikazu Hattori4, Chojiro Kojima4, Masatsune Kainosho3,5,Michio Homma1 (1Div. Bio. Sci., Grad. Sch. Sci., Nagoya Univ., 2Department
of Frontier Materials, Nagoya Institute of Technology, 3Structural Biology
Research Center, Grad. Sch. Sci., Nagoya Univ., 4Institute for Protein
Research, Osaka Univ., 5Center for Priority Areas, Tokyo Metropolitan Univ.)
In the bacterial flagellar motor, torque is generated by interactions between therotor and the stator. In the rotor, a C ring component, FliG, mainly participatesin torque generation, primarily through its C-terminal domain. In the H+-driven
motor of Escherichia coli, mutational analyses revealed that the rotor-stator
interaction is mediated by charged residues between the FliG C-terminal domain
and the MotA cytoplasmic loop region. However, in the Na+-driven motor of
Vibrio, the effects of mutations of the charged residues on the motility were very
weak. Therefore, the mechanism of torque generation in the Na+-driven motor
remains unknown. To investigate the interaction between FliG and the MotA
ortholog PomA in the Na+-driven motor, we performed solution NMR analyses.
We previously reported that drastically improved 1 H-15 N TROSY-HSQC
spectra were obtained with the N-terminally truncated FliG variant, G214-FliG(FliGC). We’ d now like to discuss the result of the 3D 1 H-13 C-15 N NMR

1B1436

酸化還元状態に依存してプロテインジスルフィドイソメラーゼの基
質結合部位ドメインの空間配置が変化する仕組み

Molecular mechanism of redox-dependent domain
rearrangement of the substrate-binding region of protein
disulfide isomerase
Kouya Inagaki1,2, Yoshinori Uekusa1,2, Yukiko Kamiya1,2, Tadashi Satoh1,
Koichi Kato1,2 (1Grad. Sch. Phar. Sci., Nagoya City Univ., 2Natl. Inst. Nat. Sci.)
Protein disulfide isomerase (PDI), a major protein in the endoplasmic reticulum,
operates as an essential folding catalyst and molecular chaperone for disulfidecontaining protein substrates. We have previously reported that b’ and a’domains of PDI exhibit an intramolecular domain rearrangement depending on
the redox state of the active site cysteine pair in a’ domain, which is coupledwith the shielding and exposure of substrate-binding hydrophobic surface
spanning across these domains. To gain insights into the molecular mechanismof this domain rearrangement, we prepared mutated PDI-b’ a’ in which acysteine pair was substituted for serine or alanine. By using fluorescenthydrophobic probe, we revealed that the serine- and alanine-substituted mutants
mimicked the reduced and oxidized forms of wild-type protein, respectively, interms of exposure of hydrophobic surface. By nuclear Overhauser effectexperiments of NMR spectroscopy, we demonstrated that the side chain of thetryptophan residue located adjacent to the a’ active site was in contact with theb’ domain in the reduced form and serine-substituted mutant, whereas thetryptophan side chain was accommodated in the active site in the oxidized state
and the corresponding site of the alanine-substituted mutant. Based on theseresults along with those obtained by X-ray crystallographic analyses, we
proposed a mechanistic model of the domain rearrangement triggered by redoxdependent flipping of the tryptophan side chain.
1B1448Transferred cross-saturation 法を用いた EB1 の CH ドメイ
ンの微小管との結合界面の特定

Microtubule-binding sites of EB1 CH domain revealed bytransferred cross-saturation experiments
Teppei Kanaba1, Ryoko Maesaki2, Tomoyuki Mori2, Yutaka Ito1, Toshio
Hakoshima2, Masaki Mishima1 (1Grad. Sch. of Sci. and Tech., TMU, 2Grad.
Sch.l of Biol. Sci., NAIST)
End-binding 1 (EB1) is a member of plus end tracking proteins which bind to
microtubule (MT) plus end and regulate MT dynamics. EB1 binds to microtubule
lattice with its N-terminal CH domain and stabilizes MT. EB1 is thought to be
autoinhibited by the interaction between CH domain and C-terminal domain.
Despite the importance of the EB1-MT interaction in regulation of MT dynamics,
the molecular details of the EB1 autoinhibition remain unclear.
By comparison between CH domain K59E mutant (low affinity interactor) data
and a competition data, we identified the binding interface of CH domain withpolymerized tubulin by transferred cross-saturation (TCS) experiments. In
addition, by chemical shift perturbation experiments, we identified the binding
interface between EB1 CH domain and C-terminal domain. Interestingly, we
found that the MT-binding sites and C-terminal domain binding sites of CH
domain are closely located, indicating that the MT-binding sites are masked by
the interaction between the C-terminal domain in an autoinhibited state. Our
chemical shift perturbation experiments suggest that the CH domain binding
region of C-terminal domain is almost identical to the SxIP motif of APC and
CAP-Gly domain of p150 glued binding region. Autoinhibition may therefore be
regulated through competition binding by such binding partners.
1B1510

再構築型生体外タンパク質合成系を用いた分子シャペロンによる凝集抑制効果の大規模解析

Global analysis of aggregation-inhibotion effects of molecularchaperones using a reconstituted cell-free translation system
Tatsuya Niwa1, Takuya Ueda2, Hideki Taguchi1 (1Grad. Sch. of
Biosci&Bioteh, Tokyo Institute of Technology, 2Grad. Sch. of Frontier
Sciences, The university of Tokyo)− S21 −

Protein folding is often hampered by protein aggregation, which can beprevented by a variety of chaperones in the cell. A dataset that evaluates whichchaperones are effective for aggregation-prone proteins would provide aninvaluable resource not only for understanding the roles of chaperones, but also
for broader applications in protein science and engineering. Previously, we
conducted comprehensive aggregation analysis of more than 3,000 Escherichia
coli proteins by using a reconstituted cell-free translation system (PUREsystem), which does not contain any chaperones (Niwa et al., PNAS, 2009). A
histogram of the solubilities revealed a clear bimodal distribution, implying thatthe many aggregation-prone proteins require chaperones to fold correctly.
Then, we comprehensively evaluated the effects of the major E. coli chaperones,
trigger factor, DnaK/DnaJ/GrpE, and GroEL/GroES, on ~800 aggregation-prone
cytosolic E. coli proteins, using the PURE system. Statistical analyses revealed
the robustness and the intriguing properties of chaperones. The DnaK and GroEL
systems drastically increased the solubilities of hundreds of proteins with weakbiases, whereas trigger factor had only a marginal effect on solubility. The
combined addition of the chaperones was effective for a subset of proteins thatwere not rescued by any single chaperone system, supporting the synergistic
effect of these chaperones. The resource can be used to investigate the propertiesof proteins of interest in terms of their solubilities and chaperone effects.
1B1522

DNA 界面での蛋白質の局所的構造変化
Local conformational changes of proteins in DNA interfaces

Ryotaro Koike1, Shuichi Takeda2, Yuichiro Maeda2, Motonori Ota1 (1Grad. Sch.
Info. Sci., Nagoya Univ., 2Struct. Biol. Res. Center, Grad. Sch. of Sci., Nagoya Univ.)
Capping protein (CP) binds to the barbed end of an actin filament and inhibits
the further polymerization of actin monomers onto the filament. The
polymerization inhibition of CP is regulated by two distinct proteins, V-1 and
CARMIL, through their interactions with CP. The regulation mechanisms of thetwo are quite different; V-1 sterically inhibits the binding of CP to actin
filament, and allosterically does CARMIL. Moreover, CARMIL can dissociatebound CP from actin filament or V-1. The crystal structure of CP/V-1 complex
locates the V-1 binding site on CP, and reveals that V-1 sterically hinders theinteraction between CP and filament. On the other hand, the regulation
mechanism of CARMIL has not been unveiled. The crystal structure of
CP/CARMIL complex indicates that the CARMIL binding site on CP is distantfrom the binding site for filament or V-1, and the binding of CARMIL to CP
induces an insignificant structural change in the V-1 binding site of CP. To
unveil the regulation mechanism of CARMIL, we focused on structuralfluctuation of CP because CARMIL can influence an essential motion in CP.
We applied AMBER, a program for molecular dynamic simulation, to structuresof free CP, CP/CARMIL and CP/V-1 complexes and examined their dynamic
properties. We discuss the properties and their differences.
1B1558

Tomoko Sunami, Hidetoshi Kono (JAEA)
To better understand the conformational changes of proteins upon DNA
binding, we carried out quantitative analyses. We first described the proteinsusing a structural alphabet library of backbone geometries created by Kolodony
et al. and then detected the conformational changes as changes in alphabets
between DNA-free and bound forms. We first divided protein surface into two
regions, DNA interface and protein surface which exclude the DNA interface
and carried out analyses. The results show that 1) the conformational changes at
DNA interfaces are more frequent than those at the other surfaces; 2) DNA
interfaces have more conformational variation even in the DNA-free form. The
latter suggests that intrinsic, conformational flexibility of DNA interface seems
to be important to adjust their conformations to DNA. In amino acidpropensities, conformationally changed regions in DNA interfaces as well as
those in the other protein surfaces show that hydrophilic and glycine residues
are more preferred compared with conformation-unchanged regions. This trend
is also found in the propensity for disordered regions, suggesting that the
intrinsic flexibility be of importance not only for DNA binding but also forinteracting with other molecules. Comparison of the distributions of structuralalphabets between DNA interfaces and the other surfaces shows that somespecific alphabets appear more often in DNA interfaces. This suggests that these
local conformations may be adjustable to DNA structure with a low energy cost.
1B1534

天然変性タンパク質の構造ゆらぎを生かした密度変化誘起型シグナ
ル伝達過程

The possible advantage of structural disorder of intrinsicallydisordered pr oteins in the new type of signaling mechanism
Nobu C. Shirai1,2, Macoto Kikuchi1,2,3 (1Graduate School of Science, Osaka
University, 2Cybermedia Center, Osaka University, 3Graduate School of
Frontier Biosciences, Osaka University)
There are questions about intrinsically disordered proteins (IDPs), why higherorganisms have much more IDPs and what the advantages of structural disorderof IDPs are. To investigate these questions, we construct a lattice gas model of
IDPs and their targets based on the following assumption about structural
disorder of IDPs: structural disorder of IDPs can be represented by effective
volume and internal entropy under crowded condition of a cell. Based on the
result of the Monte Carlo simulation, we propose a new type of signalingmechanism, which we call density-regulated signaling mechanism. By the useof the mechanism, a cell can regulate the number fraction of bound-state IDPs,
which is considered as signal intensity, by increasing or decreasing the densityof total biomolecules. We find that IDPs must have sufficiently large internal
entropy in order to realize an all-or-none signaling switch. We conclude that the
all-or-none signaling switch is an advantage of structural disorder of IDPs.
1B1546

分子動力学法による capping protein（CP）の動的構造の解析

Structural fluctuations of capping proteins analyzed bymolecular dynamics simulations

シトクローム c 酸化酵素のプロトンポンプ機構に関する分子動力学
シミュレーションによる研究

A Molecular Dynamics Study on Proton Pump Function of
Cytochrome c Oxidase
Takefumi Yamashita (Research Center for Advanced Science andTechnology, University of Tokyo)
The proton-pumping mechanism of cytochrome c oxidase (CcO) has received
broad attention not only as a biological function but also as a physical energy
conversion. Coupling the electron transfer to reduce a trapped oxygen molecule
to a water molecule, CcO transports protons across the mitochondrial membrane
to form electrochemical gradient, which is essential to produce ATP. In thiswork, we aim to clarify how the electron transfer can control the protontransport in the non-polar cavity, which is expected to be important since thisregion is surrounded by the two heme groups. In order to simulate the protontransport in CcO realistically, the multi-state empirical valence bond (MSEVB) model is adopted here. We evaluated free energy profiles for several
oxidation states to see electron transfer effects. The results clearly suggest thatthe reduction of heme a opens the gate of the proton exit channel. More detailed
results will be discussed in this presentation.
1B1610Substrate transport mechanisms in GatCAB: the smallest
unidirectional valve in subnano scale
Jiyoung Kang2, Shigehide Kuroyanagi2, Yohsuke Hagiwara2, Masaru Tateno1(1Grad. Sch. Sci., Univ. Hyogo, 2Grad. Sch. Pure and Appl. Sci., Univ.
Tsukuba)Glutamine amidotransferase CAB (GatCAB) is a crucial enzyme involved intranslational fidelity, catalyzing the glutaminase reaction to yield ammonia(NH3 or NH4+) from glutamine and the transamidase reaction to convert
the phosphorylated Glu-tRNAGln to Gln-tRNAGln. In the crystal structure of
GatCAB, these two catalytic centers are far apart, and the presence of ahydrophilic channel to dislocate the produced NH3 /NH4+ was proposed. We
investigated the transport mechanisms of GatCAB by large time scale (~1.1 μs)
molecular dynamics (MD) simulations and free energy (PMF) calculations. As aresult of the analysis, we found that the entrance of the previously-proposed
channel is closed, as observed in the crystal structure. Instead, a novelhydrophobic channel was identified; its entrance opened and closed repeatedly,
and thus acted as a gate. The calculated free energy (PMF) difference revealed
the significant preference of the newly-identified gate/channel for NH3 transport(~104-fold). In contrast, NH4+ tightly hydrogen-bonded with hydrophilic
residues for both channels, which hindered efficient transport. The opening of
this gate was modulated by Phe206, which acted as a “valve”. Note that for the
backward flow of NH3, the opening of the gate was hindered by Ala207, which
acted as a mechanistic “stopper” against the motion of the “valve”. Thus, thiscan be considered as the smallest unidirectional valve inside proteins.
[1] J. Kang, S. Kuroyanagi, Y. Hagiwara, T. Akisada, and M. Tateno: J. Chem.
Theory Comp. 8 (2012) 649.
1C1400− S22 −

抗菌ペプチドを利用した病原体検出サンドイッチ発色検出技術の開発

New colorimetric sandwich assay for detection of pathogens byusing antimicrobial peptides as detection probes
Chihiro Sakai1, Eri Hojo2, Taichi Nakazumi1, Satoshi Tomisawa1, Takashi
Kikukawa1, Yasuhiro Kumaki1, Masakatsu Kamiya1, Makoto Demura1, Keiichi
Kawano1, Ryuji Ohtsuki2, Taro Yonekita2, Naoki Morishita2, Takashi
Matsumoto2, Fumiki Morimatsu2, Tomoyasu Aizawa1 (1Grad. Sch. Life Sci.,
Hokkaido Univ., 2R&D Center, Nippon Meat Packers Inc.)
Antibody-based assays such as an enzyme-linked immunosorbent assay (ELISA)
are important techniques for rapid and efficient detection and identification ofpathogens. These methods are widely used in many applications because theantibodies for specific antigenic sites on target pathogens can be used to be
distinguished very closely related species. However, in the case of some target
pathogens, it is very difficult to develop antibodies with both high sensitivity andhigh specificity and practical pathogen detection systems.
In this study, we used antimicrobial peptides (AMPs) as candidates foralternative detection molecule, expecting that AMPs can bind to pathogens withgreater density than antibodies because of the size. In the general sandwich
ELISA with immobilized antibodies and biotin-labeled antibodies forcolorimetric detection by using streptavidin conjugated horseradish peroxidase,
we replaced the biotin-labeled antibodies with the biotin-labeled AMPs as
probes for detection. As the results of screening of AMPs, we found that some
AMPs, such as cecropin P1 and magainin 2, are suitable for the detection
molecules in our sandwich assay system. After optimization of well platematerials and blocking reagents, our new system could be used for detection of
Salmonella with high detection sensitivity and specificity.
1C1412

VanX の溶菌活性を用いたルシフェラーゼのスクリーニングの簡略化
Toward a simplified screening method using a novel VanX cell
lysis activity

Nan Wu1, Tetsuya Kamioka1, Shihori Sohya1, Tomoki Matsuda2, Takahisa
Ikegami3, Haruki Nakamura3, Yutaka Kuroda1 (1Biotechnology andDepartment Life Science, Graduate School of Engineering, Tokyo University ofAgriculture and Technology, 2Research Institute for Electronic Science,
Hokkaido University, 3Institute for Protein Research, Osaka University)
VanX belongs to the VanA gene cluster that provides Vancomycin resistance.
VanX hydrolyzes the D-Ala-D-Ala dipeptide which eventually blocks the cellwall formation (D-Ala-D-Ala dipeptide usually fuse to the C-terminal of themurein monomer, and induce the synthesis of murein chain forming the cellwall). Recently, we expressed VanX as a single gene in E.coli, and found that it
has a strong cell lysis activity. Here, we report attempts to use this novel cell
lysis activity to purify recombinant proteins from E.coli without mechanical orchemical breakage of the cell wall. As a target protein, we used Gaussia
Luciferase, which is a bioluminescent protein containing 10 cysteines forming 5
dissulfide bridges and is expected to provide an efficient reporter protein. Our
strategy is to co-express the target protein, GLuc, with VanX, so that cell
autolysis occurs, and release Gluc into the culture medium. We thus expressed
VanX and GLuc using, respectively, pET26 and pET21(Novagen) in JM109(DE3) plysS E.coli cells. Upon addition of luciferin directly into the culture
medium, the sample emitted a strong blue light indicating that VanX lysed theE.coli cells and that GLuc was released into the medium in a native form. Cell
lysis by VanX may thus provide a versatile method for recovering protein from
E.coli without a need for mechanical, chemical cell breakage or the introductionof a secretion signal sequence. We are now applying this method to thescreening of GLuc with new bioluminescent properties.
1C1424

Fluorescent Probe for the Direct Detection of Histone
Deacetylase Activity
Koushik Dhara1, Yuichiro Hori1, Reisuke Baba1, Kazuya Kikuchi1,2(1Graduate School of Engineering, Osaka University, 2Immunology Frontier
Research Center, Osaka University)
Histone deacetylases (HDACs) are important enzymes for the transcriptional
regulation of gene expression in eukaryotic cells. The reversible acetylationof lysine residues near the N-terminal of nucleosomal histones by HDACs
and histone acetyltransferases (HATs) regulates chromatin structure andtranscriptional activity. HDAC enzymes are the major targets of drug
development and many of the reports indicate that HDACs have been found to
be major targets for the various diseases such as cancer, neurological diseases
and metabolic diseases. Therefore, in terms of medical science as well as basic
biology, the detection of the HDACs enzyme activities is a key for emerging
drugs and the elucidation of epigenetic mechanism and various biologicalphenomena. Although in vitro assays for HDACs are known, the methods do notallow the direct and real-time detection of the enzymatic activities. To the bestof our knowledge, there is no report of fluorescent probes that allow the realtime determination of HDAC activity. To overcome the problem, we design anovel fluorescent probe for the direct detection of HDAC activity. Timedependent enhancement of the fluorescent intensity was observed in the enzyme
reaction with SIRT1. Deacetylated product of the probe upon enzyme reaction
was monitored by HPLC analysis. The probe showed excellent stability for along time in the aqueous buffer. This new probe constitutes a great innovation in
the field of HDAC sensing assays in a direct manner.
1C1436

RaPID システムによる Akt2 選択的環状ペプチド阻害剤の探索とその阻害ペプチドの生化学的活性の評価

Exploring cyclic peptide inhibitors against the Akt2 kinase by
the RaPID system and evaluation of their biochemical
properties
Yuuki Hayashi1, Jumpei Morimoto2, Hiroaki Suga2 (1Dept. Life Sci., Grad
Sch. Arts and Life Sci., The Univ. Tokyo, 2Dept. Chem., Grad Sch. Sci., The
Univ. Tokyo)
Human Akt isoforms, Akt1, Akt2, and Akt3, are serine/threonine kinases that
regulate cell signal transductions. Since dysregulation of Akt activity oftencauses various carcinomas, Akt has been targeted for drug discovery. Because
most of Akt inhibitors target the substrate binding sites in kinase domain, which
is highly conserved over various kinases, the inhibitors act against both Akt
isoforms and non-Akt kinases, and might cause side effects through theundesired inhibitions. Therefore it remains challenging to achieve high isoformselectivity and potency simultaneously against a specific isoform. Here, we
explored cyclic peptide inhibitors against the kinase activity of Akt2 isoform
using the RaPID (Random nonstandard Peptide Intecrated Discovery) systemthat allows us to ‘rapidly’ select high-affinity binders for Akt2 from cyclic
peptide libraries with a huge diversity (~1013 sequences). As a result, we
succeeded in selecting three peptides, Pakti-L1, -L2 and -L3, that inhibit Akt2
kinase activity with ~100 nM of IC50 values but less inhibit the kinase activityof PKA and SGK. Interestingly, despite the absence of selection pressure toeliminate other isoform-binders, Pakti-L1 shows 250- and 40-fold higher
specific inhibition against the Akt2 isoform compared to the Akt1 and Akt3
isoforms, respectively. Akt isoforms share the same amino acid sequence in thesubstrate binding sites, it suggests that Pakti-L1 binds outside of the substratebinding sites in the Akt2 and allosterically inhibits the Akt2 kinase activity.
1C1448低分子抗体の多機能化を可能とするピンポイント化学接合デザイン

Protein engineering for site-specific bioconjugation chemistry:
Construction of multiple functional low-molecular antibodies
Mitsuo Umetsu, Asami Ueda, Takeshi Nakanishi, Kentaro Hashikami, Ryutaro
Asano, Izumi Kumagai (Department of Biomolecular Engineering, Graduate
School of Engineering, Tohoku University)
IgG-type antibodies are usually produced in mammalian cell expression
systems, but the construction of a stable and large-scale production process inmammalian systems is laborious, timeconsuming, and expensive. In contrast,
antibody fragments, such as single-chain Fv (scFv) and Fab fragments, can beprepared in bacterial secretory expression systems; however, the downsizing ofantibodies has some benefits in protein expression processes, while it results in
low affinity for antigen-displayed cell surfaces by the degrease of antigenbinding valence. Here, we propose a new construction of low molecularmultivalent/bispecific antibody by conjugation of protein engineering andchemical conjugation engineering. In this method, all the lysine residues in scFv
fragment are first mutated to alternative amino acid residues by utilizing
bioinformatics and molecular evolution technique to prepare the scFv with only
one lysine residue at a specific position by bacterial expression. After the designof the lysine-mutated scFv fragment, the two scFv fragments are chemicallyconjugated via a dicarboxylic organic linker that make amide binding with thelysine in scFv; consequently, homogeneous bivalent/bispecific antibody can begenerated. Here, we show the construction of bispecific low molecular antibody
with affinity for epidermal growth factor receptor on tumor and for CD3
receptor on lymphocyte cell.
1C1510− S23 −

ハイブリッドナノセルロームの構成要素としての CBM の機能解析

Hybrid nano-cellulosome: functional analysis of various
cellulose binding module as component of artificial cellulosome

Hikaru Nakazawa1, Do-myoung Kim1, Takashi Matuyama2, Nobuhiro
Ishida2, Akinori Ikeuchi2, Izumi Kumagai1, Mitsuo Umetsu1 (1Tohoku univ.
tech., 2Toyota Central R&D lab)
Cellulose is naturally occurring plant cell wall constituent and most abundant
polysaccharides on earth. An economically viable process for converting thisbiomass to fermentable sugar is a prerequisite for the development of cellulosic
biomass-based bio-refineries for the production of food, feed, chemicals andfuels. Cellulose can be converted to fermentable sugar by microbial cellulose
degrading enzymes, so called cellulase. Cellulases are generally modular
proteins with an independent catalytic domain (CD) and a carbohydrate-binding
module (CBM) and in some bacteria, CDs with different functions are clusteredon a giant scaffold protein containing CBM, via coheisin-dockerin interaction toefficient degradation, so called cellulosome.
Recently, we proposed a new design of artificial cellulosome to enhancecellulase activity with streptavidin or streptavidin modified CdSe quantum dot
as a scaffold via biotin-avidin interaction.Activity of the cluster enzyme with
two kind of CBD indicated that depend on combination of scaffold and CBD.
Thus, selection of the promising CBM is important in this artificial cellulosome
format for efficient cellulose degradation. Currently, cellulose binding module
was belonging to carbohydolate binding module family (CBMF) 1-4, 6, 8-11,
17, 30, 37, 44, 46, 49, 63, 64, and NC.
In this study, we carried out to express CBM from each CBM family in
Escherichia coli and evaluated the kinetic parameter in cellulose binding and theactivity of the clustering enzyme with CD and these CBM on scaffold.
1C1522

エンド-1,3-βグルカナーゼの糖結合モジュールα-リピートに存在する Trp273 のラミナリン結合への寄与

Contribution of Trp273 in the α-repeat of the carbohydratebinding module of endo-1,3-β-glucanase to laminarin binding
Tomonari Tamashiro1, Yoichi Tanabe1, Kenji Kanaori2, Teikichi Ikura3,
Nobutoshi Ito3, Masayuki Oda1 (1Grad. Sch. of Life and Environ. Sci., Kyoto
Pref. Univ., 2Grad. Sch. of Sci. and Technol., Kyoto Inst. of Technol., 3Med.
Res. Inst., Tokyo Med. and Dent. Univ.)
Endo-1,3-β-glucanase from Cellulosimicrobium cellulans DK-1, is composed of
catalytic domain and carbohydrate-binding module (CBM), both of which areconnected by a Gly/Ser-rich linker. The CBM, CBM-DK, is classified as CBM
family 13, which would have a -trefoil fold displaying the pseudo-3-foldsymmetry arising from imperfect tandem α-, -, and γ-repeats. Among the three
repeats, we recently showed that α-repeat mainly contributes to the carbohydrate
binding (Glycoconj. J. 29, 77, 2012). In this study, we focused on Trp273 in αrepeat, which has critical role for the binding, together with Asp270. We
overexpressed CBM-DK, corresponding to Gly257-Leu383, and its mutants,
W273A and W273S, in E. coli with N-terminal His-tag, and purified them using
Ni-NTA affinity chromatography. The interactions with -1, 3-glucans,laminarin and laminarioligosaccharides, were analyzed using surface plasmon
resonance biosensor and isothermal titration calorimetry (ITC). The binding
affinities to laminarin determined using ITC were 1.3 × 106 M-1 for CBM-DK
wild-type, 3.7 × 104 M-1 for W273A, and 1.5 × 104 M-1 for W273S. Trp273
could participate in the carbohydrate binding through a hydrophobic interaction.
This seems to be correlated with the facts that W273A mutant could beconcentrated using the membrane filter system, while CBM-DK wild-type could
not. We prepared 15N-uniform labeled W273A sample for NMR experiments. In
the presentations, we will discuss the structural properties of CBM-DK and its
complex with laminarioligosaccharide.
1C1534

Construction of an in vitro gene screening system for
membrane proteins

Haruka Soga1, Satoshi Fujii3, Tetsuya Yomo2,3, Hajime Watanabe1, Tomoaki
Matsuura1,3 (1Dept. Biotech., Grad. Sch. Eng., Osaka Univ., 2Dept. Bioinfo.
Eng., Grad. Sch. IST, Osaka Univ., 3ERATO, JST.)In vitro molecular evolution is a method to screen for proteins with desired
properties by mimicking the Darwinian evolution entirely in vitro. So far,various methods including ribosome display, mRNA display and in vitro
compartmentalization have been developed, and successfully used to evolve thefunction of soluble proteins, however, none of these methods can deal withintegral membrane proteins. Here, we report the development of a technology
for in vitro selection and evolution of membrane proteins. It uses synthesis ofmembrane proteins by in vitro transcription-translation system (the PURE
system) within giant unilamellar vesicles (GUV). The gene encoding themembrane protein is encapsulated in GUV at a single molecular level together

with the PURE system. Subsequently, the membrane protein is synthesized andinserted into the lipid bilayer. The GUVs that display the membrane proteinwith desired properties (affinity to the ligand or high transporter activity) arescreened. We show using EmrE, a multidrug transporter from Escherichia coli,
as a model protein that this protein can be synthesized from a single DNA
molecule inside and get localized on the surface of the GUV. Furthermore, thesynthesized EmrE transported ethidium bromide in response to the proton
gradient. Finally, we show the results of an enrichment experiment, where EmrE
gene is enriched from a mixture with others.
1C1546

Orientation-controlled immobilization of pharaonis
halorhodopsin onto gold revealed by SEIRAS

Hao Guo1,2, Tetsunari Kimura1,2,3, Yuji Furutani1,2,4 (1Department ofStructural Molecular Science, the Graduate University for Advanced
Studies, 2Department of Life and Coordination-Complex Molecular Science,
Institute for Molecular Science, 3JST CREST, 4JST PRESTO)
Despite membrane proteins play an essential role in the proper functioning of
the cell, the knowledge of their molecular mechanisms remains limited. Protein
immobilization with a desired orientation is a valuable approach to mimic anatural membrane environment. However, there is no clear experimental
evidence to validate the effectiveness of the orientational control. Here we
report the immobilization of pharaonis halorhodopsin (pHR), a light-driven
chloride pump in bacterial cell membrane, onto gold by the affinity of ahistidine-tag (His-tag) to a nickel chelating nitrilotriacetic acid surface. Controlof the orientation was performed by the adsorption of pHR via the 6×His-tag
introduced into either the N- or the C-terminus. Use of the optical near-field
effect of surface-enhanced infrared absorption spectroscopy (SEIRAS),
difference between these two surface orientations can be distinguished.
Deconvolution and curve fitting of the SEIRA spectra of bound pHR suggestthat more antiparallel -sheet structure is detected in the case of N-terminus
binding. To directly corroborate our results, the binding experiments have been
repeated under identical conditions using -sheet deleted pHR mutants. The
results show that the mutated pHRs display a significant decrease of thecharacteristic peak of antiparallel -sheet structure. Thus, the full control of theorientation of pHR on gold is distinctly confirmed by SEIRAS.
1C1558

低収量生体分子の時間分解分光計測を目指した新規溶液交換・混合装置の開発

Development of novel rapid buffer-exchange system andmicrofluidic mixer for time-resolved spectroscopic study of
low-yield biomolecules
Tetsunari Kimura1,2,3, Yuji Furutani1,2,4 (1Inst. Mol. Sci., 2Dept. Struct. Mol.
Sci., Sokendai, 3CREST/JST, 4PRESTO/JST)The real-time observation of structural changes in reacting biomolecules is oneof the best approaches to clarify their molecular mechanisms. Rapid bufferexchange and rapid solution-mixing techniques, which suddenly change thebuffer conditions, have been widely used to trigger the reaction. The additional
advantage of these techniques is an easy integration into many spectroscopic
methods. However, the high consumption of samples has limited theirapplication to low-yield and rare biomolecules. In this presentation, our recent
efforts to develop two novel systems, which require much less amount ofsample than conventional systems, and our trials to investigate the structuralchanges of biomolecules, especially membrane proteins, will be reported.
1) Rapid buffer-exchange system combined with ATR-FTIR spectroscopy.
Measurements of difference FTIR spectra are very powerful to investigate thestructural changes, because only the vibrational modes that are affected bybuffer-exchange can be observed. We developed a novel rapid buffer-exchangesystem, which can exchange buffer within tens of millisecond at slowest, andthe structural changes of the samples physically adsorbed on ATR crystal arefollowed.
2) Microfluidic mixer combined with fluorescence spectroscopy. Laminar-flow
fluid mixer composed of tens of micrometer channels is designed and fabricatedby using the photolithography and PDMS patterning techniques. The mixer is
combined with fluorescence spectroscopy, which is also sensitive to thestructure.
1C1610− S24 −

溶液中の金属タンパク質の共鳴 X 線散乱法の開発

Development of resonant X-ray scattering method of
metalloproteins in solutions

Mitsuhiro Hirai1, Kazuki Takeuchi1, Ryota Kimura1, Noboru Ohta2 (1Grad.
Sch. Eng., Gunma Univ., 2SPring-8)
Small-angle X-ray scattering technique of solutions is a useful method forobserving in-situ structural information of solute particles such as radii ofgyration and size distributions by analyzing data sets in small-angle scattering
regions. In addition, recent improvements in X-ray beam brightness anddetection at third-generation synchrotron sources enables us to obtain highstatistic data with a wide real-space resolution ranging from ~0.2 nm to ~250
nm, even in standard experimental set-ups. Due to such improvements, we
successfully discussed about whole hierarchal structures of proteins [1] and lipid
aggregates [2] and those transition phenomena in detail.
However, another characteristic of synchrotron sources, namely selectivity ofincident X-ray energy, has been rarely used for solution scattering of proteins.
Therefore, we have tried to apply resonant (anomalous dispersion) small- andwide-angle X-ray scattering (A-SWAXS) to determine internal structures ofmetallo-proteins in solutions.
As metallo-proteins, myoglobin (MY) from horse skeletal muscle, hemoglobin(HM) form bovine blood, ferritin from horse spleen, and hen-egg whitelysozyme with Hg-atom isomorphous replacements were used. A-SWAXS
measurements were performed by using the BL40B2 spectrometer at SPring-8.
The detailed results and discussion will be given.
[References]
1. M. Hirai, et al., Thermochimica Acta, 532 (2012) 15.; J. Appl. Cryst., 40(2007) s184.; Biochemistry, 43 (2004) 9036.
2. T. Onai, and M. Hirai. J. Phys. Conf. Ser., 247 (2010) 012018.
1D1400マウス ES 細胞分化の単 1 細胞解析

Single-cell-based analysis of differentiation of mouse ES cells
Atsushi Maruyama1, Yuichi Wakamoto2,3, Shogo Nakamura1, Tatsuo
Michiue2, Shin-ichi Sakai4, Bayar Hexig4, Toshihiro Akaike4, Kiyoshi
Ohnuma1 (1TRI, Nagaoka Univ of Tech, 2Grad. Sch. Arts. & Sci., Univ.
Tokyo, 3Sakigake, JST, 4Grad. Sch. Biosci. & Biotech., Tokyo Inst of Tech)
A mechanism that provides stability during embryonic development remains to
be uncovered. Embryonic stem (ES) cells are a good model for developing sucha mechanism because they can be differentiated into all kinds of cells in the
body. At the last annual meeting, we reported a system to observe a single-EScell reaction to culture medium stimulation. The system consists of four parts:
Nanog-GFP reporter ES cells, which can be observed cell state by imaging, aculture medium without any unknown factors, a specially coated culture dishthat enabled us to dissociate cells without aggregation, and time-lapse
microscopy.
In this meeting, we will report the results of single-cell analysis of ES celldifferentiation. We monitored life-and-death, proliferation, and differentiationof a single ES cell during differentiation for a few days. We created individual
histories or lineage trees to analyze temporal sequences of all reproduction
events and phenotypic changes in individual cells. We also performed sibling
analysis to compare cell pairs derived from common mother cells. Single-celllevel observation of the stimulation response of ES cells will deepen our
understanding of developmental stability.
1D1412

顕微ラマン分光法による分化因子刺激後の単一細胞ダイナミクスの計測

Measurements of Single Cell Dynamics upon Stimulation witha Differentiation Factor Using Raman Micro-Spectroscopy
Sota Takanezawa1,2, Shin-ichi Morita1, Yukihiro Ozaki2, Yasushi Sako1(1Cellular informatics Lab., RIKEN, 2Grad. Sch. Sci. and Tech., Kwansei
Gakuin Univ.)
Many extracellular factors are known to induce cell differentiation. It is
important to note that the response to these factors is variable even among a
clonal population of cells. The variance of cellular response could be caused by
the difference of cellular states relating to the chemical composition in each cell.
A general method for detecting the cellular state is required to understanddiversity and individuality of cells in the differentiation pathway. Actually,
specific genes, proteins, and metabolic products are used to define the cellularstate, but these markers should be pre-determined for each specific case. In thisstudy, we confirmed whether the Raman micro-spectroscopy is effective todetect the state dynamics of single living cells upon stimulation with adifferentiation factor. Raman micro-spectroscopy is applicable in a single living
cell, and shows several advantages: i) providing information about compositionand dynamics of cellular contents, ii) non‒destructive and non‒labeling

analytical technique, iii) the high spatial resolution. MCF‒7, a breast cancer cellline, was used for a model cell. MCF-7 is differentiated by heregulin (HRG) into
mammalian grand‒like cells. The time‒course Raman spectra changes were
measured in total 56 cells with and without HRG stimulation for 2hr with a20‒min interval. For comprehensive understanding of the time‒courses, aprincipal component analysis (PCA) was carried out. In the result of PCA,
dynamics of stimulated cells were more diverse than those without stimulation.
1D14241 分子定量計測技術を用いた C. elegans 受精卵における細胞極性のモデル化

Quantitative measurement and mathematical modeling ofpolarity-protein PAR-2 in C. elegans embryos
Yukinobu Arata1, Tetsuya Kobayashi2, Michio Hiroshima1, Chan-gi Pack1,
Masashi Tachikawa3, Kenichi Nakazato3, Tatsuo Shibata4, Yasushi Sako1(1Cell. Info., ASI, Riken, 2Inst. Ind. Sci., Univ. Tokyo, 3Theo. Biol., ASI,
Riken, 4CDB, Riken)
Cell polarization is a fundamental mechanism for cell migration, cellmorphogenesis, and asymmetric cell division in animal development. In
asymmetric cell division of C. elegans zygote, the asymmetric localization ofpolarity proteins is developed by a mutual exclusion mechanism throughphosphorylation. Despite accumulated molecular knowledge, it remains
unknown how intracellular biochemical reaction rates are balanced to maintainasymmetric localization. We succeeded to study dynamics and measureconcentration of PAR-2 proteins in living C. elegans embryos using totalinternal reflection microscopy and fluorescence correlation spectroscopy.
Cortical PAR-2 proteins were present as a mixture of monomer and oligomer.
The abundance ratio of high-degree oligomer increased along the anteriorposterior axis. Combined with our molecular genetics analyses, we conclude
that dissociation rate from cortex is regulated through the PAR-2
oligmerization. Next, we determined association rate from cytoplasm to cortex.
The association rate was higher in the posterior cortical region where cortical
PAR protein accumulated, suggesting that the association rate is under thecontrol by the feedback from cortical PAR-2 proteins. Using reaction-diffusion
model determined by experimentally-obtained parameters, we reproduced
asymmetric localization of PAR-2 proteins in silico. In this conference, I willtalk about novel molecular mechanisms and results of model analysis for cell
polarization.
1D1436形態形成中の組織の力学場を推定する

Estimating forces in the growing epithelial tissues
Shuji Ishihara1,2, Kaoru Sugimura3,4 (1Grad. Sch. Arts & Sciences, Univ.
Tokyo, 2JST PRESTO, 3iCeMS, Kyoto Univ., 4RIKEN BSI)
How do mechanical forces trigger a series of deformations to shape an adultbody during morphogenesis? Though recent studies have clarified how
geometrical changes of cells are coordinated via the activity and/or localizationof force-generating molecular machineries within a cell, comprehensive
understandings of how global balance of forces control the tissue deformation
has been still elusive. One reason is lack of direct and noninvasive measurement
to quantify the forces in the cell population inside the animal body. In this study,
we developed a theoretical framework for estimating the pressure of each cell,the tension of each cell adhesion surface and stress tensor of a group of cells, byadopting Bayesian scheme of inverse problem. Responses to laser cutting andmyosin distribution agreed with the estimated tensions. By applying our method
for growing Drosophila wing, we studied how myosin-mediated cell surface
tensions resist against external stretching forces. Further, we showed how
arising balance of forces in the tissue drives hexagonal cell packing that is
responsible for stable structure of the wing. Our force estimation method will
become a powerful tool in analyzing how information for orchestrating cellularbehaviors during morphogenesis is encoded in distributions of forces within atissue.
1D1448

ショウジョウバエ形態形成におけるアポトーシスの能動的力学的寄与

Active Mechanical Role of Apoptosis in Tissue Dynamics
during Drosophila Morphogenesis
Yusuke Toyama1,2,3 (1Dep. of Biol. Sci., National University of Singapore,2
Mechanobiology Inst., National University of Singapore, 3Temasek Life
Sciences Lab.)
During the long journey from egg to adult, the number of cells is finely
controlled as the cells collect into distinct tissues that undergo patterned− S25 −

movements to form the adult body. Apoptosis is the process that eliminates cells
which are no longer biologically necessary. During the apoptotic process, thedying cell is squeezed out from the tissue by mechanical forces produced within
the cell and by forces produced within the neighboring cells. We experimentallydemonstrated that the forces that squeezed out the apoptotic cell also contributed
to tissue movements during development in Drosophila. This active mechanicalrole is not classically attributed to apoptosis.
1D1510A model of cell-division stop for the limb regeneration
Hiroshi Yoshida (Faculty of Math. Kyushu Univ.)

Cell-division stop is essential for the limb regeneration. The steepness
hypothesis was submitted as a model that explains regulation of the celldivision stop under an assumption of a gradient across cells. The Dachsous/Fat
heterodimers are now considered to substantiate the steepness hypothesis and to
be involved in regeneration in insect legs. Little is, however, known about theway Dachsous and Fat molecules are distributed during cell division. Extending
the steepness hypothesis, we here show that some condition of the distribution
ratio provides the cell with cell-division stop, enabling regeneration. We foundthat the obtained condition provides a molecular-based explanation for a varietyof limb regeneration such as distal outgrowth and intercalary regeneration. It has
been revealed that the condition can explain several results of experiments using
regeneration-dependent RNAi in cricket legs. We anticipate our result toprovide a unified view of regeneration based on molecules within the cell.
1D1522ラット培養神経回路網の長期発達過程における同期活動に関わる機能的分子の探索

Analysis of functional molecules underlying synchronous
activity during long-term development of rat cultured
neuronal networks
Daisuke Ito1, Keiko Yokoyama2, Kazutoshi Gohara2 (1Fac. Adv. Life Sci.,
Hokkaido Univ., 2Fac. Eng., Hokkaido Univ.)
Synchronous activity is a remarkable phenomenon in the electrical activity ofneuronal networks and is thought to be involved in the higher function of thenervous system, such as memory and learning. Studies using multi-electrodearrays (MEAs) have revealed that cultured neuronal networks reconstructed invitro show synchronized bursts (SBs). However, the molecular mechanisms
underlying the generation of SBs during long-term development remain unclear.
In order to clarify the mechanisms of this phenomenon at a molecular level, we
analyzed the gene expression involved in the changes in network activity. We
cultured rat cortical neurons for 1 month, and the spontaneous electrical activity
was recorded using MEAs. SBs were observed starting at approximately 2
weeks and the rate increased gradually during the culture periods. Reverse
transcription PCR analysis revealed that the housekeeping gene andtranscription factor gene creb were found to be consistently expressed during theculture period. However, the immediate early gene arc was not identified at 1-7
days in vitro (DIV), but the expression level increased up to 28 DIV. This
increase in the expression of arc resembled the increase in the SB rate. These
results suggest that the generation of SBs is correlated with the increase in
immediate early gene expression during the culture periods. Based on thesepreliminary results, we have begun to quantify the expression level of arc andinvestigate the expression of other genes and proteins involved in network
activity.
1D1534

モノアラガイの長期記憶におけるインスリンとグルコースの役割

Insulin and glucose for memory in a snail
Etsuro Ito1, Ryuichi Okada1, Mika Morikawa1, Satoshi Takigami2, Akiko
Okuta3, Manabu Sakakibara2 (1Kagawa School of Pharmaceutical Sciences,
Tokushima Bunri University, 2School of High-Technology for Human Welfare,
Tokai University, 3Graduate School of Science, Kyoto University)
The pond snail Lymnaea stagnalis is capable of learning taste aversion andconsolidating this learning into long-term memory (LTM) that is called
conditioned taste aversion (CTA). First, we examined the role of insulin in the
LTM formation of CTA. When we applied insulin to the brain, we observed along-term change in synaptic efficacy (i. e., enhancement) of the synaptic
connection between some neurons. This synaptic enhancement was blocked byapplication of an insulin receptor antibody to the brain. Injection of the insulin
receptor antibody into the snail before CTA training, while not blocking theacquisition of taste aversion learning, blocked the memory consolidation
process, thus LTM was not observed. Second, we examined the relationshipamong learning ability, starvation period and hemolymph glucose
concentration, and tested the effect of insulin on the learning score. One-day
mild starvation improves the learning score and keeps good LTM. When we
injected glucose into the abdominal cavity of mild starved snails for a shortperiod, the snails showed worse scores than the case of no injection. Further,when insulin was injected into snails with a complete bellyful of food, theglucose concentration decreased and the learning score and LTM were
improved. These results showed that learning ability depends on the starvation
period and the glucose concentration and that learning ability must be controlled
by manipulation of glucose concentration and injection of insulin in CTA
learning of Lymnaea.
1D1546

光捕捉された神経細胞内シナプス小胞群の集合ダイナミクス

Assembling dynamics of optically trapped synaptic vesicles inneuronal cell
Yusuke Ueda1,2, Suguru N. Kudoh2, Takahisa Taguchi1, Chie Hosokawa1,2(1Health Res. Inst., AIST, 2Grad. Sci. Eng., Kwansei Gakuin Univ.)
The turnover of synaptic vesicles in presynaptic terminals of neurons is essential
for information processing within the brain. For aiming artificial control towardmodulating the synaptic transmission in a neuronal network with opticaltweezers, we investigated assembling dynamics of optically trapped synaptic
vesicles in neuronal cells by fluorescence analysis. When a 1064-nm trapping
laser beam is focused on synapses of a neuronal cell labeled with a fluorescentendocytic marker, the fluorescence intensity increased gradually with the laser
irradiation time, suggesting that optical trapping force causes vesicles assemblyat the focus. The synaptic vesicle dynamics in an optical trap was evaluatedby fluorescence correlation spectroscopy. The decay time of fluorescence
autocorrelation curves increased with the trapping laser power and the laser
irradiation time, indicating that vesicle motion was restrained at the focus due tooptical trapping force. Moreover, the decay time of autocorrelation curves wasdecreased after applying the phosphatase inhibitor okadaic acid causes vesicles
to diffuse freely and the degree of decrement was related to the trapping laser
power, which will be presented and discussed.
1D1558

膜電位感受性色素・カルシウムイメージングに使える新しい超高速共焦点顕微鏡の開発

A new class of confocal microscope for a fast voltage-sensitivedye (VSD) and Ca2+ imaging
Takashi Tominaga, Yoko Tominaga (Dept. Neurophysiol., Kagawa Sch
Pharm Sci, Tokushima Buniri Univ)
Monitoring neuronal activities using imaging devices is central to ourunderstanding of brain microcircuit functions. Recent progresses in imaging
devices together with the rapidness of VSD response have allowed us to follow
the sub-millisecond timescale of neuronal activities by VSD imaging. Confocal
microscopy provides a more efficient S/N ratio in terms of spatial resolution andthus could be used to obtain better S/N ratios from the VSD signal. However,sampling rates have been difficult to achieve in millisecond timescales. Here we
describe the basic configuration of a new no-scanning type of confocal
microcopy designed for VSD imaging. The confocal optics was designed to fit astandard camera adaptor of an eqi-fluorescent microscope (e. g., BX51WI,
Olympus). The images of the subject placed under the objective lens (20x/0.95,
Olympus) were projected onto a 100 × 100 pinhole array placed at a
conjugation point. An excitation light reflected by a standard mirror unit was
projected onto the pinhole array and illuminated onto the subject. Fluorescenceemitted from the subject was then converted into an image using the MOS-type
100 × 100 pixel imager (MiCAM Ultima, Brainvision). The pinhole array andpixel imager correspond with each other in terms of pixels. Since the pinhole
array is at a fixed position, there are no mechanical disturbances. We tested themicroscope by imaging a rat hippocampal slice preparation by bulk and single
cell VSD staining of the slices, and bulk staining with a Ca2+ indicator.
1D1610De novo アセンブルによる軟体動物脳の全トランスクリプトーム解析

De novo sequencing and transcriptome analysis of themolluscan brain by deep RNA sequencing
Hisayo Sadamoto1, Hironobu Takahashi2, Taketo Okada1, Hiromichi
Kenmoku2, Yoshinori Asakawa2 (1Facul. Pharmaceut. Sci. Kagawa Camp,
Univ. Tokushima Bunri, 2Inst. Pharmacognosy, Univ. Tokushima Bunri)
The pond snail Lymnaea stagnalis is among several mollusc species that havebeen well investigated due to the simplicity of their nervous systems and large− S26 −

identifiable neurons. Nonetheless, despite the continued attention given to thephysiological characteristics of its nervous system, the genetic information of
the Lymnaea central nervous system (CNS) has not yet been fully explored. The
absence of genetic information is a large disadvantage for transcriptome
sequencing because it makes transcriptome assembly difficult. We here
performed transcriptome sequencing for Lymnaea CNS using an Illumina
Genome Analyzer IIx platform and obtained 81.9 M of 100 base pair (bp) single
end reads. For de novo assembly, five programs were used: ABySS, Velvet,
OASES, Trinity and Rnnotator. Based on a comparison of the assemblies, wechose the Rnnotator dataset for the following blast searches and gene ontology
analyses. The present dataset, 116, 355 contigs of Lymnaea transcriptome
shotgun assembly (TSA), contained longer sequences and was much larger
compared to the previously reported Lymnaea expression sequence tag (EST)established by classical Sanger sequencing. The TSA sequences were subjectedto blast analyses against several protein databases and other molluscan EST
data. As a result, the richness of the present TSA data allowed us to identify alarge number of new transcripts in Lymnaea and molluscan species.
1E1400分子動力学シミュレーションによる Hsp90 と ADP の結合自由エ
ネルギープロファイル

Free energy profile for binding of ADP to Hsp90 withmolecular dynamics simulation
Kazutomo Kawaguchi, Hiroyuki Takagi, Masashi Iwayama, Megumi
Nishimura, Hiroaki Saito, Hidemi Nagao (Inst. Sci. Eng., Kanazawa Univ.)
Binding of a ligand molecule to a protein plays a key role in the function ofmany proteins. The functional cycle of proteins is driven by the binding of someof the ligand molecules, and is inhibited by the binding of some of the otherligand molecules. Hsp90 (Heat Shock Protein 90) is one of a group of molecularchaperones required for protein folding, maturation and trafficking of client
proteins. Recent studies have shown that Hsp90 is associated with many
pathologies: cancer, virus infectious diseases, protein folding disorders andneurological diseases. Hsp90 is an attractive target for the therapies of many
pathologies. The functional cycle of Hsp90 is driven by ATP binding andhydrolysis to ADP. However, the processes of binding and hydrolysis of ATP
and dissociation of ADP are not revealed at atomic resolution. The purpose of
our study is to elucidate the free energy profile for binding of ADP to Hsp90.
We performed all-atom molecular dynamics (MD) simulations of Hsp90 with
ADP. The initial structure used in our simulations is the X-ray crystallographic
structure of the N-terminal domain of human Hsp90 in complex with ADP(PDB code: 1byq). We calculated the free energy profile for binding of ADP to
Hsp90 using the thermodynamic integration method. We will report the resultsof the free energy calculation.
1E1412

シャペロニン空洞内の変性タンパク質が受けるコンフォメーション
制限

The conformational restriction of denatured protein
encapsulated in the chaperonin cage
Fumihiro Motojima, Yuko Motojima-Miyazaki, Masasuke Yoshida (Kyoto
Sangyo Univ.)
Chaperonin assists protein folding in its closed cage. Recently, in addition to theisolation of protein into the cage to reduce a risk of aggregation, it has been
proposed that the folding rate of denatured protein is accelerated by the
restriction of conformational space in the cage. Hartl’s group suggested that theformation of disulfide-bond in the slow folding mutant of maltose binding
protein (DMMBP) resulting in more rapid folding as wild-type MBP mimics theconformational restriction in the chaperonin cage. However, on the contrary, ourrecent results showed that denatured protein encapsulated in the cage is notcompletely isolated and can even escape out of the cage.
In the last meeting, we reported that blue fluorescence protein starts its folding
from more unfolded state than its spontaneous folding, using FRET to observe
its conformational change. Here, we applied same technique to maltose binding
protein (MBP) and its slow folding mutant (DMMBP). If Hartl’s notion is true,
the distance between cysteine residues of MBP that used for disulfide-bondformation should be smaller than that of DMMBP. However, their distancechanges during folding are almost same. Therefore, the folding acceleration of
DMMBP in the chaperonin cage is not caused by disulfide-bond-like
conformational restriction, and thus our proposition of unfolding effect in thecage is more probable. We will report the comprehensive study of theconformational effect in the chaperonin cage on MBP and DMMBP at thissession.

1E1424

小角 X 線散乱によるアミロイド線維形成の初期会合プロセスの解析

Investigation of initial assembling process of fibrillation bysmall angle X-ray scattering
Eri Chatani1, Rintaro Inoue2, Koji Nishida2, Toshiji Kanaya2, Masahide
Yamamoto3 (1Dept. Chem., Grad. Sch. Sci., Kobe Univ., 2Inst. Chem. Res.,
Kyoto Univ., 3Kyoto Univ.)
Amyloid fibrils are supramolecular assemblies associated with manydegenerative diseases including Alzheimer’s, prion, and Huntington’s diseases.
It has been proposed that several smaller aggregates such as oligomers andprotofibrils are involved in the fibrillation process, but little is known about adetailed picture how these species contribute to form the mature amyloid fibrilswith a rigid cross- structure. To explore this issue, we have performed
time-resolved small angle X-ray scattering (SAXS) measurements of insulin
fibrillation combined with analyses of thioflavin T fluorescence and secondary
structure. The SAXS data were monitored every 9 seconds immediately after the
initiation of fibrillation reaction inside a quartz cell.
As a result, we have found that insulin molecules transform into a characteristic
oligomeric structure in the very early stage of lag phase. After the accumulationof oligomers, the scattering curves further increased, implying the assemblingprocess of oligomers into mature fibrils. Interestingly, a distinct type ofoligomers in terms of size, cross- content, and thioflavin T fluorescenceintensity was observed when fibrillation reaction was performed at a high salt
concentration, suggesting the presence of diverse oligomeric structures that
may contribute to a variety of fibrillation pathways and furthermore, themanifestation of polymorphism of mature amyloid fibrils.
1E1436

Distinguishing crystal-like amyloid fibrils and glass-like
amorphous aggregates from their kinetics of formation
Yuichi Yoshimura1, Yuxi Lin1, Hisashi Yagi1, Young-Ho Lee1, Hiroki
Kitayama1, Kazumasa Sakurai1, Masatomo So1, Hirotsugu Ogi2, Hironobu
Naiki3, Yuji Goto1 (1Inst. Protein Res., Osaka Univ., 2Grad. Sch. Eng. Sci.,
Osaka Univ., 3Fac. Med. Sci., Univ. Fukui)
Amyloid fibrils and amorphous aggregates are two types of aberrant aggregatesassociated with protein misfolding diseases. Although they differ inmorphology, the two forms are often treated indiscriminately. 2Microglobulin (2m), a protein responsible for dialysis-related amyloidosis,
forms amyloid fibrils or amorphous aggregates depending on the NaCl
concentration at pH 2.5. We compared the kinetics of their formation, which
was monitored by measuring thioflavin T (ThT) fluorescence, light scattering,and 8-anilino-1-naphthalenesulfonate (ANS) fluorescence. ThT fluorescence
specifically monitors amyloid fibrillation, whereas light scattering and ANS
fluorescence monitor both amyloid fibrillation and amorphous aggregation.
The amyloid fibrils formed via a nucleation-dependent mechanism in asupersaturated solution, analogous to crystallization. The lag phase of
fibrillation was reduced upon agitation with stirring or ultrasonic irradiation,and disappeared by seeding with preformed fibrils. In contrast, the glass-like
amorphous aggregates formed rapidly without a lag phase. Neither agitation nor
stirring accelerated the amorphous aggregation. Thus, by monitoring the
kinetics, we can distinguish between crystal-like amyloid fibrils and glass-like
amorphous aggregates. Solubility and supersaturation will be key factors forfurther understanding the aberrant aggregation of proteins.
1E1448細胞濃度に近い無添加細胞抽出液の調製

A method to prepare cell extract near intracellularconcentration without additional salts and buffers
Kei Fujiwara, Shin-ichiro M. Nomura (Dept. Biorobot., Tohoku Univ.)
For the aim to elucidate essential processes for synthesizing cells, we haveestablished a method to prepare additive-free cell extract having highconcentration like cells. At first, essentiality of buffers and salts for preparing
functional cell-free translation extract was verified. Exponential growth cells ofEsherichia coli were washed with double distilled water (DDW) at twice, andwere dissolved with DDW. After freezing, the cells were disrupted bysonication. S30 fraction of the obtained cell extract (DDW-S30) showedsufficient activity for cell-free protein production of GFP and luciferase.
Next, a gradual evaporating method to accumulate biological components has
been established. Here, we used vacuum desiccator to accumulate samples.
Total volume decreased 20 μl per hour on average, and 4.5-fold concentrationof BSA was obtained thorough the methods. The methods also achieved− S27 −

accumulation of GFP, alkaline phosphatase (PAP), and diluted PURE systemwith active forms. Especially, accumulation rate of PAP reached 25-fold. The
methods also succeeded to accumulate DDW-S30. The accumulated DDW-S30
showed 180 mg/ml protein concentration, which expected to contain ca. 260
mg/ml of macromolecules, and showed equivalent activity of cell-free protein
production to the DDW-S30 before accumulation. Such an accumulated DDWS30 has a potential to be a material for revealing the difference between cells
and materials, and is expected to contribute synthesizing cells from cell extract
in future.
1E1510TMAO による分子クラウディング環境への蛋白質移相自由エネル
ギーの三次元描像

Three-Dimensional Imaging of the Protein Transfer Free
Energy into the Molecular Crowding Condition with TMAO
Isseki Yu1, Kyoko Nakada1, Masataka Nagaoka2 (1Dep. of Cem. and Biol. Sci.
Aoyama Gakuin Univ., 2Grad. School of Info. Sci., Nagoya Univ.)
Some of the living organisms in the extreme environments contain variousorganic cosolvents (osmolytes) in their cells. These osmolytes protect theconformation and the function of biomolecules from environmental stresses,
such as high temperature or high pressure.
In this study, cytoplasmic conditions of the extremophiles were mimicked byaqueous solution of 2.2M TMAO (trymethilamine N-oxide: osmolyte found in deepsea fishes). The volume fraction of the TMAO as crowder was set to mimic the
typical molecular crowding condition in the cytoplasm. Under such conditions,transfer free energy (TFE: the change in Gibbs energy of the solute when it
transfers from the solvent phase into a different phase) of apomyoglobin were
calculated by Kirkwood-Buff (KB) theory using the atomic configurations of thesolvent molecules generated by MD simulations. The simulated TFE and thepreferential interaction parameter correlated favorably with experimental values.
Furthermore, 3-dimensional distribution of the TFE were revealed with ourdeveloped space-time deconvolution techniques of the KB integrals. The results
indicated that the positive TFE regions have planer shapes, and are continuously
distributed along the surface of protein. In contrast, the negative TFE regions
have spherical shapes and are scattered mainly around the side-chains [1]. The
correlation between TFE distribution and the chemical or geometric property ofthe protein surface will be discussed.
[1] Yu, I; Nakada, K.; Nagaoka, M., J. Phys. Chem. B, 2012, 116, 4080-4088.
1E1522シトクロム c に対する尿素効果の自由エネルギー解析

Free-energy analysis of urea effect on cytochrome c
Yasuhito Karino, Nobuyuki Matubayasi (Inst. Chem. Res., Kyoto Univ.)
Urea is a widely used denaturant for protein. The mechanism of the denaturation
has not been clearly determined though its effect has been discussed in bothexperimental and theoretical studies. At the moment, the driving force ofdenaturation is explained by the following two mechanisms. One is the direct
mechanism. In the direct mechanism, the denaturation is caused by the directinteraction between protein and urea. Urea is considered to interact withhydrophilic residues and backbone via the electrostatic interactions, and withlarge, heavy residues via the van der Waals interactions. The other is the indirect
mechanism. In the indirect mechanism, urea disturbs the water structure such ashydrogen bonding, and the hydrophobic core is exposed to the solvent. The
calculation of salvation free energy may provide a key for elucidating the urea
effect if the contributions of water and urea are obtained individually. In thisstudy, we calculated the solvation free energies of cytochrome c immersed inpure water and that in urea solution by using the molecular dynamics simulation
coupled with the method of energy representation. The transfer free energy isalways negative, and the transfer free energy from pure water to urea solution isfound to be strongly correlated to the van der Waals interaction between soluteand solvent molecules. The result supports the “direct” mechanism and thedominance of the van der Waals interaction in the urea effect on protein.
1E1534タンパク質の水和水の水素結合ダイナミクスとタンパク質ダイナミ
クスとの動的カップリング

Kinetics of hydrogen-bonding of protein hydration water andits dynamical coupling with protein dynamics
Hiroshi Nakagawa1, Mikio Kataoka1,2 (1Japan Atomic Energy Agency,
Quantum Beam Science Directorate, 2Nara Institute of Science andTechnology, Graduate School of Materials Science)

Unique physical properties of water are originated from the hydrogen bond. The
hydrogen bond determines the spatial network patterns and water dynamics.
Hydration water around the molecules, such as ions, polymers and biomolecules, has quite different physical properties from bulk water. The protein
hydration affects the biological function of the protein. Here, by using themolecular dynamics simulation, we show that the hydrogen bond kinetics of theprotein hydration water is strongly affected by the local bonding patterns near
the tagged hydrogen bond and that percolation of hydration water on the protein
surface essentially determines the cooperative hydrogen bond forming andbreaking dynamics. These dynamical characters of hydration water are quite
different from those of the bulk water. The hydrogen bond kinetics between
hydration water is correlated with the kinetics between the protein and hydrationwater, suggesting the dynamical coupling between them. The percolation ofhydrogen bond network would contribute to the physical properties of protein
hydration water. This hydration water dynamics induces the protein dynamicaltransition observed by neutron inelastic scattering. The dynamical coupling
between hydration water and protein should be essential for protein thermal
fluctuation and functionality.
1E1546

テラヘルツ時間領域分光を用いた天然および変性状態タンパク質における低振動スペクトルの水和および温度依存性の観測

Temperature and hydration effects of low-frequency spectra ofnative and denatured proteins studied by terahertz timedomain spectroscopy
Naoki Yamamoto1, Atsuo Tamura2, Keisuke Tominaga1,2 (1Molecular
Photoscience Research Center, Kobe University, 2Graduate School of Science,
Kobe University)
Theoretical calculations have shown that in the pico-second region proteins
exhibit collective vibrational motions. These modes can be referred as lowfrequency modes, since these modes usually appear in several wavenumber
regions, or terahertz frequency regions. It is known that these motions areusually well correlated with large conformational changes that are necessary forprotein functional expression. Until now a lot of studies focusing on hydrationand temperature dependences on protein dynamics have been done by using alarge number of spectroscopic methods since these parameters are deeply
connected to the protein function. Though it has been proved that “dynamicaltransition” is universally observed at around 200 K, little is known about theeffect of hydration. In this study we measured low-frequency dynamics ofnative and chemically denatured lysozyme to understand the effect of hydrationby using terahertz time-domain spectroscopy. Difference spectra between dried
and hydrated states indicate that above the dynamical transition temperature thelow-frequency dynamics were activated. In contrast, the differential spectra
below the dynamical transition point show complicated features. Below 25 cm-1
absorption coefficients of the difference spectra are negative, and above thiswavenumber the values increase as the wavenumber increasing. We discuss
about the interpretation of these observations and differences between the nativeand denatured states.
1E1558

誘電緩和分光法によるアルキルカルボン酸の水和特性

Hydration Properties of Alkyl Carboxylate Series by Dielectric
Relaxation Spectroscopy
Yangtian Wang, George Mogami, Tetsuichi Wazawa, Nobuyuki Morimoto,
Makoto Suzuki (Grad. Sch. Eng., Tohoku. Univ)
We measured hydration properties of sodium carboxylate series A: Cn H2n+1
COONa (n=1-5), and B: C4 H8-m (COONa)m (m=2-4) in a low concentration
range (<0.09 M) at 20.0±0.01 °C by dielectric relaxation (DR) spectroscopy inthe frequency range 0.2-26 GHz. The DR spectra were decomposed into several
Debye components and the bulk water component by Hanai mixture theory.
Their DR parameters were determined by least fitting. As results, a Debye
component with a DR frequency (fc) of ~18 GHz, which is higher than bulk
frequency (17.0 GHz at 20 °C) hence assigned as hyper-mobile water (HMW),
and another component with lower fc (5.3-8.5 GHz) assigned as constrainedwater (CW) were found around solutes of series B, while only the 5.3-8.5 GHz
component (CW) was found around series A. An error propagation analysis
assured the resolution of fc ~0.5 GHz, supporting the existence of thesecomponents distinguished from the bulk water component. By fixed volume
analysis, we found that: 1) with increasing the length of hydrophobic alkyl
group, CW increased while bulk water decreased; 2) with increasing the numberof carboxylic group, HMW increased and bulk water decreased. Also, both
HMW and CW were found around HCOONa (0.06M) at 10.0±0.01 °C. In− S28 −

addition, DR measurements of CH3COONa at different temperatures (5, 10, 15
and 20 °C) were carried out. The results showed that a decrease of temperaturegave rise to an increase of CW. The Gibbs free energy of formation of hydrationwater and the protonation effect of carboxyl group will be discussed at meeting.
1F1400サル緑感受性視物質の全反射赤外分光解析

ATR-FTIR study of monkey green-sensitive visual pigment
Kota Katayama1, Yuji Furutani1,2, Hiroo Imai3, Hideki Kandori1 (1Department ofFrontier Materials, Nagoya Institute of Technology, 2Department of Life andCoordination-Complex
Molecular
Science,
InstituteforMolecular
Science, 3Primate Research Institute, Kyoto University)
Our color vision is based on three different proteins absorbing blue, green andred lights using the same chromophore, an 11-cis retinal. Because of the
difficulty in sample preparation, there have been little structural studies on color
visual pigments. We thus started low-temperature FTIR analysis of monkey
green (MG) and red (MR) sensitive pigments, from which we revealed
important structural differences such as green-specific amide-I vibration, redspecific X-H stretch, and unique protein bound waters1, 2. It is also known thatbinding of a chloride ion contributes to the spectral red-shift in primate green
and red pigments, but no structural studies have been performed. Here we apply
attenuated total reflection (ATR) FTIR spectroscopy to MG to gain structural
information of the chloride binding site. MG proteins are attached to the ATR
cell, and difference FTIR spectra are measured between two anions among Cl-,
Br-, NO3- and I- at room temperature. The Cl- minus Br- difference spectra
coincide with the baseline, which is entirely different from those of a lightdriven chloride pump halorhodopsin. In contrast, clear difference spectra are
obtained between Cl- and I-, or Cl- and NO3-. Isotope shift for 15NO3- implies
direct nitrate binding to MG, and chloride binding accompanies formation of sheet and protonation of a carboxylate. We propose a model for the chloride
binding site on the basis of the present results.
[1] Katayama et al. Angew. Chem. Int. Ed. 49, 891 (2010).
[2] Katayama et al. Biochemistry 51, 1126 (2012).
1F1412

精製したタコロドプシンの SAXS 測定

SAXS measurements of purified octopus rhodopsin
Shingo Watanabe1, Mitsuhiro Hirai2, Tatsuo Iwasa1,3 (1CEDAR, MuroranIT, 2Grad. Sch. Eng., Gunma Univ., 3Grad. Sch. Eng., Muroran IT)
Photo-activated rhodopsin (Rh) couples with G-protein, triggers an enzymaticcascade and finally causes the depolarization of an invertebrate photoreceptor
cell. In order to investigate the photo-induced conformational changes of Rh, the
SAXS measurements were performed with solubilized octopus microvillar(MV) membrane last year and showed that the irradiation of the sample withblue light caused an increase in the particle size. In the present study, we
performed SAXS measurements using purified octopus Rh to survey the originof the change in the particle size. Rhodopsin purified by conA-sepharosecolumn was irradiated with blue light (510 nm) to convert it into acid meta-Rh.
The acid meta-Rh was then reverted to Rh by orange light irradiation (>560nm). The gyration radii of the purified Rh, that of blue light irradiated products(acid meta-Rh), and that of the further orange light irradiated products (revertedRh) were the same within the experimental error, about ca. 40 Å. The size wassmaller than that estimated from the solubilized MV experiment. The results
suggest that the increase in the particle size observed with solubilized MV may
be resulted from the presence of the other proteins.
1F1424

ナノディスク試料を用いたロドプシンと錐体視物質によるトランス
デューシン活性化効率の比較解析

Comparative analysis of transducin activation by rhodopsin
and cone pigments in nanodiscs
Keiichi Kojima, Ryo Maeda, Yasushi Imamoto, Takahiro Yamashita,
Yoshinori Shichida (Dept. Biophys., Grad. Sch. Sci., Kyoto Univ.)
Most vertebrate eyes have two types of photoreceptor cells, rods and cones.
Both cells have similar phototransduction systems, but cones show fasterand less sensitive photoresponses, suggesting that the photoresponses arecharacterized by the different molecular properties of functional proteins
between these cells. In order to elucidate the difference in responses, we focusedon the activation process of transducin (Gt) by photoactivated rhodopsin andcone pigment, which is the first step of signal transduction cascades and likely
to directly affect the signal amplifications. In the previous works, weinvestigated them by using the detergent-solubilized pigments. However,detergent possibly affects the interaction between photoactivated pigments
and Gt. In this study, we prepared nanodiscs containing bovine rhodopsin
and chicken green-sensitive cone pigment to conduct the spectroscopic
measurement in the detergent-free system. We have reported that thephotobleaching processes of rhodopsin and chicken green in nanodiscs weresimilar to those in the membrane unlike those in detergent (Kojima et al.,Annual Meeting of Biophysical Society of Japan, 2011). We first showed that
total amount of activated Gt by photobleached rhodopsin was much greater thanthat by chicken green. On the other hand, the initial rate of Gt activation byrhodopsin was only a little greater. Based on these results, we will discuss thedifference in Gt activation by rhodopsin and cone pigments, and its contribution
to the natures of photoreceptor cells.
1F1436

脊椎動物非視覚オプシン Opn5 とその類似光受容タンパク質の分子的性質の解析

Analysis of the molecular properties of vertebrate non-visual
opsins, Opn5 and Opn5-like protein
Takahiro Yamashita1, Hideyo Ohuchi2, Sayuri Tomonari3, Sari FujitaYanagibayashi1, Kazumi Sakai1, Sumihare Noji3, Yoshinori Shichida1 (1Grad.
Sch. Sci., Kyoto Univ., 2Grad. Sch. Med. Dent. Pharm. Sci., Okayama
Univ., 3Inst. Technol. Sci., Univ. Tokushima Grad.Sch.)
Opsins are the universal photoreceptive molecules that underlie the molecularbasis of visual and non-visual photoreceptions in animals and are classified into
seven distinct groups based on their amino acid sequences. Opn5 (neuropsin)
was first identified in mouse and human genomes and was found to be expressedin retina and brain. However, there was little information about the molecularproperties and the physiological functions of Opn5.
Vertebrate Opn5 genes are diversified into three subtypes. Most mammals have
only one Opn5 gene, Opn5m, and many non-mammalian vertebrates haveadditional two subtypes, Opn5-like1 and -like2 (L1 and L2). We previously
revealed that chicken Opn5m is sensitive to UV light and functions as a Gicoupled receptor. The distribution of Opn5m in the retina, pineal gland andparaventricular organ of the hypothalamus was detected by the specificantibody. Thus Opn5m is related with multiple photoreceptions in chicken [1].
Recently, we successfully obtained the active protein of chicken Opn5L2 andfound that Opn5L2 also works as a Gi-coupled UV-sensitive opsin. In addition,
we detected the expression of Opn5L2 within the retina, brain, and,
unexpectedly, adrenal gland of chicken [2]. Based on the analyses of chicken
Opn5 proteins, we discuss the relationship between the molecular properties andthe physiological function of Opn5.
[1] Yamashita et al., (2010) PNAS 107, 22084-22089. [2] Ohuchi et al, (2012)
PLoS One 7, e31534.
1F1448グロイオバクターロドプシンの光反応ダイナミクスの分光研究

Spectroscopic study of photoreaction dynamics of Groeobacterrhodopsin
Kumiko Nagata, Keiichi Inoue, Hideki Kandori (Nagoya Inst. Of Technol.)Groeobacter rhodopsin (GR) is a member of microbial rhodopsins foundin the genome of cyanobacterium PCC7421. GR pumps protons likebacteriorhodopsin (BR) and proteorhodopsin (PR), but the detailed pumping
mechanism and structural changes during the photocycle are not wellunderstood. Therefore, we started studying the dynamics of structural change of
GR. Here we conducted transient absorption (TA) and transient grating (TG)measurements of GR. GR proteins were expressed in E. coli, and TA and TG
methods were applied to the purified proteins in detergent. TG measurements
showed that GR and the O intermediate form trimer at 300 mM NaClconditions, while a portion of the O intermediates has larger volume than theothers. TG signals were dramatically changed at higher salt conditions such as 4
M NaCl. First, a new component was found after the production of the O
intermediate, which was assigned as the component to proton uptake. We
performed the quantitative TG measurements and determined Φ ΔV H+ = 36cm 3/mol. At 4 M NaCl, diffusion rate of the O intermediate was slower thanthat of GR, indicating that large conformational changes occur in the O
intermediate. From the diffusion coefficient, we estimated the size of theoligomer to be about 7 molecules. The results of gel filtration chromatography
and circular dichroism (CD) analysis were consistent with those of TG. The
observed large conformational changes in the O intermediate will be discussed
in relation to the proton-pumping function.

− S29 −

1F1510

ASR の Pro206 はレチナール構造と光反応を制御する
Pro206 of Anabaena sensory rhodopsin is responsible for theisomeric composition of retinal and photochromism

Yoshitaka Kato1, Akira Kawanabe2, Kwang-Hwan Jung3, Hideki Kandori1(1Grad. Sch. Eng., Nagoya Inst. Tech., 2Grad. Sch. Med., Osaka Univ., 3Sogang
Univ. Korea)
Anabaena Sensory Rhodopsin (ASR) is a microbial rhodopsin found in
Anabaena (Nostoc) sp. PCC7120, a freshwater cyanobacterium. ASR isbelieved to function as a photochromism sensor. Microbial rhodopsins possess
all-trans and 13-cis retinal as the chromophore, but only the former is importantfor function. In bacteriorhodopsin (BR), the stable photoproduct at the end of
the functional cycle of the all-trans form is 100% all-trans. Nevertheless, wefound that the photoreaction of ASR is completely photochromic; the stable
photoproduct of the all-trans form is 100% 13-cis, and that of the 13-cis form is100% all-trans [1]. It is intriguing that BR and ASR exhibit similar protein
architecture, but photoreactions are entirely different.
One of key amino acids may be Pro206 of ASR, because the corresponding
amino acid of other microbial rhodopsins is Asp (Asp212 in BR). In the present
study, we replaced Pro206 of ASR into 19 amino acids and examined theirmoleculer properties. Interestingly, all mutants were properly expressed andfolded, including P206R and P206K. HPLC analysis shows that the isomericcomposition between all-trans and 13-cis forms is highly amino-acid dependent,
and some mutants contain 9-cis and 11-cis forms under light conditions. P206D
contains 13-cis retinal dominantly under both dark and light conditions. We thus
conclude that Pro206 is a responsible residue for the photochromic reaction in
ASR, and the mechanism will be discussed.
[1] Kawanabe et al. J. Am. Chem. Soc. 129, 8644 (2007).
1F1522Large deformation of helix F upon formation of the M
intermediate of the azide-bound purple form of pharaonis
halorhodopsin
Taichi Nakanishi1, Soun Kanada1, Midori Murakami1, Kunio Ihara2, Tsutomu
Kouyama1 (1Grad. Sch, Sci., Univ. Nagoya, 2Center for Gene Research., Univ.
Nagoya)Halorhodopsin (HR) utilizes light energy to transport chloride ions from theextracellular side to the cytoplasmic side. When the chloride ion bound to theactive site is replaced with an azide ion, it acquires the ability to transport
protons. To clarify this proton pumping mechanism, we investigated lightinduced structural changes in pharaonis halorhodopsin (pHR) using the C2
crystal that was prepared in the presence of chloride ion. When the C2 crystal
was soaked in a solution containing azide ion, the chloride ion and one watermolecule in a cavity between the Schiff base and Thr126 were replaced with an
azide ion. When the azide-bound purple form of pHR was exposed to orange
light, an M-like yellow species containing 13-cis retinal was generated
efficiently in one of the three subunits in the asymmetric unit. This subunit has
the EF loop facing a free space, so that a profound outward movement of thecytoplasmic half of helix F was induced upon formation of the M state. This
structural change was accompanied by a large swing of the phenyl ring ofPhe259 in helix G, creating a large cavity in the cytoplasmic side of the retinal
Schiff base, where a linear cluster of water molecules were incorporated.
Simultaneously, the middle moiety of helix C distorted largely, leading to asignificant shrinkage of the primary anion binding site (site I). As a result, theazide ion that was initially located in site I was forced to move to a large cavity
created by a flip-flop motion of the side chain of Glu234 in the anion uptake
channel.
1F1534

センサリーロドプシン II-トランスデューサー複合体の比較シミュ
レーションにより明らかにされた基底状態と M 中間体の差異

Differences between the ground state and the M-intermediateof Sensory Rhodopsin II-Transducer complex revealed bycomparative simulations
Koro Nishikata1,3, Mitsunori Ikeguchi1, Akinori Kidera1,2 (1Grad. Sch. of
Nanobioscience, Yokohama City Univ., 2Research Program for Computational
Science, RIKEN, 3Bioinformatics And Systems Engineering division (BASE),
RIKEN)
The complex of sensory rhodopsin II (SRII) and its cognate transducer HtrII(SRII:HtrII/2:2) is a photosensor associated with negative phototaxis in extreme
halophiles. To examine how photoexcitation of SRII influences the overall

structure of the complex, we performed comparative molecular dynamics
simulations of the complex of SRII and truncated HtrII (residues: 1-136) fromN. pharaonis of two photochemical states, the ground state and the Mintermediate, in explicit solvent and lipid bilayer. The simulation systems
consist of each crystal structure appended with the models of the missing
residues and the N-terminal HAMP domain. The simulation results showedsignificant increases in the structural differences compared to the two crystal
structures: the outward bending motions of F-helix of SRII and the rotation of
the C-terminal region of TM2 helix. The most remarkable change was found in
the overall orientation of the two SRII molecules: the cytoplasmic side wasclosed in the ground state and open in the M-intermediate. This change wasattributed to the differences in the side-chain packing of the 4-helix bundle ofthe HtrII dimmer. We will discuss the results in detail with additional
simulations of the complex without the HAMP domain. Finally, we will proposea hypothesis for the signal transduction mechanism of SRII-HtrII system.
1F1546

Effect of potential on the photo reaction of Sensory rhodopsin
II monolayer studied by Surface Enhanced IR absorption
Spectroscopy

Kenichi Ataka, Joachim Heberle (Freie Universitaet Berlin, Fachbereich
Physik)Membrane potential is one of important factors that set out the function of many
membrane proteins. Although an importance of this factor is widely accepted,
experimental method that can access directly to the effect is limited. Here, we
introduce a method tackling this problem, in which exploit a membrane protein
monolayer tethered on a metal solid-support and surface enhanced infraredabsorption spectroscopy (SEIRAS). To reproduce highly orientated protein
layer as mimic of the cellular membrane, we employed a tactic that themembrane protein bind through recombinant histidine tags with Ni-NTA selfassembled monolayer modified solid support. After orientated binding of theprotein to the surface, full functional integrity is regained by embedding into alipid layer. SEIRAS provides specific enhancement of the signal from thetethered membrane protein, in which is difficult to detect by conventional IR
technique due to a scarce amount of monolayer as few as pico-mol/cm2.
Advantage of SEIRAS becomes most beneficial when the functional studies of
the tethered membrane proteins are performed by the application of external
stimuli (light, voltage, chemicals etc.) to initiate the reaction. Functional
difference spectroscopy between initial non-active and active state can extract
the contributions from functional groups that are involved in the reaction.
Application on the study of a photo-cycle reaction of sensory rhodopsin II underpotential control will be discussed.
1F1558In-situ 光照射固体 NMR による光受容膜蛋白質の光活性状態の解明
Light activated states of photoreceptor membrane proteins as
revealed by in-situ photo-irradiated solid-state NMR

Akira Naito1, Yuya Tomonaga1, Tetsurou Hidaka1, Yusuke Shibafuji1,
Yoshiteru Makino1, Izuru Kawamura1, Yuki Sudo2, Akimori Wada3, Takashi
Okitsu3, Naoki Kamo4 (1Graduate School of Engineering, Yokohama National
University, 2Graduate School of Science, Nagoya University, 3Kobe
Pharmaceutical University, 4Matsuyama University)
Pharaonis phoborhodopsin (ppR or sensory rhodopsin II) is a negative
phototaxis receptor of Natronomonus pharaonis and forms a 2:2 complex withthe cognate transducer (pHtrII), which transmits the photosignal into cytoplasm.
Light absorption of ppR initiates trans-cis photo-isomerization of the retinal
chromophore followed by cyclic chemical reaction consisting of several
intermediates (K, L, M and O). The M intermediate is thought to be an
active state for signal transduction. We have successfully observed the M
intermediate by using photo-irradiated solid-state NMR, because the life time of
the M-intermediate is much longer than the other intermediates. 13C NMR
signal from [20-13C] retinal-ppR and ppR/pHtrII revealed that multiple Mintermediates (M1, M2 and M3) with 13-cis, 15-anti retinal configurations
coexisted under the photo-irradiated condition. Further, since the life time of M3
state was much longer than those of the other M states, the M3 state could be
distinguished from the other M states. Carbonyl carbon signals of [1-13C]
Tyr174 in ppR also showed that multiple structures coexisted in the proteinside. Light induced structural changes were further observed in [3-13C] AlapHtrII/ppR complexes. Particularly, helix structure of the interfacial region in
pHtrII changed significantly. Detailed structure of photo-intermediate in photoreceptor membrane proteins is now possible to gain insight into the signaltransduction mechanism by using in-situ photo-irradiated solid-state NMR.

− S30 −

1G1400

Larger Intercellular Adhesion Strength Generates More
Coherent and Directional Multicellular Movement- Measurement and Simulation
Tsuyoshi Hirashima1, Takanori Iino2, Yoichiroh Hosokawa2, Masaharu
Nagayama3,4 (1Anatomy and Developmental Biology, Faculty of Medicine,
Kyoto University, 2Graduate School of Materials Science, Nara Institute of
Science and Technology, 3Research Institute for Electronic Science, Hokkaido
University, 4CREST, Japan Science and Technology Agency)
Collective cell movement is an essential process in many events of all animals,
including wound healing and embryonic development although ourunderstanding of what attributes the emergence of collective cell movement is
far from complete. We examined cellular behaviors in a monolayer sheet of askin cell under culture conditions with different Ca2+ concentration, andobserved that the frontal line of sheet moved faster in higher Ca2+
concentration. The particle image velocimetry analysis for the movement ofskin cell revealed that the cells in the leading front region moved faster andthose in the rear moved slower for the treatment of higher Ca2+ concentration
although there were no differences in the average speed of whole cell movement
for any Ca2+ treatments. We also found that the movement of cells became
more directional with longer spatial correlation in proportional to the treated
Ca2+ concentration. To know influences in the cellular level to the variation oftreated Ca2+ concentration, we quantitatively measured cellular behaviors suchas cell division rate and the strength of intercellular adhesion, and found that thestrength of intercellular adhesion more increases in higher Ca2+ concentration.
Integrating the quantitative data into a computational model, we reproduced thecoherent and directional multi-cellular behavior, indicating that the collective
cell behavior of skin cell can be induced only by the strength of intercellularadhesion.
1G1412

GPI アンカー型タンパク質の短寿命ホモダイマーはラフト組織化・機能のための最小単位であるTransient GPI-anchored protein homodimers are units for raftorganization and function
Kenichi G. N. Suzuki1, Rinshi S. Kasai1, Koichiro M. Hirosawa1, Yuri L.
Nemoto1, Munenori Ishibashi1, Yoshihiro Miwa2, Takahiro K. Fujiwara1,
Akihiro Kusumi1 (1iCeMS, Kyoto Univ., 2Dept. Pharmacol., Univ. Tsukuba)
Advanced single-molecule fluorescent imaging was applied to study the
dynamic organization of raft-associated glycosylphosphatidylinositol-anchoredprotein (GPI-APs) in the plasma membrane and their stimulation-inducedchanges. In resting cells, virtually all of the GPI-APs are mobile and continuallyform transient (~200 ms) homodimers through ectodomain protein interactions,stabilized by the presence of the GPI-anchoring chain and cholesterol (termed
homodimer rafts). Heterodimers do not form, suggesting a fundamental role forthe specific ectodomain protein interaction. Under higher physiologicalexpression conditions, homodimers coalesce to form hetero- and homo-GPI-AP
oligomer rafts by raft-based lipid interactions. Upon ligation, CD59 formed
stable oligomer rafts, containing up to four CD59 molecules, which triggered
intracellular Ca2+ responses that were dependent on GPI-anchorage andcholesterol, suggesting a key role played by transient homodimer rafts.
Transient homodimer rafts are likely one of the basic units for the organizationand function of raft domains containing GPI-APs.
1G1424and GCN2, but not PKR. Experiments using PERK- and GCN2-knockout cells
demonstrated dual contribution of PERK and GCN2 to nsPEF-induced eIF2α
phosphorylation. Moreover, nsPEF exposure yielded the elevated GADD34
expression, which is known to function as a negative feedback control for eIF2α
phosphorylation. RT-PCR analysis of a set of stress-inducible genes
demonstrated that cellular responses to nsPEFs are distinct from those induced
by previously known forms of cellular stress, such as ER stress, UV, and heat.
Taken together, these results provide new mechanistic insights into nsPEF
action and implicate the therapeutic potential of nsPEFs for stress responseassociated diseases.
1G1436弾性膜モデルを用いたゴルジ再集合過程シミュレーション

Simulation study on Golgi reassembly process based on elastic
membrane model
Masashi Tachikawa, Atsushi Mochizuki (Theoretical Biology Laboratory,
ASI, RIKEN)
Golgi apparatus is a cellular organelle which processes proteins, and sorts themfor delivery to various destinations. Golgi apparatus shows characteristic shape;a stack of flattened cisternae made of lipid membrane. In the mitotic phase, it
disperses into small vesicles, and they reassemble to form the characteristic
shape in individual daughter cells. No plausible mechanism has been proposed
to explain the characteristic shape.
In this study, we reconstruct a Golgi reassembly process by computer
simulations and propose a possible mechanism to produce the characteristic
Golgi shape. The relation between local shapes of lipid membrane and its
deformation is well characterized by elastic membrane theory. Based on it, weconstructed a bio-membrane simulator. An arbitrary shape of membrane surface
is approximately represented by a polygon, and the shape deformation issimulated using Monte Carlo method. Effects of membrane-associatedmolecules, such as deformability change, adhesion and membrane fusion, arealso considered.
Without any additional assumption, sponge-like structures are emerged throughthe simulations. It indicates that the Golgi-like layered structure is not formedby itself. We then considered several assumptions for the occurrence of themembrane fusion, and found that if the fusion occurs only between convex
membranes, a Golgi-like layered structure is observed. This fusion modification
can be realized when the distribution of fusion-promoting proteins depends on
membrane curvature.
1G1448Analysis of cell behavior near silicon based micro-scale pore
Yo Otsuka1, Shinya Murakami1, Manabu Sugimoto1, Toshiyuki Mitsui2(1Grad. Sch. Sci., Aogaku Univ, 2Assoc.Sci., Aogaku Univ)

引き起こす

The propose of our study is to understand of the cell behavior under various
conditions for example, concentration gradients created by silicon based microscale pores. In bone remodeling and fracture treatment, recruitment to sites forbone formation of osteoblasts secrete bone matrix is crucial. If we are able tocontrol the cell recruitment, it may be possible to promote the bone formation on
specific site. For this recruitment, we have created concentration gradients ofcertain substances in m-range by using micro-scale pores milled on a freestanding SiN membrane, and analyzed the possible control cell differentiation
and behavior. In addition, we have assembled the instrument to culture cellunder the microscopy and to observe to analyze cell behavior in real time. By
using this instrument, the concentration gradient of FBS induced by the 5 mdiameter pore enhances the Osteoblast motions toward the pore mouth. In thisconference, we describe the method to create concentration gradients by using
the micro-scale pores and the possibility to control of cell recruitment.

Nanosecond pulsed electric fields act as a novel cellular stressthat induces translational suppression

1G1510

ナノ秒パルス高電界は新規細胞ストレスとして作用して翻訳抑制を

Ken-ichi Yano, Keiko Morotomi-Yano (Bioelectrics Res. Center, Kumamoto Univ.)
Pulsed electric fields (PEFs) of millisecond durations are widely used for DNA
electroporation. Recent advances in electrical engineering enable the generationof ultrashort PEFs (nanosecond PEFs, nsPEFs). Contrary to conventional PEFs
for DNA electroporation, nsPEFs can directly reach intracellular components
without membrane destruction and efficiently induce apoptosis. AlthoughnsPEFs are now recognized as a unique tool in life sciences, the molecularmechanism of nsPEF action remains largely unclear. In this study, we revealed
that nsPEFs act as a novel cellular stress. Exposure of HeLa S3 cells to nsPEFs
quickly induced eIF2α phosphorylation, which is a common hallmark of stressresponses, and elicited concomitant translational suppression. We analyzed
upstream stress-responsive kinases and observed that nsPEFs activate PERK

重イオンマイクロビーム照射によるバイスタンダー応答で誘導されるシグナル伝達機構の解明

Mechanisms of signal transduction activated by heavy-ion
microbeam induced bystander responses
Masanori Tomita1, Hideki Matsumoto2, Tomoo Funayama3, Yuichiro
Yokota3, Kensuke Otsuka1, Munetoshi Maeda1,4, Yasuhiko Kobayashi3(1Radiat. Safety Res. Cent., CRIEPI, 2Biomed. Imaging Res. Cent., Univ.
Fukui, 3QuBS, JAEA, 4R&D, WERC)
Radiation-induced bystander responses are defined as responses in cells thathave not been directly traversed by radiations but are in the neighborhood ofcells that have been directly traversed by them. Bystander responses induced bylow doses of heavy-ion beams are an important problem in cancer therapy. In− S31 −

the present study, we aim to clarify the bystander signal transductions activatedby heavy-ion microbeam irradiation. After forming 5 colonies, which composedby the targeted colony in center and four untargeted colonies around it, on a dishusing normal human fibroblast WI-38 cells, a cell in the targeted colony wasirradiated with Ar-ion microbeams. Focus formation of DNA repair proteins
was examined in the cells in the untargeted colonies. Next, 5 cells within theconfluent culture were irradiated with the Ar-ion microbeams. Phosphorylationof NF-κB in the bystander cells was observed 3 h after irradiation, but was not 6
h. Phosphorylation of Akt was not only observed 3 h but also 6 h after
irradiation. On the other hand, accumulation of COX-2 was observed only 6 hafter irradiation. In addition, accumulation of COX-2 was not detected in the
broadbeam irradiated cells, whereas phosphorylation of NF-κB and Akt wereobserved. Bystander responses induced by Ar-ion microbeam irradiation were atleast partially suppressed by pretreatment with a scavenger of nitric oxide (NO).
These results suggested that NF-κB-dependent signaling pathway involving Akt
and COX-2 play an important role in the NO-mediated bystander response.
1G1522出芽酵母の遺伝子発現パターンの変化における染色体揺らぎの影響

Effects of conformational fluctuation of chromosomes inbudding yeast on the pattern of gene expression
Naoko Tokuda, Masaki Sasai (Grad. Sch. Eng., Univ. Nagoya)
Recently, conformation of all chromosomes in the interphase nucleus of budding
yeast has been inferred from the Chromosome Conformation Capture-on-Chip(4C) data (Duan et al., Nature (2010)). However, it has not yet been ascertained
how the conformational fluctuation around their mean structures affects theregulation of gene expression. To clarify this issue, we developed a dynamical
structural model of interphase chromosomes in budding yeast (Tokuda et al.,Biophys. J. (2012)). In the present paper, the effects of the conformational
fluctuation on the pattern of gene expression will be examined by using thiscoarse-grained chromosome model. Specifically, I will discuss the observed
difference in the pattern of gene expression between the yku70 esc1 mutant which
abrogates telomere anchoring and the wild-type strain (Taddei et al., Genome
Res. (2009)). In the data of Taddei et al., 32 genes are expressed at higher levels
and 28 genes are expressed at lower levels in the yku70 esc1 mutant than in thewild-type strain. We examine the reason of this misregulation by comparing thefluctuation of the chromosome conformation in the case that telomeres are notanchored to the nuclear periphery with that in the anchored case.
1G1534

人工自己複製システムのダーウィン進化

Darwinian Evolution of artificial self-replication system
Norikazu Ichihashi1,2, Tetsuya Yomo1,2,3 (1JST ERATO, 2Graduate School ofInformation Science and Technology, Osaka University, 3Graduate School of
Frontier Bioscience, Osaka University)
Darwinian evolution, the consequence of variation and natural selection, hasplayed a central role in the emergence of the complexity of living organisms. To
understand requirements for Darwinian evolution, we have constructed an
artificial self-replication system which capable of Darwinian evolution from
non-living molecules. We used an RNA encoding an RNA-dependent RNA
polymerase as a genetic molecule and mixed it with a reconstituted cell-freetranslation system. The RNA polymerase was translated from the RNA, andtranslated polymerase, in turn, replicated the original RNA. During this selfreplication process, mutations are spontaneously introduced by replication error.
In addition, to link the genotype to the phenotype, we encapsulated the reaction
into a micro-scale water-in-oil emulsion.
After about 180 rounds of repeated self-replication, the self-replication activity
increased gradually and finally reached 100-fold. Mutations were randomly
introduced into RNA, some of which became fixed in the population. The final
RNA has higher replication rate and higher competence against parasitic
replicator. These result demonstrated that in our system, more self-replicableRNA has evolved as a consequence of spontaneously mutation and natural
selection. This artificial self-replication system would be a novel platform toexperimentally investigate the possible evolutionary process of complex natureof living organisms
1G1546多点蛍光相関分光を用いた時空間解析によるグルココルチコイド受容体の核移行解析

Nuclear translocation analysis of glucocorticoid receptor by
spatiotemporal analysis with multipoint fluorescence correlation spectroscopy

Johtaro Yamamoto, Masataka Kinjo (Fuc. Adv. Life Sci., Hokkaido Univ.)
Glucocorticoid receptor (GR) is one of the nuclear receptors that localizes incytosol without ligand binding. Once the GR binds with their ligand, the GR
starts to translocate to nuclear. Finally, the GR binds to DNA as dimers, andregulates transcriptions of various genes. The nuclear translocation property of
GR is unclear yet though the dexamethasone, a ligand of GR, is widely usedsuch as steroidal anti-inflammatory drug.
In this work, the nuclear translocation rate of GR was analyzed in living cells
using a multipoint fluorescence correlation spectroscopy (MP-FCS) with single
molecule sensitivity. The system realizes simultaneous measurement offluorescence fluctuation emitted from EGFP-GR at up to three dimensionally
arbitrary seven points in living cells. The auto-correlation functions of these
fluorescence signals provide the concentrations of EGFP-GR and its diffusion
constants. In addition, the spatiotemporal cross-correlation functions provide thetraveling time of EGFP-GR between two measurement points. Analyzing thecorrelation functions between a point in cytosol and in nucleus, the nuclear
translocation rates of GR were obtained. The result was much slower than the
expected result of free diffusion, and this can be considered the result caused bythat the nuclear translocation of GR is an energy consuming process. We expect
the MP-FCS system can reveal a part of active transport mechanism. In ourpresentation, we will investigate the effect of some mutation of GR on the
nuclear translocation rate.
1G1558細胞内 FRET イメージングによる情報処理タンパク質 RAF の構
造と EGF 応答の相関解析

Correlation between the molecular conformation and EGF
response of RAF by FRET imaging in individual cells
Kayo Hibino1, Masahiro Ueda1, Yasushi Sako2 (1RIKEN, QBiC, 2RIKEN, ASI)
RAS-RAF signaling regulates various cellular responses including proliferation,differentiation, and carcinogenesis. After the stimulation of cells with epidermal
growth factor (EGF), the activated RAS recruits RAF from the cytoplasm
to the plasma membrane. At the membrane, RAF is activated throughphosphorylations by undetermined kinases and signals to downstream. Thus, theredistribution of RAF in ensemble molecules is important in the cell-fate
determination, however, this responsiveness to EGF differed in individual cells,
even though the cells were synchronized each other by serum starvation andstimulated with the saturation amount of EGF.
By using FRET-based probe of RAF, we found that the EGF response correlatedwith the ensemble conformation of RAF in individual resting cells. The cellswith an open-bias form of RAF responded to EGF and showed theredistributions of RAF remarkably, whereas the cells with closed form of RAF
hardly responded to EGF and did not show the redistribution of RAF.
By analyses of RAF with particular phosphorylation status or mutations ofphosphorylation-sites, we found that the conformation of RAF in resting cells
was modulated by the phosphorylation status of RAF. This result suggests thatthe phosphorylation status of RAF differs in individual resting cells, whichcorrelates with the EGF response in the RAS-RAF signaling. The
phosphorylation status of RAF could be affected by the cellular memory of thepast extracellular environment and the intracellular reactions.
1G16101 分子キネティクス解析により示された、ヘレギュリンと ErbB タ
ンパク質のダイナミックに変化する相互作用

Interaction between heregulin and ErbB proteins varies
dynamically shown by single-molecule kinetic analysis in living
cells
Michio Hiroshima1,2, Yuko Saeki3, Mariko Okada-Hatakeyama3, Yasushi
Sako1 (1RIKEN ASI, 2RIKEN QBiC, 3RIKEN RCAI)
Cell fate of the MCF7 human breast adenocarcinoma cells is determined by theligand-specific manner. When MCF7 cells are stimulated by epidermal growth
factor (EGF) or heregulin (HRG), proliferation or differentiation is induced,
respectively. Both EGF and HRG receptors are ErbB receptor tyrosine kinases
that transduce ligand signals to the cytoplasmic proteins thorough homo- andhetero-dimerization. The ErbB family proteins consists of four members, ErbB1
to B4, and EGF mainly associates with ErbB1, whereas HRG with B3 and B4.
By single-molecule measurement on interactions between dye-conjugated HRG
and the receptors in living MCF7 cells, we obtained kinetic parameters of both
ligand association and dissociation. The ligand-receptor association pathways of
ErbB3/4 were almost the same as that of ErbB1. A receptor predimer
accelerated the formation of a doubly-liganded signaling dimer through anintermediate formation between the first and the second HRG bindings. On the− S32 −

other hand, the first dissociation of HRG from the doubly-liganded dimers was
rapid, but the other HRG from the singly-liganded dimers was slow. The
dynamic change between the association and dissociation kinetics that produced
different affinity sites of HRG on the cell surface lead to concave-up shape of
the Scatchard plot. The biphasic kinetics indicated the existence of negative
cooperativity in the ligand-receptor interaction and enables the ErbB system todetect the extracellular HRG signals rapidly and sensitively.
1H1400

ラスター画像相互相関分光法（ccRICS）を用いた外来 DNA に対する細胞内防御機構の可視化

Visualization of intracellular defense system against exogenous
DNAs by using cross-correlation raster image correlation
spectroscopy
Akira Sasaki, Masataka Kinjo (Fac. Adv. Life Sci., Hokkaido Univ.)
Gene delivery systems with artificial vectors are widely used in the research
field of cell biology and also in the clinical field as gene therapy to promote
exogenous gene expression or inhibit production of a target protein. It is
presumed that the translocation of transfected DNA to nucleus competes withdegradation by cytoplasmic nucleases as the intracellular defense system against
exogenous DNAs, such as viral genes. Previously, the size and structure ofdegraded DNAs were monitored by fluorescence cross-correlation spectroscopy(FCCS) in solution and in the cytoplasm of living cells [1]. The result showedthat the exonuclease activity affected the transfection efficiency, and the level ofexonuclease activity in cytoplasm was different depending on the cell line.
However, such FCCS measured only one point at a time. To overcome thisdrawback of FCCS measurement, raster image correlation spectroscopy (RICS)
and cross-correlation RICS (ccRICS) are employed to investigate when andwhere nucleases work in living cells. As the probe of DNA degradation, dual
fluorescent labeled linear DNA was synthesized. RICS enabled the mapping ofdegradation state of the DNA probe in living cells. In this report, the proof ofprinciple study of the measurement system by observation of the DNA probe insolution and in living cells will be discussed. This work will contribute inunderstanding the fate of exogenous DNA, which is critical in development ofefficient vectors.
[1] Sasaki A and Kinjo M., J . Control. Release 143(1):104-111 (2010)
1H1412

Non-invasive in vivo imaging of vesicle movement in neutrophils in mouse ear
Kenji Kikushima, Sayaka Kita, Hideo Higuchi (Dept of Phys, School of
Science, The Univ. of Tokyo)
Neutrophils play an essential role in innate immune response. Observation ofneutrophil under non-invasive conditions has been an important problem to besolved. Quantum dots (QDs) are semiconductor nanoparticles that show good
photostability and strong fluorescence compared to conventional dyes. Here, we
succeeded to observe QD-labeled neutrophils in mouse ear under non-invasive
conditions up to 100 um below the surface of skin. Neutrophils began toextravasate from blood vessels upon inflammation, and locomote in interstitialspace. Most intriguingly, QDs were uptaken into the neutrophils and vesicle
movements inside the cells were able to be seen. Movement of these vesicles
was tracked at 80 frames/sec with 15 nm accuracy or in 3D space. While
vesicles in the neutrophil on the vascular wall did not show any directional
movements, vesicles in the neutrophil locomoting in the tissue were transported
actively. Movements of these vesicles were independent each other even in thesame cell, and some of them underwent as “diffuse-and-go” manner. The speedof the vesicle at “go” phase achieved about 4 um/sec, which was about threetimes faster than the velocity of a molecular motor such as kinesin or dynein in
vitro. The high speed transport of the vesicle should be important for the abilityof neutrophil to kill various microbes.

1H1424

脂質二重膜を用いた T 細胞活性化の 1 分子イメージング解析

Single molecule analysis of T cell activation with stimulatory
lipid bilayers by live cell imaging
Yuma Ito1,2, Hirofumi Oyama1,2, Kumiko Sakata-Sogawa1,2, Makio
Tokunaga1,2 (1Grad. Sch. Biosci. Biotech., Tokyo Inst. Tech., 2RCAI, RIKEN)
T cells are activated when the T cell receptor (TCR) binds to a peptide-MHC
complex. TCRs form microclusters with T cell signaling molecules on theactivated T cell surface. Although it is necessary for the initialization ofadaptive immune responses, the formation mechanism of the microclusters
remains unclear. To observe the dynamics of TCR on the living T cell surface,

we have developed a model system on planer lipid bilayers with stimulatory
antibodies. With this system we can visualize the dynamic formation of TCR
microclusters at the single-molecule level.
Using this system, here we compared the dynamics of TCR on the stimulated
and resting T cell surface. We analyzed two-dimensional motions offluorescently labeled TCR. On the activated T cell surface, the diffusion
coefficient of TCR was one fourth of that of CD45 (the receptor-like protein
tyrosine phosphatase outside of microclusters). It indicates the interaction of
TCR molecules with TCR microclusters. Contrary to expectation, TCR
molecules formed clusters also in resting T cells before activation. We
calculated the number of TCR molecules in the clusters using the fluorescenceintensity of single TCR molecules. The clusters contained 18 ± 6 molecules ofTCR. The clusters moved diffusively at whole regions on the cell surfaces, andfrequently divided and fused each other. The present result raises the possibility
that the TCR can exist as nano-scale clusters even in resting T cells before
activation and gives a fresh insight into the mechanism of T cell signaling.
1H1436

GTP 交換促進因子 Sos の生細胞一分子計測

Single molecule imaging of guanine nucleotide exchange factor
Sos in living cells
Yuki Nakamura1,2 (1RIKEN sako celluar informatics laboratory, 2Osaka
university Yanagida lab)
The small GTPase Ras is one of the essential regulators for controlling celldifferentiation and proliferation. Ras activation is induced on the plasma
membrane by the exchange of bound GDP for GTP. The guanine nucleotide
exchange factor son of sevenless (Sos) regulates this GDP/GTP exchange on Ras.
Upon stimulation with various extracellular factors including epidermal growth
factor EGF, Sos translocates from the cytoplasm to the plasma membrane.
However, spatio-temporal dynamics of the translocation and the regulation of Ras
activation have not been known for the full length of Sos molecule.
In this study, we observed single molecules of GFP-tagged Sos in living HeLa
cells using total internal reflection fluorescence microscopy. We found anincrease of the amount of Sos molecules on the plasma membrane upon EGFstimulation, which had not been detected by ensemble molecule imaging in theprevious studies. In addition to the increase of the on rate, duration of themembrane association for each single molecule increased after the stimulation.
As the result, even though the time-course and amount of Sos translocation werevaried among cells, on average, an increase of the molecular density by a factorof 2.5 was observed 5 min after the stimulation. Although, before stimulation, asmall number of Sos molecules localized on the plasma membrane with shortdurations, our result suggested that Sos molecules, associated with themembrane for long durations, are responsible for Ras-activation.
1H1448

リガンド結合したβ 2 アドレナリン受容体の短寿命ダイマーは三量体 G タンパク質のターンオーバーを加速する：2 色同時 1 分子観察による測定

Single-molecule imaging revealed acceleration of trimeric
G-protein turnover by transient dimerization of liganded
β2adrenergic receptor
Rinshi Kasai1, Takahiro Fujiwara2, Akihiro Kusumi1,2 (1Inst. For FrontierMed. Sci., Kyoto Univ., 2WPI-iCeMS, Kyoto Univ.)
Previously, we for the first time succeeded in fully characterizing the dynamic
dimer-monomer equilibrium of GPCRs, formyl peptide receptor and beta2
adrenergic receptor (AR) (Kasai, 2011 JCB & BPS meeting), including thedimer lifetime of ~100 ms for both molecules. To examine the physiological
functions of the transient GPCR dimers, we performed simultaneous dualcolor single-molecule imaging of trimeric G-protein, Gs (Gs-mGFP), andfluorescently-labeled AR in the living cell plasma membrane underphysiological conditions. Arrivals of Gs at the AR monomers and dimers and Gs
departures from them were observed at the single-molecule level, allowing us todetermine the residency time of Gs on the transient AR monomers and dimers.
Due to much higher expression levels of endogenous Gs, the Gs-mGFP
residency time (or Gs off rate) is likely to be the rate limiting step for the Gs
turnover at the AR and thus for the Gs activation at the bulk level. We foundthat, even before stimulation, G-protein is recruited to AR and its residency
times on transient AR monomers and dimers were about the same. Ligation
reduced the G-protein residency times on both transient monomers and dimers,
but the effect was more on that on dimers, suggesting that transient dimerizationof liganded AR accelerates Gs turnover.

− S33 −

1H1510

光学顕微鏡と同時観察可能な高速 AFM

High-speed AFM combined with optical microscopy
Shingo Fukuda, Takayuki Uchihashi, Toshio Ando (Depertment ofMathmatics and Physics, Grad School of Natural Science and Technology
Kanazawa Univ)
High-speed-atomic-force-microscopy (HS-AFM) can directly visualize
dynamic processes of single protein molecules at work. This new microscopy
has recently been proven to be capable of obtaining previously inaccessible
information. 1-3). It now can be routinely applied to various isolated
biomolecules. The next stage towards the evolution of HS-AFM would be theincorporation of functions by which we can gain multiple aspects of datainaccessible only with HS-AFM. In fact, with the current HS-AFM setup, it ishard to observe large objects such as live cells because the maximum scan range
is limited to a few m. Therefore it is difficult to position the AFM tip on a
specific area of a large sample. In addition, very small molecules such as
nucleotides cannot be seen, prohibiting us from observing, for example, thedynamic events of nucleotide binding and dissociation. On the other hand,
optical microscopy has a wide view and enables us to visualize small moleculesby the attachment of fluophores to them.
Thus, we have now been developing a HS-AFM system which can be mounted
on a conventional optical microscope. For this combined system, we havedeveloped a compact optical detection system, a laser tracking device, andothers. We are now using this combined HS-AFM/optical microscopy setup to
observe dynamics of live cells and single molecules.
1. M. Shibata et al., Nature Nanotechnology 5, 208-212 (2010).
2. N. Kodera, D. Yamamoto et al., Nature 468, 72-76 (2010).
3. T. Uchihashi, R. Iino et al., Science 333, 755-758 (2011).
1H1522High-speed AFM observation of intrinsically disordered
proteins (NTAIL-GFP, PNT-GFP and SIC1-GFP)
Sujit Kumar Dora1, Kodera Noriyuki1, David Bloquel2, Johnny Habchi2,
Antoine Gruet2, Sonia Longhi2, Toshio Ando1 (1Bio AFM frontier research
center, Institute of physics and mathematics, Kanazawa University, 2AFMB
Laboratory, University of Aix-Marseille)
Intrinsically disordered proteins (IDPs) are functional proteins either fully or
partly lacking stable secondary and tertiary structures under physiologicalconditions. They are involved in important biological functions such astranscription, translation, and cellular signal transduction. Despite the importantroles of ID regions, effective methods to observe their structures werepractically not available until the advent of high-speed AFM (HS-AFM). In thisreport, HS-AFM is employed to study the structure of three different ID
proteins: two measles virus protein (NTAIL and PNT) and a yeast protein SIC1,
all fused to green fluorescent protein (GFP). Successive AFM images of these
proteins on mica surface captured at 10 frames per second clearly show thepresence of two globular parts (small globule of ~1.5 nm diameter and large
globule of ~4 nm diameter) interconnected by ID regions. The measured end-toend distances of these ID regions are in excellent agreement with the respective
theoretical values predicted under the assumption that the microscopic
persistence length is the same (~1.2 nm) irrespective of different ID regions.
Thus, the value of the microscopic persistence length is a finger print of theconformation of ID regions. Further, the height distribution of globular parts
indicates the large globule (~4nm) to be GFP. The reason for the existence ofsmall globular parts is still unknown.
1H1534位相差電子顕微鏡用の帯電防止位相板

Anti-charging Phase Plates for Phase Contrast Electron
Microscopy
Kuniaki Nagayama1, Toshiyuki Itoh2, Yoko Kayama2, Zenpei Saitoh2,
Yukinori Nagatani1, Yoshihiro Arai2, Masahiro Ohara1, Noriko Kai2,
Kazuyoshi Murata1 (1National Institute for Physiological Sciences, 2Terabase
Inc.)
Phase contrast electron microscopy (EM) has a particular advantage whenapplied to biological samples that are made of light elements of which electron
scattering is weak. A thin-film type of phase plates made of amorphous carbon
has been applied to various biological samples with an expected performance ofhigh contrast. Nevertheless the phase plate EM has not gotten a foothold in the
EM community mainly due to the tedious handling of the plate. The success of
biological applications have actually been supported by a mass-consumption ofphase plate to redeem its short-life owing to the trouble of phase plate charging.
Charging has always been the biggest obstacle to the phase plate development.
Such a charging as originated from external causes, namely contaminantcharging, has been cleaned up by a treatment of wrapping contaminants withcarbon. But charging of carbon material itself after a high electron dose as
revealed as an aging effect, also known as "Berriman effect", has remained to be
fixed at last. To overcome the internal cause, the best way is known to add highconductivity to the carbon film by converting the carbon material from
amorphous to graphite one, which is robust to the electron dose and highly
conductive. We have tried several approaches to improve the conductivity anddeveloped anti-charging phase plates which guarantee better handling. We
report the mechanism behind the charging, an image deterioration model due tocharging, anti-charging technologies and applications to biological samples.
1H1546500kV LinacTEM の開発
Development of the 500kV Linac TEM

Yukinori Nagatani1, Yoshihiro Arai2, Takumi Sannomiya3, Tadao Shirai4, Ryuzo
Aihara2, Giichi Iijima2, Kuniaki Nagayama1 (1National Inst. Physiological
Sci., 2Terabase Inc., 3Tokyo Inst. Tech., 4RTS)
High Voltage Electron Microscopy (HVEM) is providing a powerful mean toobserve thick (>1 um) samples with nanometer resolutions in biology. The deep
penetration of the high voltage electron beam (> 0.5MV) is useful to reconstruct
3-dimensional structures of biological samples. HVEM is, however, quite hugeand expensive, of which cost exceeds 10 - 20 million dollars, because it isequipped with a high voltage DC accelerator which should be stored into a highpressure SF6-gas tank. We have constructed a novel kind of HVEM, which isdesigned to avoid the costly high voltage DC accelerator, 500kV Linac TEM, in
the National Institute for Physiological Sciences. The Linac (linear accelerator)
technologies have no need for the DC accelerator but need only for a smallerscaled RF accelerator. Thus the total HVEM system demands a cost smaller
than 6 million dollars and a height smaller than 4 meters. The idea applying thelinac to HVEM is not new but the technical requirements as an EM system suchas high stability (dE/E <10^-5) of the beam energy and fine emittance (<1mrad) of the beam, both of which are beyond the current accelerator
technologies, have been blocking its implementation. To satisfy therequirements, we have developed a system including a 10^-5 stabilized RF
power supply of 30kW/2.45GHz and 250 femtoseconds beam choppers with alow energy spread and a fine emittance. We propose a project of the 5MV
HVEM, by superconducting accelerators, which is to realize the 3-dimensional
nanoscale imaging of a whole cell.
1H1558

光学顕微鏡とカソードルミネッセンス顕微鏡を用いたマルチモーダ
ル細胞イメージング

Multimodal cellular imaging with light microscopy andcathodoluminescence microscopy
Hirohiko Niioka1, Taichi Furukawa2, Masayoshi Ichimiya3, Tomohiro
Nagata4, Masaaki Ashida2, Tsutomu Araki2, Mamoru Hashimoto2 (1Inst. for
NanoSci. Design, Osaka Univ., 2Eng. Sci., Osaka Univ., 3Osaka Dental
Univ., 4Tsukuba Institute for Super Materials, ULVAC Inc.)
Correlative light and electron microscopy (CLEM) allows us to observe
molecular distribution with the combination of light microscopy (LM) andelectron microscopy. Quantum dots are used for multi-labeling and the same
molecules were observed with LM via color and TEM directly [1, 2]. However,TEM provides monochrome image and we cannot see the colors but check thesize and shape of quantum dots to determine the molecular species in TEM
images.
Cathodoluminescence microscopy (CLM) has a potential to enable color
imaging of individual biomolecules with using nanophosphor labeling [3].
Cathodoluminescence (CL) is the emission of light from a material as a result ofbombardment by a beam of accelerated electrons and because of electron beam
excitation the spatial resolution of CLM reaches 10 nm order.
Rare-earth doped nanophosphors emit light via both UV and electron beam
excitation. We prepared Eu doped nanophosphors and observed the phosphors
in HeLa cells with LM and CLM. For LM imaging 254 nm light line of mercury
lamp was used. The emission wavelength of the phosphors is around 614 nm,
therefore the effect of autofluorescence is negligible. The phosphors in the cells
were observed by CLM with high-spatial resolution after thin-sectioning of thecells. This technique is, so to say, correlative light and CL microscopy.
[1] B. N. G. Giepman et al., Nat. Meth, 2, 743 (2005). [2] C. V. Rijnsoever etal., Nat. Meth, 5, 973 (2008). [3] H. Niioka et al., APEX 4, 112402 (2011).

− S34 −

1H1610

精製タンパク質の構造から in situ の構造まで：電顕を使用してFrom purified protein structures to in situ structures using EM
Naoyuli Miyazaki1,2, Mineo Iseki3, Koji Hasegawa4, Akihiro Narita5, Shinichi
Adachi6, Masakatsu Watanabe7, Kenji Iwasaki1 (1Institute for Protein Research,
Osaka University, 2National Institute for Physiological Sciences, 3Pharmaceuticalsciences, Toho University, 4AdvanceSoft Corp., 5Structural Biology ResearchCenter, Nagoya University, 6Institute of Materials Structure Science, High Energy
Accelerator Research Organization (KEK), 7The Graduate School for theCreation of New Photonics Industries)
The fullness of electron microscopy (EM) imaging techniques is not comprised
only of single particle reconstruction methods for obtaining the molecular
structure of purified symmetrical proteins at a middle range resolution. For
instance, one additional EM imaging technique is CEMOVIS (Cryo-Electron
Microscopy of Vitreous Sections), which unlike other methods enables us tovisualize sections of hydrated cells or tissues with cryo-EM. Conventional
plastic sectioning requires chemical cross-linking, dehydration and resin
embedding. These processes undoubtedly denature molecules, and any unlinked
molecules are lost through the dehydration process. Finally, we observe
specimens stained with heavy metal for high contrast. Contrary to these, in thecase of CEMOVIS, a sample is frozen in a high-pressure freezing machine suchas the Leica EM PACT2 and directly cut into vitreous cryo-sections by cryomicrotome. In this way, we can also observe the phase contrast in vitrified
samples by cryo-EM, which conveys the signals coming from the molecules in
the proteins themselves as opposed to contrast attributed to the heavy metals inplastic sections. Here, we revealed the paracrystal structure of the photosensing organelle composed mainly of photoactivated adenylyl cyclase (PAC)in Euglena using CEMOVIS. We also succeeded in showing the tetrameric
structure of PAC using single particle reconstruction in addition to thehomology modeling of PAC. Merging these collective results, the arrangementof PAC in situ has been successfully shown.
1I1400Analyzing the conservation of the folding nucleuses among theFerredoxin-like fold in their amino acids sequence
Masanari Matsuoka, Takeshi Kikuchi (Dept. Bioinf., Coll. Life Sci.,
Ritsumeikan Univ.)
While there are many studies focused on how the proteins fold by means ofMolecular Dynamics or Go-like Model simulations, we may extract thisinformation regarding the folding processes only from their amino acids
sequences, because all information on the 3D structures should be coded inthem. We have been trying to extract this kind of information based on theamino acids sequences using inter-residue average distance statistics. In thisanalysis, we construct the predicted contact map (Average Distance Map) andanalyze them to extract possible structural domains or folding nucleuses. Our
previous results for four proteins of Ferredoxin-like fold showed good
agreements with experimental data. Thus, in this study, we extend this analysis
to their homologues and observe the relationship between the conservation of
the folding nucleuses and their sequence similarities. The detailed results will be
discussed at the following conference.

1I1412RNA とタンパク質の相互作用におけるタンパク質構造変化のデー
タベース解析

Database analysis of the degree of protein conformationalchanges in RNA-protein interactions
Chihiro Kubota1, Kei Yura1,2,3 (1Graduate School of Humanities and Sciences,
Ochanomizu University, 2Center for Informational Biology, Ochanomizu
University, 3Center of Simulation Sciences, Ochanomizu University)
Conformational changes in the interactions between proteins and othermolecules are one of the basic issues in predicting the docking structures.
Previously, we have developed a method to predict RNA interface residues onproteins with relatively high accuracy and started applying the method to build
structures of RNA-binding proteins with the target RNA molecule. To build an
accurate model structures, the knowledge for the effect of conformationalchanges in proteins is required. We, therefore, studied the degree ofconformational changes in RNA-binding proteins, when the proteins bind RNA
molecules. Three-dimensional structures of proteins determined with RNA
molecules were selected from Protein Data Bank (PDB). Proteins which havemore than 90% sequence identity to the RNA-binding proteins, and of which
structures were determined without an RNA molecule, were also selected. By

sorting out the selected protein structures, we made a list of 108 RNA-binding
proteins with RNA bound and RNA non-bound forms. Of them, 77% has
RMSD of alpha carbons no more than 2.0Å, when RNA bound and non-boundforms were superimposed. Protein pairs with the RMSD more than 2.0Å showed
an overall tendency to slightly shrink into a cleft for an RNA molecule without ahuge conformational change, when the protein binds to it. Except for the
intrinsically disordered regions, the conformational changes in proteins were
found to be slight. These findings suggest that conformational changes of theproteins should play a minor role in RNA-protein interactions.
1I1424

天然変性タンパク質のデータベース IDEAL の構築

Construction of IDEAL database for the investigation of
intrinsically disordered proteins
Takayuki Amemiya1, Shigetaka Sakamoto2, Yukiko Nobe1, Seiko D.
Murakami1, Kazuo Hosoda3, Ryotaro Koike1, Hidekazu Hiroaki4, Motonori
Ota1, Satoshi Fukuchi3 (1Grad. Schl of Info. Sci., 2HOLONICS Co., Ltd., 3Fac.
Engr., Maebashi Ins. Tech., 4Grad. Schl of Pharm. Sci., Nagoya Univ.)
Considerable regions of eukaryotic proteins are thought to be intrinsicallydisordered, which lack a globular three dimensional (3D) structure inphysiological conditions. The intrinsically disordered proteins (IDPs) play
important roles in signal transduction and transcriptional regulation. The
characteristics of IDPs are contrary to the traditional view of structural biology,
where a stable 3D structure is necessary for protein function. In the past 10
years, many studies of IDPs have been carried out to contribute the accumulated
experimental information. A well-designed database of IDPs is demanded forsummarizing this information. We have developed a new database, IDEAL(IDPs with Extensive Annotations and Literature, http://www.ideal.force.cs.is.
nagoya-u. ac. jp/IDEAL/), which collects experimentally verified intrinsicallydisordered regions (IDRs). Now, we are annotating mainly human nucleus
proteins. We defined the protean segments (ProSs) as coupled folding andbinding regions which are IDRs acting as recognition elements of their partner
proteins. We have provided 209 entries including 97 entries with ProSs. In thepresentation, we will demonstrate how to use IDEAL and present someexamples of collected IDPs.
1I1436

Computational analysis of protean segments (ProSs) inintrinsically disordered proteins (IDPs)
Divya Shaji1, Takayuki Amemiya1, Satoshi Fukuchi2, Motonori Ota1
(1Graduate school of information science,Nagoya University, Japan, 2Facultyof Engineering, Maebashi Institute of Technology, Japan)
Intrinsically disordered proteins (IDPs) are proteins that lack a stable, tertiary
structure. Many regions of disordered proteins undergo disorder to ordertransitions upon binding to their protein or nucleic acid partners, known ascoupled folding and binding mechanism. We call these regions Protean
segments (ProSs). In this study, we analyzed the amino acid composition of
ProSs. A non-redundant dataset of ProSs were extracted from IDEAL, adatabase with collection of experimentally verified intrinsically disordered
proteins. The amino acid composition of ProSs was compared to those ofordered and disordered datasets. We created these datasets based on SCOP(SCOP25). The results of this analysis demonstrate that ProSs are similar incomposition to disordered residues than to ordered residues. Next, we compared
the composition of ProSs with those of interior and surface residues. For thisanalysis, we extracted a dataset of non-redundant monomeric structures from
PDB. We calculated the relative accessible surface area (rASA) of residues byusing Naccess program and classified the residues with greater than 25% of
rASA as surface and the residues with less than 25% as interior. The results ofthis analysis indicate that ProSs are similar in composition to surface residues.
Based on these results, we will discuss the physicochemical properties of ProSs.
1I1448MODIC: a novel ab inito identification system of transcription
factor binding motifs in genome DNA sequences

Masaru Tateno1, Ryo Nakaki2, Jiyoung Kang2 (1Grad. Sch. Sci., Univ.
Hyogo, 2Grad. Sch. Pure and Appl. Sci., Univ. Tsukuba)
We developed a novel program, MODIC, to identify transcription factor binding
motifs (TFBMs) in genome DNA sequences, which involves a new algorithm
for identification of gaps in TFBMs. In MODIC, we defined a new information
entropy to conduct the statistical comparison of two types of frequency
distributions with respect to the TFBM candidates in the target DNA sequences
and the non-candidates in the background sequence, with the latter generated by− S35 −

utilizing the intergenic sequences. Our identification algorithm is based on the
maximum information entropy principle, employing the above-mentioned
information entropy. We tested our system employing several sets of ChIP-seq
data concerning mammalian genomes as well as yeast ChIP-chip data, and it
was revealed that the result of MODIC is the best among the available programs(MDscan, MEME, Weeder, DME, and BioProspector) [1]. Our system does notneed any other biological information except for gene positions, and does notneed tuning of the parameter set for each TFBM and pre-treatment/editing of thetarget DNA sequences. Thus, MODIC is also expected to be applicable togenome DNA sequences to accurately identify unknown TFBMs as well as
known ones.
[1] Nakaki, R., Kang, J., and Tateno, M., Nucleic Acids Research, in press.
1I1510Interaction of DNA molecules with nanopore studied byfluorescence microscopy
Genki Ando1, Kazuteru Yamada2, Toshiyuki Mitsui1,2 (1Grad. Mat.Sci., Univ.
Aoyama, 2Phys., Univ. Aoyama)
Silicon based solid state nanopore devises have shown the capability to monitor
the translocation of single DNA molecule and expected for future low-cost
DNA sequencing at a single molecule level. However, relatively long DNA
molecules, for example Lambda-phage DNA near 16.3 m in length can easily
clogatnanopore and end the translocation experiments because the clogging
DNA molecules at nanopore strongly interact with the other DNA's and cannot
be removed by reversing the polarity of external bias voltages across thenanopore membrane. We have studied these clogging events carefully by using
by fluorescence microscopy. We have found the chances for single DNA
clogging at pore under the various experimental conditions. We have also
investigated the possibility to remove the clogging DNA by pulsing electricfields at nanopore. Recent trends of nanopore related researches are thetranslocation experiments through electricallymodifiednanopores. We alsofabricated nanopores with gold gate and observed the effects of pulsing gate
voltages on clogging DNA molecules at pore. Finally, we will discuss themechanism of the DNA clogging and the clogging DNA removal from
nanopore.
1I1522

診断用 DNA 計算に向けた核酸応答型 DNA 合成システムの構築

Construction of a nucleic-acid-responsive DNA synthesis
system for diagnostic DNA-based computing
Ken Komiya, Asako Kobayashi, Masayuki Yamamura (Interdis. Grad. Sch. of
Sci. and Engi., Tokyo Tech.)
The ability of a single-stranded DNA (ssDNA) to bind specifically to its
complement makes DNA a useful switch for synthetic biomolecular systems,
consisting of various reactions triggered by DNA hybridization, and allows
cascading of such DNA-based reactions for the purpose of information
processing. We previously reported a DNA state machine that implements
successive state transitions via the cascaded DNA synthesis, according to acomputational program encoded with a single ssDNA. This DNA-based
computing system is highly versatile in principle, and thus, is expected to be
applied to computational, biological and medical tasks. However, for achieving
information processing on molecular distribution, including RNA transcription
pattern, simple cascading of DNA-based reactions is not enough. Additional
machinery to convert information of distribution into the form suitable forsequential processing of the DNA state machine is required.
In the present study, we propose a nucleic-acid-responsive DNA synthesis
system that generates distinct ssDNA species at sufficient intervals in response
to different nucleic acid species. Conversion of nucleic acids distribution is
implemented via toehold-mediated strand exchange reaction, followed by
ssDNA generation reaction utilizing a DNA polymerase and a nicking
endonuclease. The proposed system could be applied to diagnostic DNA-based
computing, where biomolecules autonomously determine the pathological stateof their surrounding environment based on distribution of nucleic acidbiomarkers.
1I1534

DNA scaffold logic: logic operation on molecular inputs using
FRET cascades
Takahiro Nishimura1, Yusuke Ogura1, Hirotsugu Yamamoto2, Kenji
Yamada3, Jun Tanida3 (1Osaka University, 2The University ofTokushima, 3Osaka University)

useful in biological applications. To construct scalable DNA logic circuits withsimple DNA reactions, we propose an effective method called DNA scaffold
logic. In the DNA scaffold logic, a logic operation on molecular inputs isachieved by formation of FRET paths on a DNA scaffold and the result isobtained as the cascaded FRET signal. FRET signaling contributes to simplify
the DNA reactions for logic operations, to concentrate the signal path into a
nanometer region, and to accelerate the response time of molecule-based logic
circuits. The FRET signaling path is regulated according to the input molecules.
If a set of input molecules satisfies the given logic condition, the FRET path isformed and the fluorescence molecule emitting the output signal is excited.
Experimentally, we demonstrated a complete set of Boolean logic functions(AND, OR, NOT) and logic expressions represented by several Booleanfunctions by using multi-step FRET. In numerical simulation, we confirmed that
additional time for increasing extra layers approaches asymptotically to zero.
This result suggests that the method is useful for construction of multi-layered
circuits with fast response.
1I1546

寄生虫による接着性宿主細胞への侵入過程観察のためのマイクロ加
工デバイスの作製

Observation of parasite invasion with the micro-fabricateddeviceTetsuhiko Teshima1, Hiroaki Onoe1, Kaori Kuribayashi-Shigetomi1, Hiroka
Aonuma2, Hirotaka Kanuka2, Shoji Takeuchi1 (1The Institue of Industrial
Science, 2Jikei University School of Medicine)
Cytological studies for infectious microorganism have garnered much attention
for detailed analysis of their behavior and function. Especially, it is important toobserve the invasion of obligate intracellular pathogens, which gives us muchinformation about morphological changes and expressed protein. With
conventional methods, however, it is difficult to capture pathogen invasion into
adherent cells and to confirm whether parasites are located inside cells or not. In
this study, we propose microflap system to realize multiple-angle observation ofadherent cells in order to detect boundary membrane of parasites and host cells.
Fabricated devices are composed of alginate hydrogel, Parylene, Chromium andPermalloy layer. After removing the alginate layer, the microflaps were released
from glass substrates and responded to magnetic field. Permalloy layer
generates lifting torque, so that inclination angles increased with increasing
external magnetic fields. As a model of microbial infection, we used a parasite,
Toxoplasma gondii, and its host HFF cells. During changing the inclination,
cells remained attached to the surface of microflaps and cellular membrane
could be clearly observed. Parasites approached to HFF cells and microflaps
were inclined in the desired orientation. We could optimize the angle andobserve time-scale invasion. Thus, we clearly observed how parasites invaded
through membrane and judged completed infection. This method is widely
applicable to the analysis of interaction between infectious microbes and hostcells.
1I1558

金属還元菌 Shewanella における微生物呼吸代謝の電気化学的制御

Electrochemical regulation of bacterial respiratory activity of
Shewanella, a dissimilatory metal reducing bacteria
Shoichi Matsuda1, Huan Liu3, Shuji Nakanishi2, Kazuhito Hashimoto1,2,3(1Grad. Sch. Engr., Univ. Tokyo, 2RCAST, Univ. Tokyo, 3ERATO/JST)
Dissimilatory metal reducing bacteria, Shewanella is well known to express ctype cytochromes abundantly on the outer membrane. The bacteria facilitatesefficient electron transfer through the outer membrane cytochromes(OMCs) byextracellular electron transfer mechanism. Interestingly, they can utilize solidstate metal oxides as sole electron acceptor for the respiration. The bacterial
respiratory activity can be investigated electrochemically by providing anelectrode of an appropriate potential as the sole electron acceptor, which allows
the capture of respiratory electrons and reversible tuning of the electron energy
level of the solid-state electron acceptor. We investigated the electrode potential
dependence of the microbial respiratory activity with/without malonic acid, achemical inhibitor for TCA cycle. At the negative potential region where OMCs
exist as the reduced state, microbial current was drastically decreased by theaddition of the TCA cycle inhibitor. In contrast, at the positive potential region
where OMCs exist as the oxidized state, the addition of the inhibitor had noeffect on the microbial current. These results indicated that TCA cycle activityof Shewanella changed depending on the redox state of OMCs. Thus, it was
revealed that TCA cycle activity of Shewanella can be electrochemically gated.

DNA logic circuits handling molecular information based on DNA reactions are− S36 −

1I1610

Spatiotemporal control of cell motility in nano- to micro-scale
topographical environment
Hiromi Miyoshi1, Hiroyoshi Aoki1, Jungmyoung Ju1, Sang Min Lee2, Jong
Soo Ko2, Yutaka Yamagata1 (1ASI, RIKEN, 2Grad. Sch. Mech. Eng., Pusan
National Univ.)
The extracellular matrix (ECM) topography is known to affect various cellfunctions, such as cell motility and proliferation. The ECM consists ofhierarchical organization over different length scales from nanometers tohundreds of micrometers. However, in most of the previous studies, the effectsof the ECM topography on the cell functions have been analyzed only focusing
on a specific scale in each study. Our aim here is to clarify how cells sense thehierarchical inputs from the ECM topography and determine motile behavior bya systematic analysis using micro/nano-topographical extracellularenvironments. Silicon micropillar array and polyacrylonitrile nanofibrous
structures were used as the extracellular environments. Fibroblasts having
bundled actin filaments and discrete cell-substrate adhesions, and fish epidermal
keratocytes having actin meshwork and continuous adhesions were used for the
analysis. In the micropillar array, cells had a tendency to wrap the pillars.
Keratocytes extended very few protrusion, whereas, fibroblasts formed lamellar
and filopodial protrusions into the gaps between the pillars. In the nanofibrous
structures, both fibroblasts and keratocytes extended protrusions along thenanofibers. We assumed that the cell motile behavior depending on the scale ofthe extracellular environments will comes from the dynamic flexibility of theactin cytoskeleton, including actin network bending, actin reorganization, andthe dynamic connection between the actin network and adhesion complex.

− S37 −

2A1400

クライオ電子線トモグラフィーを用いた細胞中骨格タンパク質およびモータータンパク質の非侵襲的構造解析

Observation of intracellular cytoskeleton and motor protein in
the non-invasive cells with electron cryo-tomography
Shinji Aramaki1, Kazuhiro Aoyama2,3, Kota Mayanagi4, Takuo Yasunaga1(1Dept. of Bioscience and Bioinformatics, Grad. School of Computer Sci. andSys. Eng., Kyushu Inst. of Tech., 2FEI Company Japan Ltd., Application
Lab., 3Grad. School of Frontier Biosciences, Osaka Univ., 4Medical Inst. of
Bioregulation, Kyushu Univ.)
Most life is composed of protein, and water, and their localizations change
depending on functions, types, and environments. Usually proteins are producednear nucleus. However, their localization is very highly regulated. It is realized
by not only diffusion which is passive transportation but also active
transportation. Actin, and microtubules called cytoskeleton, and myosin,kinesin, and dynein called motor proteins are known as important players of theintracellular transportation.
In these days motility of motor proteins and interaction between motor proteins,and cytoskeleton proteins have been revealed by in vivo single-molecule
analysis. Moreover actin filament and motor domain of cytoplasmic dynein are
difficult to structural analysis due to large their molecular complex size and sotheir structural understandings has not suggested in vivo interaction between
motor proteins and cytoskeletons. Hence we set out to reveal structure of
intracellular transportation.
We observed ultrastructure of the intracellular architecture by electron cryotomography, which can realize observation of intact cell as three-dimensional
objects, because cells are vitrified by quickly freezing by liquid ethane, and sosamples are not affected by chemical regents. Hence, it would give us intracellular
structural images without any artifacts. This powerful technique will give us amajor breakthrough of elucidating intracellular protein-protein interactions.
2A1412クライオ電子線トモグラフィーを用いたクラミドモナス軸糸外腕ダ
イニンにおけるメタロチオネイン標識 LC2 の検出

Detection of metallothionein-tagged LC2 on chlamydomonas
axonemal ODA by electron cryo-tomography
Mingyue Jin1,4, Haru-aki Yanagisawa2, Kotaro Koyasako1,4, Ritsu Kamiya2,
Kota Mayanagi3,4, Takuo Yasunaga1,4 (1Dep. Biosci. Bioinf., Inst. Tech.
Kyushu, 2Dep. Biol. Sci., Grad. Sch. Sci., Univ. Tokyo, 3Div. Struct. Biol., Med.
Inst. Bioreg., Univ. Kyushu, 4JST)
Axonemal structures including dynein molecules have been reported atrelatively high resolution. However, due to the limitation of cryo-EM,
individual dynein subunits could not discriminate in all reported axonemal outerdynein arm (ODA) structures. Thus, our group has introduced a genetic tag ofmurine metallothionein (mMT2), a heavy metal binding protein. To improve theexpression level, mMT2 was exchanged to C. reinhardtii codon usage andreborn as CrMT2. We fused CrMT2 to ODA subunits, light chain 2 (LC2),
which is known to be an essential element for ODA's assembly on the axoneme.
In this study, we successfully expressed CrMT2-tagged LC2 to rescue ODAs on
the axoneme. LC2-2xCrMT2-expressed or wild type axonemes were treatedwith CdCl2 and their 3D structures were obtained by cryo-ET. Unlike wild type,linearly aligned punctate high densities were found in LC2-2xCrMT2transformed axonemes alone. In addition, these high densities have a 24-nm
periodicity identical to that of wild type ODA molecules on the axoneme. We
concluded that individual punctate high densities identified on the CrMT2tagged axoneme must be from Cd-CrMT2. Furthermore, the Cd-CrMT2
location on the ODA was measured from three orientations. Based on these data,
we decided precise LC2 localization in the axonemal ODA. This is the first time
to visualize a genetic heavy metal labels on the axoneme by cryo-ET. Taken
together, the CrMT2 adapted to C. reinhardtii will be a promising method todetect subunits of huge protein complex in situ.
2A1424

クライオ電子顕微鏡法とメタロチオネインラベルを用いた軸糸構造の解明

Elucidation of Axoneme architecture by cryo-electron
microscopy and metallothionein labeling
Reiko Chijimatsu1, Mingyue Jin1,2, Takuo Yasunaga1,2 (1Kyushu Institute ofTechnology, 2JST)
Motile cilia/flagella are driven by dynein motor proteins that form the inner andouter rows of arms attached to the outer doublet microtubules. Chlamydomonas
outer arm dynein has three heavy chain motor units (α, , γ). Previous works
reported that γ heavy chain contains LC4 like calmodulin, but its detailed
location are unknown. Thus we used cryo-EM and single particle analysis to
identify LC4’s detailed location.
The Chlamydomonas strains used in this study are wild type and F11 whose
LC4 is fused to Mt (heavy metal-binding protein). Cells were cultured in TAP
medium with a light/dark cycle of 12h before crude dynein was extracted from
cells. Crude dynein was set on ice with 10M Cd2+ overnight and dialyzed in
two types of ionic strength (0.6M KCl, 50mM K-acetate) before quick freezing
and observations. We also prepared crude dynein from F11 and confirmed thatit had Mt-fused one, by electrophoresis. We observed two samples, crude
dynein prepared from wild-type and F11 and compared them. As a result, when
we observed crude dynein extract prepared from F11, high density wasobserved near dynein heavy chain-like particles. We thus conclude that this highdensity is Mt binding Cd and the position of LC4 should be determined.
Interestingly, in only lower ionic strength (50mM K-acetate), the filamentous
protein on 24nm cycle was observed. Due to the periodicity, this protein can beimportant for axoneme assembly. We’ll identify the location of LC4 precisely
and what is the filamentous protein above-mentioned.
2A1436

電子線トモグラフィー法により明らかになった、LIS1 による細胞質ダイニンの運動活性制御メカニズム

Regulation of cytoplasmic dynein-motility on microtubules by
LIS1 revealed by electron-computed tomography
Shiori Toba1, Kotaro Koyasako2,3, Shinji Hirotsune1, Takuo Yasunaga2,3,4(1Dept. Genetic Disease Research, Osaka City Univ. Grad. Sch. ofMedicine, 2Dept. Bioscience & Bioinformatics, Faculty of Computer Scienceand Systems Engineering, Kyushu Inst. Tech., 3JST-SENTAN, 4JST-CREST)
Cytoplasmic dynein is a microtubule-based and minus end-directed motor
protein. Striking feature of the dynein is that its carries out diverse transport
activities through interactions with the numerous accessory molecules. LIS1 is
one of those dynein-associated proteins, and its regulatory role on the dynein
has been intensively investigated. Although the dynein is critically regulated by
LIS1, the underlying mechanism remains largely unknown.
Here, we have uncovered three dimensional structure of the dynein bound to
microtubules and nucleotide-induced structural changes by electron-computed
tomography (ECT) analysis. The typical features of the dynein motor domainsuch as a ring-like structure were successfully visualized from ECT of single
particle. ECT allows us to interpret the flexible and huge structure of the dyneinmicrotubule complex reflecting working conditions in cells.
Furthermore, we disclosed the orientation of the two motor domains of thedynein and the underlying molecular mechanism by which LIS1 regulates
dynein motility. Surprisingly, LIS1 handcuffs the structure of dynein in a nonucleotide state regardless of nucleotide conditions through its simultaneousbinding to two motor domains. These structural characterizations were further
confirmed by cryo-electron microscopy coupled with hierarchical cluster
analysis and terminal specific gold particle-labeling of LIS1. We propose thatthe dynein-LIS1 complex is used as an immotile fastener of microtubules, whichis essential for dynein-dependent cellular function.
2A1448ヌクレオチド状態に依存したダイニン分子の形態変化

Nucleotide-dependent morphological change of cytoplasmic
dyneinMuneyoshi Ichikawa, Kei Saito, Takayuki Torisawa, Keitaro Shibata, Yuta
Watanabe, Tomonori Hata, Yoko Toyoshima (Grad. Sch. of Arts & Sci., Univ. ofTokyo)
Cytoplasmic dynein moves on microtubules toward the minus-end by the ATPhydrolysis-driven structural change and is involved in diverse cellular activities,
including cell division, cargo transport, and neural development. Cytoplasmic
dynein is a homodimer of two heavy chains and forms a two-headed structure.
The structure of head domain of cytoplasmic dynein has been extensively
studied. Previous electron microscopy and FRET analysis have revealed thestructural change of the linker orientation relative to the AAA ring depending on
the nucleotide state. However, the structural basis of how dynein molecule uses
two heads during the walk is still unveiled.
Here, we examined the structural change of full length cytoplasmic dynein in anelectron microscopy. Negatively stained porcine brain dynein molecules
appeared in both open (Y-shaped) and closed (φ -shaped) structures aspreviously reported (Amos 1989). The two heads were separated in Y-shaped− S38 −

structure, and the two heads were stacked together in φ-shaped structure. In thepresence of AMPPNP, Y-shaped dynein was predominant (Y-shaped, 62.7% /φ -shaped, 24. 6%). In contrast, in an ADP-Vi condition, which mimics the
ADP-Pi state of the catalytic cycle, dynein molecule tended to form aclosed conformation (Y-shaped, 26.0% / φ-shaped, 53.4%). We constructed
His-tagged recombinant human dynein heavy chains and performed a sitespecific Ni-NTA-gold labeling. Combined with single particle analysis, theorientation of the two heads in φ-shaped structure will be discussed.
2A1510

頭部間の直接的相互作用は細胞質ダイニンの連続的な運動に必要ない

A direct interaction between dynein two heads is not necessary
for processive movements of dynein
Keitaro Shibata, Yui Utsumi, Takayuki Torisawa, Yoko Toyoshima (Dept.
Life Sci., Grad. Sch. Arts and Sci., Univ. Tokyo)
Cytoplasmic dynein is a two-headed motor protein which moves toward theminus end of microtubules. Recently, the crystal structure of dynein motor
domain has been reported and understanding of the motor machinery has
progressed. However, how dynein uses two heads during its processive
movement on microtubules is not clear yet.
Previously, we reported that dynein moved on zinc-induced tubulin polymers(zinc-sheets) which include only one protofilament to be utilized by dynein
movement, suggesting that dynein moves on zinc-sheets by inchworm
mechanism.
Here, to elucidate whether a direct interaction between the two heads isnecessary for its processive movement, we prepared an artificial dimer: thedynein motor domains are dimerized by an anti-parallel actinin rod known tohave a high persistence length. EM images of this actinin-dimers revealed thatthe two heads keep a distance of ~25 nm by the rigid rod. A single molecules of
the actinin-dimer moved both on microtubules and on zinc-sheets processively.
Our results suggest that a direct interaction between dynein two heads is notnecessary for the processive movement of dynein.
2A1522

外部負荷存在下でのヒト細胞質ダイニンのメカノケミカルサイクル

The mechanochemical cycle of human cytoplasmic dynein
under external force
Taketoshi Kambara1, Yoshiaki Tani1, Motoshi Kaya1, Tomohiro Shima2,
Hideo Higuchi1 (1Grad. Sch. Sci., Univ. Tokyo, 2QBiC, Riken)
Dynein is a molecular motor that moves toward the minus-end of microtubules.
Cytoplasmic dynein play roles in positioning the Golgi complex and otherorganelles in cells, movement of chromosomes, and positioning the mitotic
spindles during mitosis. Here, we used optical trapping to show the single
molecule properties and the effect of load on the mechanochemical cycle ofhuman cytoplasmic dynein. We expressed truncated C-terminal motor domainof dynein using baculovirus expression system. The double-headed motor
domain was responsible for producing a high force of ~6 pN with a predominant
step size of 8 nm. An unbinding force of single-headed dynein was measured invarious nucleotide conditions. Dynein with the biotin-tag was attached toavidin-coated polystyrene beads, and the bead was trapped by optical tweezers.
The external load was imposed by moving the stage. An unbinding force
measurement indicates that dynein-microtubule binding is weak for the
ADP/vanadate state and strong for the nucleotide-free, AMPPNP and ADP
states. The unbinding force was weaker when dynein was pushed toward theminus end of microtubule in the strongly bound state, while it was independentof loading direction in the weakly bound state. Analysis with a model revealed
that the dissociation rate of dynein from microtubules increases as the force
increases. Our results suggest that force plays an important role in themechanochemical cycle of dynein to ensure the increasing of a probability forrear-head detachment with strain.
2A1534

Rotation of microtubules driven by Tetrahymena 22S outerarm dynein and its sub-particles
Shin Yamaguchi, Yoko Toyoshima, Junichiro Yajima (Dept Life Sciences,
Graduate School of Arts and Sciences, Univ. of Tokyo.)
Axonemal dynein is the molecular motor that provides motive force in cilia.
Tetrahymena 22S outer arm dynein, which is localized between the outerdoublet microtubules (MTs), consists of three different heavy chains, termed α, and γ, and is decomposed into two-headed γ sub-particle and one-headed α
sub-particle by chymotryptic digestion. Previous study showed that these subparticles had motility, yet it has not been clear how the three-headed structure of

22S dynein affected its force generation. To decipher the contribution of eachheads to the 22S dynein activity, we examined the motility of the 22S dynein
and its sub-particles, using three-dimensional tracking of a quantum dot
attached to MTs in an in vitro motility assay. As a result, we found that surfaceattached 22S dynein and its sub-particles drove not only on-axis MT sliding, butalso clockwise MT rotation around its axis. We estimated rotational pitch byfitting the xy-position of a quantum dot with a sine function. The pitch of 22S
dynein and γ sub-particle had different ATP dependency, whereas the pitch of
α sub-particle was constant. At 10 M ATP, each dyneins showed almost same
pitch. These corkscrewing motions of MTs have not been seen for both 22S
dynein and its sub-particles, and it demonstrates that these dyneins produce
torsional forces as well as axial sliding forces. Our results suggest that torquegeneration by the three-headed 22S dynein is not due to the cooperation between
each of the three heads, but due to the intrinsic property of individual heads.
2A1546哺乳類細胞質ダイニンが一方向性を獲得する機構について

The mechanism of the transition from diffusion to directedmovement in mammalian cytoplasmic dynein
Takayuki Torisawa1, Furuta Ken'ya2, Muneyoshi Ichikawa1, Yoko
Toyoshima1 (1Dept Life Sciences, Graduate School of Arts and Sciences, theUniv of Tokyo, 2Bio ICT lab, NICT)
Cytoplasmic dynein is a minus-end-directed molecular motor involving invarious cellular functions. Mammalian cytoplasmic dynein has been reported to
exhibit unidirectional movements for several micrometers in vivo (Kobayashi,2009). However, some in vitro studies have reported that single molecules ofcytoplasmic dynein/dynactin complex showed diffusive movements along
microtubules (Ross, 2006; Miura, 2010), and a recent study has demonstrated
that an mRNP complex bound by a few dyneins displayed diffusive movement(Amrute-Nayak, 2012). To reveal the mechanism of the transition fromdiffusion to directed movement in cytoplasmic dynein, we designed
recombinant dyneins using HEK293 cell expressing system. An artificiallydimerized, tail-truncated human cytoplasmic dynein 1 (GST+380 kD) showed
unidirectional movement, whereas full-length dyneins purified from the same
system diffused along microtubules. Furthermore, using several otherrecombinant dyneins that have different head-to-head distances between two
motor head, we discovered that the dynein with longer head-to-head distancecontained larger diffusive component. We also observed that multiple fulllength dyneins bound to Qdot moved unidirectionally along microtubules, while
single dyneins carrying Qdot exhibited diffusive movement. These observations
imply that the directed movement of dynein is influenced strongly by the headto-head distances and the motor number, leading to the possibility of dynein
regulation by these factors in the cell.
2A1558

骨格筋形成過程における細胞質ダイニンの分布変化

The dynamic change of the distribution of cytoplasmic dynein
during skeletal muscle differentiation process
Takuya Kobayashi, Motoshi Kaya, Hideo Higuchi (Department of physics,
Graduate school of science, The university of Tokyo)
Skeletal muscles are thought to be rigid structure, but it must be always
maintained. In non-muscle cells, cytoplasmic dynein is bearing important roles
for intracellular maintenance. However, the roles of dynein are not understoodin skeletal muscles. In order to understand the role of cytoplasmic dynein, wedeveloped the in vivo fluorescence imaging of mouse skeletal muscle whichmolecular structures are visualized by using the gene transfer of GFP-fusedproteins. Microtubules and cytoplasmic dyneins in in vivo muscles weresuccessfully visualized by expressing GFP-Tubulin and dynein IC74-GFP with
confocal microscope. The distribution and dynamics of vesicles transported bycytoplasmic dyneins in living muscle differed from non-muscle cells, and thesevesicles did not move along the microtubule in living muscle. Meanwhile,
dynein IC74-GFP were stably expressed in the myoblast cell line C2C12 and,vesicle transports were observed in myoblast cell and myotube. The dyneinbinding vesicles in the myoblast stage distributed as similar as in non-muscle
cells. However, in myotube, the vesicles mainly moved along longitudinal axis
and the speed of the vesicles was decreased to 20% of myoblast stage. Thus, thedistribution and dynamics of vesicle transport by cytoplasmic dyneinsremarkably changed during the muscle differentiate process.
2A1610− S39 −

ダイニンアダプター Bicaudal-D2 の細胞周期依存的核膜局在の分子機構

Molecular mechanism of cell-cycle dependent nuclear envelope
localization of Bicaudal-D2
Takashi Murayama, Hiroki Ota, Takashi Sakurai (Dept. Pharmacol.,Juntendo Univ. Sch. Med.)
Bicaudal-D is an evolutionarily conserved dynein adaptor and involved in Rab6dependent cargo transport. In mammals, there are two subtypes of Bicaudal-D, i.
e., BICD1 and BICD2. Recent study has shown that BICD2, but not BICD1, islocalized to the nuclear envelope (NE) in a G2-specific manner via interaction
with RanBP2, a protein of nuclear pore complexes, and may play an importantrole in nuclear positioning during mitotic entry. However, it remains unclear (1)how G2-specific NE localization of BICD2 is regulated and (2) why BICD1 is
NOT localized to NE. To address these questions, we expressed various
truncated and chimeric BICD1/BICD2 mutants and tested their NE localizationin HeLa cells. Bicaudal-D has three coiled-coil domains (CC1-CC3) and linkers(L1 and L2) between them. BICD2 is reported to interact with RanBP2 at the Cterminal CC3 domain. Consistent with the previous study, full-length BICD2
was localized to NE only in G2 phase (cyclin B1 positive), whereas full-length
BICD1 was not localized to NE throughout the cell cycle. BICD2 lacking the Nterminal CC1 domain showed NE localization in both G2 and non-G2 (cyclin
B1 negative) phases. Surprisingly, BICD1 lacking CC1 was also localized to NE
in both G2 and non-G2 phases. The BICD1 chimeric mutant having CC1 and L1of BICD2 showed G2-specific NE localization. These findings suggest that CC1functions as inhibitory domain for NE localization and CC1 and L1 domains
confer G2-specific disinhibiton of CC1 in BICD2.
2B1400

単一アミノ酸ポテンシャル力場の改良と短鎖ペプチドの構造予測へ

to Vanden-Eijnden and Heynman (J. Chem. Phys. 128, 2008, 061103). To
facilitate path sampling, we further combine the OM method with our replica
exchange strategy (Fujisaki, Shiga, Kidera, in preparation), and analyze theresulting huge path space in terms of transition rates and transition state
ensembles.
2B1424

蛋白質系のシミュレーションの緩和モード解析

Relaxation mode analysis for simulations of protein systems
Ayori Mitsutake, Toshiki Nagai, Hiroshi Takano (Keio Univ.)
Classical molecular dynamics simulations are a popular method for describing
the structure, dynamics, and function of biomolecules in microscopic detail. As
longer and larger MD simulations are performed, it is more important to develop
methods to extract the “essential”modes from the trajectory. Relaxation mode
analysis (RMA) has been developed to investigate “dynamic” properties ofhomopolymer systems while principal component analysis (PCA) is one of the
popular methods for analyzing the static properties of fluctuations of structures.
In the case of liner polymers, the relaxation phenomena have been studied
systematically in terms of the relaxation modes and rates by using RMA. We
have applied RMA to a biomolecular system (J. Chem. Phys. 135, 164102(2011)). As a biomolecular system, we choose the system of Met-enkephalin ingas phase because it is known to have several meta-stable or stableconformations and is therefore suited for the verification of the validity of
RMA. We perform a Monte Carlo simulation of the system and apply PCA and
RMA. Here, we show the effectiveness of RMA by comparing these results with
RMA and PCA.

の応用
2B1436

Improvement of the Single Amino Acid Potential Force Field
and the Application to Structure Prediction of Short Peptides
Michio Iwaoka, Kenichi Dedachi, Taku Shimosato, Toshiya Minezaki (Tokai
Univ)

A multi-body potential for normal mode analysis of protein
structure
Bhaskar Dasgupta, Narutoshi Kamiya, Haruki Nakamura, Akira Kinjo(Institute for Protein Research, Osaka University)

The Ramachandran-type potential energy surfaces calculated for single aminoacid units in water are in good agreement with the statistical structures of thecorresponding amino acid residues in proteins. Based on this interesting
property of proteins, we recently developed a new all-atom force field called theSingle Amino Acid Potential force field (SAAPFF) for molecular simulation for
polypeptide molecules. In this paper, we report on improvement of the SAAPFF
and the application to structure prediction of short peptides. The classicalcanonical Monte Carlo simulation was carried out by using the improved
SAAPFF for Met-enkephalin, a short peptide with five amino acid residues, andchignolin, a small protein with ten amino acid residues. Diverse structures wereobtained for Met-enkephalin at 300 K in water, while three representative folded
structures, one corresponding to the native-like structure with three nativehydrogen bonds, were obtained for chignolin. The results were compared to themolecular dynamics simulations using a conventional AMBER force field with
the Generalized Born solvent model. The SAAPFF was found to be useful for
conformational sampling for short peptides. In addition, a protocol of the
SAAP-MC simulation followed by structural clustering and ab initio calculation
was suggested to be an effective strategy toward prediction of the native folded
structure.

To describe native protein dynamics normal mode analysis (NMA) of an elastic
network model (ENM) is widely accepted. In ENM, the energy of a native
protein is modeled as a sum of energy of all pairs of atoms, each connected by a
Hookean spring. However, in this model protein-solvent interactions are
ignored. Therefore, solvent-exposed atoms sometimes exhibit unrealisticallyhigh fluctuations. To model the effect of solvent on protein here we propose
differential contact number model (DCNM), which is based on the physicalintuition of phase separation between hydrophobic and hydrophilic atoms. The
hydrophobicity of an atom is defined in terms of contact number of an atom. In
the objective energy function the difference in the contact numbers ofneighboring atoms are restrained to their native value. To evaluate the DCNM
against the ENM, we compared the dynamics of 19 pairs of proteins, each ofwhich shows a conformational change between open and closed forms. We
observed that the DCNM yields high fluctuation for a less number of atoms thanthe ENM. From the first 30 modes we observed that the DCNM exhibited
greater overlap with the conformational change than the ENM and on average
the modes obtained from the DCNM are more collective than the ENM.

2B1412

オンサーガー・マハラップ作用を用いたペプチド系のパスサンプリ
ングPath sampling for small peptide systems using the OnsagerMachlup action method
Hiroshi Fujisaki1,2, Yasuhiro Matsunaga2, Akinori Kidera2,3 (1Nippon
Medical School, 2RIKEN, 3Yokohama City University)
Proteins often accompany conformational changes when they function in cells,
and its characterization is one of the most important subjects in biophysics. The
ability of theoretical and computational methods based on molecular dynamics
simulation has been increasing mainly due to the advance of hardware, it is,however, still not feasible to simulate and clarify a biological role ofconformational changes of large proteins. To address this time-scale problem
from a different point of view, we proposed to use a statistical approach basedon the Onsager-Machlup (OM) action (J. Chem. Phys. 132, 2010, 134101),
where a path, not a configuration, is the most fundamental object to be studied,and its distribution is described by a path-integral representation using the OM
action. In the present study, we apply this formalism to small peptide systems,
including alanine dipeptide, chignolin, and WW domain, in implicit solvent
where a hessian matrix is implicitly evaluated using the numerical scheme due

2B1448アクチン線維へのコフィリンの協同的結合の統計力学モデルの作成

A theoretical model of cooperative binding of cofilin to actin
filaments
Shotaro Sakakibara2, Kimihide Hayakawa1, Hitoshi Tatsumi2, Masahiro
Sokabe1,2 (1FIRST Research Center for Innovative Nanobiodevice, Univ.
Nagoya, 2Grad. Sch. Med., Univ. Nagoya)Actin dynamics is regulated by a large number of actin binding proteins. Cofilin,
one of these actin binding proteins, severs actin filaments and presumably playsa central role in the regulation of the actin dynamics. Biochemical and electron
microscopic studies suggest that cofilin binds to the actin filament in acooperative manner, and electron microscopic analyses have revealed that thetwist of the actin filament is increased when the filament is fully decorated bycofilin. Here, we propose a simple theoretical model inspired from the Isingmodel in statistical physics. The model assumes that the actin filament is anarray of actin protomers, and that each protomer has only two states “0” or “1”,where “0” denotes the non-twisted state and “1” the twisted-state. The model
also assumes that binding of a cofilin to an actin protomer (i th) induces changes
in the state (from 0 to 1) in the neighboring actin protomers (i-1 th and i+1th),
resulting in an acceleration of cofilin binding to these protomers. By fitting thetheoretical curve calculated with this model to the experimentally obtained
relationship (percentage of cofilin binding protomers in actin filaments vs.

− S40 −

cofilin concentration), we estimated the binding energy of cofilin to actin
filaments at ca -7 kT and the energy required for twisting the neighboring
protomer at ca 3.5 kT.
2B1510

リゾチーム分子中のアミロド性ぺプチド

Amyloidogenic Peptides from Hen Lysozyme
Hideki Tachibana1,3, Masao Fujisawa1,3, Ryohei Kono2,3, Minoru Kato4 (1Sch
Biol-Oriented Sci Tech, Kinki Univ, 2Wakayama Med Univ, 3High Press Prot
Res Center, Kinki Univ, 4Coll Pharm Sci, Ritsumeikan Univ)
Amyloid protofibril-like assembly spontaneously forms from hen lysozyme
disulfide-deficient variant protein termed 0SS. Four peptide regions protectedfrom the access of solvent in the 0SS fibrillar state have been identified with
NMR-detected hydrogen/deuterium exchange measurements. These regions
have high predicted beta-aggregation propensity, and most probably constitutethe core of 0SS fibril. Here, the fibrillation of one of these peptides,Y52-R61,
was investigated in detail. When monitored with thioflavine T fluorescence theY52-R61 peptide facilely form amlyoid fibrils under mildly acidic solution
conditions, but not under neutral or alkaline conditions, which indicates that thepresence of small amount of excess positive charge facilitates amyloidfibrillation of this peptide. Atomic force microscopy observation showed that
the Y52-R61 peptide fibrils were thin at the initial stage of fibrillation whenobtained from dilute peptide solutions, and grew thick with prolonged
incubation period by bundle formation. MMPBSA calculation on two moleculesof the Y52-R61 peptides showed that an anti-parallel beta structure is morestable than a parallel one by about 40 kJ, which agrees with our previous finding
with IR measurement that the anti-parallel beta structure predominantly exists in
the 0SS amyloid-like fibrils.
2B1522

Effects of raft components on the membrane-mediatedaggregation of IAPP
Kenji Sasahara (Kobe University)
Human islet amyloid polypeptide (IAPP) is a 37-resudue peptide which is the
major constituent of amyloid fibrils deposited in the pancreatic islets with type
II diabetes mellitus (T2DM). IAPP is synthesized in pancreatic beta-cells andco-secreted with insulin. Numerous studies have shown that the interactions
between IAPP and lipid membranes play an important role in the pathogenesisof T2DM, in which the lipid membranes of beta-cell are the target of IAPP
toxicity. Despite recent efforts toward biophysical characterization of the IAPPlipid interactions, the effects of IAPP aggregation on the physiologicalproperties and organization of lipid membranes have not been wellcharacterized, which is of critical importance in further understanding of thepathogenesis of T2DM. In the present study, amyloid aggregation of IAPP was
induced on the supported lipid bilayers (DOPC/DOPS/raft components) toinvestigate the effects of raft components on its aggregate formation and theassociated membrane damage. Microscopic observations indicated that IAPP
aggregation is significantly accelerated on the membrane including the raftcomponents, and that the aggregates are subsequently converted to the needlelike fibrillar aggregates during the prolonged incubation, which is promoted forthe rafts-containing membranes. Furthermore, the conversion of IAPP tofibrillar forms was microscopically found associated with the membrane
damage.

2B1534タンパク質が生体内レドックス環境に依存して異なる凝集体を形成するメカニズムとその病理学的意義

Redox environment is an intracellular factor to operatedistinct pathways for protein aggregation
Yoshiaki Furukawa (Dept. of Chemistry, Keio Univ.)
Redox control of the thiol-disulfide status is critical for functioning of many
proteins, and Cu,Zn-superoxide dismutase (SOD1) is one of such proteins inwhich formation of an intramolecular disulfide bond is required for folding into
its native structure. Indeed, abnormal reduction of a disulfide bond in SOD1
triggers the formation of insoluble aggregates in vitro. In addition, aggregationof mutant SOD1 proteins is a pathological hallmark in a familial form of
amyotrophic lateral sclerosis (fALS); therefore, a thiol-disulfide status of SOD1
should be correctly regulated inside cells. Given that SOD1 exists in various
intracellular organelles, however, it remains obscure how their distinct redox
environment affects misfolding pathways of SOD1. To investigate effects of
intracellular redox environment on SOD1 misfolding, fALS-mutant SOD1s
were expressed in E. coli, BL21 and SHuffleTM. BL21 offers strongly reducing

environment of the cytoplasm, while the cytoplasm in SHuffleTM is moreoxidizing by deleting cytoplasmic reductases keeping cysteines in their thiolstate. In both cells, fALS-mutant SOD1s were obtained as insoluble pellets butwith distinct properties; a disulfide bond of SOD1 was either completely
reduced in BL21 or abnormally formed between SOD1 molecules in
SHuffleTM. Depending upon the intracellular redox environment where SOD1
resides, therefore, fALS-mutant SOD1 is considered to be misfolded into
aggregates with distinct properties, which would be relevant in the pathologicalheterogeneity in fALS cases.
2B1546

銅シャペロンに依存しない SOD1 酵素の活性化制御メカニズム

A copper chaperone-independent mechanism for activation ofCu, Zn-superoxide dismutase
Yasuyuki Sakurai, Yoshiaki Furukawa (Dept. of Chem, Keio Univ.)
Cu,Zn-superoxide dismutase (SOD1) is an antioxidant enzyme that removes
superoxide anion radicals, and is widely conserved among aerobic organisms. In
order for SOD1 to gain catalytic activity, two post-translational processes are
known to be necessary: binding of a copper ion and formation of an
intramolecular disulfide bond. In eukaryotes, the copper chaperone CCS plays apivotal role in the activation of cytoplasmic SOD1 by delivering the catalytic
copper ion and introducing the intramolecular disulfide bond to SOD1. A ccsknockout mouse, however, exhibits residual SOD1 activity, implying that a
CCS-independent pathway is also operative for SOD1 activation.
To get insight into the mechanism of this CCS-independent pathway, we havefocused upon the activation of Escherichia coli periplasmic SOD1 (SODC),
which occurs independently of CCS, as the E. coli genome contains no CCS
homologue. We have confirmed that an intramolecular disulfide bond as well as
a bound copper ion is essential for catalytic activity of SODC and also foundthat the canonical binding of copper/zinc ions in SODC requires the formationof a disulfide bond. During the activation of SODC in E. coli cells, therefore,
metal binding is considered to be preceded by formation of a disulfide bond.
This mechanism appears to fit well with the oxidizing environment of thebacterial periplasm. We thus propose a regulatory role of the disulfide formation
both in the CCS-dependent and independent activation of SOD1 enzymes.
2B1558タンパク質凝集体の形態を制御する「凝集後修飾」のメカニズム

A mechanism controlling the morphologies of protein
aggregates by “post-aggregation oxidation”
Yasushi Mitomi1, Takao Nomura1, Masaru Kurosawa2, Nobuyuki Nukina2,
Yoshiaki Furukawa1 (1Dept. of Chem, Keio Univ., 2RIKEN Brain Science
Institute)
Misfolding of a protein molecule often leads to the formation of insoluble
aggregates and is also characterized as a pathological hallmark inneurodegenerative diseases. One of the important factors regulating theaggregation of proteins is a post-translational modification; indeed, abnormal
modifications are often identified in the aggregate-consisting proteins. Actually,
however, it remains quite obscure when and how proteins are modified in thecourse of the aggregation.
To reveal an unprecedented role of modifications in protein aggregation, we focus
upon a protein, huntingtin (HTT), which has been known to form insoluble
aggregates in a neurodegenerative disorder, Huntington disease (HD). By
examining aggregation of recombinant HTT proteins, we have for the first time
identified oxidation of a Met residue in HTT during the aggregation in vitro. The
Met residue has also been found to oxidize in pathological HTT aggregates
purified from HD-model mice. A trace amount of contaminated copper ions aswell as a physiological concentration of hydrogen peroxide oxidized a Met
residue in HTT, and more interestingly, this Met oxidation occurred only in theaggregated HTT but not in the soluble state. Furthermore, we have found that the
Met oxidation creates additional interactions among HTT aggregates and alters
those overall morphologies. Protein aggregates can thus be a target of oxidative
modifications, and we propose that such a “post-aggregation” modification is arelevant factor to regulate properties of protein aggregates.
2B1610ジスルフィド結合の組換えによる不溶性 SOD1 オリゴマーの新たな形成メカニズム−筋萎縮性側索硬化症における分子病理変化

Destabilization of SOD1 facilitates abnormal scrambling of its
disulfide bond in the familial form of amyotrophic lateralsclerosis
Keisuke Toichi, Yoshiaki Furukawa (Dept. of Chem, Keio Univ.)− S41 −

Dominant mutations in Cu,Zn-superoxide dismutase (SOD1) are a cause of afamilial form of amyotrophic lateral sclerosis (fALS). Wild-type SOD1
possesses four Cys residues of total, among which Cys 57 and 146 form a highly
conserved intra-molecular disulfide bond. In contrast, mutant SOD1 proteins in
fALS-model mice have been known to be cross-linked via inter-moleculardisulfide bonds and form insoluble oligomers. A thiol-disulfide status in SOD1
will thus play a regulatory role in determining its folding/misfolding pathways;
however, it remains unknown how pathogenic mutations affect the thioldisulfide status in SOD1.
Here, we show that the structural destabilization of SOD1 scrambles a disulfide
bond among four Cys residues in an SOD1 molecule. Destabilization of wildtype SOD1 with a chaotropic reagent produced an SOD1 protein withalternative electrophoretic mobility, and a peptide-mapping analysis using
MALDI-TOF mass spectrometry revealed that a disulfide bond was shuffledamong four Cys residues in SOD1. Furthermore, we have found that fALSlinked mutations facilitate the disulfide-scrambling process and also reproduce
the formation of insoluble oligomers cross-linked via inter-molecular disulfide
bonds. Based upon our results, therefore, scrambling of the conserved disulfide
bond in SOD1 will be a key molecular event to describe the pathologicalchanges of mutant SOD1 in fALS cases.
2C1400A Brownian particle undergoing reversible binding in asurface: exact theoretical results and an application in single
molecule biophysics
Ziya Kalay (Institute for Integrated Cell-Material Sciences, Kyoto University)
Despite the apparent need to study reversible reactions between molecules
confined to a two-dimensional space such as the cell membrane, exact solutionsof the diffusion equation, or Green's functions, have not been reported. Here we
present exact analytical Green's functions for a Brownian particle reversibly
reacting with a fixed reaction center in a finite two-dimensional region withreflecting or absorbing boundaries, considering either a spherically symmetricinitial distribution or a particle that is initially bound. We show that theseGreen's functions can be used to predict the effect of measurement uncertainties
on the outcome of single-particle/molecule-tracking experiments in whichmolecular interactions are investigated. Hence, we bridge the gap between
previously known solutions in one dimension and three dimensions, and provide
an example of how theoretical results can be used to predict experimentally
accessible quantities in single molecule biophysics.

2C1412Single-particle-level simulation reveals effects of molecular
crowding on biochemical signaling response
Kazunari Kaizu, Koichi Takahashi (Laboratory for Biochemical Simulation,
RIKEN Quantitative Biology Center (QBiC))
Signal transduction pathway is a sophisticated information processing system
consisting of biochemical reactions. Although the reactions occur in a cellwhich is significantly different from ideal conditions assumed in conventional
biochemical theories, effect of “cellular environment”, such as clustering,compartmentalization and macromolecular crowding, on biochemical reaction
networks is poorly understood. In this study, molecular crowding effects on
dynamics of biochemical reactions and signal transduction pathways were
quantitatively determined by particle-level simulations. We have previously
shown that re-binding of kinases to substrates can remarkably increase theprocessivity of dual-phosphorylation reactions and change the response
characteristics. To quantify the significance in vivo, we simulated diffusioncontrolled reactions in crowded medium and measured re-binding probabilities
at the single-particle level. As a result, re-binding was highly enhanced bymolecular crowding and its time dependence was nonlinearly changed at
microscopic level even though the kinetics at macroscopic level followed theconventional equations in dilute media. The rate law revised on the basis ofthese calculations enables the quantitative modeling and analysis of biochemical
networks in intracellular environments with crowding. In particular, the results
were applied to the MEK-ERK system and validated with experimental
measurements.

2C14242 つのヒートショック反応系における 反応機構の比較

Conflicted dissociation constant in two reaction mechanisms
for heat shock response
Masayo Inoue1, Ala Trusina2, Namiko Mitarai2, Kim Sneppen2 (1CMC, Osaka
Univ., 2CMOL, NBI)

Cellular homeostasis is essential for proper protein folding and function. Heatshock, sudden increase of temperature, causes protein unfolding and misfolding
and can result in cell death. To counteract the heat shock, cells upregulate
production of chaperons and proteases.
The heat shock response is one of the stress responses observed in most livingthings. Interestingly, the protein sequence of most chaperones and proteases arewell conserved from bacteria to humans. It is, however, unclear if the features ofheat shock response are also preserved at the level of the regulatory network.
In this study, we focus on two different mechanisms for heat shock response
widely observed in microorganisms. The production rate of chaperon isregulated with a negative feedback loop in each system, however, the directionof regulations is opposite, i.e., the production of chaperon is activated in one
system but inhibited in the other.
In this study, we constructed a model for each system and obtained a reasonable
response in the production rate of chaperon. We also showed that acorresponding parameter have to satisfy a conflicted condition. This condition
based on the difference of activation/inhibition direction in the regulation
mechanisms.
2C1436

Mathematical analysis for vascular and spot patterns by auxin
and PIN dynamics in plant development
Yoshinori Hayakawa1,2, Atsushi Mochizuki1,2 (1Tokyo Institute ofTechnology, 2Wako Inst., Riken)
Inhomogeneous distribution of auxin is essential factor in various differentiation
processes of plant development. Auxin distribution is generated by auxin efflux
carrier protein called PINFORMED (PIN) and the interaction between thesemolecules has been discussed for long time. However, the mechanism of the
dynamics generated by relationship between auxin and PIN are still unknown.
In this study, we analyzed pattern formation of auxin distribution mediated bypolarization of PIN using mathematical methods. We developed several
different models for interaction between auxin and PIN on 2-dimentional
hexagonal cellular lattice, (1) Auxin flux model, (2) Auxin-dependent PIN
degradation model and so on.
We analyzed these models by numerical simulation using different parametervalues and initial patterns. We found that there are three different types ofdynamical behaviors from the view of pattern formation, (a) Homogeneous, (b)Passage and (c) Spot formation. In numerical simulation, these patterns appear
in different parameter and initial conditions. We introduce mathematicalanalysis using approximation of 1-dimensional periodic space. We analyzed
stabilities of three patterns (a) to (c), and determined condition of parameters forgenerating these patterns. These results suggest possibility that plants use
different mechanisms of interaction between auxin and PIN for passage and spot
formation. We are examining other different types of interaction between auxin
and PIN orientation.
2C1448Modeling wave propagation dynamics in MDCK woundhealing assay
Yusuke Sawabu1, Masaharu Nagayama2, Takashi Miura3, Hiroyuki Kitahata4(1Inst. of Nat. Sci. and Tech., Kanazawa Univ., 2Res. Inst. for Elect. Sci.
Hokkaido Univ., 3Kyoto Univ. Grad. Sch. Of Med., 4Dept. of Phys., Chiba
Univ., Grad. Sch. of Sci.)
Wound healing assay is frequently utilized as an example of collective cell
migration. We observe wave-like movement of cells in scratch assay of
confluent MDCK cell sheet. A region of fast-moving cells, which is 10-20 cells
wide, moved backward from the wound front, which is similar to propagation of
a compression wave in a elastic body or a traffic jam. At first we formulate a
one-dimensional model which assumes that the velocity of a cell is proportional
to the distance between neighboring cell, but the model did not reproduce thewave propagation phenomenon. We could mathematically show that under suchassumption no parameter set can reproduce the wave propagation. Next weformulate a one dimensional model which assumes that the polarity of each cellchanges according to the distance between neighboring cells, and cells moveaccording to the cell polarity. This model successfully reproduced the wave
propagation phenomenon during the wound healing assay. We also formulate
two-dimensional system which utilize the same type of cell-cell interaction.
Connection between these cells are defined in the beginning using Voronoi
diagram, which do not change during simulation. we confirmed twodimensional wave propagation observed in one-dimentional system.
Experiments are underway to quantitatively measure the interaction between thecells.

− S42 −

they provide a deeper understanding of the intrinsic properties of cardiac cells.
2C1510

CA モデルによる多細胞性シアノバクテリアの形態形成ダイナミク
スの解析

Analysis of the internal dynamics for pattern formation in
multi cellular cyanobacteria by CA model
Jun-ichi Ishihara1,2, Masashi Tachikawa2, Hideo Iwasaki1, Atsushi
Mochizuki2 (1Grad.Sci.Eng., Waseda Univ., 2Wako Inst., RIKEN)
The multicellular cyanobacteria, Anabaena sp. PCC 7120, differentiates cells
named heterocysts for nitrogen fixation at every ~10 cells under nitrogendeprived culture. This is known as one of the simplest pattern formation, but adetailed mechanism has not yet been validated theoretically.
To address this question, we made a 1-dimensional cellular automaton (CA)model for the pattern formation, including stochastic cell cycle processing, cell
division and differentiation. And a differentiated cell suppresses differentiationof neighboring cells. In the CA model, we considered that the division and/or
differentiation probability may depend on time after cell birth, “cell age”. Someparameters were quantitatively determined from cell-lineage analysis made by
our group.
To evaluate our models, we compared the distribution of heterocyst interval
between experimental data under microscope and computer simulation data by
CA models. As a result, respective features in steady-state of simulation dataagreed with that of the experimental data if cell division and differentiation
depended on cell age in CA model. These result suggested that the probability ofcell differentiation and division connected to cell age was very importantcondition. Moreover, pattern formation in transitional state of experimental dataagreed with that of simulation data if cell differentiated independently of cell
age in CA model.
We also succeeded in solving the distribution of heterocyst interval of CA
model by Marcov process including cell age transition.
2C1522Modeling the mammalian circadian clock
Craig Jolley, Hiroki Ueda (RIKEN Center for Developmental Biology,
Laboratory for Systems Biology)
Life generally runs on a schedule. The earth's rotation creates daily changes inlight, temperature, and other environmental conditions, and many organisms
respond to this by synchronizing their behavior and physiology with the
day/night rhythm. We have developed a simplified model of the circadian clock
system in mammals that captures many of its key features and can beparameterized by fitting to cellular-level measurements of biological rhythms.
By using Monte Carlo sampling of the parameter-fitting landscape, we havebeen able to create an ensemble of data-fitted models and use tools fromstatistical mechanics to assess the system's robustness to parameter variations.
We are currently using this model to explore the effects of external perturbations
on the cellular clock, which can be important for tissue-level synchronizationand entrainment to environmental signals.

2C1534

Determining the ratio of longitudinal to transverse
conductivity in a cardiac tissue culture through applicationof FFP

Marcel Hoerning1, Seiji Tagaki2, Kenichi Yoshikawa3,4 (1RIKEN, Center for
Developmental Biology, Physical Biology Unit, 2Hokkaido University,
Research Institute for Electronic Science, 3Doshisha University, Life andMedical Sciences, 4Kyoto University, Department of Physics)
Tachycardia and fibrillation are potentially fatal arrhythmias associated with theformation of rotating spiral waves in the heart. Low-energy far-field pacing(FFP) has recently been proposed as a superior therapy, and it has been foundthat the formation of such sites can lead to the termination of undesired states in
the heart and the restoration of normal beating. In this study, we investigate aparticular aspect of this method. Here, we seek to determine how the activationsite density depends on the applied electric field through in vitro experiments
carried out on neonatal rat cardiac tissue cultures. The results indicate that theactivation site density increases exponentially as a function of the intracellular
conductivity and the level of cell isotropy. Additionally, we report numericalresults obtained from bidomain simulations that are quantitatively consistent
with our experimental results. Also, we derive an intuitive analytical frameworkthat describes the activation site density and provides useful information for
determining the ratio of longitudinal to transverse conductivity in a cardiac
tissue culture. The obtained results should be useful in the development of an
actual therapeutic method based on low-energy far-field pacing. In addition,

2C1546

気道上皮細胞における Cl-分泌の数理モデル

Mathematical model of Cl- secretion in airway epithelial cells
Naomi
Niisato2,3,
Yoshinori
Marunaka2,3Kouhei
Sasamoto1,
1
2( Undergraduate Student, Kyoto Prefectural Univ. Med., Molecular Cell
Physiology, Kyoto Prefectural Univ. Med., 3Japan Institute for Food Educationand Health, Heian Jogakuin (St. Agnes') Univ.)
Transcellular Cl- secretion in airway epithelial cells followed by paracellular
Na+ secretion from the basolateral space to the apical one increases osmoticpressure in the apical space, which induces water secretion. Therefore,transcellular Cl- secretion plays a crucial role in prevention form infection ofbacteria and virus etc, by producing water secretion into the apical spacecovering airway epithelial cells.
Even using various techniques, we could not exactly get ideas how transcellular
Cl- secretion varies in its magnitude and time course due to modification ofactivity of Cl- channels and transporters located at the apical and basolateral
membranes. In the present study, we try to clarify how transcellular Clsecretion varies in its magnitude and time course when activity of Cl- channels
and transporters located at the apical and basolateral membranes changes using
mathematical simulation with three parameters.
(1) the entry step; Cl- into the intracellular space across the basolateral
membrane(2) the secretion step; Cl- from the intracellular space across the apicalmembrane(3) the recycle step; Cl- across the basolateral membraneThis novel method reported in the present study provides us how activity of Clchannels and transporters located at the apical and basolateral membranes
changes and understandings on roles of intracellular signaling in transcellular
Cl- secretion.
2C1558

エラーカタストロフィーによる HIV-1 擬種集団の自壊過程に関する研究

A Study on a Self-Destruction Process of a HIV-1 Quasispecies
Population through the Error Catastrophe
Kouji Harada (Toyohashi Univ. of Tech. Dept. of Comp. Sci. and Eng.)
The present study examines possibility of a new AIDS treatment without using
anti-HIV-1 drugs to avoid drug resistance. It is well-known that HIV-1 isfrequently-mutated. Our idea for the new treatment is by inducing excess
mutations to HIV-1 genome, to drive HIV-1 population to self-destruction.
Namely, we will use the HIV-1's remarkable character of its mutating at highrate as an underhanded way. Our study proposes a novel HIV-1 mathematicalmodel considered viral kinetic processes such as mutation, replication, infection
and an action of a mutagen to control HIV-1 mutation rate. In terms of HIV-1
population, our model considers a quasi species formed by four different traits.
Fast and slow replicators and either type can take viable and defective form.
Through some numerical simulations of the model we will present that
increasing HIV-1 mutation rate causes HIV-1 population to the error
catastrophe.
2C1610Characterization of Death and Division Process in Synthetic
Bacterial Population with Antibiotic Treatment

Takashi Nozoe1, Reiko Okura1, Yuichi Wakamoto2,3 (1Grad. Sch. Arts andSci., Univ. Tokyo, 2Research Center for Complex Systems Biology, Univ ofTokyo, 3JST PRESTO)
We previously succeeded in observing non-genetic adaptation in antibiotic
exposure by using a synthetic E. coli strain, expressing antibiotic resistant geneas fusion with fluorescent protein. Generally, phenotypic adaptation requires
cell-to-cell variation of fitness, which is defined as difference between division
rate and death rate. So, there are three possibilities on how phenotypic
adaptation occurs in lethal environment; (i) growth rate varies among cells
while death rate not, (ii) death rate varies while division rate not, and (iii) bothrates vary. To test which scenario would be probable, we estimated, as a firststep, temporal transitions in division frequency and death frequency, whichwere defined against a whole population. Time-lapse observation on a
microfluidics device enabled us to directly observe growth, division and deathin antibiotic exposure at single-cell level, followed by acquisition of time-seriesof division and death events. Consequently, it was clarified that death frequency
decreased in antibiotic exposure while division frequency was almost constant− S43 −

at lower frequency than before antibiotic addition. These results could support
that fluctuation of the antibiotic resistant gene expression level might cause cellto-cell variation of death rate in antibiotic exposure whereas division rate might
be independent of the expression level of the resistant gene. This consideration
would be useful for further analysis of the relation between gene expression
noise and phenotypic adaptation.
2D1412クライオ電子線トモグラフィーに対する超分解能技術

Super-resolution cryo-electron tomography
Ryuzo Azuma1,2, Takuo Yasunaga1,2 (1Grad. Sch. of Comp. Sc. & Eng., Dept.
of Biosc. & Bioinfo., Kyushu Inst. of Tech., 2JST)
We have been investigating a tomographic method that enables a 3D
visualization of computationally reconstructed object from electron micrographsof biological samples prepared under cryo-conditions that severely require lowamount of total electron dose. The key issue was to resolve phase and amplitude(P/A) moieties separately from intensities of these electron micrographs,
thereby complement missing information from finite tilt series sampling. This
problem was also concerned with how to manage super-resolution (SR) issues
arising from discretized data structures recorded as 2D-pixels. We are now
tackling these problems from the respects of recovering z-axis resolution
technique as proposed by Hoppe, combined with the “mechanical” tilting (notby beam tilting [1].) Also we incorporate iterative restoration of the P/A parts,
by referring to the method of Grechberg and Saxton [2]. The SR issue turned out
to crucially dependent on the method of phase image reconstruction from its
auto-correlations [3], provided that the development of 3D-version underinvestigation was successful. Also the Histogram matching and modified Sayre
methods may help to improve the performance of SR technique [3]. The basic
concept, theory, and characterization by simulations will be presented. Also we
acknowledge the method will be implemented as a software tool participated in
Eos program package.
[1] W. Hoppe, Z. Naturforsch, A26, 1155 (1971.)[2] R. W. Grechberg & W. O. Saxton, Optik, 35(2), 237-46 (1972.)[3] V. Elser, J Opt Soc Am A, 20(1) (2003.)
2D1424

定量位相顕微鏡による生細胞におけるナノスケールの形状変化の非

ultrasound irradiation is also known to accelerate the cell differentiation.
However, the mechanism of these fracture healing and cell differentiation are
not known. Therefore, we have observed the alteration of the cell interior in vivo
where ultrasound is irradiated by using Raman spectroscopy. In this study, we
have fabricated the “ultrasound probe” for our experiment. This needle shapeprobe allows us to measure the effects of ultrasound irradiation on one cell unit
and control the ultrasound intensity near 24mW/cm2 with its frequency at 1.5
MHz. We will discuss the results of the alterations of the cell and their analysisof Raman spectrum measurements before and after the ultrasound irradiations
on the cell.
2D1448脂肪酸と 7-ヒドロキシ -4-メチルクマリンを用いた質量と蛍光測定によるアンモニアの二次元検出

Two dimensional ammonia sensing by measurement of massand fluorescence using 7-hydroxy-4-methylcoumarin film andfatty acidYuta Ando, Masahiro Terashita, Ryohei Matsueda, Yutaka Tsujiuchi(Department of Material Science and Engineering, Akita University)
Two-dimensional sensing method for highly detecting of an organic compoundin atmosphere is demonstrated in this report. As a simple model case, anammonia sensing is exhibited, by measurement of mass with QCM sensor
system and measurement of fluorescent spectrum of composite film of 7hydroxy-4-methylcoumarin and fatty acid on a QCM Sensor unit. The
fluorescent dye used here, 7-hydroxy-4-methylcoumarin, has an appropriate pH
dependency of changing its emission light wavelength. Therefore, in this study,
detecting of ammonia by measuring of emitted light at different wavelength isconducted. And mass change measurement is also conducted. In the previous
conference, our first attempt of construction of ammonia sensor, using two
types of phospholipid, dimyristoyl-phosphatidylcoline (DMPC) or soya PC(phosphatidylcholine contained in soya beans), comparison for applying them tothe composite film with 7-hydroxy-4-methylcoumarin was reported. In thisreport, we demonstrated an improved attempt using different mol cular
combination in the composite film on sensor unit. A set of reproducible results
will be exhibited that the ammonia sensing with high precision that has not been
observed before.

染色ビデオレートイメージング

Label-free and video-rate imaging of live cell membrane motionin the nanometer-scale by quantitative phase microscope
Toyohiko Yamauchi1, Takashi Sakurai2, Hidenao Iwai1, Yutaka Yamashita1(1Hamamatsu Photonics, 2EIIRIS, Toyohashi Univ. of Tech.)Video-rate surface topography of live cultured cells was realized by our newly
developed microscope called "quantitative phase microscopy"(QPM). QPM isan optical interference microscope which shines white light onto the cells,
probes weak reflection light from cell membrane and visualizes threedimensional morphology of cell surface. QPM, which utilizes optical phaseresolved interferometric technique, is able to quantify nanometer-scale verticaldisplacement of the reflecting interface, such as cell membrane.
As a preliminary application study, we compared spontaneous membranemotions of HeLa cells (derived from cervix cancer) and INS-1 cells (derived
from rat insulinoma). Both samples were measured at two different
temperatures (36 °C and 24 °C). Surface topography of every cell was obtained
at 21 frames/sec. and the vertical motions of the cell membrane were analyzed
by the mean-square-displacement (MSD). We have also demonstrated a timelapse (2 sec. intervals) measurement for 10 minutes and even after themeasurement, the cells lost no activity in terms of the amplitude of theirspontaneous membrane motion. The HeLa and INS-1 cells showed significant
difference in terms of membrane motion. For example, MSD(τ=10sec) = 630
nm2 in HeLa cells while MSD(τ=10sec) = 3200 nm2 in INS-1 cells (both at
36 °C). We believe that our low-invasive and three-dimensional microscopic
technology could be a unique tool for further biophysical studies.
2D1436

ラマン分光を用いた超音波照射による細胞への影響の評価

Evaluation of Ultrasound Irradiation effects on Cells by using
Raman Spectroscopy
Manabu Sugimoto1, Yo Otsuka1, Shinya Murakami1, Toshiyuki Mitsui2(1Grad. Sci Eng., Aogaku Univ, 2Assoc Prof, Aogaku Univ)

2D1510インビトロ心毒性予測のための心筋細胞ネットワークの時間的細胞外電位ゆらぎと空間的伝達速度ゆらぎの評価

Evaluation of temporal fluctuation and spatial fluctuation on
cardiomyocyte network for in vitro predictive cardiotoxicity
measurement
Tomoyo Hamada, Fumimasa Nomura, Tomoyuki Kaneko, Kenji Yasuda(Institute of Biomaterials and Bioengineering, Tokyo Medical and Dental
University)
Current guidelines for drug safety recommend the preclinical studies to topredict lethal arrhythmia potential especially torsade de pointes (TdPs) bymeasuring the in vivo drug-induced QT interval prolongation. However, recent
studies show that those guidelines have limitations because they do not cover
the pro-arryhthmic potential of drugs induced fatal ventricular tachycardia (VT)or fibrillation (VF) in the absence of QT prolongation. Hence, we haveexamined the most effective risk index to be able to replace from conventional
QT prolongation. First, the temporal fluctuation of the field potential duration(FPD) of human embryonic stem (hES) cell derived cardiomyocyte clusters in
on-chip microelectrode array (MEA) system was measured, and increased by
the positive compounds even though in the absence of QT prolongation, but notby the negative compounds. Next, the spatial fluctuation of the conduction
velocity (CV) in the lined-up hES cardiomyocyte network on a MEA chip byusing agarose-microchambers was measured, and showed that the spatial
fluctuation of CV was increased even by terfenadine, which was difficult to
detect the risk in the traditional in vitro assays. The results suggest that both thetemporal fluctuation of FPD and the spatial fluctuation of CV are good indicesto evaluate the potential risks of pro-arrythmia including VT/VF and further forunderstanding the mechanism of pro-arrhythmia.
2D1522

We evaluate the effects of ultrasound irradiation on the cell’s interior by using
Raman spectroscopy. Recently, the ultrasound irradiation is known to promote aborn growth and used for the treatment of born fracture healing. In cell unit,− S44 −SACLA における非結晶粒子の低温 X 線回折イメージング実験
Cryogenic Coherent X-ray Diffraction Imaging of
non-crystalline particles at SACLAMasayoshi Nakasako1,2, Yuki Takayama1,2, Tomotaka Oroguchi1,2, Yuki
Sekiguchi1,2, Masaki Yamamoto2, Koji Yonekura2, Takaaki Hikima2, Saori
Maki-Yonekura2, Yukio Takahashi3, Akihiro Suzuki3, Sachihiro Matsunaga4,
Shoichi Kato4, Takahiko Hoshi5 (1Department of Physics, Keio University, 2RIKEN
SPring-8 Center, RIKEN Harima Institute, 3Department of Precision Science andTechnology, Graduate School of Engineering, Osaka University, 4Department ofApplied Biological Science, Faculty of Science and Technology, Tokyo University of
Science, 5Kohzu Precision Co.,Ltd.)
Coherent X-ray diffraction imaging (CXDI) experiments of non-crystalline
particles were performed at 66 K using our diffraction apparatus, named
KOTOBUKI, at BL3 of SACLA on March and April. In the preparation ofsamples, suspension of non-crystalline particles at high concentration was
dropped on thin silicon-nitride or carbon membrane using a humidity controlled
sample preparation chamber. Samples were set in the liquid nitrogen pot insidethe diffractometer and moved synchronously with the XFEL pulse. The hit-rateof XFEL pulse to sample particles was 40-60%. Diffraction patterns from nanomaterials with the maximum dimensions of 100-300 nm were retrieved by using
only the combinational use of hybrid-input-output and shrink-wrap algorithms.
2D1534

A protocol for structure analysis of non-crystalline particles
with X-ray free electron laser
Tomotaka Oroguchi, Masayoshi Nakasako (Department of Physics, Faculty of
Science and Technology, Keio University)

X-ray free electron laser facilities provides coherent and intense X-ray pulse in
the range of 1011-1012 photons/m2/pulse. Taking the cross section in Thomson
scattering into consideration, the X-ray intensity is still smaller than thatrequested to carry out single molecular diffraction experiments for biologicalsupramolecular assemblies such as ribosome that has a molecular mass of a few
mega Dalton. Therefore, the single particle structure analyses of proteins withthe molecular mass are still formidable task. Here we propose a protocol tovisualize the structure of molecular assemblies with a molecular mass of morethan a few mega Dalton by the combinational application of the coherent X-ray
diffraction imaging method and the three-dimensional reconstruction algorithm.
The details and limitations of the protocol are going to be reported based on thesimulation for the structure analysis at 0. 8 nm for 50S ribosome particles
embedded in vitreous ice plates of 500×500×50 nm3.
2D1546

イオンコンダクタンス顕微鏡による細胞膜揺らぎの測定

Fluctuation of cell membrane investigated by ion conductancemicroscopy
Yusuke Mizutani2, Satoshi Ichikawa1, Zen Ishikura1, Takaharu Okajima1(1Grad. Sch. Inform. Sci. Tech., Hokkaido Univ., 2Grad. Sch. Life Sci.,
Hokkaido Univ.)
The cell membrane thermally fluctuates and actively moves. Since themembrane contains various types of membrane proteins and interacts thecytoskeleton such as actin filaments, understanding the dynamic behaviors of
the cell membrane is key to elucidate how various cell functions are regulated.
In this study, we report the direct measurement of the fluctuation of living cell
membrane by scanning ion conductance microscopy (SICM). As an electricallycharged micropipette tip approaches a sample surface in an oppositely charged
bath of electrolyte, the ion current steeply decreases in proximity to the surface[1]. Therefore, the SICM has a potential to precisely measure the position of thesurface in a noncontact region. We used a commercial ICM system (XE-Bio,
Park Systems, Korea) combined an inverted optical microscopy (Ti-U, Nikon)
to map the spatiotemporal ion current of epithelial MDCK cells which wereconfluent on culture dish. It was observed that the ion current-distance curves
largely changed depending on cell modifications such as a disruption ofcytoskeleton and a chemical fixation of cell. We found that the disruption ofactin filaments facilitated a change in the fluctuation of cell membrane. The
averaged amplitude of cell membrane fluctuation could be estimated in different
cell samples. [1] P. K. Hansma et al., Science 243, 641-643 (1989). Y. E.
Korchev et al., Biophys. J 73, 653-658, (1997). Y. E. Korchev et al., Biophys.
J, 78, 451-457, (2000).
2D1558Relationship between Intercellular Adhesion Strength andCommunication Detected by Femtosecond Laser-induced
Impulsive Force

Takanori Iino1, Man Hagiyama2, Tadahide Furuno3, Akihiko Ito2, Yoichiroh
Hosokawa1 (1Nara Institute of Science and Technology, 2Kinki University,
3
Aichi Gakuin University)
We have indicated that femtosecond laser-induced impulsive force can beavailable as an external force to break the cell adhesion[1]. Furthermore, we havedeveloped a method to estimate the intercellular adhesion strength using
femtosecond laser-induced impulsive force and atomic force microscopy[1,2].
Now, we are interested in a relationship between the adhesion strength andintercellular communication. In this work, we investigated the communication
between a neuron and a mast cell as propagation of Ca2+ wave between them,simultaneously with the intercellular adhesion strength.
Mast cells (from mouse) and neurons resulting from differentiation induction ofneuroblastoma (Neuro2a) were prepared. By depositing the mast cells on theneurons and culturing them together (co-culture), the mast cells were adhered to
the neurites. After they were co-cultured for 16 hrs, the communication between
the neuron and the mast cell was investigated to monitor the propagation of
Ca2+ wave, in which the neuron was activated by loading the impulsive force tofocus the femtosecond laser (780 nm, 250 fs) near the neuron through an
objective lens (10x, NA=0.25). After that, the intercellular adhesion strength
was estimated as described previously[1]. In the presentation, the relationshipwill be discussed on the basis of these results.
[1] I. Takanori et. al., The 48th Annual Meeting, 3p211 (2010).
[2] H. Yoichiroh et al., Proc. Natl. Acad. Sci. USA, 108, 1777 (2011).
2E1400

AFM を用いたホロミオグロビンの一分子力学計測によるアン
フォールディング経路の探索

Mechanical unfolding pathways of holo-myoglobin explored by
AFM-based single molecule force spectroscopy
Aya Yoshida, Masaru Kawakami (Materials Sci, JAIST)
We have applied Single Molecule Force Spectroscopy (SMFS) using atomic
force microscopy (AFM) technique to characterize the unfolding pathway of
myoglobin. A recombinant chimera polyprotein of holo-myoglobin with titin
I27 [(I27)7-Mb-(I27)2] was engineered, in which a Heptameric I27 concatamer
serves as a molecular handle for the manipulation of the polyprotein by an AFM
cantilever via non-specific adhesion. SMFS results showed that the mechanicalunfolding of myoglobin occurs at forces well below that of I27. Thisobservation agrees with the previous reports that all α -helical proteins aremechanically weaker than their all -sheet counterparts. Close inspection ofeach unfolding force curve of myoglobin revealed that the mechanical unfolding
trajectory of myoglobin can be categorized into several patterns. One patterncontains only one unfolding peak with a full-molecular length elongation upon
unfolding, indicating a catastrophic unfolding, i.e. myoglobin molecules unfold
in all-or-none fashion. Other patterns include complex force peaks following thefirst major unfolding peak. They appeared with diversity of unfolding force andelongation length, suggesting that mechanical unfolding of myoglobin occurs ina three-(or more) state fashion through mechanically stable intermediate (s).
These results indicate that the mechanical unfolding of myoglobin has multiple
complex pathways, due to the bound heme molecule which is mechanicallystabilizing the tertiary structure of myoglobin.
2E1412走査型プローブ顕微鏡を用いた癌抑制因子 p53 の一分子構造解析

Single-molecule structural analysis of tumor suppressor p53
using scanning probe microscopes
Seiya Takahashi1, Yasuyuki Sainoo1, Risa Kashima2, Takashi Tokino3,
Tadahiro Komeda1, Satoshi Takahashi1, Kiyoto Kamagata1 (1IMRAM,
Tohoku Univ., 2Univ. California, 3Res. Inst. Front. Med., Sapporo Med. Univ.
Sch. Med.)
p53 binds specific sites of DNA tightly with hopping and/or sliding motions
along the DNA, which is the initial stage of transcriptional regulation of target
genes. However, the detailed mechanism of the DNA-binding based on the
structural and dynamical characterization of p53 remains unclear. A singlemolecule characterization is required to further understand the DNA-binding
mechanism, because ensemble experimental methods cannot determine
heterogeneity of its structure with flexible disordered regions. Here, we
performed structural analysis of p53 at a single-molecule level using two
scanning probe microscopys; atomic force microscopy (AFM) and scanningtunnel microscopy (STM). p53 molecules were attached to gold surface, andthen individual molecules on the surface were characterized under a dry
condition using AFM. The volume histogram of these molecules showed theexistence of monomers and oligomers such as dimers, tetramers and higher-− S45 −

order aggregations. These structures were determined at a nanometer resolution.
In the tetramer, small spheres were observed, which were spatially separated
each other. Because their size is similar to that of the DNA binding domain, they
were assigned to the DNA binding domains connected to a tetramerization
domain with flexible linkers. Furthermore, we confirmed that p53 molecules on
the surface can be imaged using STM. We will discuss the result of further
statistical analysis of single-molecule structures and the conformational change
against specific and non-specific binding to DNA.
2E1424塩基性条件下でのヒトガレクチン 1 のβガラクトシド結合能力

β-Galactoside-binding activity of human galectin-1 at basic pH
Hirotsugu Hiramatsu, Tomohide Nishino, Hideo Takeuchi (Graduate Schoolof Pharmaceutical Sciences, Tohoku University)
Human galectin-1 (hGal-1) is a soluble, -galactoside-binding lectin. hGal-1
takes part in physiological functions such as cell adhesion and intercellularsignal transduction by binding glycoconjugates on cell surfaces. In this study,
the -galactoside-binding (lectin) activity of hGal-1 was evaluated from thehGal-1 fraction bound to lactose gel (Y) and the lactose-binding constant insolution (Kb) at pH 7-10 by using fluorescence spectroscopy. Y showeddependence on the hGal-1 concentration, NaCl concentration, and pH. Y
decreased at pH >7.5 in the absence of NaCl, while it was constant in thepresence of 150 mM NaCl. On the other hand, Kb was found to be independentof pH and the NaCl concentration. Analysis of the S-H Raman band intensity atvaried pH suggested that the average pKa of six Cys residues of hGal-1 was 9.6± 0.1. The apparent pH dependence of Y is explained by Coulombic repulsion
among hGal-1 molecules on the lactose gel surface. At basic pH, the Cys (andother) residues of hGal-1 will be deprotonated to gain negative charges, which
would prevent hGal-1 from binding to the gel excessively. The Coulombic
effect is supported by the lack of the pH dependence in the presence of 150 mM
NaCl. In conclusion, the lactose-binding ability of hGal-1 does not decrease atbasic pH, despite that the amount of hGal-1 bound to the gel decreases at basic
pH in the absence of NaCl. This result suggests that Y is not a good indicator of
the lectin activity of galectins, which contain many Cys residues.
2E1436

A Molecular Mechanism of Induced-fit of U1A Protein
Ikuo Kurisaki1,2, Masayoshi Takayanagi1,2, Masataka Nagaoka1,2 (1Grad.
Sch. Info. Sci. Univ. Nagoya, 2CREST, JST)The induced-fit mechanism of U1A protein upon RNA-binding has been aparadigm of protein-RNA molecular recognition. However, the molecularmechanism still remains not fully understood in atomic level. To address the
problem, we performed molecular dynamics (MD) simulations starting with
apo-U1A and U1A-RNA complex structure. We compared the conformation
distribution between apo-U1A and U1A-RNA system. The result indicates that
the RNA-binding to U1A maintains the RNA-binding form of U1A. Amongsnapshot structures in apo-U1A system, we obtained a plausible intermediate
structure at the initial step of induced-fit, termed the open-unfolded form [1].
This finding is consistent with previous conjectures for intermediate structure
upon induced-fit [2,3]; (1) the C-terminal region partially unfolds and (2) the Cterminal region reorients under the inter-molecular electrostatic interaction. We
performed the MD simulations starting from the complex of U1A in the openunfolded form and RNA, resulting in the structure relaxation of U1A into theRNA-binding form [4]. The obtained results can be promising clues to clarify
the molecular mechanism of induced-fit of U1A at the atomic level, leading toestablishment of a full understanding of the protein-RNA molecular recognition
mechanism.
[1] Kurisaki, Takayanagi and Nagaoka, J. Phys. Chem. Lett., submitted for
publication.
[2] Tang and Nilsson, J. Biophys., 77(3), 1284-1305 (1999)[3] Williamson, Nat. Struct. Biol., 7(10), 834-837 (2000)[4] Kurisaki, Takayanagi and Nagaoka, in preparation.
2E1448Single mutation on a loop alters the key dynamics of the core,
but not the average structure
Akihiro Maeno1, Sunilkumar P.N.1, Yuji Wada2, Eiji Ohmae2, Shin-ichi Tate2,
Kazuyuki Akasaka1 (1HPPRC, Kinki Univ., 2Grad. Sch. Sci., Univ. Hiroshima)
Evolution of a protein occurs often by mutations on the peripheral region of theprotein structure, which cause functional changes. To find the molecular originof the surface mutation on protein function, we studied the effect of a single
mutation in a loop in E-coli DHFR, G67V, on the structure and dynamics in asite-specific manner by using NMR spectroscopy on the 15N-uniformly labeledwild-type and the G67V mutant.
In a binary complex, DHFR: folate, the G67V mutation caused structuralchanges only localized around the mutation site, but caused no change in the
average structure in the rest of the protein, the major core part including theactive sites. On the other hand, the G67V mutation showed definite changes inconformational fluctuation in the remote core region of the protein including theactive sites. In accord with the finding, distinct changes were observed in spin
relaxation dispersion in the G67V mutant in the ternary complex, DHFR:
NADPH:folate, which explains the observation that the hydride transfer rate isdecreased by the same mutation. The present finding shows that a surface
mutation, causing practically no change in the average structure of the protein,can cause a change in the crucial dynamics for function. The result may explain
why surface mutations prevail in protein evolution.
2E1510

ヒト主要組織適合複合体の安定化に置ける揺らぎの重要性について

The effect of flexibility on the stability of Human Leucocyte
Antigen
Saeko Yanaka1, Kenjji Sugase2, Takamasa Ueno3, Kouhei Tsumoto1 (1Med.
Genome Sci., Grad. Sch. Frontier Sci., the Univ. of Tokyo, 2Suntory Institue for
Bioorganic research, 3Center for AIDS Research, Kumamoto Univ.)
In the first step of human infection defense, human leucocyte antigens (HLA)
present foreign peptides, which are recognized by cytotoxic T lymphocytes(CTL), resulting in the activation of CTLs to kill infected cells. Previous CD
experiments have revealed that the thermal stability of HLA in cells is closely
related to the CTL activity, and considerably depends upon the presented
peptides. Although many crystal structures of HLA with different peptides havebeen determined, they are almost identical to each other. DSC experiments haveshown that denaturation enthalpy cannot explain the HLA stability, whilesuggesting the structural difference in solution. Therefore, the stabilization
mechanism of HLA is still unclear. Here we have studied the stabilization
mechanism from the viewpoint of flexibility, using NMR relaxation dispersion
spectroscopy. We focused on two HLAs presenting peptides, VY8-5A, andRY11-8A, which have the same sequence except that RY11-8A has three
additional amino acids in the N-terminus. VY8-5A is known to activate CTL
much longer than RY11-8A. In previous CD measurements it is shown that
HLA in complex with VY8-5A has a higher temperature midpoint. NMR
relaxation dispersion experiments revealed that the peptide recognition site in
HLA is fluctuating in solution. Interestingly, HLA with VY8-5A, which is more
thermostable, is more flexible, and is prone to form low-populated minor
conformation. This suggests that the flexibility is critical for the HLA stability.
2E1522

Cavity-dependent dynamics of c-Myb R2R3 revealed by highpressure fluorescence and NMR spectroscopy
Satomi Inaba1, Akihiro Maeno2, Hisayuki Morii3, Harumi Fukada4, Kazuyuki
Akasaka2, Masayuki Oda1 (1Grad. Sch. of Life and Environ. Sci., Kyoto Pref.
Univ., 2High Pressure Protein Res. Center, Kinki Univ., 3Biomed. Res. Inst.,
Natl. Inst. Adv. Ind. Sci. Technol., 4Grad. Sch. of Life and Environ. Sci., Osaka
Pref. Univ.)
The c-Myb DNA-binding domain consists of three imperfect tandem repeats(designed R1, R2, and R3), and the last two repeats, R2 and R3, are necessary
for the specific DNA-binding. Each repeat has three conserved Trp residues,
which are involved in the hydrophobic core. The ternary structure of each repeat
is similar, but R2 has a larger internal cavity. The cavity-filling mutation,
V103L, reduced the specific DNA-binding, simultaneously reducing its
conformational fluctuation and hydration, indicating that the cavity-dependent
dynamics is closely related to the function. In this work, we studied the cavitydependent dynamics of c-Myb R2R3 in more detail, using high-pressure Trp
fluorescence (up to 700 MPa) and high-pressure 1H one-dimensional and 15N/1
H two-dimensional NMR spectroscopy (up to 250 MPa), along with differential
scanning calorimetry. The maximum fluorescence emission of the wild-type
R2R3 shifted from 332 to 346 nm at 700 MPa, while the cavity-filling mutant,
V103L, shifted from 332 to 340 nm, showing that the cavity significantly
contributes to the hydration, while the full unfolding of R2R3 was not yet
attained even at 700 MPa. The high-pressure NMR experiments reveal moredetails of the cavity-dependent dynamics and stability of R2R3, in particular thedifferent behavior and cooperativity of the two repeats, R2 and R3.
2E1534− S46 −

単純化 BPTI に融合したペプチド系タグ配列を用いたアミノ酸溶解性の測定

Analysis of amino acid contributions to protein solubility using
short peptide tags fused to a simplified BPTI variant
Yutaka Kuroda1, Alam, M. Khan1, Mohammad, M. Islam1,2 (1Dept of Biotechand Life Sci, TUAT, 2Dept of Mol Biol and Biochem, Chittagon University)
Protein solubility is usually characterized in terms of a hydrophobicity scale,
which refers to the free energy of transfer of a molecule from an aqueous to a
nonpolar solution and is not a "solubility propensity scale" per se. Using a "hostguest" approach, we measured the effects of short poly-amino-acid tags (guests)
on the solubility of a host protein, a simplified bovine pancreatic trypsin
inhibitor (BPTI), to which they were fused at the C-terminus. We analyzed 10
amino acid types, representing the full range of biophysical properties (acidic,
basic, polar, and hydrophobic). As anticipated, positively charged residues
significantly increased the solubility of the model protein, at both pH 4.7 and
8. 7, whereas very hydrophobic poly-Ile markedly reduced the solubility of
BPTI. Poly-Asp and poly-Glu barely affected BPTI solubility at pH 4.7, but
induced an eight to ten-fold increase at pH 8.7, attributable to the ionization of
their side chains. Although Pro is the most soluble amino acid, poly-Pro did notaffect the protein's solubility. The effects of the other tags on BPTI solubility
ranged from none to an eight-fold increase. These observations suggest that asolubility propensity scale could eventually allow the calculation of apolypeptide's relative solubility from its amino acid sequence.
2E1546

Protein Aggregation Kinetics Using Short Amino acid Peptide
Tags
Monsur A. Khan1, Mohammad M. Islam1,2, Yutaka Kuroda1 (1Tokyo
University of Agriculture and Technology, 2University of Chittagong,
Bangladesh)Protein aggregation is becoming an important issue in protein chemistry ranging
from truly academic to biotechnological applications. Understanding thesolubility and/or aggregation tendency is a prerequisite for proper use of aprotein. Here we report the aggregation kinetics of protein using short artificial
poly peptide tags containing five amino acids added to the C terminus of asimplified bovine pancreatic trypsin inhibitor (BPTI) variant. We measured theconcentration of the sample of BPTI variants in supernatant after different
incubation period (20min, 6hrs, 12hrs, 24hrs and 48hrs) and starting withdifferent initial protein concentrations ranging form 0.25mg/ml to 20.0mg/ml.
We observed that precipitation occurs promptly when BPTI was dissolved at aconcentration above a maximum protein concentration, which we named“maximum transient solubility (MTS)”. Samples with protein concentration
below the MTS but above a lower critical concentration, which we named“maximum mid-term solubility (MMTS)”, decreased upon long incubation
period. On the other hand, samples with concentrations lower than the MMTS
remained unchanged up to 48hrs of incubation. These results provide insights
into the understanding of protein aggregation kinetics.
2E1558

Protein specific partial charges by Ab-initio fragment
molecular orbital method for more accurate protein
simulations
Le Chang, Shoji Takada (Grad. Sch. Sci., Kyoto Univ.)
Reliability of atomistic molecular dynamics simulations largely depends on
accuracy of the force fields used. Among force field parameters, partial charges
are one of the most important and hard-to-decide parameters. Normally, partial
charges are determined for small building-blocks, often single amino acids, andthey are used in proteins for simplicity. However, chemical moiety of an aminoacid depends on its environment and even the same type of amino acid can havedifferent electronic states. Thus, if one can determine partial charges for each
atom in the target protein environment, they could be more accurate.
In the past, it was impossible to solve the electronic Schrödinger equations forlarge biomolecules. Recently, the fragment molecular orbital (FMO) method
makes the ab-initio quantum mechanical calculation for the energy of protein
size system possible. In this work, the partial atomic charges for protein native
conformation is obtained from FMO calculation. From the new partial atomic
charges, we observe some interesting features of the protein electrostatic
characteristics. Besides, the binding free energies of some ligands to proteins arecalculated from the molecular dynamic simulations using new partial atomic
charges. The free energies of protein-ligand binding based on the new partial
atomic charges are compared for several ligands with those by the standard
AMBER force field, giving some promising results.

2E1610Regulatory Mechanism of the fluorescence emission fromGreen Fluorescent Protein Chromophore
Jumpei Torii, Shoji Yamashita, Etsuko Nishimoto (Institute of Biophysics,
Faculty of Agriculture, Graduate school of Kyushu University)
Green fluorescent protein (GFP) has been widely used in the biological studies
as a marker of gene expressions, protein localizations, and protein-protein
interactions. The chromophore of GFP shows high quantum efficiency (80%),
its radiation mechanism however is yet unclarified. In this study thefluorescence intensity of p-hydroxybenzylidene dimethylimidazolinone (pHBDI) as an analogue of GFP chromophore was measured at both 296 K and 77
K. While p-HBDI showed strong fluorescence at 77 K, little fluorescence wasobserved at 296 K. This result suggests that twisting motion of p-HBDI in the
singlet excited state competes with fluorescence emission. To estimate theactivation energy of this motion, which is speculated to cause the photo induced
E-Z isomerisation of p-HBDI and its fluorescence quenching at the same time,
the quantum yield of S65T-GFP was measured in the temperature ranging from296 K to 340 K. In the lower temperature range, higher activation energy wasobserved than in the higher temperature range. This result indicates that when
GFP folds properly, twisting motion of GFP chromophore is suppressed becauseof its high activation energy, however this barrier gradually diminishes with therelaxation of tertiary structure, which results in fluorescence quenching. For
further investigation into the radiation mechanism of GFP chromophore,temperature and viscosity dependence of p-HBDI fluorescence intensity, CD
spectrum of S65T-GFP, and the fluorescence lifetimes of both p-HBDI and
S65T-GFP were measured respectively.
2F1400全原子量子化学計算によるレチナールタンパク質の吸収波長制御機
構の解析

Full-Quantum Chemical Calculations of the Absorption
Maxima of Retinal Proteins
Tomohiko Hayashi1, Azuma Matsuura2, Hiroyuki Sato2, Minoru Sakurai1(1Center for Biological Resources and Informatics, Tokyo Institute ofTechnology, 2Fujitsu Laboratories, Ltd.)A retinal protein consists of a seven-transmembrane protein (called “opsin”) anda retinal chromophore covalently bound to a Lys residue via protonated Schiffbase linkage. The absorprion maxima (λmax) of the chromophore is 610 nm in
the gas phase but blue-shifts by 42~102 nm in the opsin. Such a shift is hencereferred to as the “opsin shift”. The elucidation of the origin of the opsin shift
has been an important subject not only in photobiology but also computational
chemistry in recent decades. Although there have been reported a variety ofquantum chemical calculations on the opsin shift, its precise physical origin isstill a challenging open question. In this study, the λ max values of thebacteriorhodopsin (bR) and pharaonis phoborhodopsin (ppR) are calculatedusing our recently developed method that enables us to treat the whole protein
quantum-chemically. We show that such a full quantum chemical calculation
reproduce the opsin shift with an error of less than ±0.04 eV. To decompose theopsin shift into the contributions from each amino acid, we also apply the same
calculation for 226 different bR Gly mutants and 217 different ppR Gly mutants.
Finally, we discuss the origin of the opsin shift from the following four factors,
1) contribution of the structural change of the chromophore accompanied by its
transfer from the gas state to the protein interior, 2) contribution of each aminoacid, 3) contribution from the backbone structure of the protein and 4) theirsynergistic effect.
2F1412プロテオロドプシンにおける細胞質側ループの変異がもたらす遠隔的波長制御メカニズム

Mechanism of color change of proteorhodopsin by mutations
at distant cytoplasmic loop
Yuya Ozaki1, Rei Abe-Yoshizumi1, Susumu Yoshizawa2, Kazuhiro Kogure2,
Hideki Kandori1 (1Grad. Sch. Eng., Nagoya Inst. Tech, 2AORI, Univ. Tokyo)
It has been believed that the amino acids near the retinal chromophore are only
responsible for color tuning of rhodopsins. However, we found that replacementof Ala178 with Arg in the E-F loop of green-absorbing proteorhodopsin (GPR)
exhibits spectral red-shift and the pKa increase of the Schiff base counterion1,
which was not observed for bacteriorhodopsin (BR) 2. Distant mutation effect
was also observed for Ala115 in the C-D loop of GPR. It is likely that the loop
regions contain unique structure in GPR, disruption of which causes large-scale
rearrangement of α-helices.

− S47 −

Here we studied similar mutation effect for blue-absorbing PR (BPR).
Thousands of PR are classified into GPR (λmax~520 nm) and BPR (λmax~490
nm). GPR and BPR possess Leu and Gln at position 105, and L/Q GPR and Q/L
BPR absorb blue and green lights, respectively (L/Q switch) 3. In case of BPR,
distant mutation effect was only observed for the C-D loop, not E-F loop,
suggesting that large-scale structural rearrangement is absent for the E-F loop of
BPR. In case of GPR, distant mutation effects of the C-D and E-F loops were
not additive, suggesting that common structure around the retinal chromophore
is formed. Molecular mechanism of mutation effect in the cytoplasmic loops
will be discussed on the basis of the present results.
1. Yoshitsugu et al. Angew. Chem. Int. Ed. 47, 3923 (2008).
2. Yoshitsugu et al. Biochemistry. 48, 4324 (2009).
3. Man et al. EMBO J. 22. 1725 (2007).
2F1424

キ メ ラ タ ン パ ク 質 を 用 い た Rhodobacter capsulatus 由 来

Photoactive Yellow Protein の相互作用部位の解明
Analysis of interaction sites on the Photoactive Yellow Proteinof Rhodobacter capsulatus with chimeric proteins
Mayu Shimad, Yoichi Yamazaki, Hironari Kamikubo, Mariko Yamaguchi,
Mikio Kataoka (Grad. Sch. Mat. Sci., NAIST)
Photoactive Yellow Protein (PYP) is a photoreceptor protein that absorbs blue
light with p-coumaric acid as a chromophore. We identified the target protein of
PYP named PBP from Rhodobacter capsulatus (Rc-PYP). PBP interacts with
Rc-PYP in light dependent manner. This is the only target protein of PYP
homologues revealed so far. Rc-PYP shows no substantial conformation
changes upon absorption of light. The observation raised a serious question how
PBP recognizes the photolyzed state of Rc-PYP. In order to clarify themechanism of light dependent interaction of PBP with Rc-PYP, we aimed to
identify the interaction sites on Rc-PYP by using chimeric mutants between RcPYP and Halorhodspira halophila PYP (Hh-PYP). Rc-PYP and Hh-PYP are
separated into four segments considering secondary structure and amino acidconservation. The first block contains N-terminal 30 residues, the second blockcontains 31st -57th residues, the third block contains 58th - 86th and the fourth
block contains remaining residues. Chimeric proteins were designed to replacesome of four segments into a parent protein. Binding ability of PBP was verified
spectroscopically, since the complex formation brings distinguishable spectral
shift. The complex formation was also examined by photocycle kinetics the size
exclusion chromatography. As a result, chimeric proteins having the second
block of Hh-PYP depressed binding ability. While chimeric proteins having theother block(s) of Hh-PYP did not bind PBP. The further analyses are required to
identify interaction sites.
2F1436FTIR study of Aureochrome that possesses LOV domain at the
C-terminus
Shota Ito1, You Zhang1, Tatsuya Iwata2, Osamu Hisatomi3, Fumio Takahashi4,
Hironao Kataoka5, Hideki Kandori1 (1Nagoya Inst. Tech, 2Ctr. Fost. Yng.
Innov., Nagoya Inst. Tech, 3Grad. Sch. Sci., Osaka Univ, 4PRESTO, JST,
5
Botanical Gardens, Tohoku Univ)
LOV domain is a blue-light sensor that binds an FMN. In many blue-lightsensors, LOV domain is located at the N-terminal side. For example,
phototropin possesses two LOV domains at the N-terminal side and regulates
serine-threonine kinase domain at the C-terminal side1. By means of lightinduced difference FTIR spectroscopy, we have studied photoactivation
mechanism of LOV domains in phototropin2.Aureochrome, a novel photosensor
protein from Vaucheria frigida, possesses a LOV domain at the C-terminus,
while bZIP domain is located at the N-terminal side3.It is reported that the bZIP
domain of aureochrome can recognize a DNA sequence (TGACGT), when its
LOV domain is photoactivated. An interesting question is how signaltransductions in LOV domains differ between the N- and C-termini?
In this study, we measured light-induced structural changes of aureochrome(bZip-LOV) by low-temperature FTIR spectroscopy, where temperaturedependent structural changes were observed at 150, 200 and 295 K. The
obtained spectra were compared with those of an isolated LOV domain, andthose with DNA. We also measured the mutants of Phe298 and Gln317, which
are located at the -sheet near the FMN and identified as the important residues
for the structural changes of LOV domain in phototropins2. Molecularmechanism of the light-switch in aureochrome will be discussed.
1. Briggs, R, W., et al. Trends Plant Sci. 7, 204-210 (2002).
2. Iwata, T., et al. BIOPHYSICS 7, 89-98 (2011).
3. Takahashi, F., et al. Proc. Natl. Acid. Sci. USA 104, 19625-19630 (2007).

2F1448

LOV ドメインを鋳型としたレドックス感受性蛍光タンパク質の開発
Construction of LOV-based redox-sensitive fluorescentproteins

Yukiko Ono1, Tatsuya Iwata2, Hideki Kandori1 (1Nagoya Inst. Of
Technol., 2Ctr. Fost. Yng. Innov. Res., Nagoya Inst. Tech.,)LOV domain is a blue-light sensor domain that was first found in phototropin.
LOV domain binds a flavin mononucleotide (FMN) as a chromophore, and its
sensor function is initiated by the adduct formation with an S-H group of Cys. It
was proposed that the C/A-based mutant LOV domains (iLOV) could be better
fluorescent probe than GFP because of smaller size [1]. In such case, highly
fluorescent iLOV proteins are desired, where the oxidized FMN emits green
light. It should be however noted that FMN is a redox active cofactor, andphotoreduction of the oxidized form causes florescence quenching. In otherwords, this fact suggests that redox-sensitive fluorescent proteins may bedesigned from the C/A mutants of LOV domains. The purpose of this research is
to construct redox-sensitive probes using random mutagenesis into Arabidopsis
phot2-LOV2 C426A mutant. The candidate plasmids were transformed into
JM109 E. coli cells. The bacteria were grown in liquid media in the dark, andthe fluorescence quenching by light illumination was used as a screening
method to find desired proteins. Consequently, we found a mutant showing
faster photoreduction and lower fluorescence level compared to C426A.
Another mutant showed slower photoreduction than C426A. Interestingly, themutated amino acid residues were distant from FMN. Highly redox-sensitive
fluorescent proteins are searched by this method, and the molecular propertiesof the obtained mutant proteins will be discussed.
[1] Chapman S et al. PNAS USA 105, 20038 (2008)
2F1510クラミドモナス由来の全長フォトトロピンが示す光反応ダイナミク
スの時間分解検出

Time-resolved study on photoreaction dynamics of full-length
phototropin from Chlamydomonas reinhardtii
Yusuke Nakasone1, Koji Okajima2, Yusuke Aihara3, Akira Nagatani3, Satoru
Tokutomi2, Masahide Terazima1 (1Grad. Chem. Sci, Kyoto Univ., 2Grad. Sci,
Osaka Prefecture Univ., 3Grad. Biol. Sci, Kyoto Univ.)
Phototropin (phot) is a blue-light dependent kinase which regulates
phototropism of higher plants. It contains two LOV domains (LOV1 and LOV2)
as light sensor domains and adduct formations between their chromophores andCys residues upon light illumination are essential for the signal transduction. So
far, biophysical studies of phot has been restricted to shorter fragments such as
the LOV domains, LOV2-linker, and the LOV1-LOV2 tandem protein, becauseof the difficulty of purification of the full-length protein. Kinase activation ofphot has been studied in vitro by autophosphorylation assays, which, however,
did not provide any information about the signal transfer to the kinase after theactivation of LOV domains. In this study, we used full-length Chlamydomonas
phototropin (Cr-phot), which regulates sexual differentiation. In order tounderstand the mechanism of the kinase activation, we have investigated thereaction dynamics of full-length Cr-phot by using the transient grating (TG)method. The TG signal showed that the adduct formation was completed within
few microseconds, which is similar to the case of shorter constructs.
Additionally, we have detected global conformational changes of protein moiety
in milliseconds time scale as a decrease of diffusion coefficient. CD
measurements also showed that the secondary structure was partially changed
upon light illumination. We will discuss the photochemistry of intact protein on
the basis of these findings.
2F1522

N 末アミノ酸残基の有無は AppA BLUF ドメインの保存されたト
リプトファン残基の位置に影響しない

N-Terminal Truncation Does Not Affect the Location of aConserved Tryptophan in the BLUF Domain of AppA from
Rhodobacter sphaeroide
Masashi Unno1, Yuuki Tsukiji1, Kenshuke Kubota1, Shinji Masuda2,3(1Depart. Chem. Appl. Chem., Saga University, 2Center for BioRes. & Inform.,
Tokyo Inst. Tech., 3PRSTO, JST)The flavin-binding BLUF domains are a class of blue-light receptors, and AppA
is a representative of this family. Although the crystal and solution structures ofseveral BLUF domains have already been obtained, there is a key uncertainty− S48 −

regarding the position of a functionally important tryptophan (Trp104 in AppA).
In the first crystal structure of an N-terminally truncated BLUF domain of
AppA133 (residues 17-133), Trp104 was found in close proximity to flavin(Trpin), whereas in a subsequent structure with an intact N-terminus AppA126(residues 1-126), Trp104 was exposed to the solvent (Trpout). A recent study
compared spectroscopic properties of AppA126 and AppA133 and claimed that
the Trpin conformation is an artifact of N-terminal truncation in AppA133. In thisstudy, we compared the flavin vibrational spectra of AppA126 and AppA133 byusing near-infrared excited Raman spectroscopy. In addition, the conformations
as well as the environments of Trp104 were directly monitored by ultraviolet
resonance Raman spectroscopy. These studies demonstrate that the N-terminal
truncation does not induce the conformational switch between Trpin and Trpout.
2F1534

Substrate-Dependent Measurement Revealed Different
Protein Conformations on Photoactivation and DNA Repairof E. coli CPD Photolyase
I Made Mahaputra Wijaya1, Yu Zhang1, Junpei Yamamoto2, Kenichi
Hitomi3, Shigenori Iwai2, Elizabeth D. Getzoff3, Hideki Kandori1 (1Nagoya
Institute of Technology, 2Osaka University, 3The Scripps Research Institute, La
Jolla, USA)
Photolyases (PHRs) are flavoproteins that repair UV-induced damaged DNA by
the use of near UV/blue light. PHR that repair cyclobutane pyrimidine dimers(CPDs) and (6-4) photoproducts are called CPD-PHR and (6-4) PHR,
respectively. In order to study protein-mediated chemical reactions on PHRs,
light-induced difference FTIR spectroscopy was applied. Previously, we havesuccessfully measured FTIR difference spectra of the light-induced activation
and DNA repair of X. laevis (6-4) PHR [1] and E. coli CPD-PHR [2].
CPD-PHR can repair not only cyclobutane thymine dimer, but also cyclobutane
cytidine dimer and dimers from combination thymine-cytidine and vice versa.
In the present study, we introduced those different dimer compositions as DNA
substrates and observed FTIR light-induced activation and DNA repair spectraof CPD-PHR. From these substrate-dependent measurements, we successfully
observed different signals of peptide backbone, α-helices, and -sheets of the
enzyme, suggesting different protein conformations occurred during
photoactivation and photorepair, which depend on the substrates. In addition,
we also successfully observed difference spectra on binding of each different
substrate to CPD-PHR enzyme. Enzyme conformations difference ofphotoactivation, photorepair, and substrate-enzyme binding will be discussed on
the basis of the spectral comparison among each substrate.
[1] Zhang et al. Biochemistry 50, 3591 (2011)[2] Wijaya et al. to be submitted
2F1546

アニオンラジカル型の(6-4)光回復酵素による DNA 修復

Repair of damaged DNA by the anion radical form of (6-4)
photolyase
Daichi Yamada1, Yu Zhang1, Tatsuya Iwata2, Junpei Yamamoto3, Kenichi
Hitomi4, Shigenori Iwai3, Elizabeth Getzoff4, Hideki Kandori1 (1Nagoya Inst.
Tech., 2Ctr. Fost. Yng. Innov. Res., Nagoya Inst. Tech., 3Grad. Sch. Eng. Sci.,
Osaka Univ., 4The Scripps Res. Inst. USA)
Photolyases (PHRs) are repair enzymes of UV‒damaged DNA by utilizingnear‒UV/blue lights, which maintain genetic integrity by reverting UV‒induced
photoproducts into normal bases. The FAD chromophore of PHRs has four
different redox states: oxidized (FADox), anion radical (FAD·‒), neutral radical(FADH·) and fully reduced (FADH‒) forms, and the enzymatically active stateof PHR is FADH‒. Mechanism of photoactivation from FADox to FADH‒ byproton‒transfer coupled electron transfer through FAD·‒ and FADH· isintriguing, and we recently obtained difference FTIR spectra among four redox
states of Xenopus (6‒4) PHR by selecting temperature and illumination
conditions [1]. According to the FTIR analysis, the protein structure of the
FAD·‒ form trapped at 200 K is very similar to that of the FADH‒ form,suggesting that FAD·‒ might have the DNA repair activity. To test thishypothesis, we attempted to stabilize the FAD·‒ state at room temperature, byintroducing the N392C mutant that mimics insect cryptochromes andcyanobacterial cryptochrome mutant [2, 3]. The FAD·‒ form is observed even atroom temperature, and FTIR spectroscopy detects possible photorepair signalsof (6‒4) photoproduct by the FAD·‒ form. Activation and repair mechanism of
the N392C mutation of (6‒4) PHR will be discussed on the basis of the present
spectroscopic observations.
[1] Yamada D. et al, submitted.
[2] Kao Y.-T. et al, J. Am. Chem. Soc. 130, 7695‒7701 (2008).

[3] Iwata T. et al, Biochemistry 49, 8882‒8891 (2010).
2F1558光センサータンパク質 UVR8 の解離・回復反応カイネティックスの測定

The study of the dissociation and recovery reaction kinetics forphoto-sensor protein UVR8
Takaaki Miyamori1, Yusuke Nakasone1, Kenichi Hitomi2, John M. Christie3,
Elizabeth D. Getzoff2, Masahide Terazima1 (1Grad. Sci. Chem., Univ.
Kyoto, 2Scripps Research Inst., 3Univ. Glasgow)
Plants can perceive and respond to UV-B in order to protect them from UV-light.
UVR8 from Arabidopsis has been found as a UV-light sensor protein, whichmediates gene expression responses to establish the UV protection. Dimers of
UVR8 are dominant under dark condition in the living cells. Doubly hydrogenbonded salt bridges, which link R286 with D107 and R146 with E182 is thought
to make the dimer stable. Upon UV-B light illumination, the dimer dissociates
into the monomer, and this dissociation reaction is believed to be important forthe biological function. Generally, light sensor proteins possess specificchromophore molecules to absorb light and initiate photochemical reactions for
signal transduction. Interestingly, however, the UVR8 protein employs Trp
residues, particularly W285 to sense the environmental light condition instead of
having such specific chromophores. We investigate the kinetics of its dissociation
reaction by the time-resolved transient grating method (TG method). In order toexamine the importance of above key residues (R286, R146, W285), we
performed the same kinetic measurements for the following mutants; R146A/
R286A and W285F. Furthermore, the dark recovery reaction (dimerization) was
studied by the circular dichroism method (CD method).
2G1400

膜融合におけるストーク構造からの変形：分子動力学シミュレーション

Morphology change of fusion stalk studied by molecular
dynamics simulation
Shuhei Kawamoto, Wataru Shinoda (Nano. AIST)The life cycle of living organisms involves membrane fusion. In eukaryotes,
membrane fusion is ubiquitous. As a plausible fusion pathway, the stalk-pore
pathway has been identified and explored theoretically. However, stability oftransiently appeared morphologies and driving force of the morphology change
are not clear yet. We have investigated the fusion pore formation processfrom the stalk structure by coarse-grained molecular dynamics simulations. We
found that the stalk was stabilized when the lipid membranes containphosphatidylethanolamine (PE), which are more likely found around the stalk.
By applying tension selectively on the proximal leaflet, we observed theprogress of pore formation. The energy required for the fusion pore formation isestimated as about 50 kB T, which is close to that obtained from the previous
theoretical study.
2G1412蛍光検出と電気計測による膜タンパク質計測に向けた脂質膜チャン
バアレイ

Lipid bilayer chamber array toward fluorescent andelectrophysiological measurement of membrane proteins
Taishi Tonooka1, Ryuji Kawano2, Koji Sato1, Toshihisa Osaki2, Shoji
Takeuchi1 (1Institute of Industrial Science, The University of Tokyo,
2
Kanagawa Academy of Science and Technology)
Artificial lipid bilayer chamber, which is a small chamber sealed with lipid
bilayer, is an attractive tool for membrane protein analysis because of its purity.
Previous studies of artificial lipid bilayer chambers for membrane protein
analysis had been focused on two main streams: one was for fluorescentmeasurement, the other for electrophysiological measurement. Recently, thedevices for two-way measurement of fluorescent imaging andelectrophysiological recording have been developed. These devices allow us tocombine the information of channel conformational change resulting inelectrical signals and molecular interaction between channels and ligands.
Therefore, they have potential to demonstrate novel membrane protein
properties. Here, we propose an array system leading to multi-channel two-way
measurement as a next challenge for high-throughput membrane protein
analysis. In this study, we successfully demonstrated the function of the poreforming membrane protein, α-hemolysin, by electrophysiological measurement,
and succeeded in Ca2+ ion concentration measurement in the chambers by using
Ca2+ ion fluorescent indicators. We believe that this technique will be a strong
tool for basic research or drug screening of membrane proteins.

− S49 −

2G1424

蛋白質毒素ライセニンがスフィンゴミエリンを含む脂質膜中に誘起するポア形成： 単一 GUV 法による研究

Single Giant Unilamellar Vesicle Method Reveals LyseninInduced Pore Formation in Lipid Membranes Containing
Sphingomyelin
Jahangir Md. Alam1, Toshihide Kobayashi2, Masahito Yamazaki1 (1Int.
Biosci. Sec., Grad. Sch. Sci. Tech., Shizuoka University, 2Lipid Bio. Lab.,RIKEN)
Lysenin is a sphingomyelin (SM) -binding pore-forming toxin. To reveal
the interaction of lysenin with lipid membranes, we investigated lysenininduced membrane permeation of a fluorescent probe, calcein, throughdioleoylphosphatidylcholine (DOPC) /SM, DOPC/SM/cholesterol (chol), andSM/chol membranes, using the single giant unilamellar vesicle (GUV) method1.
The membrane permeability coefficient of calcein increased over time to reach amaximum, steady value, Ps, which persisted for a long time (100-500 s),
indicating that pore concentration increases over time and finally reaches its
steady value. The Ps values increased as the SM/lysenin ratio decreased, and at
low concentrations of lysenin the Ps values of SM/DOPC/chol (42/30/28) GUVs were much larger than those of SM/DOPC (58/42) -GUVs. The
dependence of Ps on the SM/lysenin ratio for these membranes was almost thesame as that of the fraction of sodium dodecyl sulphate (SDS)-resistant lysenin
oligomers. On the other hand, lysenin formed pores in GUVs of SM/chol(60/40) membrane in homogeneous liquid-ordered phase, indicating that thephase boundary is not necessary for the pore formation. The Ps values of
SM/chol (60/40)-GUVs were smaller than those of SM/DOPC/chol (42/30/28)GUVs even though the SDS-resistant oligomer fractions was similar for both themembranes, suggesting that there are irreversible oligomers (or pre-pores)
before conversion to pores. Based on these results, we discuss the elementary
processes of lysenin-induced pore formation.
1. Biochemistry, in press
2G1436

人工細胞ベシクルにおける脂質ドメインへの化合物局在を介したドメインサイズと膜崩壊率の制御

Localization of bulky-molecules in raft-like domains on phaseseparated liposomes: Control of domain size and bursting rateof liposome
Miho Yanagisawa1, Damien Baigl2, Kenichi Yoshikawa3 (1Grad. Sch. Sci.,
Kyushu Univ., 2Depart. Chem., Ecole Normale Superieure, 3Depart. Biomed.
Info., Doshisya Univ.)
It is established that the cell-sized liposomes composed of a saturated lipid
DPPC, an unsaturated lipid DOPC, and cholesterol (Chol) show phaseseparation below the transition temperature, and form domain structures likelipids rafts [1]. These micro-domains grow up to mono-domain, so as tominimize interfacial instability. By applying the phase separation of lipids, wereport two control methods, i.e., a size-control of domains by the addition ofpoly(ethylene glycol)-conjugated Chol (PEG-Chol) [2], and a rate-control of
UV-induced bursting of liposomes by the addition of Azo-TAB molecules [3].
As for the domain-size control, dispersed micro-domains <1m were observedin the presence of PEG-Chol above a critical composition. The transition fromglobal- to micro-segregation is interpreted in terms of the competition between
steric repulsive interaction between the bulky-head groups of PEG-Chol and thecost in line energy along the domain boundaries. On the other hand, the additionof Azo-TAB into the membrane expands it and induces bursting of liposomes
due to the UV irradiation. Furthermore, the bursting rate was controlled by thearea fraction of domains rich in DPPC. These achievements lead us to further
understanding the lipid raft structure from physicochemical view.
[1] M. Yanagisawa et al., 2007 Biophys. J. 92:115.
[2] M. Yanagisawa, et al., 2012 Soft matter, 8:488.
[3] A. Diguet, et al., 2012 J. Am. Chem. Soc., 134:4898.
2G1448

PI4P と脂質膜曲率による Kes1 ステロール輸送活性の制御
Regulation of Kes1 sterol transport activity by PI4P and lipid
membrane curvature

Hirokazu Yokoyama1, Masaki Wakabayashi1, Yasushi Ishihama1, Minoru
Nakano2 (1Graduate School of Pharmaceutical Sciences, Kyoto
University, 2Graduate School of Medicine and Pharmaceutical Sciences,
University of Toyama)

Kes1 is one of seven members of the yeast Oxysterol binding protein homolog(Osh) family. This protein transfers sterols between plasma membrane and theGolgi, and regulates Golgi secretory function. However, these detailed
mechanisms have not been well understood. In this work, we investigated theeffect of lipid membrane state (lipid composition and membrane curvature) on
Kes1 activities (sterol extraction and transport) by fluorescence resonance energy
transfer. Especially we focused on phosphatidylinositol 4-phosphate (PI4P)
because PI4P is considered to stimulate Kes1 sterol transport activity. Effects ofmembrane curvature were evaluated by using liposomes with different size. For
planar membranes, while two anionic lipids, phosphatidylinositol andphosphatidylserine, had little effect on the Kes1 activities, PI4P was found toaffect on the Kes1 activities depending on PI4P amount: Although a small amount(1 mol%) of PI4P stimulated sterol extraction and transport by Kes1, larger
amount (~10 mol%) of PI4P inhibited the Kes1 activities. This sterol extraction
inhibitory effect of PI4P was dependent on the membrane curvature. That is, even
1 mol% of PI4P inhibited the sterol extraction from highly curved membranes,suggesting that the membrane curvature regulates the effect of PI4P. Thesefindings suggest a possibility that PI4P and the membrane curvature areregulating factors of Kes1 biological function.
2G1510リン脂質輸送タンパク質 Sec14 の脂質輸送メカニズムの解明

Elucidation of lipid transfer mechanism of phospholipid
transfer protein Sec14
Chisato Takahashi1, Makiko Yamada1, Masaki Wakabayashi1, Yasushi
Ishihama1, Minoru Nakano2 (1Grad. Sch. Pharm., Kyoto Univ., 2Grad. Sch.
Pharm., Toyama Univ.)
Sec14, a major yeast phosphatidylinositol/phosphatidylcholine transfer protein(PITP) regulates lipid membrane trafficking from the trans-Golgi network.
However, its detailed mechanism remains to be elucidated. In this work, weinvestigated PITP-membrane interactions by surface plasmon resonance (SPR)
and fluorescence spectroscopy. Sec14 WT and two mutants, which have been
designed to ablate their phosphatidylcholine (PC)-binding ability (Sec14 II) andphosphatidylinositol (PI)-binding ability (Sec14 AD) were used and compared.
In the fluorescence spectroscopy, capture of pyrene-labelled PC by PITPs fromlarge unilamellar vesicles (LUVs) was detected by the decrease in the pyrene
excimer/monomer intensity ratio. The results showed that Sec14 WT and its
mutants, including PC-binding mutant (Sec14 II), have PC transfer activity. In
the presence of PI, however, PC transfer activity for Sec14 II was reduced,
suggesting that Sec14 II has higher affinity for PI.
Binding behavior of PITPs to immobilized LUVs was evaluated by SPR
method. Sec14 II exhibited higher binding to PC membrane than Sec14 WT and
AD, which have intact PC binding pocket. On the other hand, membranebinding of Sec14 II was decreased in the presence of PI, Ligand for this mutant.
These findings that Sec14 represents reduced affinity to the membranes that
contain ligands for the protein suggest the ingenious lipid transfer mechanism,
by which Sec14 dissociates from the membrane when it captures PC or PI in
appropriate position of the binding pocket.
2G1522

ゲル、アミノ酸、水素化アモルファスシリコンを用いた光制御イオン
伝導整流素子Photo-controlled ion conductive rectification element using gel,amino acids and hydrogenated amorphous silicon film
Takaaki Ichikawa1, Hiroki Suzuki1, Ryohei Matsueda1, Hiroshi Masumoto2,
Takashi Goto3, Yutaka Tsujiuchi1 (1Department of Material Science andEngineering, Akita University, 2Center for Interdisciplinary Research, Tohoku
University, 3Institute for Materials Research, Tohoku University)
Ionic conduction, in solution as well as in intermediate states between a liquidand a solid, such as gels, is a phenomenon key to inter-conversion between lightenergy, chemical energy, and electric energy. As such, this phenomenon isessential for devices that store, generate, and convert electric energy, including
batteries, photovoltaics, capacitors, and actuators. While not dismissing theseapplications, this article focuses on ionic conduction in nontoxic materials to
discuss the creation of elements coated with multi-layer functional thin-films
using a combination of different technologies, such as the rectification propertyof ionic conduction, photo-induction control, and the use of wavelength
conversion materials, and the potential application of these materials tobiological tissue. Amino acid is the elementally element of bio molecule, has
potential of diversity to electro chemical device. Our group has been working
with amino acids dispersed in agarose gel, and with semi-conductive material,hydrogenated amorphous silicon (a-Si: H) film. In previous report, ion− S50 −

conductive behavior of neutral, acidic, or basic amino acid dispersed in agarose
gel are characterized. In this conference, we report an attempt of measuring aphoto-controlled effect of a-Si: H film on ion conducting behavior of biomolecules in detail, comparing several hydrogen concentrations that used inprocess gas for preparation of a-Si:H.
2G1534

高速 AFM による CFTR チャネルの動態観察

Single molecular observation of CFTR channels by high speed
AFM
Hayato Yamashita1, Kazuhiro Mio2, Muneyo Mio2, Takayuki Uchihashi3,
Masato Yasui1, Toshio Ando3, Yoshiro Sohma1,4 (1Pharmacol., Keio Univ.
Med. Sch., 2AIST, 3Dept of Physics, Kanazawa Univ., 4Dalton Cardiovas. Res.
Cen., Univ. Missouri-Columbia)
Cystic Fibrosis Transmembrane conductance Regulator (CFTR) chloride
channel, a member of ABC transporter superfamily, gates following ATPdependent conformational changes of the nucleotide binding domains.
Mutations in the CFTR gene cause cystic fibrosis in which salt, water andprotein transports are defective in various tissues. Channel function of CFTR
has been mainly studied by measuring ionic current going through the poreusing the patch-clamp technique, which has given us many important findings
about CFTR dysfunction. On the other hand, recent advances in X-ray
crystallography provide atomic-level structures for several bacterial andmammalian ABC transports. However, neither the electro-physiology nor the
crystal structure can give us the information about the molecular dynamic
processes of CFTR proteins.
In this study, we applied the high speed atomic force microscopy (HS-AFM) to
image dynamic structural changes and interactions occurring in individual
CFTR molecules. The HS-AFM visualized a dimeric formation of DMMsolubilized, purified WT-CFTR molecules attached on the stage over sideways.
Next we attempted to observe the PKA-dependent phosphorylation process ofRegulatory (R)-domain in CFTR. With applying a solution containing PKA
catalytic subunits, we confirmed many small molecules appeared around CFTR
and occasionally bound to the bottom of CFTR molecules where R-domain
was supposed to be located. Now we are attempting to visualize thephosphorylation-induced conformational change of the CFTR R-domain.
2G1546

KcsA チャネルの細胞内ドメインは不活性化に影響を与える
The KcsA channel cytoplasmic domain effects on theinactivation gating

Minako Hirano1,2, Yukiko Onishi2, Daichi Okuno2, Toru Ide1,2 (1GPI, 2RIKEN)The KcsA channel is a representative potassium channel that is activated bychanges in pH. Recently, we found that the cytoplasmic domain (CPD) acts as a
pH-sensor by observing significant confirmation changes there in response to
pH. These changes can influence the opening and closing of the KcsA channel.
A selectivity filter region within the KcsA channel is also known to regulate
gating by forming activation and inactivation states. However, it is not clear
how the CPD and the selectivity filter region coordinate. We therefore made amutant channel that has the wild-type filter and a CPD with all its negative
charges neutralized. We found that this mutant had high activity independent of
pH and no inactivation gating, indicating removal of negative charges in the
CPD causes removal of inactivation. These results suggest that the CPD
primarily regulates activation gating, and conformational changes of the CPD
effects on the inactivation gating.
2G1558原子間力顕微鏡によるカリウムイオンチャネル KcsA の脂質膜中でのゲート開閉構造の直接観察

Direct Observation of Ion Entryway of Potassium Channel
KcsA in Lipid Bilayer by Atomic Force Microscopy
Ayumi Sumino1, Takashi Sumikama2, Masayuki Iwamoto2, Takahisa Dewa1,3,
Shigetoshi Oiki2 (1Grad. Sch. Eng., Nagoya Inst. Tech., 2Deprt. of Mol.
Physiol. and Biophys., Univ. of Fukui Fac. Med. Sci., 3JST-PRESTO)
The crystallography has provided high resolution structure of channel proteins,
whereas they are mostly in the absence of the lipid bilayer, and even some parts
are remained unsolved. For the open conformation of the KcsA potassiumchannel, structure of the intracellular entryway is still elusive, even though it iscritical to understand the ion permeation mechanism. Here, we examined thenative transmembrane structure of the KcsA channel embedded in themembrane using atomic force microscopy (AFM). To observe the ion entryway
from the intracellular side, the cytoplasmic domain (CPD) was truncated. In the

open conformation, the individual subunits in the tetramer with the pore open at
the center were resolved. The measured longitudinal length is longer than that of
crystallography, which indicates that AFM successfully captured the unresolved
structure of the terminal parts. The crystallographycally-missed terminal helix(bulge helix) was grafted to the pore entrance using the molecular dynamics
simulation, which demonstrated that the bulge helices fluctuate vigorously. The
single-channel currents for the CPD-truncated channel exhibited fluctuating
currents, indicating that the helices chop up the steady ionic currents. In thisstudy an integrated approach, involving AFM measurements, MD simulationand single-channel current recordings, revealed the dynamic feature of the ion
entryway of the KcsA channel.
2G1610

全反射赤外分光法による KcsA のイオン選択フィルターの振動解析

The Vibrational Analysis of the Selectivity Filter of KcsA byusing ATR-FTIR Spectroscopy
Yuji Furutani1,2, Hirofumi Shimizu3, Yusuke Asai1, Tetsuya Fukuda1,
Shigetoshi Oiki3, Hideki Kandori1 (1Grad. Sch. Tech., Nagoya Inst. Tech., 2Inst.
Mol. Sci., 3Facul. Med. Sci., Univ. Fukui)
KcsA is a potassium ion channel, which selectively permeates K+ ions. The Xray structure of KcsA showed that the carbonyl oxygens of the selectivity filter(TVGYG) interacts with K+ or Na+ ions. Distribution of ions in the selectivity
filter and interactions between ions and carbonyl oxygens are crucial tounderstand the role of the selective filter in KcsA channel. To address thisquestion, we used attenuated total reflection Fourier-transform infrared (ATRFTIR) spectroscopy which has been recently utilized for the study of the ionprotein interaction1.
Two solutions containing potassium (x mM; x =3, 10, 20, 50, 200) and sodium(200-x mM) cations or only sodium cation (200 mM) were alternately flowed
through the KcsA/liposome samples on the ATR cell, which provided ionexchange induced difference FTIR spectra at pH 7. The up-shift of the amide I
vibration upon binding of potassium ions were observed, which means that the
carbonyl groups in the amide groups in the KcsA(K+) has weaker interactions
than those in the KcsA(Na+). From the spectral comparison of the wild-type andthe Y78F mutant proteins, we concluded that the amide-I vibrations originate
from the C=O groups in the selectivity filter2.
(1) Y. Furutani, T. Murata, H. Kandori, J. Am. Chem. Soc. 133 (9), 2860-3,
2011.
(2) Y. Furutani, H. Shimizu, Y. Asai, T. Fukuda, S. Oiki, H. Kandori, submitted.
2H1400

中性子散乱による心筋症関連トロポニン変異体のダイナミクス測定

Dynamics of cardiomyopathy-causing mutant of troponinobserved by neutron scattering
Tatsuhito Matsuo1, Francesca Natali3, Giuseppe Zaccai2,3, Satoru Fujiwara1(1QuBS, JAEA, 2CNRS, 3ILL)
Troponin (Tn) is a protein that consists of three subunits (C, I, and T) andregulates the muscle contraction depending on the intracellular Ca2+
concentration. K247R mutation of TnT is known to cause the familial
hypertrophic cardiomyopathy. For understanding the molecular mechanism ofpathogenesis of cardiomyopathy, it is important to elucidate the relationshipbetween differences in dynamics (thermal fluctuations of a protein) and functionof troponin caused by mutation because dynamics of a protein is crucial for its
function.
To detect possible changes in dynamics of troponin caused by mutation, elastic
incoherent neutron scattering experiments were carried out on solution samplesof the wild type (80 mg/ml), and K247R mutant (40 mg/ml) in the D2O buffer
containing Ca2+, at the thermal neutron backscattering spectrometer IN13 in theInstitut Laue-Langevin (Grenoble, France), at temperatures between 280 K and
292 K with an interval of 3 K.
Root mean square displacements (rmsd) of hydrogen atoms in the protein wereobtained from the spectra at each temperature. From the slope of the rmsd withrespect to the temperature, force constants (<k>), which reflect the resilience ofthe protein, were calculated. The < k > values for the wild type and K247R
mutant were 0. 077 ± 0. 035 N/m and 0. 046 ± 0. 026 N/m (mean ± s. d.),respectively. This suggests that the disease-causing mutant is more flexible than
the wild type. The large flexibility might modulate Ca2+ signal transmission
mechanism, leading to the functional aberration.
2H1412− S51 −

スピンラベル ESR 距離測定による細いフィラメントのアクチン、
トロポミオシン、トロポニンの動的構造解析

Structural dynamics of actin, tropomyosin, and troponin in thethin filament as studied by distance measurements using spinlabeling ESR
Keisuke Ueda1,2, Chenchao Zhao1, Akie Yamamoto1, Takayasu Somiya1,
Tomoki Aihara1,3, Shoji Ueki4, Masao Miki5, Toshiaki Arata1 (1Grad. Sch.
Sci., Osaka Univ., 2Inst. Prot. Res., Osaka Univ., 3Harima Inst., Riken,
4
Tokushima-Bunri Univ., 5Univ. Fukui)
We can measure the distance between spin labels on protein complex on
nanometer scale by cw and pulse ESR (1-3). Using pulse ESR for reconstituted
thin filament we previously found that the regulatory-switch region of TnI
moves by ~4 nm between TnC and presumably actin (4). The heart beating iscontroled by phosphorylation of cTnI. Recently we measured the interspin
distance between cTnC and regulatory-switch region of cTnI by cw-ESR, andfound that cTnI binding to cTnC was loosened by PKA or PKC phosphorylation
which may cause Ca2+ desensitization. We next examined how tropomyosin(Tm) and actin undergo structural changes in the thin filament in response toCa2+. The side-chain mobility of a spin label on the 1-284 a.a. of Tm showed
unexpectedly no Ca2+ effect (5). The distance between Cys374 and Mn2+ within
actin monomer also increased by ~0.2 nm when actin filament was decorated
with Tm-Tn with and without Ca2+. Then, 14 N-based spin label bound to
Cys374 of each actin whereas 15N-label bound to a cysteine at several positionsof Tm. Distance was successfully determined by fitting the cw-spectrum
obtained after 14N-label spectrum of excess actin was cancelled out for distinct
spectral characteristics of 14N- and 15N-labels. Ca2+ effects on the distance wererestricted at the N-C joints of Tm. More spin-labeling points are needed for final
conclusion. 1.Ueki et al. Biochem. 44 411(’05). 2. Nakamura et al. JMB 348
127(’05). 3. Abe et al. AMR 42 473(’12). 4. Aihara et al. JBC 285 10671(’10).
5. Ueda et al. Biophys J 100 2432(’11).
2H1424

アクチン周りのハイパーモバイル水の形成とその熱力学的考察

Formation of hyper-mobile water around actin and its
thermodunamic considerations
Makoto Suzuki, George Mogami, Tetsuichi Wazawa, Asato Imao, Noriyoshi
Ishida (Tohoku Univ. Grad. Sch. Eng.)This paper introduces recent studies on the hyper-mobile water around actin in Gand F- forms. Microwave dielectric relaxation spectroscopy (DRS). The DRS
measurements showed that F-actin exhibits dual hydration properties as astructure-maker and a structure-breaker. Thus, in addition to the water moleculeswith lowered rotational mobility that form a constrained hydration shell aroundthe F-actin, there exist water molecules with rotational relaxation frequencies
higher than those of bulk water, which are here referred to as hyper-mobile water(HMW). HMW was detected in G-actin solution, and significantly increased uponpolymerization into F-actin. Formation of HMW was investigated by measuring
the temperature dependence of the dispersion amplitudes of HMW andconstrained water. The van’t Hoff enthalpies of those water from the bulk were
estimated. The results indicate that formation of HMW is endothermic and that ofconstrained water is exothermic. Isothermal titration calorimetry experiments
were performed to measure the exothermic heat of depolymerization of F-actin in
G-buffer. These results will be reported at the meeting.
2H1436

アクチン様トレッドミリングモータが満たすべき最小条件

Minimum requirements for the actin-like treadmilling motor
system
Akihiro Narita (Grad. Sch. Sci., Nagoya Univ.)
Actin is one of the most abundant proteins in eukaryote cells, which forms adouble stranded filament. The actin filament is not only a main component of
the cytoskeleton, but also acts as a motor protein which moves toward one
specific end, the barbed end, driven by polymerization at the barbed end anddepolymerization at the other end, the pointed end, without any associatedproteins. This motor activity is referred to as “treadmilling” and it represents thesimplest motor system known, consisting of only one 42 kDa protein, actin. The
mechanisms of molecular motors are not fully understood, although many
motors have been identified, including myosins, kinesins, dyneins and F1ATPase. Therefore, knowing the minimum requirements of the actin-like motor
system may help us to understand not only actin-like motors but also many othermotors. We clarified there are three requirements for the treadmilling motor
system by computer simulations: 1) Nucleotide binding and ATPase activity inthe filament; (2) Polarity in the rates of polymerization and depolymerization
between the two ends; and (3) The dependence of the subunit-subunit
interactions on the bound nucleotide. These requirements are simple and thisknowledge should facilitate the development of artificial molecular motor
systems in the future.
2H14483 次元トラッキングによる in vitro アクチンフィラメントモーティ
リティーアッセイ

Three-dimensional tracking of gliding actin filament in an invitro motility assay
Tatsuya Naganawa, Togo Shimozawa, Tomoko Masaike, Takayuki Nishizaka(Gakushuin Univ.)
In an in vitro motility assay of actin filament (AF) and skeletal myosin system, it
has been reported that the AF rotated around filament axis during its sliding
motion. However, the previous two researches had reported the rotation in
oposite direction by each detection method and assay system (Nishizaka et al.,Nature; 1993; Beausang et al., Biophys J., 2008). Thus, the mechanisms of the
AF rotational motion have been controversial. On the other hand, microtubule
rotation accompanied with sliding motion was revealed by three-dimensional
Prismatic Optical Tracking, termed tPOT (Yajima et al., Nat. Struct.Mol.Biol.,2008). Applying tPOT to AF sliding assay expects to contribute for further
information about the AF rotation machanisms.
Actin and heavy meromyosin were purified from rabbit skeletal muscles.
To apply tPOT to AF gliding assay, avidin conjugated quatum-dots (QD)
were sparsely attached to the AF containing ˜10 % of Cys-374 biotinylated actin(QD-AF), and the QD-AF was stablized and visualized by rhodamine phalloidin(RhPh).
Similar sliding velocities of QD-AF (6.3 µm s-1, n = 8) and AF labeled with
only RhPh (6.2 µm s-1 , n = 5) (ATP 2 mM , 25 °C) suggested that the attached
QD did not disturb intact sliding motion. We measured three-dimesional
discplacement of QD in sliding QD-AF in various conditions, and the possible
mechanisms of the AF rotation will be discussed at the meeting.
2H1510Ca2+に依存しない熱パルスによる心筋細胞の On-Off 制御

Ca2+-independent on-off regulation of a cardiomyocyte bymicroscopic heat pulses
Kotaro Oyama1, Akari Mizuno1, Seine Shintani1, Hideki Itoh1, Takahiro
Serizawa1, Norio Fukuda2, Madoka Suzuki3,4, Shin’ ichi Ishiwata1,3,4 (1Sch.
Adv. Sci. Eng., Waseda Univ., 2Dept. Cell Physiol., Jikei Univ. Sch.
Med., 3Org. Univ. Res. Initiatives, Waseda Univ., 4WABIOS, Waseda Univ.)
Laser irradiation on neural and cardiac tissues has developed into a novel
technique of non-invasive stimulation. The physical parameters that induce
contraction of cardiomyocytes and a heart are under discussion, because the laser
irradiation triggers various reactions, such as photochemical and photothermal.
Here we focused on the laser-induced local temperature changes. We
demonstrated previously that a microscopic heat pulse (ΔT = 0.2°C for 2 sec)
induces a Ca2+ burst in cancer cells (HeLa cells) at body temperature (Tseeb, V.,et al., HFSP J. , 2009). The mechanism is thought to be similar to that of rapid
cooling contracture of skeletal and cardiac muscles. In this study, we show that a
microscopic heat pulse (ΔT = 5°C for 0.5 sec) generated by a focused infraredlaser induces the contraction of rat adult cardiomyocyte at basal temperature of
36°C. ΔT required to induce the contraction of cardiomyocyte at 36°C was lower
than at 25°C. Contrary to a regular muscle contraction, Ca2+ transients were notdetected during the contraction. Also, we induced the contraction of skinned
cardiomyocytes by heat pulse in Ca2+-free solution. These results indicate that aheat pulse regulates the contraction without the involvement of Ca2+ dynamics,i.e., the microheating stimulation skips several steps relating to Ca2+ signaling in
the excitation-contraction coupling and directly activates actomyosin interaction.
Our method may be useful for stimulating the beating of failing hearts without
exacerbating abnormal Ca2+ dynamics.
2H1522

ミオシン分子の非線形弾性とフィラメントの弾性変形を考慮した筋収縮のシミュレーション

The Effects of Nonlinear Elasticity of Myosin Molecules on
Muscle Contraction Studied by Numerical Simulation
Keita Miyamoto, Kazuo Sasaki (Dept. Appl. Phys., Sch. Eng., Tohoku Univ.)
The elasticity of myosin molecules is responsible for the force (tension)
generation in muscle contraction. In traditional theories of muscle contraction,
researchers have assumed that the displacement of a myosin head relative to the
myosin filament is proportional to the force applied to the head along thefilament. However, a recent experiment by Kaya and Higuchi [M. Kaya and H.

− S52 −

Higuchi, Science 329, 686 (2010)] clearly showed that the elasticity of myosin
is strongly nonlinear. In a previous work (Biophysical Society Meeting in
Himeji, 2011) we studied the quick response of tension to the length change of amuscle fiber and the force-velocity relation in isotonic contraction based on thetheory proposed by Duke [T.A.J. Duke, Proc. Natl. Acad. Sci. USA 96, 2770
(1999)] with the linear elasticity of myosin replaced by a nonlinear elasticity. It
turned out that we have to set the (nonlinear) elastic constant of myosin
molecule to about one forth of the value obtained by Kaya and Higuchi toaccount for the experimental results. A plausible reason for the discrepancy isthat actin and myosin filaments are treated as rigid rods in our model as in theDuke model. In the present work, we will carry out numerical simulations taking
account of the elasticity of actin and myosin filaments as well as the nonlinear
elasticity of myosin molecules to reveal whether we can resolve theinconsistency between the values of elastic constant in the simulation and theexperiment.
2H1534

高塩濃度溶液中における熱変性したミオシン分子のミオシンフィラ
メントへの付着Adhesion of heat-denatured myosin molecules to myosin
filament at high salt concentration
Masato Shimada1, Eisuke Takai1, Daisuke Ejima2, Kentaro Shiraki1 (1Facul. ofPure and Appl. Sci., Univ. of Tsukuba., 2Institute for Innovation, Ajinomoto Co.
Inc.)
Myosin filament is formed from myosin molecules mainly by electrostaticinteraction. Thus, myosin filament disassociates in salt solutions at above 0.3 M.
However, it may be questioned whether the interaction is completely damped out
even at the high salt concentration. To explore the question, we investigated the
myosin filaments at various salt concentrations by dynamic light scattering (DLS)
and transmission electron microscopy (TEM). Myosin from porcine was welldissolved in 1.0 M NaCl solution. Then the myosin solution was diluted to various
salts concentration. The myosin filament with hydrodynamic radius of 200 nm wasdissolved to myosin molecules with that of 60 nm monitored by DLS, which was
not fully disassociated with increasing temperature and salt concentration. The sizeof myosin filament gradually increased with heating at above 55 °C . Interestingly,
the heat-denature myosin molecules adhere onto myosin filaments observed by
TEM. These data suggest that the adhesion above the denaturation temperature of
myosin decreased with increasing salt concentration through hydrophobic
interaction. This experimental system could quantitatively analyze the kinetics ofmyosin filament-myosin molecule interaction.
2H1546

ラット幼若心筋細胞内サルコメア集団の自励振動(SPOC)特性

Auto-oscillation (SPOC) properties of sarcomeres in rat
neonetal cardiomyocytes
Seine Shintani1, Kotaro Oyama1,2, Norio Fukuda2, Shin'ichi Ishiwata1,3 (1Sch. Adv.
Sci. Eng., Waseda Univ., 2Dept. Cell Physiol., Jikei Univ. Sch. Med., 3WABIOS,
Waseda Univ.)
Auto-oscillations of sarcomere length (SL) occur at intermediate activation
conditions in cardiac and skeletal muscles. The waveform of sarcomeric autooscillation usually consists of slow shortening and quick lengthening. We havetermed this phenomenon SPOC. SPOC has properties of auto-oscillation at
single sarcomere level and synchronous dynamics of sarcomeres, e.g.,
propagation of SPOC wave. Therefore, observation of SPOC allows us tounderstand the dynamic properties of sarcomeres. In order to fully understandthe molecular mechanism of SPOC, we examined neonatal rat cardiomyocytes
expressing GFP-α-actinin and visualized the motions of sarcomeres. First, we
successfully induced SPOC in the neonatal myocytes with the intact innermembrane system following treatment with ionomycin by controlling thecytoplasmic Ca2+ concentrations. And we have termed this phenomenon CellSPOC. Our analysis of the Cell-SPOC revealed that the SPOC period of SL
oscillations (in the range of 0.2 s to 1 s) increased with the increase in theamplitude of SL oscillations. As the amplitude became smaller, the shortening
velocity tended to become larger, especially in the range of 0.2 to 0.5 s of the
SPOC period. We also found that the oscillation properties of the Cell-SPOC
and those of intact cells activated by electrical stimulation became similar as theoscillation period approaches to 0.2 s from 1 s. Based on these results, we will
discuss the similarities and differences between the Cell-SPOC and theoscillations of intact cells.
2H1558横紋筋サルコメアのフィラメント格子の安定性

Stability of Myofilament Lattice in Striated Muscle Sarcomere
Shigeru Takemori1, Masako Kimura2, Maki Yamaguchi1, Tetsuo Ohno1,
Naoya Nakahara1, Shunnya Yokomizo3 (1Jikei Univ. Sch. Med., 2Kagawa
Nutrition Univ., 3Tokai Univ. Graduate Sch. Physical Education)
Removal of cell membrane from striated muscle cells to prepare skinned muscle
specimens not only increases inter-myofilament spacing but also destabilize
lattice structure of myofilaments. Takemori et al (2007) revealed that thisdestabilization is accompanied by diffusion of macromolecular solutes out ofinter-myofibrillar space. Therefore, we expected that exogenous macromoleculesthat penetrate into inter-myofibrillar space may recover the stability. Adopting a
set of myosin layer lines on the two dimensional X-ray diffraction patterns(obtained at BL-6A of KEK and at BL-45XU of SPring8) as one of the indicatorsof lattice stability, we found that 3% polyethylene glycol molecules (Mw less than3, 360) that penetrate into myofilament lattice restore myosin layer lines ofskinned specimens of striated (rabbit skeletal and rat cardiac) muscles.
Polyethylene glycol also dehydrated the skinned specimens to restore
physiological inter-filament spacing. Since the dehydrating effect mainly depends
on the concentration of hydrophobic -CH2 - unit in the solution (Kimura andTakemori, 2008), we consider this stabilizing effect of polyethylene glycol tosupport our hypothesis that construction of myofilament lattice involves
structured water that serves as free-energy sink for contractile interaction. From
this view point, cause of differential radial stiffness of relaxed and rigor muscle
specimens are reconsidered.
2H1610

羽ばたくマルハナバチ中で拮抗する 2 種の飛翔筋の超高速 X 線回折像同時記録

Simultaneous ultrafast X-ray recordings of actions of two
antagonistic flight muscles during wingbeat of live bumblebee
Hiroyuki Iwamoto, Naoto Yagi (SPring-8, JASRI)
In bees and other insects, their flight muscles drive their wings indirectly by
deforming the thoracic exoskeleton (indirect flight muscle). The deformation iscaused by alternating force generation by two antagonistic flight muscles (dorsal
longitudinal muscle, DLM, and dorsoventral muscle, DVM). Here we studied
how the actions of these two antagonistic muscles are coordinated bysimultaneously recording the X-ray diffraction patterns from these muscles
during wingbeat. Intense X-ray beams from the BL40XU high-flux beamline ofSPring-8 were irradiated from the side of the thorax of a live bumblebee, where
the two antagonistic muscles cross. The diffraction patterns were recorded at
5,000 frames/s by using a fast CMOS video camera. A second fast CMOS video
camera recorded its wingbeat. The frames of the two video cameras weresynchronized in a master-slave connection. Besides the strong equatorialreflections, the 1st myosin meridional reflection (1MM) and the 2nd troponin
off-meridional reflection (2TN, reporting myosin binding to actin) were clearlyrecognized for both DLM and DVM after summing the frames for ~5 wingbeat
cycles. The intensities of these reflections, as well as the lattice spacing (d10),changed basically in an anti-phase fashion for DLM and DVM but not always ina sinusoidal manner as generally assumed. Further analyses are underway andthey will give more detailed information about the timing of action of the twoantagonistic flight muscles during wingbeat.
2I1400海洋性ビブリオ菌の周毛性べん毛形成の抑制に関与する DnaJ モ
チーフを持った新規遺伝子の解析

A novel gene of dnaJ family plays a role in the suppression offlagellation in Vibrio alginolyticus
Takehiko Nishigaki1, Maya Kitaoka1, Kunio Ihara2, Noriko Nishioka1, Seiji
Kojima1, Michio Honma1 (1Grad. Sch. Sci., Univ. Nagoya, 2Gene., Univ. Nagoya)
Bacterial flagella are formed in various numbers and locations in different kindsof bacteria. A Vibrio alginolyticus strain VIO5 that lacks lateral flagella andpossesses wild-type flagella, has a single polar flagellum by expressing FlhF
and FlhG. FlhF positively regulates the number and location of flagella, while
FlhG does so negatively. The ΔflhFG strain, derived from VIO5, has mostly no
flagellum, but a small population of the ΔflhFG cells have some peritrichous
flagella and only slightly form the motility ring on the soft-agar plate. In ourlaboratory, the suppressor mutant recovering motility on the soft-agar plate wasisolated from the ΔflhFG strain (named ΔflhFG-sup). The ΔflhFG-sup cells
were peritrichously flagellated. We identified the sup mutation by wholegenome sequence, on the unknown gene which has a DnaJ motif and was
specific in Vibrio. We named this gene sflA (suppressor of Δ flhFG). We
showed that the ΔflhFG strain expressing mutant SflA from plasmid recovered
motility and biosynthesis of flagellin, and the Δ flhFG-sup strain expressing− S53 −

wild-type SflA from plasmid repressed motility and biosynthesis of flagellin. In
addition, the Δ flhFG Δ sflA triple deletion mutant represented the same
phenotype as the ΔflhFG-sup strain. Thus, we concluded that mutation in sflA is
responsible for suppressor phenotype of the ΔflhFG-sup strain. We propose thatsflA was a novel gene involved in the flagellation of V. alginolyticus.
2I1412バクテリアⅣ型線毛の粘着脱離動力学

Unbinding Dynamics of Type IV Pili
Tomonari Sumi1,2, Rahul Marathe2, Stefan Klumpp2 (1Dept. Comp. Sci. Eng.,
Toyohashi Univ. Tech., 2Dept. Theo. & Bio-syst., Max Planck Inst. Colloids &Interfaces)
Type IV pilus is a highly dynamic fiber supporting adhesion, surface motility,and gene transfer. To the best of our knowledge, type IV pilus is the strongest
linear motor that generates considerable strong force larger than 100pN. In thisstudy, we present a simple stochastic model for rupture dynamics of pilus
binding to a bead manipulated by laser tweezers. Theoretical analysis of singlemolecular observation data reveals that bacteria use a switching mechanism
between high velocity retraction and low velocity one to reduce mean rupture
rate of pilus binding to a bead under higher external forces. We show that theauto-regulation-like system that seems to be effectively working as a tension
control mechanism can be constructed by introducing two independentcompeting motors for pilus retraction and elongation without feedback controlof the tension.
2I1424

細菌べん毛特異的シャペロン FliT の C 末αヘリックの役割

Role of the C-terminal α-helix of FliT chaperone in the exportof its cognate substrate FliD
Tohru Minamino1, Miki Kinoshita1,2, Noritaka Hara1, Katsumi Imada2,
Keiichi Namba1,3 (1Grad. Sch. Frontier Biosci., Osaka Univ., 2Grad. Sch. Sci.,
Osaka Univ., 3QBiC RIKEN)
For construction of the bacterial flagellum, which is responsible for motility,
most of the flagellar proteins are exported into the distal end of the growing
structure by the flagellar export apparatus. The export apparatus consists of anexport gate complex made of six membrane proteins and a water-soluble
ATPase complex consisting of FliH, FliI, and FliJ.
FliT, which is a substrate-chaperone responsible for the filament-capping
protein FliD, consists of four α-helices, α1, α2, α3 and α4. The core of themolecule is formed by an anti-parallel α-helical bundle composed of α1, α2 andα 3. The FliT-FliD complex binds to the FliI ATPase complex throughinteractions of the core domain of FliT with FliD, FliI and FliJ in the cytoplasm
and then is transferred from the ATPase complex to the export gate. It has beenshown that the C-terminal α4 helix binds to a hydrophobic cleft formed by the
α2 and α3 helices to regulate the binding affinity of FliT for FliI and FliJ. But
it remains unknown how FliD enters the gate.
To clarify a specific role of the C-terminal α4 helix of FliT in protein export, weinvestigated how the FliT-FliD complex interacts with the export gate. We showthat an interaction between FliT and an export gate protein FlhA is required for
efficient FliD export. We also show that a highly conserved Tyr106 residue inthe α 4 helix of FliT is critical for the interaction with FlhA. These results
suggest that the FliT-FlhA interaction efficiently transfers FliD to the export
gate to promote its protein export.
2I1436

べん毛輸送装置近傍の局所 pH に対する FliI ATPase の効果

Effect of FliI ATPase on local pH around the bacterial flagellar
protein export apparatus
Yusuke V. Morimoto1,2, Nobunori Kami-ike1, Tomoko Miyata1, Keiichi
Namba1,2, Tohru Minamino1 (1Grad. Sch. Frontier Biosci., Osaka
Univ., 2QBiC, RIKEN)Membrane-imbedded molecular machines utilize proton motive force (PMF)
across the biological membrane for their biological activities. The bacterial
flagellum is a biological nanomachine consisting of a rotary motor and afilamentous helical propeller. Most of flagellar proteins are transported to the
distal end of the growing structure for their self-assembly by the flagellar
protein export apparatus. The export apparatus consists of a water-soluble
ATPase complex consisting of FliH, FliI, and FliJ, and a membrane-imbedded
export gate. Recently, it has been shown that the export gate is a proton-protein
antiporter that uses the two components of PMF, Δψ and ΔpH for different stepsof the protein export process, and a specific interaction between the ATPase
complex and the gate switches the export gate into a highly efficient Δψ-driven

export apparatus, but it remains unknown how it occurs.
To clarify the energy transduction mechanism used in flagellar protein export,
we measured local pH around the export apparatus of wild-type cells and a FliHFliI bypass mutant using a ratiometric pH indicator, pHluorin(M153R), fused to
FliG, a rotor component of the flagellar motor. The local pH near the export gateof wild-type cells was significantly lower than that of the bypass mutant. The
FliI catalytic mutations increased the local pH to the FliH-FliI bypass mutant
level. These results suggest that ATP hydrolysis by FliI ATPase is closely
linked to proton translocation through the export gate.
2I1448Na+駆動型べん毛モーター固定子タンパク質 PomA の細胞質ループ領域の示差走査熱量測定を用いた性質検討

Characterization of cytoplasmic loop of PomA, Na+-driven
flagellar stator protein, using differential scanning calorimetry
Shiori Kobayashi1, Rei Abe-Yoshizumi1,2, Mizuki Gohara1, Seiji Kojima1,
Michio Homma1 (1Division of Biological Science, Graduate School of Science,
Nagoya University, 2Department of Frontier Materials, Nagoya Institute ofTechnology)
Vibrio alginolyticus has a single polar flagellum that is driven by rotary motor
powered by the Na+-motive force. The motor is consisted of the stator and therotor, and it is believed that the interaction between stator and rotor that coupledwith ion-flux through the stator generates torque. From previous our study, wedemonstrated that the structure of C-terminal domain of FliG is important forinteraction between stator and rotor using differential scanning calorimetry(DSC). Next, we focused on the stator protein PomA, which is thought tointeract with rotor protein FliG in Vibrio. It has been shown that the cytoplasmic
loop domain of either H+-driven E. coli MotA or Na+-driven Vibrio PomA is
closely participated in the rotor-stator interaction. To characterize this domain
of Na+ -driven Vibrio PomA (PomA-loop), we tried to over-produce therecombinant PomA-loop with His-tag in E. coli and purify it by affinity
chromatography, but it was insoluble. So, we made a fusion with a small solubletag (GB1) and we could handle and characterize the soluble recombinant proteinof PomA (Q54-D148 or Q54-E253) fused with GB1. We characterized thepurified proteins by DSC and compared the thermal stability between the PomA
fragments. Furthermore, we are planning to introduce mutations in the PomA
cytoplasmic region which are predicted to affect the interaction between statorand rotor and to measure the mutant proteins by DSC. We want to discuss thethermodynamic property of the cytoplasmic loop.
2I1510

プラグを欠失した Na+駆動型べん毛モーター固定子複合体の精製・再構成系の構築

Purification and reconstitution of the plug-deleted Na+-driven
stator complex from Vibrio alginolyticus
Tetsuya Oba, Seiji Kojima, Michio Homma (Division of Biological Science,
Graduate school of science,Nagoya University)
The polar flagellar motor of Vibrio alginolyticus is driven by Na+ motive force.
This motor couples Na+ influx through the stator to torque generation. It has
been known that PomA, PomB, MotX and MotY are required for rotation of theVibrio motor. PomA and PomB are integral membrane proteins that form the
PomA4/PomB2 stator complex, and MotX and MotY are periplasmic proteinsassociated with the basal body to form the T ring structure. The PomA/B
complex functions as Na+ channel in the stator. However, quantitative analysis
of Na+ permeation through the PomA/PomB complex is technically challenging
because of the difficulty to purify and reconstitute intact complex. Recently, weinvestigated the plug region of PomB that participates in Na+ flux regulation.
When the stator with plug-deleted PomB (PomA/BΔL) was over-expressed thegrowth rate of cells was decreased without motility impairment. We speculated
that plug-deleted PomA/B complex mimics open state of the stator channel, sothat massive Na+ influx caused growth impairment. Therefore, we attempted to
overproduce and purify the open-state mimicking PomA/BΔL complex and thenreconstitute it to proteoliposome. Using the detergent Cymal-5, we could isolate
relatively pure complex with appropriate molecular size. Currently we are trying
to establish the method to reconstitute the purified complex into liposome andby using radioactive Na+ (22Na+), to detect Na+ uptake activity of
PomA/PomBΔL complex.
2I1522− S54 −

蛍光相関分光法（FCS）を用いたべん毛モーターを構成するタンパ
ク質間の相互作用解析

Analysis of interactions between rotor proteins of flagellar

motor using Fluorescence Correlation Spectroscopy
Takaaki Kishi, Seiji Kojima, Michio Homma (Division of Biological Science,
Graduate School of Science, Nagoya University, Chikusa-ku, Nagoya, Japan.)Vibrio alginolyticus has a single polar flagellum responsible for motility by
utilizing sodium motive force. The flagellar motor consists of rotor and stator. It
is thought that rotor-stator interaction that couples to Na+ flux through the stator
generates torque. However, the direct interaction between rotor and stator has
not been detected clearly in vitro yet, presumably because such an interaction is
weak and transient. Here, to establish a new method to detect interactions
between rotor and stator, we used Fluorescence Correlation Spectroscopy(FCS). FCS can measure diffusion time of the fluorescently labeled particles in
the confocal area. If a protein is bound to the labeled one, then diffusion timeincreases, so that we can detect protein-protein interactions. Last year we havefound that some kinds of detergents still allowed us to detect membrane protein
interactions by the FCS analyses. Also, the purification method of the
PomA/PomB stator complex that lacks the linker region of PomB (named
PomA/BΔL) was established. Using PomA/BΔL and the rotor protein FliG, we
tried to detect the direct interaction between rotor and stator by FCS. FliF, a
component of rotor, form the MS ring structure and serve as a platform of rotor.
To detect FliG-PomA interaction, MS ring might be necessary to mimic the invivo rotor structure. However overproduction of Vibrio FliF alone in E. coli did
not produce MS ring. Now we co-express FliF with the flagellar export
apparatus FlhA to facilitate formation of MS ring.
2I1534Na+駆動型べん毛モーターの固定子と回転子間における荷電残基変異による協調作用の解析

Synergetic effects of the mutations of charged residues between
the rotor and the stator in the Na+-driven flagellar motor
Norihiro Takekawa, Seiji Kojima, Michio Homma (Div. of Biol. Sci., Grad.
Sch. of Sci., Nagoya Univ.)
Some bacteria have flagella for their motility. At the base of the flagellum, there
exists the reversible rotary motor. The flagellar motor is consisted of two parts,
rotor and stator. Essential components for torque generation in the rotor is
located at the C-ring composed of FliG, FliM, and FliN, and the stator iscomposed of the membrane proteins MotA and MotB in H+ -driven flagellar
motor and PomA and PomB in Na+ -driven flagellar motor that forms ionchannel structure. In the H+ -driven motor of E. coli, the interaction between
conserved charged residues of FliG and MotA is important for generation of thetorque of the motor. However, the importance of such interactions had been
unclear in the Na+-driven motor. In this study, we systematically made mutants
whose charged residues of FliG and PomA were replaced to uncharged or
charge-reversed ones, and examined motility of those mutants. We found thatsome FliG/PomA double mutations gave the synergetic effects or suppression
on the motor rotation. Our results suggest that interactions between charged
residues of FliG (K284, R301, D308, and D309) and those of PomA (R88, K89,
E96, and E97) are important for force generation of Na+-driven flagellar motor.
Especially the suppression effect between FliG-K284E and PomA-E97K was
only seen in the Na+-driven motor, may show more robust rotor-stator interface
due to the Na+-type specific interaction as suggested in the previous study.
2I1546

光分解されたセリンへの大腸菌の応答計測

Measurement of cellular response of single E. coli to thephotoreleased serine
Takashi Sagawa1, Hajime Fukuoka2, Yuichi Inoue2, Hiroto Takahashi2,
Takahiro Muraoka2, Kazushi Kinbara2, Akihiko Isjijima2 (1Grad. Sch. Life
Sci., Tohoku Univ., 2IMRAM, Tohoku Univ.)
A rotational direction of flagellar motors of E.coli is regulated by binding of
intracellular signaling protein, CheY-P. By the recognition of serine,
intracellular concentration of the CheY-P is decreased and the rotational
direction of the motor is kept in CCW direction.
In this study, we investigated the cellular response of the E.coli to the locallyapplied chemotactic signal, using caged-serine. By photoreleased serine, themotor was switched back to CCW direction within sub-second delay andcontinued the CCW rotation for several seconds. From the sub-second delay andthe CCW duration, the upward and downward concentration of serine wasestimated, respectively. The upward concentration of serine was higher than thatof downward. This result suggested that it is necessarily to refill the CheY-P torecover the signal transduction.
To investigate the detail of response of motors to the concentration of CheY-P,
dual motor analysis was preceded. When the rotational direction of motor near

receptor changed CCW to CW, serine was photoreleased. The motor near
receptor was switched back to CCW direction within sub-second delay.
However, the motor far from the receptor kept CCW rotation. Therefore, thesignaling of CheY-P molecules could not reach to the end of the cell by the
inhibition of CheY-P production.
These results suggested that the signaling molecules diffused fromchemoreceptors and the sufficient concentration change of CheY-P in entire cellshould be required to transmit the signal from the receptor to the end of the cell.
2I1558CheZ 極局在の有無における大腸菌細胞内シグナル伝達の計測

Propagation of intracellular signaling molecule in the presence
and absence of polar localization of CheZ in a single E. coli cell
Hajime Fukuoka1, Takashi Sagawa2, Yuichi Inoue1, Hiroto Takahashi1,
Akihiko Ishijima1 (1IMRAM, Tohoku Univ., 2Grad. Sch. life Sci., Tohoku
Univ.)In chemotaxis system, the extracellular signals are detected by receptors and aretransduced to an intracellular signaling molecule, phospholylated CheY (CheYP), at the cell pole. The binding of CheY-P to the flagellar motor switches
rotational direction of it from counterclockwise (CCW) to clockwise (CW). By
measuring switching delay between 2 motors, we previously proposed anincrease and decrease in the CheY-P concentration are propagated wave-likely
from receptor patch to the opposite cell pole. However, it is not understood how
such dynamic propagation is generated. In this study, to understand thesignaling process by CheY-P, we measured the switching delay of CCWto-CW and CW-to-CCW switching individually with and without polar
localization of CheZ, which dephosphorylates CheY-P. For the measurement in
the absence of CheZ localization, we constructed a mutant cell lacking
CheAshort, which binds CheZ in receptor patch. Regardless of CheZ localization,
the switching delay correlated with the relative positions of 2 motors fromreceptors in CCW-to-CW switching. However, in CW-to-CCW switching,switching delay correlated with the positions of 2 motors only in the presence of
CheZ localization. These results indicate the polar localization of CheZ iscritical for the directed propagation of the decrease of CheY-P concentration.
Now we are measuring chemotactic response in the presence and absence of
CheZ localization to understand the role of its localization in chemotaxis. We
would like to discuss our results in annual meeting.
2I1610大腸菌の遊泳速度に及ぼすγ線照射の効果

Effect of gamma ray irradiation on the swimming speed ofEscherichia coli
Eriko Fujimoto1, Masakazu Furuta2, Tatsuo Atsumi3, Mikio Kato2 (1Osaka
Prefecture University School of Science, 2Osaka Prefecture University
Graduate School of Science, 3Gifu University of Medical Science)
The effects of ionizing radiation on bacteria are generally evaluated using adose-dependent survival ratio based on the colony-forming ability and mutation
rate of the cells. To date, the mutagenic damage to cellular DNA induced byradiation has been extensively investigated, primarily by using methodologies
that estimate the degree of DNA damage in specimens following bulk
irradiation. However, the effects of irradiation on cellular machinery in situ
remain unclear.
Recently, we applied a micrometer-sized proton beam, generated by a Pelletron
accelerator through a tapered glass capillary, to a single cell of Escherichia coli
in liquid media to evaluate the effect of irradiation on the flagellar motor andcell division. We observed an unexpected robustness of the bacterial flagellar
motor against irradiation from a high-energy (2 MeV) proton beam; the average
ion fluence required to stop the flagellar motor of the tethered cells was 2.0 ×
1012 protons/cm2 (approximately 60 kGy).
In the present study, we applied a γ ray from 60Co (up to 8 kGy) to E. coli cells
in liquid media, and measured their swimming speed using a microscope. The
results indicated that the swimming speed remained unaltered after the lethal
gamma ray dose was administered to the cells; however, the ratio of the cells
adhering to the glass substrate (not swimming freely) increased by theirradiation. Moreover, the inhibition of protein synthesis by kanamycin did notalter the swimming speed, but increased the ratio of the stuck cells.

− S55 −

3A0912

F1-ATPase と繊毛軸糸の機能に関わる構造変化
Motions in F1-ATPase and ciliary axonemes that drive
functions

Tomoko Masaike1, Koji Ikegami2, Rinako Nakayama1, Mitsutoshi Setou2,
Takayuki Nishizaka1 (1Department of Physics, Gakushuin University, 2Department
of Cell Biology and Anatomy, Hamamatsu University School of Medicine)
Our goal is to visualise how motions and functions of components are coupledin various hierarchy levels of biological systems under optical microscopes.
As a model of enzymes, we chose F1-ATPase. Cooperative ATPase activity atthree catalytic  subunits is known to be greatly accelerated by the presence ofthe central shaft subunit γ. To investigate involvement of physical rotation of γ
in conformational states of , orientation of the C-terminal helix of  was
measured using advanced TIRF microscopy, while γ was forcibly rotated using
magnetic tweezers. We found that conformational changes of  are dependent
on both orientation of γ and direction of its rotation. Thus the interplay between
 and γ indeed contributes to conformational states and thus chemical states ofthe enzyme during efficient rotary catalysis.
The next target is an organelle. A fluorescent bead was attached to the axonemal
tip of a tracheal cilium of a mouse, and beating motions were recorded under athree-dimensional tracking microscopy. We obtained 3-D coordinates oftrajectories and fitted them with spherical shells. We found multiple phases ofvelocities along the spherical shells during strokes. Moreover, counterclockwise
rotational motions and a difference of height between effective and recovery
strokes were quantitatively indicated. Cilia may follow different trajectories insynchronised effective and recovery strokes to avoid drag of the opposite stroke
and maintain effective fluid flow for elimination of harmful materials.
3A0924高速暗視野照明による金ナノロッドの方向検出システムを用いた

F1-ATPase の回転の検出
Detection of rotation of F1-ATPase using high-speed
orientational detection of gold nanorod
Sawako Enoki1,2, Ryota Iino1,2, Hiroyuki Noji1,2 (1Grad. Sch. Engineering.,
Univ. Tokyo, 2CREST)
The scattered light from single gold nanorod is strongly polarized along the axisof the rod and can be used for the detection of its orientation. For theorientational detection, the gold nanorod would be a better probe than afluorescent dye, because its high scattering intensity made it possible thedetection at high rate for a long time without photobleaching. Furthermore, gold
nanorod would be better than gold nanoparticle, because the orientationalchange that causes only very small lateral displacement of the attached probe
can be detected. At last annual meeting, we reported two dark-field imaging
systems based on objective-type evanescent and backscattering illuminations todetect the orientation of nanorod by analyzing the scattering intensities. Here,
we tried defocus imaging of scattering of gold nanorod based on objective-typebackscattering illumination. We tested defocus imaging of nanorod byobserving the rotation of molecular rotary motor, F1-ATPase. By analyzing thescattering pattern from gold nanorod, we could detect the orientation of the gold
nanorod quantitatively at high speed (10 s) and at high angle resolution. There
are several advantages for defocus imaging compared to previously reported
imaging systems. The angle can be directly determined from the image andangle range was wide (0-360 degree), while those of previously reported
systems are narrow (0-90 degree). We will apply this imaging system toobservation of domain motion of proteins.
3A0936

UTP を基質とした時の F1-ATPase の回転運動

Characterization of UTP driven rotation of F1-ATPase
Hidenobu Arai, Rikiya Watanabe, Hiroyuki Noji (Grad. applchem., Univ.
Tokyo)
F1-ATPase (F1) is a rotary motor protein driven by ATP hydrolysis. In our earlywork, we examined the competency of various nucleotides to drive the rotationof F1. While ATP, GTP, and ITP have been shown to support the rotation, we
have not found evidence of UTP-driven rotation despite of the hydrolyzablity of
UTP. In this study, we again attempted to verify the incompetency of UTP todrive the rotation by using very pure UTP that did not contain detectable ATP(<0.0025 %). At various UTP concentrations, we observed the anticlockwise
rotation of F1. From the Michaelis-Menten analysis, the rate constant of UTP
binding was determined as 104 M-1s-1, which is 1,000 times smaller than that ofATP. Thus, the rotation is not attributable to the possible ATP contamination,

showing that UTP drives the rotation of F1. We measured the rotary torque of
UTP rotation from the viscous coefficient (Γ)and the angular velocity of therotation probe (ω); N=Γω. The estimated torque was around 40 pNnm as sameas the ATP-driven rotation. To confirm this, we also conduced a buffer
exchange experiment from ATP to UTP to compare the angular velocity of
individual F 1. molecules in the presence of ATP or UTP. No obvious difference
was found. These results showed that F1 efficiently convets the hydrolysis
energy of UTP into mechanical wok even though UTP is a very low affinity
substrate. The present work suggests that the base moiety of nucleotides isdispensable for the chemomechanical coupling of F1.
3A0948Single Molecule Analysis of Inhibitory Pausing States of V1ATPase
Naciye Esma Tirtom1, Yoshihiro Nishikawa2, Daichi Okuno3, Masahiro
Nakano4, Ken Yokoyama5, Hiroyuki Noji1 (1Department of Applied Chemistry,
School of Engineering, University of Tokyo, 2Dept. of Biotechnology, Osaka
University, 3RIKEN, Osaka, Japan, 4The Institute of Scientific and Industrial
Research, 5Dept. of Biolmolecular Sciences, Kyoto Sangyo University)
V1-ATPase, the hydrophilic domain of V-ATPase, is a rotary motor fueled by
ATP hydrolysis. Some biochemical studies indicated a strongly-inhibited stateof V1 -ATPase, which we analyzed in detail at single molecule level, in thisstudy. Here we showed that Thermus thermophilus V1-ATPase had two types ofinhibitory pauses interrupting continuous rotation: a short pause (SP, 4.2 s) that
occurred frequently during rotation and a long inhibitory pause (LP, >30 min)that terminated all active rotation.Both pauses occurred at the same angle with
ATP binding and hydrolysis. Kinetic analysis revealed that the time constants ofinactivation into and activation from SP were too short to correspond tobiochemically predicted ADP inhibition, suggesting that SP is a newly
identified inhibitory state of V1. The time constant of inactivation into LP was
17 min, consistent with one of the two time constants governing the inactivation
process observed in bulk ATPase assay. When forcibly rotated in forward
direction by using magnetic field, V1 in LP resumed active rotation. Solution
ADP suppressed the probability of mechanical activation, suggesting thatmechanical rotation enhanced inhibitory ADP release. These features werehighly consistent with mechanical activation of ADP-inhibited F1, suggestingthat LP represents the ADP-inhibited state of V1. Mechanical activation largely
depended on the direction and angular displacement of forced rotation, implyingthat V1-ATPase rotation modulates the off rate of ADP.
3A1010F1-ATPase のトルク伝達における DELSEED ループの役割

The role of DELSEED loop in torque-transmission of F1ATPase
Kazuma Koyasu1, Rikiya Watanabe1, Mizue Tanigawara2, Hiroyuki Noji1(1Department of Applied Chemistry, School of Engineering, The University ofTokyo, 2Graduate School of Frontier Biosciences, Osaka University)
F1 -ATPase is an ATP-driven rotary motor that generates torque at the statorrotor interface. The torque-generating unit, , largely changes its conformation,by swinging the C-terminal domain inwardly upon ATP binding. The protruding
loop of the C-terminal domain, so called DELSEED loop (D-loop) forms a large
contact interface with the rotor subunit. From these observations, D-loop has
been supposed to have a crucial role in the torque transmission of F1. In ourrecent work, we analyzed the rotation behaviors of the mutant F1 of which Dloop residues were all substituted to glycine in order to disrupt the specificinteraction with the rotor and rigidity of the loop (Tanigawara et al. submitted).
Surprisingly, the mutant has shown active rotation although it halved the torque.
In the present study, we attempted to identify which reaction step is impaired by
the glycine substitution. For this purpose, we measured the angular dependencyof the equilibrium constant and reaction rate of ATP binding and hydrolysis
steps by conducting the single-molecule stall-release experiment (Watanabe etal. NCB 2012). It was shown that the angular dependency of ATP binding was
reduced to 60% while that of hydrolysis was not changed. These results suggestthat D-loop has a crucial role in the force transmission from the catalytic
reaction center to the rotor subunit in ATP-binding process but not in hydrolysis
step.
3A1022− S56 −

F1-ATPase の回転子γサブユニットに有限のトルク発生に必要な残基はない

None of the rotor residues of F1-ATPase are essential for
torque generation

Ryohei Chiwata1, Tomoya Kawakami1, Ayako Kohori1, Shou Furuike2,
Katsuyuki Shiroguchi3, Kazuo Sutoh1, Masasuke Yoshida4, Kazuhiko Jr.
Kinosita1 (1Dept. Physics, Fac. Sci. Eng., Waseda Univ., 2Fac. Physics, OsakaMed. Col., 3Dept. Chem. and Chem. Biol., Harvard Univ., 4Dept. of Mol.
Biosci., Kyoto Sangyo Univ.)
F1-ATPase is a rotary motor in which the central γ subunit rotates inside a statorring made of α33 subunits. ATP hydrolysis in the  subunits generates torquefor γ rotation by a mechanism not fully understood. The γ rotor consists of a
globular domain that protrudes from the stator and a slender shaft that penetrates
the α33 ring. The shaft is an anti-parallel coiled coil of the N- and C- terminal
helices of γ. Previously we have deleted either or both of these terminal helices
genetically. Surprisingly, all mutants rotated in the correct direction, showingthat the shaft portion is dispensable. Here we inquire if the rest of the γ rotor, theglobular protrusion that accounts for ~70 % of the γ residues, is also
dispensable. Keeping the N- and C-terminal helices that constitute the shaft, wehave replaced the middle ~200 residues with a short helix-turn-helix motif
borrowed from a different protein. Furthermore, 14 residues of the remaining Nand C- terminal helices have been replaced with unrelated residues such that theresulting construct and the previous shaft-truncated mutants (ΔC36 and ΔN29)
that generated ~half the wild-type torque do not share common residues. This
construct retained a high ATPase activity and rotated fast in the correct
direction, generating a sizable torque. We conclude that none of the residues of
the rotor are needed for the generation of finite torque.
3A1034

F1-ATPase のシリンダーから中心軸を引き抜く力の測定

UNBINDING FORCE MEASUREMENTS OF THE SHAFTFROM THE CYLINDER OF F1-ATPase
Tatsuya Naito, Kaoru Okada, Tomoko Masaike, Takayuki Nishizaka (Dept
Phys, Gakushuin Univ.)
F1-ATPase is a rotary motor protein in which the central γ subunit rotates inside
the cylinder made of α33 subunits. We investigated interactions between γ andthe cylinder by unbinding γ in the direction of the optical axis. Threedimensional single-particle tracking using a wedge prism has been combinedwith optical tweezers to quantify molecular scale 3-D force in F1-ATPase. We
placed the trap center 200-300 nm above a bead attached to γ, and the spring
constant of the trap was increased by adjusting laser power. Here we report
improvements of the experimental setup. First we adjusted the height of thetrapping center every time just before unbinding to accommodate the trapping
force within the range that it is proportional to the displacement. Second we
attempted to ensure that γ is actually dismantled from the cylinder: Specificbiotin-avidin binding of γ and beads were confirmed. Moreover the glass
surface was modified with Ni-NTA to achieve specific binding of F1-ATPase.
Then we set up a system to check whether the fluorescently labeled cylinder
remained on the glass surface after unbinding. As a result, the averageunbinding force was 7.7 ± 5.0 pN (n=30) at 100 nM ATP. In the course of theexperiment, we noticed that the sample plane apparently displaced in the Zdirection as laser power was increased. We plan to show differences inunbinding force depending on chemical states during catalysis.
3A1046

リポソーム膜中に再構成した好熱菌由来 FoF1- ATP 合成酵素による ATP 駆動 H+輸送の定量

Quantification of ATP-driven H+ transport by thermophilic Bacillus
PS3 FoF1-ATP synthase reconstituted in a liposomal membrane

Yuzo Kasuya1, Naoki Soga1, Toshiharu Suzuki2, Masasuke Yoshida2,
Kazuhiko Kinosita1 (1Dept. Phys., Facl. Sci. Eng., Waseda Univ., 2Dept. Mol.
Bio., Facl. Life Sci., Kyoto Sangyo Univ.)
FoF1 ATP synthase is composed of a water-soluble F1 that has three catalytic
sites for ATP synthesis/hydrolysis and a membrane embedded Fo that acts as a
H+ channel. The FoF1 synthesizes ATP using H+ transport through the Fo andpumps H+ reverse direction using ATP hydrolysis. ATP hydrolysis and resultant
subunit rotation in F1 have been well characterized by various single molecule
techniques. The transport of H+ through Fo, in contrast, has been difficult toobserve in single molecules, and bulk assays have in most cases qualitative
rather than quantitative.
Here, we measured the H+ pump activity of Fo F1 from thermophilic Bacillus
PS3 (TFo F1) by an ensemble assay in a spectrophotometer and a singleliposome assay under a microscope. To visualize the H+ transport through the
TFoF1 into liposomes driven by ATP hydrolysis, a fluorescent dye pH-rodo, of
which the fluorescence intensity increases upon lowering of pH, was conjugatedto the phospholipids. The dye-labeled liposomes containing ~one TFo F1 on

average responded to the addition of ATP by gradually increasing thefluorescence. We estimated the initial H+ pump activity of TFoF1 by calibrating
the buffering capacity; Vmax was in the order of 600 H+ sec-1 in ensemble and800 H+ sec-1 in single-liposome assays, with Km for ATP of 20 M in bothassays for the starting pH of 8.0 and at room temperature. Now we are stilltrying to determine the H+ pump activity more precise.
3A1058

マイコプラズマモービレゴーストのステップ検出

Detection of steps of Mycoplasma mobile gliding ghost
Yoshiaki Kinosita1, Daisuke Nakane2,3, Kana Mizutani1, Makoto Miyata2,
Takayuki Nishizaka1 (1Dept. phys., Gakushuin Univ, 2Dept. Biol., Osaka City
Univ, 3Present address: Mol. Microbiol. and Immunol., Grad. Sch. Biomed.
Sci.,Nagasaki Univ)
Mycoplasma mobile is a pathogenic flask-shaped bacterium about 0.8 µm longand has prominent gliding activity, on the glass surface coated with sialic acids
at velocities of 2.5 µm/sec. Previous studies showed that the gliding motion isdriven by many legs which repeatedly catch and release sialic acids on the glass
surface, accompanied by ATP hydrolysis. To comprehend a unique mechanismof the gliding machinery, the step size and the kinetics per one ATPase cycle
should be clarified. Nakane et al. have shown that the living M. mobile takes 85nm step during gliding. Next challenge is to elucidate the kinetics of ATPhydrolysis in the stepwise motion. Here we performed the improved motility
assay by using “gliding ghosts” of which the cell membrane was permeabilized
by triton X-100. The ghosts allow us to control the gliding speed by modifying
ATP concentrations in solution. The cell membrane was stained with Cy3 sothat we could observe the gliding motility at the time resolution of 2 ms without
a huge probe . Furthermore, to reduce the number of the legs contributing thegliding motility, free sialic acids was added to the assay buffer. Through these
improvements we successfully observed the stepwise motion of the ghosts. A
pairwise distance analysis showed that the M. mobile gliding ghosts took 60100 nm steps, which are consistent with that of the living cell. MichaelisMenten constant and Vmax were 59 µM and 2.5 µm/sec.Now we are analyzing
the dwell time.
3B0900

原核生物由来ナトリウムチャネルにおける C 末端 4 ヘリックスバ
ンドルを用いたゲーティング制御

The cytosolic C-terminal four-helix bundle regulates the gatingof prokaryotic sodium channel
Katsumasa Irie, Takushi Shimomura, Yoshinori Fujiyoshi (CeSPI., Univ. Nagoya)
Voltage-gated sodium channels play essential roles in many important
physiological processes, including electric signalling and muscle contraction.
Therefore their activation and inactivation are needed to be strictly regulated.
Here, we show that the cytosolic C-terminal region of NavSulP, a prokaryotic
voltage-gated sodium channel cloned from Sulfitobacter pontiacus, accelerates
the inactivation rate and keeps the voltage dependent activation positively. The
crystal structure of the C-terminal region of NavSulP grafted into the Cterminus of NaK channel at 3.2 Å resolution revealed that the C-terminal region
forms a four-helix bundle. Point mutations of the residues of the four-helix
bundle destabilised channel tetramer and reduced the inactivation rate. The fourhelix bundle was directly connected to the inner helix of the pore domain. The
increase of the rigidity of the inner helix with the glycine-to-alanine mutation
also reduced the inactivation rate, but did not destabilize the tetrameric channel.
Moreover, all of these mutants shifted the voltage dependent activation
negatively, which indicates that the four-helix bundle suppress the opening of
the pore domain. These findings suggest that the NavSulP four-helix bundle
plays important roles not only in stabilising the tetramer, but also in regulating
the channel gating by the restriction of the conformational change of the inner
helix. The four-helix bundle could reduce excess ion flux after activation andalso suppress accidental ion flux even in the resting state.
3B0912[NiFe]ヒドロゲナーゼ成熟化因子 HypE，HypF の X 線結晶構造解析

X-ray structural analysis of HypE and HypF, maturation
factors for [NiFe]-hydrogenases
Yasuhito Shomura1,2, Yoshiki Higuchi1,2 (1Grad. Sch. Life Sci., Univ.
Hyogo, 2RIKEN/SPring-8 Center)
As a remarkable structural feature of hydrogenase active sites, [NiFe] hydrogenases harbor one carbonyl and two cyano ligands, where HypE and− S57 −

HypF are involved in the biosynthesis of the nitrile group as a precursor of the
cyano groups. HypF catalyzes S-carbamoylation of the C-terminal cysteine of
HypE via three steps using carbamoylphosphate and ATP, producing two
unstable intermediates: carbamate and carbamoyladenylate. It remains unclear
how the consecutive reactions occur without the loss of unstable intermediates
during the proposed reaction scheme. We have recently determined the crystal
structures of full-length HypF both alone and in complex with HypE at
resolutions of 2.0 and 2.6 Å, respectively. Three catalytic sites of the structuresof the HypF nucleotide- and phosphate-bound forms have been identified, witheach site connected via channels inside the protein. This finding suggests thatthe first two consecutive reactions occur without the release of carbamate or
carbamoyladenylate from the enzyme. The structure of HypF in complex with
HypE revealed that HypF can associate with HypE without disturbing its
homodimeric interaction and that the binding manner allows the C-terminal
Cys351 of HypE to access the S-carbamoylation active site in HypF, suggestingthat the third step can also proceed without the release of carbamoyladenylate.
3B0924

Dynamic structural and antigen binding analyses of antibody
single-chain Fvs
Yusuke Tanaka1, Hiroshi Sekiguchi2, Yuji C. Sasaki3, Takachika Azuma4,
Masayuki Oda1 (1Grad. Sch. of Life and Environ. Sci., Kyoto Pref. Univ., 2Jpn.
Syn. Rad. Res. Inst., 3Grad. Sch. of Fron. and Sci., Univ. of Tokyo., 4Res. Ins.
for Biol. Sci., Tokyo Univ. of Sci.)
We generated single-chain Fvs (scFvs) of anti-(4-hydroxy-3-nitrophenyl)acetyl(NP) antibodies, N1G9 and C6, which are germline and affinity maturated IgG1
antibodies, to analyze the change of structural fluctuation upon the antigen
binding and through the affinity maturation. We overexpressed the scFvs in E.
coli, and refolded by stepwise-dialysis method, followed by purification using
NP-conjugated column. The scFv-antigen interactions were analyzed using
surface plasmon resonance biosensor, Biacore, in which NP conjugated proteins
were immobilized on sensor chip. The equilibrium association constant of C6
scFv for NP was about 10 times higher than that of N1G9 scFv, mainly due tothe decreased dissociation rate constant. The kinetic parameters were similar to
those of corresponding Fab and intact antibody, indicating that the scFvs are
refolded correctly. The structural fluctuations of scFvs were measured using
diffracted X-ray tracking (DXT) system at single-molecule level. Upon thebinding of NP-ε-amino caproicacid, the fluctuations were decreased in either
N1G9 or C6 scFv, indicating that the antigen binding stabilizes the scFv
structure. In comparison of N1G9 and C6 scFvs in the absence of antigen, thefluctuation of C6 was lower than that of N1G9, which could be correlated withthe different antigen binding mechanism such as “induced-fit” and “lock-andkey” types. On the advantage of scFv, whose amino acid could be mutated, we
try to obtain more quantitative and site-specific information.
3B0936

PYP の光構造変化の機械的な制御

Mechanical control of light-induced protein conformationalchange of photoactive yellow protein
Yasushi Imamoto, Take Matsuyama, Yoshinori Shichida (Grad. Sch. Sci.,
Kyoto Univ.)
Proteins are formed from polypeptides by folding into the specificconformations, by which the physiological roles of the proteins are determined.
The structure of the protein closely correlates with the physiological activity,
and the activity of the protein is regulated by conformational change. For
example, the receptor proteins alter their conformations in response to thestimuli to activate the cellular transduction system. Thus, if the protein
conformation is artificially regulated, one would control the protein activity.
Here we tried to establish the technique to regulate the protein activity bychanging the conformation.
Photoactive yellow protein (PYP), a bacterial blue-light sensor protein, was usedas the model protein. Two amino acid residues present in the surface of PYP
molecule were replaced by cysteines and crosslinked by an azobenzene linker,
whose length is reversibly changed by trans/cis photoisomerization. We foundthat the velocity of photocycle was varied by 10-100 times by the isomerization ofazobenzene when Met100 loop and pCA loop were crosslinked. Since the global
protein conformational change involving Met100 loop takes place in thephotocycle, it is likely that the protein conformational changes were perturbed by
the isomerization of azobenzene linker. This technique is possibly applied toswitch the enzymatic activity of the protein by light.

3B0948αカテニンによるアクトミオシン収縮の阻害

Inhibition of actomyosin contractility by α-catenin,
a component of adherens junctions
Shuya Ishii1, Takashi Ohki1, Hiroaki Kubota1, Shin'ichi Ishiwata1,2,3 (1Dept.
of Phys., Faculty of Sci. and Eng., Waseda Univ., 2Adv. Res. Inst. for Scie. andEng., Waseda Univ., 3WABIOS,Waseda Univ.)
α -catenin is known as a linker protein between cell adhesion and the actin
cytoskeleton. It has been believed that monomer of α-catenin forms a complex
with -catenin and E-cadherin in cell-cell adherens junctions and plays anessential role as an anchor, directly or indirectly, of E-cadherin-mediatedadherens junctions and cytoskeletal F-actin networks. Dimer of α-catenin, which
is formed by the dissociation from the complex, binds to F-actin with M affinity
and bundles it, locally inhibiting the Arp2/3 complex. This reorganization of actin
dynamics decreases membrane activity at sites of cell-cell contact and stabilizes
cadherin-mediated cell-cell adhesion. In addition, inhibition studies of myosin
ATPase activity suggested that the intracellular actomyosin contractile force also
contributes to the assembly and extension of adherens junctions. In present study,
we report that α-catenin inhibits actomyosin contraction in the dose-dependent
manner, which was characterized by an in vitro F-actin gliding assay with myosin
II HMM. Furthermore, no direct significant association between α-catenin andmyosin II was observed in immunoprecipitation assay, indicating that α-catenin
may compete with myosin II for binding to actin, resulting in a decrease in thenumber of myosin heads able to propel actin filaments, or may indirectly, throughan actin filament, inhibit myosin activity. Herein, we will discuss how α-catenininhibits actomyosin contraction and the implications of that in maintaining
adherens junctions.
3B1010ダイニンモータードメインの溶液中の分子動力学シミュレーション

Molecular dynamics simulations of dynein motor domain in
explicit water
Narutoshi Kamiya1, Tadaaki Mashimo2, Yu Takano1, Takahide Kon1, Genji
Kurisu1, Haruki Nakamura1 (1Institute for Protein Research, Osaka Univ.,
2
BIRC, AIST)
Dyneins are large microtubule motor proteins that play important roles invarious biological movements. Cytoplasmic dynein is responsible for cell
division, cell migration and other basic cellular functions. The motor domain ofdynein consists of a ring-shaped six ATPases called AAA+ modules. Recently,
an ADP-bound high-resolution structures of cytoplasmic dynein have revealed
the organization of the motor domain that comprises the AAA+ ring, the linker,
stalk/strut and C sequence (PDB IDs = 3vkh, 3vkg). However, the highresolution structure of an ATP-bound dynein remains unclear. Here, we carriedout molecular dynamics (MD) simulations of both ADP and ATP-bound forms
to examine their structures and dynamics.
We built initial structures for MD as following. A higher resolution structure(3vkg), which is a truncation mutant, was chosen. Then, we modeled missing
residues and added a truncated domain from the wild type structure (3vkh). FourADP molecules were placed to their original positions in the ADP bound form.
One of the ADP molecules, bound to the main ATPase site (AAA1), wasreplaced to ATP in the ATP bound form. A rectangular water box was placedaround dynein. Finally, the systems consisted of approximately one million
atoms. Electrostatic interactions were treated with zero-dipole summation
method, and their computation was accelerated by using GPGPU.
We will discuss the effect of ATP on the structure and dynamics of dynein bycomparing the trajectories between the ADP- and ATP-bound forms.
3B1022

A computational Investigation into the MHC-I Recognition
Mechanism of MIR2 from Kaposi's Sarcoma-Associated
Herpesvirus
Pai-Chi Li1,2, Naoyuki Miyashita3, Satoshi Ishido1, Yuji Sugita2 (1RIKEN
Research Center for Allergy and Immunology, 2RIKEN Advanced Science
Institute, 3RIKEN Quantitative Biology Center)
Kaposi's sarcoma-associated herpesvirus, a human tumor virus, encodes two
membrane-bound E3 ubiquitin ligases, modulator of immune recognition 1(MIR1) and MIR2, to evade host immune system through the ubiquitinationmediated endocytosis and lysosomal degradation of the proteins involved in
immune recognition. MIR2 downregulates the surface expression of MHC I(major histocompatibilty complex class I), co-stimulatory proteins (B7-2,
ICAM-1), and platelet endothelial cell adhesion molecule 1 (PECAM-1). Recent− S58 −

experiments results have shown that the palmitoylation of MIR2 Cys146 isessential for MHC I and PECAM-1 downregulation. This observation raises
questions about the function of palmitoyl Cys146 (Cyp146), such as Cyp146 iswhether located at the MIR2/MHC-1 and MIR2/PECAM-1 contact interfaces.
To gain insight into the molecular basis of MHC-1 recognition by MIR2 and therole of Cyp146 of MIR2, we performed a series of molecular dynamic
simulations. We first performed the replica-exchange molecular dynamics
simulations in an implicit membrane/solvent environment to predict thestructure of the transmembrane region of MIR2, then simulated the interaction
between MHC-1 and MIR2. We then estimated the association energy ofMIR2/MHC-1 complex by performing all-atom molecular dynamics
simulations in the explicit membrane/solvent environment. Finally, therecognition mechanism for MIR2/MHC-1 is proposed and compared with thatfor MIR2/B7-2.
3B1034タンパク質構造変化経路予測法の新規開発

A Novel Method to simulate protein conformational change
upon ligand binding
Koichi Tamura, Shigehiko Hayashi (Grad. Sch. Sci., Univ. Kyoto)
Proteins undergo large conformational change when they interact with ligands.
Whereas all-atom molecular dynamics simulations offer detailed description of
the systems, its capability is limited to several hundreds of nanoseconds
timescale, much lower than the actual timescale of the transitions. To enforce
conformational change within the accessible simulation timescale, one can
employ external forces to bring systems to end states. Especially, force probing,or steered molecular dynamics(SMD) and targeted molecular dynamics(TMD)
technique is often applied to investigate the transition paths. In SMD, one haveto determine direction of the applied forces somewhat intuitively, making it
difficult to use the method within complex system. TMD requires explicit end
state's knowledge to construct biasing forces, which indicates it's non-predictive
nature. Thus, both methods have some deficiencies. In this work, we present a
novel method, which based on the linear response theory, to study protein's
complex conformational change upon ligand binding. The new method, which
we call linear responce path following(LRPF), has some features not containedin the method described above; external forces are calculated nonintuitively, andit doesn't require any end state's information. The technique was succeffuly
applied to comformational change of yeast calmodulin N terminal domain. The
detailed transition pathway is discussed.
3B1046

時計タンパク質 KaiA-KaiC 相互作用の ESR 解析

Interactions between cyanobacterial clock proteins KaiA and
KaiC revealed by ESR analysis
Kentaro Ishii1, Toshiaki Arata2, Masahiro Ishiura1 (1Center for gene research
Nagoya Univ., 2Grad. Sch. of Science, Osaka Univ.)
The circadian clock is an endogenous biological mechanism that generates daily
cycles in gene expression, physiological, and behavioral activities (circadianrhythms). Many organisms from cyanobacteria to eukaryotes have circadianclocks, which enable the organisms to predict environmental alterations during
the day. Cyanobacteria are the simplest organisms that have the circadian clock.
The cyanobacterial circadian clock can be reconstituted in vitro from three clock
proteins, KaiA, KaiB and KaiC, in the presence of ATP. A series of reactions
and interactions among Kai proteins generate circadian oscillations such as
oscillations in the phosphorylation level and ATPase activity of KaiC andcomplex formation among Kai proteins. Here, we investigated interactions
between KaiA and KaiC by the spin labeling-electron spin resonance ( SDSLESR) method. SDSL-ESR spectroscopy can provide information on the locationand environment of an individual residue within a protein. We labeled eachcystein (Cys) residue of Cys-substituted mutants of KaiC and KaiA derived
from the thermophilic cyanobacterium Thermosynechococcus elongatus withspin labels, performed ESR analyses, and identified specific residues involvedin KaiA-KaiC interactions. We also found that KaiA induces structural changes
in the base part of the C-terminal tail of KaiC on its interaction with KaiC.
3B1058ATP を中間体とするシアノバクテリア概日時計蛋白質 KaiC の新規脱リン酸化機構

Autodephosphorylation of cyanobacterial circadian clock
protein KaiC occurs via formation of ATP as an intermediate
Taeko Ohkawa-Nishiwaki, Takao Kondo (Div. Biol. Sci., Grad. Schl. Sci.,
Nagoya Univ.)

The cyanobacterial circadian oscillator can be reconstituted in vitro; mixing
three clock proteins KaiA, KaiB, and KaiC with ATP results in an oscillation of
KaiC phosphorylation with a periodicity of approximately 24 h. The hexameric
ATPase KaiC hydrolyzes ATP bound at subunit interfaces. KaiC also exhibits
autokinase and autophosphatase activities, the latter of which is particularly
noteworthy because KaiC is phylogenetically distinct from typical protein
phosphatases. To examine this activity, we performed autodephosphorylation
assays using 32P-labeled KaiC. We detected the transient formation of [32P]ATPpreceding the accumulation of 32 Pi. Together with kinetic analyses, our data
demonstrate that KaiC undergoes dephosphorylation via a mechanism thatdiffers from those of conventional protein phosphatases. A phosphate group at a
phosphorylation site is first transferred to KaiC-bound ADP to form ATP as anintermediate, which can be regarded as a reversal of the autophosphorylation
reaction. Subsequently, the ATP molecule is hydrolyzed to form inorganic
phosphate. The ATPase activity of KaiC has two critical roles for
autodephosphorylation: one is the degradation of the intermediate, and the otheris providing ADP, a phosphate acceptor for the reversal of the autokinase
reaction. We propose that KaiC phosphorylation rhythm is sustained byperiodical shift in the equilibrium of the intersubunit phosphate transfer reaction
between KaiC-bound nucleotides and phosphorylation sites.
3B1110

シアノバクテリアの時計タンパク質 KaiC の ATPase に備わる分
子内フィードバック制御

Intramolecular feedback regulation of cyanobacterial KaiC
ATPaseAtsushi Mukaiyama1,2, Yasuhiro Onoue1,2, Masato Osako1,2, Takao Kondo1,2,
Shuji Akiyama3 (1Grad. Sch. of Sci., Nagoya Univ., 2CREST/JST, 3IMS,
SOKENDAI)
Circadian clock is an endogenous timing device in various organisms to adapt
their biochemical and physiological processes to daily alterations in external
environments. In cyanobacterium Synechococcus elongatus PCC 7942,
circadian clock is composed of three proteins termed KaiA, KaiB, and KaiC.
KaiA stimulates the auto-phosphorylation of KaiC, whereas KaiB attenuates theeffect of KaiA, resulting in the auto-dephosphorylation of KaiC. In vitro
incubation of the three Kai proteins with ATP generates phosphorylation cycleof KaiC with a period of approximately 24h.
Previous studies demonstrate that the frequency of the phosphorylation cycle isshown to be linearly correlated with ATPase activity of KaiC alone. In addition,
our biophysical analyses have shown a conformational ticking of KaiC in
solution, which reveals that KaiC expands and contracts its C-terminal ring
during circadian oscillation in order to assemble/disassemble with KaiA andKaiB. These observations suggest that KaiC plays a key role as a circadianpacemaker.
ATPase activity of KaiC is extremely low compared to those for other ATPases,
and temperature-compensated. Low and temperature-compensated ATPase
activity seems to be under negative feedback regulation, but experimental
evidences were not sufficient to support this hypothesis. For this purpose, wedeveloped a method to examine the negative feedback regulation of KaiC
ATPase. We present the results obtained by this method.
3C0900

DNA/RNA 結合タンパク質 TDP-43 が特異的な塩基配列を認識するメカニズム

Tandem-like fusion of two heterologous RNA-recognition
motifs in TDP-43 enhances the specificity to its target
nucleotide sequence
Yo Suzuki, Yoshiaki Furukawa (Dept. of Chem, Keio Univ.)
The RNA-recognition motif (RRM) is the most abundant RNA-binding domain
in higher vertebrates and present in about 1 % of human genes. Typically, RRM
consists of six strongly conserved secondary structures, αα, in which thefour -strands form a single anti-parallel -sheet and the two α-helices packtightly on one side of it. Interestingly, in spite of such a simple structuralorganization of RRM, each protein achieves its distinct specificity toward thetarget nucleotide sequence. Given that most of RRM-containing proteinscomprise tandem copies of RRM, such multiplicities of RRM in a single protein
may enhance the binding affinity and/or the specificity to the target RNAs;
however, roles of multiple RRMs in the binding of nucleotides remain obscure.
To find functional cooperativity among multiple RRMs in the protein, we havenoted a DNA/RNA-binding protein, TDP-43, which contains two tandem copies
of RRM (RRM1 and RRM2). In addition to RRM1 and RRM2 in isolate, a
TDP-43 fragment spanning from RRM1 and RRM2 (RRM1-2) was prepared. A− S59 −

convenient method to evaluate the DNA/RNA binding of those RRMs was firstdeveloped; thereby, ability to bind DNA/RNA was detected in RRM1 andRRM1-2 but was completely absent in RRM2. Compared to RRM1 alone,furthermore, RRM1-2 was found to more exclusively bind the TG-repeatedsingle-stranded DNA among other repeated sequences. We will thus discuss anunprecedented mechanism of how tandem-like organization of multiple RRMs
contributes to the specific recognition of the target nucleotides.
3C0912

不安定な二次構造をつくる DNA のハイブリダイゼーション速度

Hybridization Rates of DNA Strands with Unstable Selffolded Secondary Structures
Hiroaki Hata1, Akira Suyama1,2 (1Grad. Sch. Sci., Univ. Tokyo, 2Grad. Sch.
Arts and Sci., Univ. Tokyo)
In this work, we investigated the effects of self-folding and single-strand basestacking of ssDNA strands on hybridization rates. Hybridization rates of 47
pairs of complementary DNA strands were measured by using DNA
microarrays. These DNA sequences have a uniform length, a uniform melting
temperature and unstable self-folded structures. We found that the upper-boundof apparent hybridization rates proportionally increased with the probability ofboth complementary ssDNA strands being in random coil, while for the mostsequences with the similar random coil probability the hybridization rate varied
significantly from sequence to sequence. The large variation of hybridization
rates was found to be related with the strength (Ssbs) of single-strand basestacking formation in ssDNA. The hybridization rates for sequences with larger
Ssbs were more reduced compared to the upper-bound. From the experimental
results, we have constructed a hybridization reaction model describing that selffolded and base-stacked states of ssDNA reduce hybridization rates.
3C0924

長鎖 DNA の 1 分子内折り畳みはカチオン性ポリマー凝縮剤の長さによってモードが変わる

Folding of a Single Giant Duplex DNA Chain Expresses Contrastive
Behaviors depending on the Length of Cationic Polymer
Tatsuo Akitaya1, Norio Hazemoto2, Toshio Kanbe3, Makoto Demura4,
Hideaki Yamaguchi1, Koji Kubo5, Anatoly Zinchenko5, Shizuaki Murata5,
Kenichi Yoshikawa6 (1Fac. Pharm., Meijo Univ., 2Grad. Sch. Pharm. Sci.,
Nagoya City Univ., 3Sch. Med., Nagoya Univ., 4Grad. Sch. Life Sci., Hokkaido
Univ., 5Grad. Sch. Env. Study, Nagoya Univ., 6Grad. Sch. Life Med. Sci.,
Doshisha Univ.)
Folding transitions typically take place on duplex DNA that is sufficiently
longer than the persistent length of about 50 nm, that is, 150 base pairs. On
folding transition of a long duplex DNA chain induced by poly-L-lysine (PLL),
we found that weak interaction induces an ON/OFF switch, whereas strong
interaction causes gradual change. T4 bacteriophage genomic DNA composedof 166 kbp with a contour length of 57 m was used for fluorescencemicroscopy. Tetramer and 100-mer PLL were mixed with T4 DNA solution andobserved the effect on folding of a single giant DNA chain, respectively. To
visualize DNA, DAPI was used to stain DNA molecules. The large-scale
structure of T4 bacteriophage DNA in aqueous solution was directly observedwith fluorescence microscopy. Fluorescence images were recorded on DVD
through a high-sensitivity EB-CCD camera (Hamamatsu Photonics). The effectsof coexisting salts on the folding of T4 DNA induced with tetramer and 100mer PLL, concerning with an ON/OFF manner of folding and precise structureof condensed DNA. Coexisting salt inhibited the ON/OFF folding of T4
genomic DNA chain by tetramer PLL, but enhanced continuous folding by 100mer PLL. Precise structure of folded structure was analyzed with TEM andζ -potential. Folding analysis were also demonstrated for nonspecific DNAbinding protein HMG-1, 2 and specific protein STPR. Mechanisms andbiological behavior of discrete (ON/OFF) / continuous change in large-scale
structure of genomic DNA will be discussed.
3C0936

カリウムイオン依存的な四重鎖構造形成によって活性がオンになる
インテリジェントリボザイムの創製

Development of intelligent ribozyme whose activity switches onin response to K+ via quadruplex formation
Yudai Yamaoki1,2, Tsukasa Mashima1,2, Yu Sakurai3, Yukari Hara3, Takashi
Nagata1,2, Masato Katahira1,2 (1Inst. Adv. Energy, Kyoto Univ., 2Grad. Sch.
Energy Sci., Kyoto Univ., 3Grad. Sch. Nanobio., Yokohama City Univ.)

parallel quadruplex structure in response to increased K+ concentration. We
divided hammerhead ribozyme (HR) into the 5´ and 3´ portions (5´HR and3´HR) within its catalytic core and inserted this R11 via linkers composed ofuracil residues. The idea is to render a switch to a HR so that it turns on its
catalytic activity upon increase of K+ concentration. This molecular would beinactive outside the cell (~5 mM K+) but turns on its activity by entering the cell(~100 mM K+). Our first designed ribozyme, however, had some small but nonnegligible activity under low K+ concentration. The purpose of this study is tofurther suppress the ribozyme activity at low K+ concentration, bring out
highest possible activity at high K+ concentration, and make the on/off
difference more distinct.
First, we optimized the lengths of the uracil linkers. Among the designed
ribozymes, ‘5´HR-UUU-R11-UU-3´HR’ exhibited the highest activity
under high K+ concentration. Its activity was enhanced by ca. 4-fold as K+
concentration was increased. Next, we introduced the complementary strand(CS) against the R11 and examined the correlation between its length and theactivity. Presence of 5-mer CS showed ca. 0.3-fold lower activity than without
the CS at low K+ concentration. As a result it turned out that the enhancement of
the activity by increasing K+ concentration reached as high as ca. 6-fold.
3C0948

翻訳伸長におけるリボソームと tRNA の挙動−粗視化分子シミュ
レーションによる解析

Dynamical motions of tRNA and ribosome complex duringtranslation elongation studied by coarse-grained molecularsimulations
Naoto Hori, Shoji Takada (Grad. Sch. Sci., Kyoto Univ.)
In the final step of the translation-elongation cycle, the complex of two tRNAand mRNA molecules need to be advanced by exactly one codon in theribosome. To realize this movement, the two ribosomal subunits are thought tobe largely rearranged by the so-called ratchet-like rotation. Experiments havealso shown that the tRNAs form hybrid state and cofactor EF-G is essential toefficient translocation. Although many experiments, including high-resolution
structural studies, have been performed, the entire mechanism is still unclear
and we were motivated to examine how these multiple factors involved in thetranslocation.
To reveal the molecular mechanisms, we performed coarse-grained (CG)molecular dynamics (MD) simulations. In the CG model, each nucleotide isrepresented as three beads and interactions are modeled with a structure-based
potential, parameters of which were determined by fluctuation matching method
to achieve higher accuracy. First we investigated the difference of tRNA
motions between before and after the subunit rotation. In simulations, upperparts of both A-site and P-site tRNAs fluctuate between canonical(A/A and P/P)and hybrid(A/P and P/E) states. Both MD trajectories and free energy profile
obtained by umbrella sampling show that the hybrid states are more stabilized in
the rotated conformation. Next we further focused on relations among the intersubunit motion, fluctuation of the tRNAs, conformational change of EF-G, andtRNA-mRNA movement.
3C1010

A structural basis for the antibiotic resistance conferred by an
A1408G mutation in 16S rRNA
Jiro Kondo (Fac. Sci. Tech., Sophia University)
Antibiotic resistance continues to be a major public health threat. An A1408G
mutation at the A site of 16S rRNA confers high-level resistance toaminoglycosides with a 6’-NH3+ group on ring I, but moderate resistance tothose with a 6’-OH group such as geneticin. In order to gain insights into theantibiotic resistance, crystal structures of the mutant A site with and without
geneticin have been solved in the present study. As observed in the wild-typebacterial A site, geneticin specifically binds to and stabilizes an "on" state of themutant A site. Its ring I with a 6'-OH group forms a pseudo pair with G1408.
Obviously, ring I with a 6’- NH3+ group cannot form the identical pseudo pair,because the NH3+ group repels N1-H or N2-H of G1408. In the absence ofgeneticin, the mutant A site takes an "off" state, which is identical to one ofseveral "off" state conformations reported for the wild-type A site. The
structural similarities observed for both the "off" and "on" states of the wildtype and A1408G-mutant A sites suggest that the A1408G mutation does not
drastically disturb the function of the A site, where a cognate tRNA isdiscriminated from near-cognate tRNAs. Indeed, it has been reported that theA1408G mutation confers only a little reduction of the fitness of bacteria in theabsence of antibiotics.

R11, an RNA that has r (GGAGGAGGAGG) sequence, forms a compact− S60 −

3C1022

Q β replicase を構成する翻訳因子の RNA 合成における役割
Non-canonical functions of translational factors as RNA
replication cofactors

Daijiro Takeshita, Kozo Tomita (Biomed. Res. Inst., AIST)Q virus infects E. coli and replicates its genomic RNA by Q replicase, atetrameric protein complex of the virus-encoded catalytic RdRp (-subunit), andthe host-donated translational elongation factors EF-Tu, EF-Ts and ribosomal
protein S1. The core Q replicase, comprising the -subunit, EF-Tu and EF-Ts,
has RNA polymerization activity. The functions of EF-Tu and EF-Ts in Qreplicase for RNA polymerization, beyond their established functions in protein
synthesis, have remained elusive. We present six structures of the core Q
replicase complexed with RNAs, either with or without nucleotides (and/or
nucleotide analogs). The structures represent the initiation stage and fiveelongation stages of RNA polymerization. At the initiation stage, the 3’ terminal adenosine of the template RNA is required for de novo initiation, andacts as a platform for the construction of a stable initiation complex. Thus, Qvirus acquired a nucleic acid element at the 3’-end of its genomic RNA for
efficient replication of its own genome. At the elongation stages, EF-Tu in Qreplicase assists in the separation of the dsRNA of the template and the growing
RNAs, and acts as a modulator for processive RNA polymerization. Separationof the dsRNA formed by the template and the growing RNA is required for the
exponential amplification the own genomic RNA in infected cells. The present
study has now revealed the novel function of translational factors as RNA
replication cofactors, beyond their established function in protein synthesis.
3C1034

抗 HIV 宿主因子 APOBEC3G の位置依存的デアミネーション反応機構の解明Elucidation of the location-dependent deamination reaction
mechanism of an anti-HIV factor, APOBEC3G
Ayako Furukawa1,2, Kenji Sugase2, Ryo Morishita3, Taashi Nagata1, Akifumi
Takaori4, Akihide Ryo5, Masato Katahira1 (1Inst. of Adv. Energy, Kyoto
Univ., 2Bioorg. Res. Inst., Suntory Found. Life Sci., 3CellFree Sci., 4Grad. Sch.
Med., Kyoto Univ., 5Grad. Sch. Med., Yokohama City Univ.)
APOBEC3G (A3G) deaminates cytidines in the minus-strand of the HIV DNA
so that the HIV replication can ultimately be inhibited. A3G preferentiallydeaminates the third cytidine residue of a CCC sequence in a single-strand DNA(ssDNA). In order to obtain insight into the mechanism of the deamination
reaction by A3G, we developed a real-time NMR method that can analyze thecytidine deamination reaction quantitatively. We chased the intensity changes of
the cytidine peaks to be deaminated after addition of A3G to ssDNA solution.
The real-time NMR experiments revealed that the deamination rates were fasterfor cytidines located closer to the 5’ end than the 3’ end, indicating that the A3G
deamination reaction depends on the CCC-location.
Subsequently, to understand how A3G exerts the location-dependent
deamination activity, we fitted the real-time NMR data to newly constructedkinetic models. In the model 1, A3G catalyzes at the same rates, but slides at
two different rates, ks1 and ks2, in back and forth. In the model 2, A3G slides onan ssDNA back and forth with the same sliding rate, but catalyzes at two
different rates kcat1 and kcat2, corresponding to the catalytic reactions in which
A3G enters to CCC from 3’ and 5’, respectively. The experimental data fit wellto the both models, and the quality of fitting was identical to each other.
However, model 2 yielded biologically more reasonable kcat values than model
1. Therefore, we concluded that the location-dependent deamination activity canbe explained by model 2.
3C1046

大腸菌非六量体型 DNA ヘリカーゼ UvrD は多量体で DNA を巻
き戻す

we performed simultaneous single-molecule visualization of DNA unwinding
events driven by the helicase and association/dissociation events between thehelicase and DNA. Kinetic analysis of the data suggests that DNA unwinding
can be completed in a few seconds after two or more helicases are bound to
DNA, which strongly supports the model that an oligomer is the active form of
the helicase. In addition, the analysis reveals that both of the dissociation andassociation rates increase as the number of helicases that are bound to DNA
increases. Thus, we can draw a new kinetic scheme of the DNA‒helicase
interaction in which not only a dimeric helicase but also a trimeric helicase canunwind DNA.
3C1058Zhuohao Yang, Ryo Iizuka, Takashi Funatsu (Grad. Sch. of Pharm. Sci., The
Univ. of Tokyo)
The SecM protein contains an arrest sequence at the C-terminus, which interacts
with the ribosomal tunnel to induce the translation arrest. It is thought that thenascent SecM chain out of the ribosomal exit tunnel is pulled by the protein
export machinery to release the translation arrest. However, this has never been
fully substantiated. Then, we tried to release the translation arrest by pulling thearrested complex with optical tweezers.
The biotinylated HaloTag protein containing SecM arrest sequence at the Cterminus was synthesized using PURE system and C68 ribosome to preparearrested ribosome-HaloTag complex. The complex was immobilized onto theglass surface via biotin-streptavidin linkage. An oligonucleotide labeled withdigoxigenin was designated to hybridize to the extension loop on 30S subunit ofC68 ribosome. A bead coated with anti-digoxigenin antibody was conjugated tothe oligonucleotide, and used for optical trapping of the ribosomal complex.
First, we measured the rupture force for the interaction between ribosome andthe arrest sequence by moving the piezo stage at a constant rate. The rupture
force was estimated to be 20 pN. On the other hand, the rupture force of thearrested ribosome-HaloTag complex on mRNA lacking stop codon showed 22
pN. These results suggest that <20 pN of external force is required to release thetranslation arrest. Now, we are examining the relationship between the lifetimeof translation arrest and the applying force to evaluate the interaction between
ribosome and SecM arrest sequence.
3C1110The probability of double-strand breaks in genome-sized DNA
decreases markedly as the DNA concentration increases
Shunsuke Shimobayashi1, Takafumi Iwaki2, Toshiaki Mori3, Kenichi
Yoshikawa1,4 (1Grad. Sch. of Sci., kyoto Univ., 2Fukui Inst., Kyoto
Univ., 3Rad. Res. Cent., Osaka Pref. Univ., 4Grad. Sch. of Life and Med. Sci.,
Doshisha Univ.)
DNA double-strand breaks (DSBs) represent a serious source of damage for all
living things. Despite this fact, the processes that lead to their production have notyet been clearly understood, and there is no established theory that can accountfor the statistics of their production, in particular, the number of DSBs per basepair per unit Gy, here denoted by P1. This is the most important parameter forevaluating the degree of risk posed by DSBs. Here, using the single-molecule
observation method with giant DNAmolecules (166 kbp), we evaluate the number
of DSBs caused by gamma-ray irradiation. We find that P1 is nearly inversely
proportional to the DNA concentration above a certain threshold DNA
concentration. A simple model that accounts for the marked decrease of P1. showsthat it is necessary to consider the characteristics of giant DNA molecules assemiflexible polymers to understand the mechanism of DSBs.
3D0900

膜配向した PS II の ENDOR 法による Mn クラスター周辺のプ
ロトンの位置の決定

Single-molecule visualization of a non-hexameric helicase
reveals active roles of its oligomeric forms in DNA unwinding
Hiroaki Yokota, Yoshie Harada (iCeMS, Kyoto Univ.)
Escherichia coli UvrD protein is a non-hexameric superfamily I DNA helicase
which plays a crucial role in nucleotide excision repair and methyl-directedmismatch repair. We reported that the helicase takes the form of a dimer or atrimer when bound to duplex DNA with a ssDNA tail (12, 20 or 40 nt) in theabsence of nucleotide or in the presence of ATPγS. Here we have extended theprevious study to provide a clear answer to the question whether the helicase
unwinds DNA in the form of a monomer or an oligomer in the presence of ATP,

リボソーム-SecM 翻訳アレスト配列間相互作用の 1 分子顕微解析

Single-molecule force measurement for the interaction
between ribosome and SecM arrest sequence

Determine the location of protons surrounding Mn cluster of
PS II by proton matrix ENDOR using oriented PS II
membrane
Hiroki Nagashima, Hiroyuki Mino (Division of Material Science, Graduate
School of Science, Nagoya University)
In oxygenic photosynthesis, light driven water-splitting is catalyzed by the Mn
Cluster (Mn4 O5 Ca) in photosystem II (PS II). There are five different redox
states, labeled Sn (n=0-4). By absorbing one photon, S state advanced higher
oxidation state. Molecular oxygen is released during the transition of S4 to S0
states. Recently, the crystal structure of PS II was revealed with 1.9 &Aring− S61 −

resolutions, including the location of the water molecules, amino acid andoxygen atoms coordinated to Mn cluster.
S2 multiline signal is the well-characterized EPR signal, arising from Mncluster. In this work, S2 multiline signal was investigated by proton matrix
ENDOR. Six pairs of ENDOR signals have been reported previously. In order todetermine the locations of protons proximity to Mn cluster, the oriented PS II
membranes were used for the measurements. The hyperfine splitting with 4.0
MHz changed to that with 3.6 MHz at the angle of 0°C between membrane
normal n and the external magnetic field H0. In addition, the new hyperfine
splitting with 1.7 MHz was detected. These signals were simulated by the dipole
interactions between the electron spin on each Mn and nuclear spin ofsurrounding protons. Based on X-ray crystal structure, the locations of the
protons for these ENDOR signals were assigned.
3D0912

Factors that differentiate the H-bond strengths of water near
the Schiff bases in bacteriorhodopsin and Anabaena sensory
rhodopsin
Hiroshi Ishikita1,2, Keisuke Saito1, Hideki Kandori3 (1Career-Path, Kyoto
U, 2JST PRESTO, 3Nagoya Inst Tech)
Bacteriorhodopsin (BR) functions as a light-driven proton pump, whereas
Anabaena sensory rhodopsin (ASR) is believed to function as a photosensor,
despite the high similarity in their protein sequences. In Fourier transform
infrared (FTIR) spectroscopic studies, the lowest O-D stretch for D2 O wasobserved at ~2200 cm-1 in BR but was significantly higher in ASR (>2500
cm-1), which was previously attributed to a water molecule near the Schiff base
(W402) that is H-bonded to Asp85 in BR and Asp75 in ASR. We investigated
the factors that differentiate the lowest O-D stretches of W402 in BR and ASR.
Quantum mechanical/molecular mechanical (QM/MM) calculations reproduced
the H-bond geometries of the crystal structures and the calculated O-D
stretching frequencies were corroborated by the FTIR band assignments. The
potential-energy profiles indicate that the smaller O-D stretching frequency in
BR originates from the significantly higher pKa (Asp85) in BR relative to thepKa(Asp75) in ASR, which were calculated to be 1.5 and -5.1, respectively. The
difference is mostly due to the influences of Ala53, Arg82, Glu194-Glu204, andAsp212 on pKa(Asp85) in BR, and the corresponding residues Ser47, Arg72,
Ser188-Asp198, and Pro206 on pKa (Asp75) in ASR. Because these residues
participate in proton transfer pathways in BR but not in ASR, the presence of astrongly H-bonded water molecule near the Schiff base ultimately results fromthe proton-pumping activity in BR.
3D0924BLUF ドメインの光反応における水素結合環境の変化

Structural changes of hydrogen-bonding environment upon
the photoreaction of the BLUF domains
Tatsuya Iwata1, Shota Ito2, Mineo Iseki3, Masakatsu Watanabe4, Hideki
Kandori2 (1Cent. Fost. Young Innov. Res., NITech, 2Dept. Front. Mater.,
NITech, 3Faculty Pharm. Sci., Toho Univ., 4Grad. Sch. Creat. New Photo.
Ind.)
Of flavin-binding photoreceptors, BLUF (sensor of blue-light using FAD)
domain shows a unique photoreaction: no apparent change in the chemical
structure of FAD between unphotolyzed and red-shifted intermediate states. It
has been reported that the conserved glutamine and tyrosine residues nearby
FAD play crucial roles in the formation of the intermediate state. Hydrogenbonding environment of FAD and amino acid residues are important for thephotoreaction of BLUF. To investigate the hydrogen-bonding environment of
FAD and these residues, we measured the BLUF domain of AppA from
Rhodobacter sphaeroides (AppA-BLUF) by FTIR spectroscopy.
In the deuterated samples, the bands at 2416 (-) and 2029 (+) cm-1 were
influenced by the Tyr-D4 and 13C -labeled AppA-BLUF. Thus, we concluded
that these signals originate form O-D stretch of Tyr21 in the unphotolyzed andintermediate states, respectively. The corresponding frequencies of O-H
stretches are ~3300 and ~2800 cm-1. O-H group of Tyr21 forms moderatehydrogen bond in the unphotolyzed state and forms stronger hydrogen bond in
the red-shifted intermediate state.
Measurement of 13C-labeled, 13C, 15N-labeld, α-15N-Gln labeled samples
revealed no formation of C=N stretch and disappearance of C=O stretch. This
result shows that the Keto-enol tautomerism reaction does not take place in the
Gln63 contrary to the previous proposal. From these data, possible model for thephotoreaction of BLUF domain will be proposed.
3D0936

励起移動を記述する変分マスター方程式の改良および PSII への適用

Improvement of variational master equation describing
excitation energy transfer, and its application to PSII
Yuta Fujihashi, Akihiro Kimura (Department of Physics, Graduate School of
Science, Nagoya University)
The excitation energy transfer (EET) is an essential process in the lightharvesting system. EET interaction J between Chlorophylls (Chl) and coupling
λ between Chl and protein are comparable in the light-harvesting system.
Recently, Nazir et al. constructed theory of quantum master equation (QME)
based on variational principle in order to adequately describe EET that bothinteractions are comparable. We improved theory of Nazir et al. They
determined the variational parameter by the free energy minimization based on
the Bogoliubov inequality. We used the generalized Bogoliubov inequality
derived by Decoster instead of original one for free energy minimization. Thisinequality can provide value close to the true free energy than the original
Bogoliubov inequality. We estimated rate constants of EET by the QME withvariational parameter determined by this procedure. Our calculation of rate of λdependence by our theory was closer to the result by the non-perturbative
numerical approach than that by theory of Nazir et al.
We investigated extent of the delocalized excited state within Photosystem II(PSII) by using the character of perturbation terms of above theory. From ourresults, it is found that at 300 K excited states are localized unless J is at least 90
cm-1. This result suggests that many of excited states of Chls in PSII are
localized at 300 K unlike the theoretical model of Renger et al. which were
determined to extent of the delocalized excited state in PSII based on only
magnitude relationship between J and λ.
3D0948光化学系 II コア複合体での非光化学的消光の可能性

Possible non-photochemical quenching mechanism withinpohotosystem II core complex
Yutaka Shibata1, Shunsuke Nishi2, Keisuke Kawakami3, Jian-Ren Shen4, Thomas
Renger5 (1Grad. Sch. Sci., Tohoku Univ., 2Grad. Sch. Sci., Nagoya Univ., 3Adv. Res.
Inst., Osaka City Univ., 4Grad. Sch. Sci., Okayama Univ., 5JKU Linz)
Non-photochemical quenching (NPQ) has been known to be an importantmechanism to protect the phososystems from the damage by high light. The
peripheral antennae of photosystem II (PS II) have been considered to play amajor role in NPQ. On the other hand, there has been few study which proposed
an NPQ component operating within the PS II core complex. We have studied
the ultrafast fluorescence dynamics of PS II to characterize the “red chlorophylls(Red Chls)”, which have the excited-state energies lower than the primary
donor. Although the thermal fluctuation ensures the up-hill energy transfer fromRed Chl to the primary donor, they seem to have no productive contribution toincrease the light-harvesting efficiency. Since the exciton energy is accumulated
into Red Chl, transient formation of a quencher in vicinity to Red Chl can be aneffective NPQ pathway within the PS II core complex. In order to validate theabove scenario, the molecular identity of Red Chl is indispensable. Recent
theoretical modeling based on the structure of PS II has offered a great
opportunity to clarify the exciton energy flow in PS II. We have combined ourultrafast fluorescence studies of PS II with the theoretical modeling andassigned the Chl29 (nomenclature of Loll et al. 2005) to Red Chl. Chl29 was
found to be located close to ChlZ, whose oxidized form was suggested tooperate as a transient quencher. Carotenoid molecules also lie proximally,
implying their quenching roles. The quenching mechanism through Chl29 will
be discussed in the presentation.
3D1010低温光化学系 II における多重光還元 Mn4 CaO5 Hx クラスター分
布: 時間平均 XRD 構造の照射 X 線量依存性

X-ray dose dependence of time-averaged XRD structure ofmulti-photoreduced Mn4CaO5Hx clusters in photosystem II at
low temperature
Masami Kusunoki (Department of Physics, School of Science Technology,
Meiji University)
The XRD structure of a light-driven water-oxidation enzyme, photosysytem II(PSII), was very recently revealed its electron density image at resolution of 1.9
Å. However, it is critically inconsistent with EXAFS data in the dark stable S1state. To evaluate the x-ray radiation damage, we present the potoreduction
kinetics for the X-ray photoelectric effect followed by the photoionization
processes leading to multiple photoreductions of the water-splitting Mn4CaO5cluster, which is directly proportional to the X-ray dose. This kinetics is basedon the fact that solvated photoelectrons can rapidly diffuse into and out from themanganese cluster through H-bonding water channel(s) to realize a quasi-− S62 −

steady distribution under x-ray irradiation. This theory was applied to analyze
two relevant photoreduction data by x-ray absorption near edge spectroscopy(XANES) at the manganese K-edge of PSII samples at low temperatures. Thus,
we could establish the universal Eedge vs K1t relationship of OEC in PSII
together with the reduction rate constants, {Kj, j=0, -1, . . , -5}, as well as themost-likely manganese valence models of the Mn4 clusters in thesephotoreduced Sj -states, to show that the 1.9 Å. resolution XRD image of the
Mn4CaO5 cluster in PSII crystal would be a result of integrated contributions
mainly from S0 and S-1 states. Using UDFT/B3LYP method, we also show Xray dose dependence of its time averaged atomic structure is in line with theobserved Mn4CaO5(H2O)4 structure.
3D1022光化学系 II 酸素発生系 S2状態における Mn 間磁気的相互作用の解明

Determination of magnetic couplings of S2 state Mn-cluster in
Photosystem II studied by PELDOR measurement
Mizue Asada1, Hiroki Nagashima1, Faisal Hammad Mekky Koua2, Jian-Ren
Shen2, Hiroyuki Mino1 (1Grad. Sch. Sci., Univ. Nagoya, 2Grad. Sch. Natl. Sci.
& Tech., Univ. Okayama)
Oxygen evolution is carried out by Mn-cluster in Photosystem II (PS II). Mncluster is consisted of four Mn, five O and one Ca, and is oxidized through a cycleof five redox states labeled Sn (n = 0-4). In the S2 state, an EPR signal, called S2
multiline signal, was detected at g = 2 with about 60 mT width. It is derived frommix valent Mn ions with one Mn(III) and three Mn(IV), coupled with exchangeinteractions. Recently, the PS II structure with 1.9 Å resolutions was revealed by
X-ray analysis [1]. However, the magnetic structure of the Mn-cluster has not
associated to the molecular structure derived by X-ray crystallography.
Pulsed electron-electron double resonance (PELDOR) is a well-establishedmethod for measuring distance between electronic spins by detecting dipole
interactions. We measured the PELDOR signals between YD and Mn-cluster in
S2 state in the spinach and T. vulcanus PS II membranes. Based on theanisotropy of the PELDOR signal in the oriented PS II, the spin projections ofeach Mn atom were determined by using coordinates in the crystal structure.
The results show that the Mn ion coordinated by H332, D342 and E189, has avalence of Mn (III) in the S2 state. The signs of the exchange interactions
between four Mn atoms were determined.
[1] Umena Y. et al. Nature, 473, 55-U65 (2011)
3D1034

光化学系 II におけるキノン電子受容体の酸化還元電位制御機構

Mechanism of controlling the redox potentials of the quinone
electron acceptors in photosystem II
Ryota Ashizawa1, Takuya Iwasa1, Miwa Sugiura2, Takumi Noguchi1 (1Divisionof Material Science, Graduate School of Science, Nagoya University, 2Division ofMaterial Science, Graduate School of Science, Nagoya University)
Photosystem II (PS II) in plants and cyanobacteria withdraws electrons fromwater using light energy and transfer them to the primary quinone electron
accepter QA and then to the secondary quinone acceptor QB. Although thechemical identity of QA and QB is both plastoquinone-9 (PQ-9), the redox
potential of QB is higher than that of QA, facilitating an electron flow from QA to
QB. The interactions of PQ with proteins should play a major role indetermining the redox potentials of these quinone electron acceptors; however,the details remain to be clarified. In this study, we have investigated themechanism of controlling the redox potentials of QA and QB using density
functional theory calculations and Fourier transform infrared spectroscopy.
Energy levels of neutral and anion forms of various H-bonded complexes of PQ
with water or amino acids as H-bond donors were calculated and the redox
potentials were estimated. It was shown that the redox potential of PQ linearly
increased with the number of H-bond by +165 mV/H-bond at ε = 4.9. Thus, thehigher redox potential of QB than QA by ~+80 mV can be attributed to more Hbonds in QB (three H-bonds) compared with QA (two H-bonds) as have been
revealed by X-ray crystallography. We also calculated the frequencies and the
13C-isotope shifts of the C=O stretching vibrations of PQ in the model H-bondcomplexes. The calculated frequencies were compared with the experimental
ones in relevance to the H-bond interactions of QA and QB in PSII proteins.

electron transfer (ET) reaction in large systems by using the fragment molecularorbital (FMO) method. With use of the molecular orbitals (MO) from the FMOLCMO method [2] that are properly delocalized over the entire system, we
compute the electronic coupling energy of ETs by the generalized MullikenHush and the bridge Green function methods. Moreover, the tunneling current
analysis is carried out at the fragment resolution, which has advantages bothconceptual and computational.
In this study [3], we have applied our approach to the ETs in photosynthetic
reaction center of Blastochloris viridis. For the ET from menaquinone toubiquinone, a major pathway via the Fe2+ ion and a histidine ligand (His L190)
is observed. When the metal ion is replaced by a Zn2+, another pathway
involving two His ligands (His M217 and His L190) opens. These two His
residues take up the central role when the metal ion is depleted. The computed
values of electronic coupling are found to be consistent with experiments.
[1] H. Nishioka and K. Ando, J. Chem. Phys. 134 (2011) 204109. [2] S.
Tsuneyuki et al., Chem. Phys. Lett. 476 (2009) 104. [3] H. Kitoh-Nishioka and
K. Ando, submitted.
3D1058光合成細菌 Rhodopseudomonas の遺伝子発現パターンの電気化
学制御

Electrochemical regulation of gene expression profiles of
Rhodopseudomonas, a photosynthesis bacterium
Yue Lu1, Syoichi Matsuda1, Shuji Nakanishi2, Kazuhito Hashimoto1 (1The
Univesity of Tokyo, School of Engineering, Department of Applied
Chemistry, 2Research Center for Advanced Science and Technology)
Photosynthetic bacteria are able to synthesize organic compounds directly from
CO2 and water by using energy from sun light. The amounts of CO2 reduced bysuch microorganism are so considerable that can significantly contribute to thecarbon cycle on the earth. Recently, production of useful organics or bioenergy
from CO2 by photosynthesis microorganisms has attracted a lot of attentions.
Most of the studies conducted so far are based on molecular biological
techniques such as construction of various mutants. Although these methods arevery powerful, they also possess a couple of problems with respect to safety andcost. We are trying to enhance CO2 fixation ability of wild types ofphotosynthetic bacterium via electrochemical tuning of the intracellular redox
environment which deeply involved in expressions of genes related tophotosynthesis. As a first step, we successfully regulate the expression level ofgenes encoding chlorophyll, which is an extremely important biomolecule
allowing bacteria to absorb light energy, of model bacteria Rhodopseudomonas
by electrochemical method.
3E0900

核膜孔複合体構造変化による核移行制御機構の構造基盤

Structural basis for nuclear import regulated by molecularrearrangements in the nuclear pore complex
Junya Kobayashi1, Yoshiyuki Matsuura1,2 (1Div. Biol. Sci., Grad. Sch. Sci.,
Nagoya Univ., 2Strl. Biol. R. Ctr., Grad., Nagoya Univ.)
In all eukaryotes, precise regulation of the bidirectional transport ofmacromolecules between the nucleus and the cytoplasm is essential for manycellular processes, including gene expression, signal transduction, cell cycle
progression and differentiation. Nuclear transport occurs through nuclear porecomplexes (NPCs) that form aqueous channels across the nuclear envelope.
Altering the structure of NPCs is emerging as a novel mechanism to control
nuclear transport. In this study, we focus on a nuclear import pathway that isregulated by a cell-cycle dependent interaction between a nuclear import
receptor and a nucleoporin. Here we report crystal structures of the import
receptor in isolation, and also in complex with either its import cargoes or the
nucleoporin or RanGTP. Our new structures establish the structural basis forrecognition of a novel nuclear localization signal (NLS), cargo-displacement by
RanGTP, and import regulation triggered by cell-cycle dependent NPC
rearrangements.
3E0912

NADH シトクロム b5還元酵素超高分解能結晶構造解析による水素と外殻電子の観察3D1046光合成バクテリア反応中心の電子移動経路解析

Electron Transfer Pathway Analysis in Bacterial
Photosynthetic Reaction Center
Hirotaka Kitoh-Nishioka, Koji Ando (Grad. Sch. Sci. Kyoto Univ.)
We have recently developed a novel approach [1] to describe long-distance

Visualizing hydrogens and outer-shell electrons of NADH
cytochrome b5 reductase by ultra-high resolution
crystallography
Kiyofumi Takaba1, Kazuki Takeda1, Masayuki Kosugi1, Taro Tamada2, Ryota
Kuroki2, Kunio Miki1 (1Grad. Sch. Sci., Univ. Kyoto, 2JAEA)− S63 −

NADH cytochrome b5 reductace (b5R) is an enzyme transferring electrons from
NADH to cytochrome b5, and it has an FAD molecule in the active center as a
cofactor for reduction. To understand the molecular mechanism of b5R, it isindispensable to determine the accurate position of hydrogen atoms and the
distribution of the outer-shell electrons. Ultra-high resolution X-ray
crystallography better than 1Å resolution enables us to visualize such details of
distribution of electrons. We determined an oxidized structure of porcine b5R at
0.78Å resolution by ultra-high resolution crystallography with high energy Xrays from SPring-8. The model structure includes about 90% of all hydrogenatoms. Considering the resolution-dependent feature of atomic scatteringfactors, some corrections in the reciprocal space was performed, which
produced clear visualization of the electrons of hydrogens of FAD and theresidues interacting with FAD, and outer-shell electrons such as lone pairs ofcarbonyl oxygens and imino nitrogens and covalent bond electrons of thesegroups in the electron density map. These detailed electron distribution revealed
the hydrogen bond network in the active center of b5R, which provided
significant clues for understanding its ability in transferring electrons.
3E0924最新の各種固体 NMR 法を用いた絹の構造解析

Silk Structure studied by Newly-developed Solid-state NMR
Keiko Okushita, Koji Yazawa, Akihiro Aoki, Tetsuo Asakura (Grad. Sch. ofEng., Tokyo Univ. Agr. Tech.)
Recently, silk fibroins from silkworms have been used as excellent biomaterials
as well as excellent textiles. In order to develop such an excellent biomaterial, thedetailed structural information is required. Newly-developed solid-state NMR has
been applied to clarify the silk fibroin structure. Ultra-high field NMR (930 MHz)
coupled with ultra high speed MAS probe (80 kHz speed) have been used toclarify the inter-molecular structures of silk model peptides through 1 H solidstate NMR. In addition, 13C labeling of silk fiber and 13C solid-state NMR suchas DARR or PDSD has been also used for studying the inter-molecular structure.
3E0936

Interaction of human calcitonin with curcumin as an inhibitorof fibrillation as revealed by NMR and docking simulation
Ken Takeuchi1, Hikari Watanabe1, Javkhlantugs Namsrai1, Hiroshi Hirota2,
Akira Naito1 (1Yokohama National University Graduate School ofEngineering, 2Riken)
Human calcitonin (hCT) is known to form an amyloid fibril precipitate inconcentrated aqueous solution and is associated with medullary carcinoma of
the thyroid, although calcitonin (CT) is a peptide thyroid hormone in mammals,
and plays a central role in calcium-phosphorus metabolism. Therefore, CT has
been studied not only for therapeutic use with long stability but also as a model
peptide to clarify the fibrillation process and mechanism of amyloid fibril
formation. In the TOCSY NMR experiments, the lyophilized preparation weredissolved in 20 mM phosphate buffer containing the 100 mM salt at pH 7.4 andadded DMSO with the volume ratio be 15%. A freeze-dried sample wasdissolved in this solution. TOCSY NMR was measured right after the mixing of
the sample with the solution. Next, we measured TOCSY spectrum in the
addition of 1.1 mM of Curcumin. It was noted that a peak of Phe16 did notdisappear in the spectrum. This result indicates that the core part of hCT does
not form fibril. It is therefore expected that Phe16 signal will disappear after the
fibril formation. Thus, it is concluded that curcumin interacts with hCT toinhibit the fibril formation. The interactions of curcumin tautomers with hCT
peptide were revealed by using docking simulation. The structures of curcumintautomers and hCT were first determined in water solution and used for the
initial conformations of the docking simulation until the stabilized structures
were obtained. Detailed simulation results will be presented in the meeting.
3E0948

単粒子クライオ電子顕微鏡解析によるサポウイルスキャプシドの 8-Å 構造
8-Å structure of sapovirus capsid by single particle electron
cryomicroscopy
Naoyuki Miyazaki1, David Taylor1, Grant Houseman2, Kosuke Murakami2,
Kuniaki Nagayama1, Kazuhiko Katayama2, Kazuyoshi Murata1 (1National
Institute for Physiological Sciences, 2National Institute of Infectious Diseases)
The caliciviruses are a family of small non-enveloped viruses of 27 to 40 nm indiameter, and can be classified into five genera: Vesivirus, Lagovirus,
Norovirus, Sapovirus and Nebovirus. Among them, noroviruses and sapoviruses
are well known to cause gastroenteritis in a range of mammals, including
humans. However, study of the sapoviruses is far behind compared withnoroviruses. For example, the capsid structures of noroviruses and vesiviruseshave been determined at atomic resolution, while no structure is available forsapoviruses. Therefore, we determined the capsid structure of the sapovirus atabout 8-Å resolution by conventional cryo-electron microscopy and single
particle reconstruction to get structural insights into the host specificity.
Furthermore, we performed homology modeling with the cryo-EM map. The
sequence of the capsid protein of sapovirus was aligned with the relatedsequences of noroviruses and vesiviruses by using information of predictedsecondary structures and electron microscopically determined threedimensional structures due to their low sequence similarities. Then, homology
model was created and refined after fitting into the cryo-EM map. To get moreaccurate model, we have tried to improve the resolution by Zernike phase
contrast cryo-electron microscopy with a direct detection device (DDD) detector
for electrons. These progresses will be discussed.
3E1010

Structural analysis of the 26S proteasome by cryo-electron
microscopy and Single-Particle Analysis
Zhuo Wang1, Yasuo Okuma1, Daisuke Kasuya2, Kaoru Mitsuoka3, Yasushi
Saeki4, Yasunaga Takuo1 (1Department of Bioscience and Bioinformatics,
Faculty of Computer Science and Systems Engineering, Kyushu Institute ofTechnology, 2Biomedicinal Information Research Center, Japan Biologycal
Information Consortium (JBIC), 3Biomedicinal Information Research Center,
National Institute of Advanced Industrial Science and Technology, 4Laboratoryof Protein Metabolism, Tokyo Metropolitan Institute of Medical Science)
The 26S proteasome is the key enzyme in eukaryotic homeostasis and it plays animportant role by undertaking highly controlled degradation of a wide range ofproteins, including cellular regulators such as those controlling cell-cycle
progression and apoptosis. The 26S proteasome is composed of one 20S core
particle (CP) and two 19S regulatory particles (RPs). According to fragile natureand dynamics of the 26S proteasome, RPs cannot bind to the CP tightly, whichmakes structural analysis of the 26S proteasome more difficult. Although thebasic organization of the RP in the Schizosaccharomyces pombe 26S
proteasome has been determined with a 9. 1 resolution by cryo-electron
microscopy (cryo-EM), the detailed localization of the RP subunits have notrevealed yet. In our study, for preventing RPs from releasing, 10% glycerol has
been added in the specimen and micrographs were acquired by cryo-EM at theliquid helium temperature, Thus it was found that the photograms of Rpn10eliminated mutants have low contrast and so their ‘picking up’ was moredifficult than usual. To solve this problem, CTF compensation using Wiener
filter has been used in image processing considering spatial frequencydependent SN ratio. After that, Rpn10-eliminated mutant particles have beenvisible and easily picked up only RP-bound CPs in the photograms. The 3D
maps of Rpn10-eliminated mutant by single-particle analysis after classification
and averaging will be reported here.
3E1022大気圧電子顕微鏡（ASEM）によるタンパク質微結晶とマイコプラ
ズマの液中観察

Direct electron microscopy of protein crystals andMycoplasma cells in solution using the Atmospheric SEM
Yuusuke Maruyama1, Tatsuhiko Ebihara1, Daisuke Nakane2, Hidetoshi
Nishiyama3, Takayuki Nishizaka4, Miki Senda5, Kazuhiro Mio1, Mitsuo Suga3,
Toshiya Senda1, Makoto Miyata2, Chikara Sato1 (1AIST, 2Univ. Osaka
City, 3JEOL, 4Univ. Gakushuin, 5JBIC)
High-resolution, quick visualization of biological sample in solution is an
important theme, especially in the field of diagnosis and screening. Protein
microcrystals and Mycoplasmas are the samples not easy to observe by optical
microscopy. The nanometer-to-sub-nanometer resolution range of electron
microscopy (EM) would be appropriate for this; however, to avoid electron
scattering, usual SEM (Scanning EM) and TEM (Transmission EM) require theelectron pathway, including sample holder, to be essentially vacuum. This
necessitates the time-consuming pretreatment of biological samples forpreservation in extremely low pressure. In response to this difficulty, we havedeveloped the advanced version of SEM, an atomospherice SEM (ASEM). Thisis an electron microscope with an inverted configuration, allowing samples to beviewed in open atmosphere. This system can be applied not only to examine
cells and tissues, but also non-biological samples; salt crystal growth, nanoparticle self-organization, electrochemical deposition, and the changes
occurring during of solid / liquid phase transition of metal (1. We applied thismicroscopy to protein microcrystals and Mycoplasma, an important creature in
medicine and in biology(2. [1] Ultramicroscopy 111, 1650-58, 2011. [2] BBRC
417, 1213-18, 2012.

− S64 −

3E1034

粗視化 Go モデルを用いた Ferredoxine-like fold タンパクのフォールディング機構の解析

Analyses of folding processes on the Ferredokin-like foldproteins by means of a coarse grained Go model
Masatake Sugita, Takeshi Kikuchi (Dept. Bioinf., Coll. Life Sci., Ritsumeikan
Univ.)
It has been recognized that analysis of proteins with a same native topology isefficient strategy for comprehending protein folding mechanisms. In particular,
proteins sharing Ferredoxin-like fold are widely studied experimentally. It has
been reported that these proteins exhibit various φ value patterns. These profiles
are interpreted that stability of the two competing foldons regulates folding
mechanisms. We are currently studying Ferredoxine-like fold proteins byanalyzing amino acid sequence information. Our data also indicate that asignificant segment for a folding is different among Ferredoxin-like foldproteins. For detailed elucidation of folding mechanisms with higher resolution
including extensive free energy profiles, we have to conduct simulation
approach. In this study, we analyze the folding processes of Ferredoxin-like foldproteins using coarse grained Go model. We use protein S6, U1A and ADA2h
as target proteins. While we couldn’t distinguish individual folding pathway
using a topology-only model, we discern the difference of heights of free energy
barrier. On the other hand, we got inherent folding process respect to each target
protein using the model including sequence colors.
3E1046

異なる参照状態を用いた統計ポテンシャルによるタンパク質モデル

from the electrostatic interactions are reduced. Moreover, the number of replicas
can be decreased by exchanging the electrostatic charges only of solute atoms.
Consequently, the CREM simulations realize effective samplings in theconformational space with small number of replicas. I will show theeffectiveness of this method through applications to a amyloid-beta fragment.
3E1110

レプリカ交換インターフェースプログラム(REIN)

Replica-exchange interface program (REIN)
Naoyuki Miyashita1,2,3, Suyong Re4, Yuji Sugita1,2,3,4 (1RIKEN QBiC, 2RIKEN
CSRC, 3RIKEN AICS, 4RIKEN ASI)
In the recent decade, replica-exchange molecular dynamics (REMD) method [1]has attracted much attention as an enhanced conformational sampling method.
In REMD, ensembles consist of non-interacting copies (replicas) of an original
system with different conditions. The pair of replicas are exchanged every few
steps by metropolis criteria to enhance the sampling efficiency.
We have developed replica-exchange Interface program (REIN) to facilitate
REMD simulations. Currently, the multi-dimensional REMD can be performed[2] using the highly parallelized conventional MD (NAMD2 [3] and MARBLE[4]). The other conventional MD programs can be easily combined as well.
REIN is also tested for use in supercomputer systems, enabling us to simulate
substrate binding or conformational changes in large proteins with highprecision. The detail of program and some recent applications will be presented.
[1] Y. Sugita and Y. Okamoto, Chem. Phys. Lett., 314,141(1999)[2] Y. Sugita, A. Kitao, and Y. Okamoto, J. Chem. Phys., 113, 6042 (2000)[3] J. C. Philip et al., J. Comp. Chem., 26, 1781 (2005)[4] M. Ikeguchi, J. Comp. Chem., 25, 529 (2004)

構造の最適化

Optimization of protein model structures according tostatistical potentials with different reference states
Matsuyuki Shirota1,2, Kengo Kinoshita1,2,3 (1GSIS, Tohoku Univ., 2ToMMo,
Tohoku Univ., 3IDAC, Tohoku Univ.)
Prediction of the 3D structure of a protein molecule from its amino acidsequence is an important challenge in computational biophysics. One of thebottlenecks is to select the most native-like structure among the model structuresgenerated in the prediction process. Statistical potentials derived from protein
structure database are widely used for this purpose. In principle, they aredeveloped by counting the conformations in the database of native protein
structures under the assumption that frequently observed conformations areenergetically favorable ones. To quantify the significance of the counts ofconformations in the database, the reference state, a virtual state in which all
atomic interactions are random, is required. Previously, we demonstrated thatthe definition of the reference state of distance-dependent atomic pair statisticalpotentials can be theoretically variable and in practice statistical potentials withvarious reference states show different advantages, such as specificity for thenative structures or the correlation to the native-likeness. To further explore theproblem of reference states, here we developed an optimization method ofprotein model structures under several statistical potentials based on distinct
reference states. In addition, we also employed several statistical potentials in a
linear combination to improve the performance. Our results showed that various
reference states have different advantages in both model evaluation and model
optimization.
3E1058

3F0900

サポーテット膜を用いた膜タンパク質の顕微鏡下でのイオン輸送活性計測

Evaluation of ion-pump activity of membrane proteins reconstituted on supported membrane under optical microscope
Jyunan Ki, Rikiya Watanabe, Kazuhito Tabata, Hiroyuki Noji (Department ofApplied Chem., Univ. Tokyo)
Supported membrane is the experimental platform in which a lipid bilayer istethered on a solid surface. Owing to the mechanical stability, it allows us toinvestigate physicochemical properties of membranes and also membraneproteins reconstituted in the membrane. In this study, to enhance the versatilityof this method, we developed a novel supported membrane system that enables
to measure the ion-pump activity of membrane proteins under an optical
microscope. As a model ion pump, we reconstituted FoF1-ATP synthase (FoF1)
in liposomes. The supported membrane was prepared on a glass surface fromthe reconstituted liposome with a buffer containing a pH indicator dye (pHrodo)
and caged-ATP. By introducing the evanescent illumination of 405 nm light for
uncaging of ATP from the bottom of the glass surface, ATP was supplied onlyin the illumination area. The local pH of the membrane-surface space wasmonitored with the fluorescent signal of pHrodo excited with the evanescentillumination of 532 nm. Only after the illumination for uncaging, the intensity ofpHrodo evidently increased, suggesting the acidification between themembrane-surface space. Furthermore, the degree of the acidification was
proportional to the amount of uncaged ATP. These results showed that thissystems allowed us to successfully monitor the H+ -pumping activity of Fo F1
reconstituted in the supported membrane.

クーロンレプリカ交換法を用いた A βフラグメントに関する研究

Studies on a Aβ fragment by the Coulomb replica-exchange
methodSatoru Itoh1,2, Hisashi Okumura1,2 (1Institute for Molecular Science, 2The
Graduate University for Advanced Studies)
Effective samplings in the conformational space are necessary to predict thenative structures of proteins. The replica-exchange method (REM) is one of themost well-known methods among the generalized-ensemble algorithms which
realize effective samplings in the conformational space for biomolecularsystems. However, REM has a difficulty for large systems that many replicas
are needed to realize such effective samplings. In other words, huge amount ofcomputer time is required in the replica-exchange method for the large systems.
In order to overcome this difficulty, we proposed a new replica-exchange
method, which we refer to as the Coulomb replica-exchange method (CREM),
recently. In this CREM, electrostatic charges are exchanged among replicas
although the temperatures are exchanged in conventional replica-exchange
method. By exchanging the electrostatic charges, the free-energy barrier come

3F0912

Catalytic activity of MsbA reconstituted in nanodisc particles
is modulated by remote interactions with the bilayer

Takeaki Kawai1, Jose Caaveiro1,2, Toyomasa Katagiri3, Hisashi Tadakuma1, Takuya
Ueda1, Kouhei Tsumoto1,2,4 (1Grad. Sch. of Front. Sci., Univ. of Tokyo, 2Inst. Med.
Sci., Univ. of Tokyo, 3Inst. Gen. Res., Univ. of Tokushima, 4Dep. Chem. Bio., Univ. ofTokyo)
ATP-binding cassette (ABC) transporters couple hydrolysis of ATP with
vectorial transport across the cell membrane. We have reconstituted ABC
transporter MsbA in nanodiscs of various sizes and lipid compositions to test
whether ATPase activity is modulated by the properties of the bilayer. ATPhydrolysis rates, Michaelis-Menten parameters, and dissociation constants ofsubstrate analog ATP-γ-S demonstrated that physicochemical properties of thebilayer modulated binding and ATPase activity. This is remarkable when
considering that the catalytic unit is located ~50 angstrom from thetransmembrane region. Our results validated the use of nanodiscs as an effective
tool to reconstitute MsbA in an active catalytic state, and highlighted the close− S65 −

relationship between otherwise distant transmembrane and ATPase modules.
3F0924

線虫イネキシン 6 ギャップ結合チャネルの単離と機能特性

Isolation and characterization of C. elegans innexin-6 gap
junction channels
Atsunori Oshima1, Tomohiro Matsuzawa2, Kouki Nishikawa1, Yoshinori
Fujiyoshi1 (1CeSPI, Nagoya Univ., 2Grad. Sch. of Sci., Kyoto Univ.)
Invertebrates possess gap junction channels composed of innexins that evolved
independently from vertebrate connexins. Only limited information is, however,available on the innexin channel structure. Here we successfully expressed andpurified C. elegans innexin-6 (INX-6, 45kDa) gap junction channels, andcharacterized the structural properties and channel permeability using electron
microscopy and microinjection of fluorescent dye tracers, respectively. The
INX-6 proteins formed gap junction plaques with a loosely packed hexagonallattice. Channel distance, based on the reflection spots of the INX-6 gap junction
plaques, was longer than that of connexin-26 (Cx26, 26 kDa). Thin-section
electron microscopy revealed that the INX-6 junction was thicker than the Cx26
junction. The purified INX-6 channels in dodecylmaltoside solution generally
ran slower than Cx26 channels in gel filtration analysis, suggesting that the
purified INX-6 channels were mostly hemichannels, which was supported byelectron microscopy and native gel electrophoresis. Dye transfer experiments
showed that 3-kDa dextran-Texas red and 10-kDa dextran-Texas red passed
through INX-6 channels at rates of approximately 50% and 10%, respectively,
while no significant amounts passed through connexin-43 channels. These
results have implications regarding the dimensions and overall structure of the
INX-6 channels, and provide insight into the wider pore diameter of INX-6
channels compared with that of connexin channels.
3F0936

ATR-FTIR 法を用いた KcsA 野生型と不活化しない変異体に対するカチオン誘起構造変化の赤外分光分析

Cation-induced structural changes in the WT and noninactivating mutant KcsA studied by ATR-FTIR
Chikako Muramatsu1, Masayo Iwaki1, Tetsuya Fukuda1, Yusuke Asai1, Yuji
Furutani1,2, Hideki Kandori1 (1Grad. Sch. Tech., Nagoya Inst. Tech., 2Inst.
Mol. Sci.,)
The bacterial potassium channel KcsA is a membrane protein complex ofhomotetramer. The channel is gated by high concentration of intracellular
protons, and transports cations through the filter that permeates K+selectively
over Na+. In the wild type (WT) KcsA, the filter can be in both open andinactivated (collapsed) structures depending on ionic/pH conditions, whereas
the filter remains open in the non-inactivating mutants such as E71A, E71V and
F103A. In this study, we investigate the mechanism of ion selectivity coupledwith gating process using attenuated total reflection Fourier transform infrared(ATR-FTIR) spectroscopy. WT and mutant proteins of KcsA of Streptmyces
lividans were expressed in E. coli, and purified proteins were reconstituted inliposomes and deposited on an ATR-prism. K+ - or Na+ -induced difference
FTIR spectra were measured by switching perfusion buffers with and without
cation. At pH 7, the gate of the WT was closed and the selectivity filter waspresumably open. K+-induced spectrum showed different IR features from Na+induced one, indicating different ionic interactions of K+ and Na+ to the protein.
The IR spectra obtained at pH 4 were different from those at pH 7. The
corresponding spectra measured in the non-inactivating mutants were comparedto those in the WT. Conformational changes in peptides upon the binding of
cations to KcsA are discussed based on the obtained IR data in the light ofatomic structures available from X-ray crystallography.
3F0948黄色ブドウ球菌の 2 成分性膜孔形成毒素の分子機構

Molecular basis of staphylococcal bi-component pore forming
toxin
Yoshikazu Tanaka1, Daichi Yamashita2, Keitaro Yamashita2, Yuka Kawai2,
Jun Kaneko3, Noriko Tomita4, Makoto Ohta4, Yoshiyuki Kamio5, Min Yao1,
Isao Tanaka1 (1Fac. of Adv. Life Sci., Hokkaido Univ., 2Grad. Schl. of Life Sci.,
Hokkaido Univ., 3Grad. Schl. of Agri. Sci., Tohoku Univ., 4Inst. of Fluid Sci.,
Tohoku Univ., 5Grad. Schl. of Life Sci., Tohoku Univ.)
Pathogenic bacteria secrete various pore-forming toxins (PFTs) to kill host cells.
PFTs are expressed as water-soluble monomeric proteins that oligomerize into
nonlytic prepore intermediates on the membrane of the target cells, and thenform bilayer-spanning pores.
Staphylococcus aureus, a ubiquitous and pernicious human pathogen, secretes

several PFTs including α -hemolysin (α HL), γ -hemolysin (γ HL), leukocidin(LUK), and Panton-Valentine leukocidin (PVL). α HL consists of a single
polypeptide, whereas the others are bi-component PFTs that require thesynergistic association of a class F component and a class S component.
Extensive experiments have been carried out for more than two decades, and the
crystal structures of the monomeric forms of bi-component PFTs have been
determined. However, the structures of the prepore and pore forms have notbeen reported at atomic resolution, which has hindered detailed discussion of
the complicated molecular mechanism.
In the present study, we determined the crystal structure of the prepore and pore
form of bi-component PFT, γHL. This is the first report of the crystal structureof a heterocomponent -barrel-type transmembrane protein. This is also the first
PFT of which all of monomer-, prepore-, and pore-form structures have been
determined by X-ray crystallography. Based on the structural differences
between pore and monomer forms in combination with biological data
accumulated over the past two decades, we propose a mechanism of pore
formation by PFTs.
3F1010分子動力学法によるグラミシジン A を含んだ脂質二重層膜の構造と圧力特性

Structure and lateral pressure profile of lipid bilayer
containing gramicidin A by molecular dynamics simulation
Hiroaki Saito, Masashi Iwayama, Megumi Nishimura, Hiroyuki Takagi,
Kazutomo Kawaguchi, Hidemi Nagao (Kanazawa University)
The molecular dynamics (MD) simulations of DMPC (diC14:0-PC) and DSPC
(di18:0-PC) lipid bilayers in the absence and presence of gramicidin A (GA)peptide dimer were carried out to investigate the GA effect on these membrane
structure and the lateral structure. The observed hydrophobic thickness andorder parameter increased, and the gauche conformation reduced in the presenceof GA, and these results are consistent with the experimental measurements.
Due to the hydrophobic mismatch between the GA and surrounding lipid
bilayer, the hydrophobic thickness of the DSPC membrane largely decreased to
fit the length of hydrophobic core region of the GA peptide dimer, resulting in a
distortion of the membrane surface around the GA. The surface tension aroundthe polar headgroup region decreased due to the decrease of electrostaticinteraction between headgroups. Since the DSPC lipid bilayer has a largerhydrophobic length, the larger positive lateral pressure appears around the GA
channel entrance region in the membrane. The larger lateral pressure in thehydrophobic region of the DSPC/GA membrane results in the reduction of poreradius around the GA channel entrance, suggesting the decrease of ion
accessibility to the GA channel entrance.
3F1022代謝型グルタミン酸受容体の膜貫通領域における 1 アミノ酸変異がアゴニストをインバースアゴニストに変える

Single amino acid substitution in the transmembrane domainof metabotropic glutamate receptor changes an agonist into an
inverse agonist
Masataka Yanagawa, Takahiro Yamashita, Yoshinori Shichida (Departmentof Biophysics, Graduate School of Science, Kyoto University)
Metabotropic glutamate receptors (mGluRs) are prototypical family 3 G
protein-coupled receptors (GPCRs) that function as a constitutive homo dimer.
They have a large extracellular ligand-binding domain (ECD) on the N-terminalside of seven-transmembrane domain (TMD). Upon binding of an agonist, anintersubunit relocation of the dimeric ECDs induces a mutual rearrangement of
TMDs, which was observed by our previous fluorescence resonance energy
transfer (FRET) analyses. However, the conformational change within each
protomer of mGluR is largely unknown. Here we report that a single amino acidmutation at a threonine residue conserved in helix VI of mGluRs caused
elevation of a basal activity, whereas the glutamate binding to the mutant
partially suppressed the basal activity. Namely, original “agonist” works as an“inverse agonist” on this mutant. The comparative FRET analysis of wild-type
and the mutant demonstrated that the glutamate-dependent dimeric
rearrangement of the mutant is similar to that of wild-type. Therefore, alterationof the ligand character in the mutant should be originated from a conformationalchange within each protomer. Double mutation analyses revealed that anadditional mutation in helix VII restored the “agonist”-dependent increase of theactivity. In conclusion, the proper packing between helices VI and VII aroundthe threonine residue within each protomer is a key determinant of the activationof mGluRs.

− S66 −

3F1034

non-detergent sulfobetaine (NDSB)による G 蛋白質共役型受容体 (GPCR)の熱安定化
Thermal stabilization of G protein-coupled receptor by nondetergent sulfobetaines (NDSBs)

Toshihide Yamai, Layla Takahashi, Masato Nakajima, Naoki Yamashita,
Takeshi Ishii, Shigeki Takeda, Kaori Wakamatsu (Gunma Univ.)
Structural analyses of G protein-coupled receptors (GPCRs) are expected tofacilitate drug development since more than a half of pharmaceutical drugs
target at GPCRs. Thermal stabilization of GPCRs will be beneficial for the
structural analyses because GPCRs generally heat labile.
Non-detergent sulfobetaines (NDSBs) are known to stabilize many proteins, andwe have found that NDSBs, in particular NDSB-256, potently stabilize m2
muscarinic acetylcholine receptor (m2-R). Here we analyzed the mechanisms
whereby NDSB-256 stabilizes m2-R.
The apparent dissociation constant Kd of NDSB-256 and m2-R, as determinedby the NDSB-256 concentration dependence of the denaturation time constantof m2R at 50 degree, was smaller than the inhibition constant Ki of NDSB-256,
as determined by competitive inhibition of [3H]QNB-binding to m2R. Theseobservations suggest that NDSB-256 stabilizes m2R by two mechanisms:
binding to the orthosteric site and stabilization of the whole protein.
Because the orthosteric sites of many amine receptors (including m2R) containboth an acidic aspartic acid residue and aromatic residues, amine receptors are
expected to be stabilized in general by NDSB-256, which has both anammonium moiety and an aromatic ring.
3F1046

固体 NMR を用いた熱および圧力により誘起されるバクテリオロド
プシンの構造と運動性変化の解析

Thermal and Pressure induced structural and dynamics
changes of bacteriorhodopsin as studied by solid-state NMR
Izuru Kawamura1, Miyako Horigome1, Hirohide Nishikawa1, Kana Tajima1,
Takashi Okitsu2, Akimori Wada2, Satoru Tuzi3, Tatsuo Iwasa4, Akira Naito1(1Grad. Sch. Eng., Yokohama Natl. Univ., 2Kobe Pharm. Univ., 3Univ.
Hyogo, 4Muroran Inst. Tech.)
Bacteriorhodopsin (BR) has a light-driven proton pump function triggered byphotoisomerization of retinal. In the dark adapted BR, the retinal configuration
coexists as a mixture of all-trans and 13-cis, 15-syn states with the isomericratio close to 1. We have revealed that two major NMR signals of [1-13C]Tyrlabeled BR is assigned to the protein structures with two retinal states in the dark
at room temperature by the solid-state NMR experiments. In this study, thermal
and pressure effects on structure of BR were investigated by solid-state NMR.
Overall peak intensity of [1-13C]Tyr-BR was higher at a lower temperature andthe plot of signal intensity showed a discontinuity at 260K. It is indicated thatdynamical transition of protein occurred at the freezing temperature of bulk
water. The ratio of relative signal intensity of two major peaks of [1-13C]TyrBR at 213K obviously increased compared with that at room temperature. In
contrast, the relative signal intensity of [20-13 C] retinal-BR did not change
significantly at 213K. This result was attributed to structural change fromdisorder to α-helix at a lower temperature. Next, we applied pressure to BR
using fast sample spinning in NMR. The signal intensity of 13-cis retinal at 22.0
ppm was increased as the pressure increased. The signal of Tyr185
corresponding to 13-cis retinal was increased more pronouncedly. It is indicatedthat the isomerization from all-trans to 13-cis, 15-syn state and change ofprotein state from all-trans to 13-cis were occurred under the pressure condition.
3F1058OBSERVATION OF TRANSMEMBRANE PROTEIN BY
HIGH SPEED ATOMIC FORCE MICROSCOPY:BACTERIORHODOPSIN D85S MUTANT, A CHLORIDE
PUMP

Maxime Ewald1, Mikihiro Shibata2, Takayuki Uchihashi1,3, Hideki Kandori4,
Toshio Ando1,3 (1School of Mathematics & Physics, Institute of Science &Engineering, Kanazawa University, 2Department of Neurobiology, Duke
University Medical Center, 3Bio-AFM Frontier Research Center, College of
Science and Engineering, Kanazawa University, 4Department of Frontier
Materials, Nagoya Institute of Technology)
Bacteriorhodopsin (BR) is a light-driven proton pump that is found in
halobacterium salinarum. Photoisomerization of the all-trans retinal
chromophore, covalently attached to Lys216 through a protonated Schiff base,to the 13-cis, 15-anti configuration initiates ion translocation across the cell

membrane, and establishes an electrochemical gradient for ATP synthesis andother energy requiring in membrane processes. It is well known that somehelices on the cytoplasmic surface change their conformation during the proton
pumping. We have recently succeeded in visualizing the photo-induced
conformational changes of the D96N mutant using high-speed atomic force
microscopy (AFM).1 On the other hand, it is known that substituting serine for
Asp85 converts bR to a halorhodopsin-like chloride pump, actively transporting
chloride ions in the direction opposite from the proton pump. 2 The crystal
structure of the halide-bound and halide-free states of D85S reveals that helicalmovements occur on the extracellular side of the protein. Here we have applied
high-speed AFM to directly visualize the conformational change of D85S
mutant activated by illumination. The AFM movies showed that the moleculeschange their conformation only on the extracellular surface as expected and thismolecular motion seemed completely different from that observed on thecytoplasmic surface for the proton pumping WT and D96N mutant. 1. M.
Shibata et al, Nature Nanotechnology 5, 208-212(2010). 2. I.V Kalaidzidis and
A.D Kaulen, FEBS Lett., 418, 239-242 (1997).
3G0900

好熱性 Meiothermus ruber H328 株が産生放出する高耐性ケラ
チン分解性プロテアーゼ複合体のフリーズレプリカ電子顕微鏡観察

Freeze-replica observation of keratinolytic protease complex
produced and released from Meiothermus ruber H328
Kazunori Kawasaki1, Maachi Kataoka2, Keiji Nomura2, Yasushi Shigeri1,
Kunihiko Watanabe2 (1AIST, 2Grad. Sch. Kyoto Pref. Univ.)
A moderately thermophilic bacterium Meiothermus ruber H328 produces
peerlessly strong keratinolytic protease. Cultivation of strain H328 with intact
chicken feather induces degradation of more than 55% of total keratin proteins(Matsui et al., Appl Microbiol Biotechnol 82, 941-950, 2009). Moreover, thekeratinolytic activity of the protease is extraordinarily stable against
denaturizing agents: detergents such as SDS and organic solvents such asacetone, methanol and ethanol. Molecular structural basis for the high activity
and stability of the protease is the subject of the current study.
Supernatant of medium after cultivation of strain H328 with 3% (w/v) chickenfeather at 60°C for 37hr was concentrated with ultrafiltration (MWCO 500kDa),
and passed through gel-filtration column (Sephacryl S-1000). The first-peakfraction from the column was applied to 20-60% sucrose density gradientultracentrifugation, and fraction with keratinolytic activity recovered fromaround 45% sucrose was examined with transmission electron microscopy.
Observation by negative staining method showed the fraction contained
particles of about 100nm diameter. Images obtained using freeze-fracture
replica method, on the other hand, revealed fractured hydrophobic faces ofmembrane fragments about 100nm wide, those bearing a high density of
intramembrane particles of several nanometers wide. These morphologicalevidence suggested the keratinolytic protease would be a membrane-bound,possibly an integral membrane protein.
3G0912

Effects of model peptides for late embryogenesis abundant(LEA) proteins on the thermal properties of liposomes

Takao Furuki, Minoru Sakurai (Center for Biol. Resources and Informatics,
Tokyo Inst. Tech.)
LEA proteins are one of the key molecules to acquire desiccation tolerance
abilities for plant species and animals. The primary structure of almost all of
LEA proteins from animals discovered so far consists more or less in repeat of
the 11-mer motif sequence characteristic to its origin. Previously, we reported
that the 22-mer peptides with two tandem repeats of the 11-mer motif such as
AKDGTKEKAGE (PvLEA-22) had conformational and thermal natures insupport of several proposed roles of LEA proteins in desiccated cells1. Theinterest is now in the interactions between the model peptide and cellular
membrane, particularily in the dried states. Here effects of the 22-mer peptide,
PvLEA-22, were studied on the thermal properties of liposomes as a model ofcellular membrane.
Results of dried DPPC liposomes will be outlined. By DSC measurements, inthe presence of PvLEA-22, the gel-to-liquid crystalline transition temperature
Tm was found to be clearly lowered as compared to pure liposome. In FT-IR
spectra of the tested dry liposomes on heating, the peak position of the CH2
stretching vibration in hydrocarbon chains shifted monotonously from ca. 2850
cm-1 to 2854 cm-1. The P=O stretching band shifts from pure liposome aresuggestive of the direct interactions between the 22-mer peptide and themembrane polar headgroups. PvLEA-22 suppressed the desiccation-induced
aggregation of liposomes. Taken together, PvLEA-22 is capable of playing as arole of protectant for dried liposomes.

− S67 −

1. T. Furuki et al. Biochemistry 50, 7093-7103 (2011).
3G0924

微細パターン化モデル生体膜における膜結合タンパク質の濃縮の空間的制御

Spatially controlled accumulation of membrane-bound protein
in a micro-patterned model biological membrane
Fumiko Okada1, Kenichi Morigaki1,2 (1Grad. Sch. Agr., Univ. Kobe, 2Res.
Cent. Env. Genomi., Univ. Kobe)
Lipid rafts play a key role to regulate many cellar functions by recruiting
membrane proteins. In a model system, liquid-ordered (lo) phases have beenused as a model of lipid raft. We previously developed a micro-patterned model
membrane on a solid support that can control the size and spatial distribution oflo phases (1). The model membranes comprised polymerized and fluid lipid
bilayers. They induced phase separation of lo and liquid-ordered (ld) domains ina designed geometry because ld domains preferentially localized at thepolymeric bilayer boundaries. The controlled phase separation should provide ameans to purposefully concentrate membrane-bound proteins in defined
regions. In the present work, we evaluated the accumulation of membranebound proteins in spatially defined areas. We used cholera toxin subunit B(CTB) as a model protein. The degree of CTB accumulation was correlated withthe progress of phase separation, the ratio of polymeric/ fluid lipid bilayers, thegeometries of polymerized bilayer regions, and the composition of fluid lipid
bilayer. Spatially controlled accumulation of proteins should provide a novel
approach to study lipid raft by separating membrane-bound proteins according
to their affinities to lipid rafts. This technique should also pave a way towardsensitive bioassay and biosensor owing to the fact that protein molecules areaccumulated in pre-defined regions.
(1) Okazaki et al. Langmuir 2010, 26, 4126
3G0936

圧力摂動熱量法によるジミリストイルホスファチジルコリン二分子膜の緩和挙動の解明への試み

An attempt to reveal the relaxation behavior of dimyristoylphosphatidylcholine bilayer by pressure perturbation calorimetry
Nobutake Tamai, Sayuri Kakibe, Saeko Tanaka, Masaki Goto, Hitoshi
Matsuki (Dept. Life System, Inst. Technol. & Sci., Univ. of Tokushima)
Pressure perturbation calorimetry (PPC) is a relatively new calorimetrictechnique, which allows us to directly quantify the thermal expansion
coefficient for solute molecules and colloidal particles in solution. In a PPC
scan, the flow of the heat that occurs when a small isothermal pressure change(~ 5 bar) is applied to a sample solution is recorded as a function of time, and the
expansion coefficient can be obtained from the total heat quantity transferred
into the sample solution during the relaxation process by applying several
thermodynamic relations. Therefore, the temperature dependence of the thermal
coefficient is easily obtained by performing a series of PPC scans at different
temperatures. Usually, this technique is used to study thermotropic volume
behavior of solutes and dispersed particles and to estimate the relative volume
changes of them with thermotropic physical transformations, such first-orderphase transitions and conformational changes, on the basis of the temperaturedependence of the thermal expansion coefficient. On the other hand, the timedependence of the heat flow measured in a PPC scan can provide us with theinformation about the relaxation dynamics of solutes and dispersed particles. On
the basis of this idea, we have recently started the investigation on the relaxation
process of a phospholipid bilayer membrane using the PPC technique. In thispresentation, we will report on our recent results about the relaxation behavior
of dimyristoylphosphatidylcholine bilayer membrane.
3G0948リン脂質二重膜の圧力誘起指組み構造形成：疎水鎖長依存性と形成
限界

Pressure-induced interdigitation of phospholipid bilayer
membranes: dependence of acyl-chain length and limitation of
the formation
Hitoshi Matsuki, Masaki Goto, Nobutake Tamai (Institute of Technology and
Science, The University of Tokushima)
Bilayer membranes of diacylphosphatidylcholines (CnPC) are known to induce
the interdigitated gel phase under high pressure. Since the interdigitated gel
phase is extended into the low-pressure region with increasing the acyl-chain
length, it is expected that the bilayers of CnPC with longer acyl chains induce
the interdigitated gel phase under atmospheric pressure. In this study, weinvestigated the bilayer interdigitation of CnPC with long acyl chains (Cn = 1922). The temperature-pressure phase diagrams of four CnPC bilayers wereconstructed by differential scanning calorimetry and high-pressure lighttransmittance technique. We observed the interdigitation of the C19PC, C20PC
and C21PC bilayers under high pressure, and the interdigitation was induced atlower pressures with the chain elongation. On the other hand, the C22PC bilayer
showed the peculiar behavior: there exist two transitions under atmosphericpressure while one transition under high pressure. In order to confirm what the
low-temperature phase of the C22PC bilayer is like, we performed
measurements of the small angle neutron scattering and fluorescence
spectroscopy under atmospheric pressure. These results proved that the phase is
the interdigitated one. Therefore, it turned out that the maximum acyl-chain
length required for the pressure-induced interdigitation is 21. Present results
definitely indicate that the bilayer interdigitation is governed by thehydrophobic interaction as well as the hydrophilic interaction of the PC
molecule in the bilayer.
3G1010均一でないリン脂質単分子膜における崩壊現象について

Collapse of nonuniform phospholipid monolayers
Masahiro Hibino, Ken Hashimoto, Takuya Fujisawa (Div. Appl. Sci., MuroranInst. Technol.)The behavior in the stochastic collapse of lipid monolayers of a mixture of
dipalmitoylphosphatidylcholine and palmitoyloleoylphosphatidylglycerol, amodel system of pulmonary surfactant assembly, has been studied extensively
by using experimental techniques and theoretical models. The monolayers at the
air-water interface appear to be phase separated under compression and retain
the continuous liquid-expanded phase network surrounding islands ofcondensed phase even at a surface pressure approaching 70 mN/m. When thetwo-dimensional monolayers are compressed beyond the equilibrium surfacepressure, they collapse and assume a three-dimensional formation. However,these phenomena have been observed under a condition of uniform monolayers.
In fact, monolayer is not always uniform at the interface. We can rarely observethat the areas with the coexistence of LE-C phases are contiguous to each other,but distinct from each other by using fluorescence microscopy. The nonuniform
surface density in the monolayer has an influence on the collapse. Here we
investigate the collapse of compressed nonuniform binary phospholipid
monolayers using Langmuir-isotherms, fluorescence microscopy and atomic
force microscopy. The monolayers at the air-water interface underwent fast
collapse by folding within 100 ms or slow collapse by the formation of
Baumkuchen-like structure at high surface pressure. They were composed of
bilayers that remained suspended beneath the film.
3G1022脂質分子の電荷が引き起こす膜構造変化：2 次元相分離と 3 次元曲率

Charge-induced transition in membrane mesoscopic
structures: lateral domains and vesicular shapes
Hiroki Himeno, Tsutomu Hamada, Masahiro Takagi (1Shool of Materials
Science, Japan Advanced Institute of Science and Technology)
Living cells exhibit various 2D and 3D structural dynamics of lipid membranes.
These structural changes are recognized as “membrane dynamics”. The 2D
membrane structures are represented by phase separation named “lipid raft”.
The 3D membrane structures are morphological changes, such as endocytosis
and intercellular vesicular traffic. The intrinsic properties of constituent lipids
should play an important role on such membrane dynamics.
Giant unilamellar vesicles (GUVs) are often used as a model of biologicalmembranes, because they are large enough to be observed directly by optical
microscope. Previously, using GUVs, we succeeded in reproducing 2D and 3D
membrane dynamics such as phase separation (Hamada et al., Soft Matter, 7,220-224, 2011) and vesicular formation (Hamada et a., J. Am. Chem. Soc., 132,
10528-10532, 2010) by changing the molecular structure of membrane
constituents.
To advance our understanding, we here focused on the effect of electric chargesof lipid molecules, which is one of intrinsic properties of membrane lipids, on
2D (lateral phase separation) and 3D (membrane curvature) membranemesoscopic structures.
3G1034− S68 −

油中水滴エマルション遠心沈降法を用いたミクロスフェア内包型
ジャイアントベシクル

Microsphere-containing giant vesicles prepared by water-inoil emulsion centrifugation method

Yuno Natsume1, Taro Toyota1,2 (1Grad. Art. Sci., Univ. Tokyo, 2JST PRESTO)
In cells, biological macromolecules are contained at a volume fraction of 20-30
vol %. Recently, this situation has much drawn attention as a crucial factor of
the crowding effect of the cytosol. To construct cell models, giant vesicles(GVs) confining macromolecules, microspheres, or both have been adopted.
However, in the conventional methods such as the natural swelling method, theinner state (concentration, number density and so on) of macromolecules or
microspheres is scarcely controlled and non-uniform among the GVs. To form
microsphere-containing GVs as desired, we employed the water-in-oil (W/O)
emulsion centrifugation method reported by Pautot et al. (Langmuir 2003, 19,2870), because this method is based on W/O emulsion droplet as a template of
GV. By using polystyrene microspheres (diameter = 1 m) dispersions of
2.0‒25 vol % to form W/O emulsions in liquid paraffin, we obtained GVs that
encapsulate microspheres with volume fractions in the range of 0‒50 vol % andtheir diameters were in range of 4‒40 m. Although the distribution of thevolume fraction of GVs was broad, it should be noted that the highest volume
fraction of GV was higher than that of W/O emulsion droplets. It is theadvantage that we can achieve GVs confining microspheres at high number
density rather than that of microspheres dispersion we prepared initially. This
technique can be applied to the GV encapsulation of other microspheres ormacromolecules that exhibit entropy-driven effects, such as the crowding effect.
3G1046

オイルフリー GUV に封入された分子演算システム RTRACS

Molecular computing system RTRACS encapsulated in oilfree giant unilamellar vesicle
Koh-ichiroh Shohda, Tadashi Sugawara, Akira Suyama (Grad. Sch. Arts Sci.,
Univ. Tokyo)
RTRACS(Reverse-transcription-and-TRanscription-based
Autonomous
Computing System) is a molecular computing system in which RNA molecules
work as input and output.1,2 RTRACS is a reaction network system which
consists of modularized molecular logic gates containing the Boolean gate suchas AND, OR, etc. We intend to construct a giant unilamellar vesicle (GUV)
encapsulating RTRACS as a minimal model of living cell. At present, the best
preparation method of GUV is the w/o emulsion centrifuge method.3 However
the GUV prepared with the centrifuge method potentially contains the oil whichsurrounded the water droplet in the w/o emulsion. The oil changes themembrane properties, such as thickness, permeability, dynamics, etc. Hence wedeveloped an oil-free GUV preparation method based on the lipid film gentle
hydration method in the presence of mono- and di-valent metal cations. The
inner water pool of GUV is stained with TMRA-dextran (M.W. 10,000) as a
volume marker. The FAM-labeled molecular beacon probe which hybridizes
with the output RNA molecule of RTRACS was used as a reaction marker. The
dual-labeled GUV in which RTRACS runs was analyzed by optical microscopy
and flow cytometry.
[1] N. Nitta, and A. Suyama Lect. Note. Comput. Sc., 2004, 2943, 203-212.
[2] M. Takinoue, D. Kiga, K. Shohda, and A. Suyama Phys. Rev. E, 2008, 78,
041921.
[3] S. Pautot, B. J. Frisken, and D. A. Weitz Langmuir, 2003, 19, 2870-2879.
3G1058低い pH が誘起する DOPS/MO 膜の液晶相からキュービック相への相転移の初期過程

Initial Step of Low pH-Induced Lamellar to Bicontinuous Cubic
Phase Transition in Dioleoylphosphatidylserine/Monoolein
Toshihiko Oka1, Tomoki Takahashi1, Taka-aki Tsuboi1, Masahito Yamazaki2(1Fac. Sci., Shizuoka Univ., 2Grad. Sch. Sci. Tech., Shizuoka Univ.)
The modulation of electrostatic interactions due to surface charges of lipid
membranes induces transitions between the Lα phase and the inverse
bicontinuous cubic (QII) phase [1]. Using time-resolved small angle X-ray
scattering (TR-SAXS) and a home-made rapid mixing method, we investigatedthe kinetics of low pH-induced Lα to QIID phase transition in MLVs ofdioleoylphosphatidylserine (DOPS)/monoolein(MO) in the presence of PEG6K, and found that the HII phase appeared after the pH change (the initial step),
and then the HII phase slowly converted into the QIID phase (the second step)[2]. However, we could not follow the initial step due to the limited timeresolution of the method.
In this report, we investigated the initial step of the low pH-induced Lα to QIID
phase transition in DOPS/MO in the absence of PEG-6K using TR-SAXS with astopped flow apparatus. We succeeded in following structural changes in themembranes after 100 ms of the mixing of DOPS/MO-MLV in a neutral buffer

with a low pH buffer with a time-resolution of 100 ms. We observed that at theinitial step the peak intensity of the Lα phase gradually decreased but at the sametime that of the HII phase gradually increased, indicating that the Lα phasedirectly converts into the HII phase without formation of any intermediates. The
rate constant of the initial step greatly depended on final pH and DOPS
concentrations in DOPS/MO membranes.
[1] Adv. Planar Lipid Bilayers Liposome, 9, 163, 2009; Langmuir, 24, 3400,
2008, [2] J. Chem. Phys. 134, 145102, 2011
3G1110X 線及び電子線回折法を用いた皮膚角層の構造解析

Breakthrough for Unresolved Structural Problems in Skin
Function by Combined Use of X-ray and Electron Diffraction
Methods
Hiromitsu Nakazawa1, Ichiro Hatta2, Satoru Kato1 (1Sch. Sci. Tech. kwansei
Gakuin Univ., 2Nagoya Industrial Science Research Inst.)
The outermost layer of the human skin, stratum corneum (SC), consists ofterminally differentiated keratinocytes and highly ordered intercellular lipid
layers. The SC serves as a physicochemical barrier between the inner body andthe outer external environment. Notwithstanding the extensive studies on
structure of intercellular lipids which plays a crucial role for the skin barrier
properties, owing to lack of the structural evidence under functioning lot ofunresolved problem are left open. Using X-ray and Electron diffraction methods
we deepened understanding the healthy skin state and developing the
percutaneous drugs.
Both synchrotron X-ray diffraction (XD) and electron diffraction (ED) methods
are the very powerful tool to analyze the intercellular lipid organization in SC.
The XD has higher resolution than the ED, but requires invasive operation
because a large amount of skin sample is necessary to obtain reliable data. On
the other hand, the ED makes it possible to analyze the structures of the SC
noninvasively, but radiation damage by electron beam must be suppressed.
By best use of these methods we will report here a fundamental structuralchange of SC in the skin permeation of chemical agents, the effect oftemperature in the skin permeation, and the regional structural differences on
the body. We will present these results and future perspective for the SC
structural study.
3H0900

GTP 結合状態と GDP 結合状態の微小管分子構造における大きな
構造変化

Large Conformational Changes in Tubulin in the GTP- and GDPStates Microtubules Observed by Cryo Electron Microscopy
Hiroaki Yajima1, Toshihiko Ogura2, Ryo Nitta1, Yasushi Okada1, Chikara
Sato2, Nobutaka Hirokawa1 (1Grad. Sch. Med., Univ. Tokyo, 2Biomedical
Research Inst., AIST)
Microtubules are dynamic polymers that stochastically switch between growing
and shrinking phases and their dynamics is regulated by GTP hydrolysis by tubulin, but its mechanism remains elusive because high-resolution microtubule
structures have only been revealed for the GDP-bound state. Here we solved the
cryo-EM structure of microtubule stabilized with a GTP analogue guanylyl 5'-α,-methylenediphosphonate (GMPCPP) at 8. 8 Å resolution by developing anovel cryo-EM image reconstruction algorithm. In contrast to the crystal
structures of GTP-bound tubulin relatives such as γ -tubulin and bacterial
tubulins, significant changes were detected between GTP- and GDP-boundstates at the contacts between tubulins both along the protofilament and between
neighboring protofilaments, contributing to the stability of the microtubule
lattice. These findings are consistent with the structural plasticity or latticemodel, and suggest the structural basis not only for the regulatory mechanism of
microtubule dynamics, but also for the recognition of the nucleotide state of
microtubule by several microtubule-binding proteins, such as EB1 or kinesin.
3H0912

紡錘体は一方の極から他方へ構造変化を伝搬することで対称形状を維持する

Meiotic spindles maintain the symmetrical shape by propagating structural changes to the opposite side
Kazuya Suzuki1, Jun Takagi1, Takeshi Itabashi1, Shin'ichi Ishiwata1,2 (1Dept.
Phys., Adv Sci. Eng., Waseda Univ., 2WABIOS)
A microtubule-based spindle is designed for proper chromosome segregation.
Recent studies have demonstrated that bipolar spindle formation requires theforce balance sustained by molecular motors. However, it remains unclear− S69 −

whether and how the mechanical properties of the two spindle poles are
symmetrically balanced. Here, we quantitatively measured the mechanicalstiffness, the microtubule density, and the response to the deformation of spindle
poles by micromanipulation techniques and 3D analysis. To directly deform thespindle self-assembled in Xenopus egg extract, we inserted two glass microneedles into a pole region and widened it perpendicularly to the pole-to-poleaxis. We found that the stiffness and the microtubule density in the manipulatedpole region reduced upon widening. Surprisingly, this reduction propagated tothe other side, which resulted in the formation of a symmetrical defocusedbarrel-like shape of the unmanipulated pole region. On the other hand, it is
known that inhibition of the dynein function causes the defocusing of poleregions, such that the barrel-shaped structure is formed. We compressed one
side by the micro-needles and found that the stiffness and the microtubule
density in the compressed region increased, and this effect propagated to theother side, resulting in the bipolar shape formation. Our results suggest that thesymmetrical spindle shape is maintained by propagating the structural change
from one side to the other via the communication through the central region.
3H0924

高圧負荷によって誘導されるクラミドモナス非運動性変異株鞭毛の屈曲運動

Resurrection of flagellar bending movements inchlamydomonas paralyzed mutants at high pressure
Toshiki Yagi1, Masayoshi Nishiyama2 (1Grad. Sch. Medicine, Univ.
Tokyo, 2The Hakubi Center, Kyoto Univ.)
Application of hydrostatic pressure modulates the activities of organisms. To
examine the effect of the pressure on ciliary and flagellar motility, here weobserved the flagellar movements of chlamydomonas under high-pressure
conditions. Wild type cell movements were inhibited by the application ofpressure; the swimming velocity and the percentage of moving cells decreased
with the pressure increased, and the movements stopped under the pressure of>80 MPa at 25 °C. Surprising, however, paralyzed mutants lacking the central
structures of the flagellum (central pair microtubule: CP, or radial spokes: RS)
displayed vigorous beating under the pressure of 40 to 60 MPa, but the
movements stopped under the pressure of > 80 MPa at 25 ° C. Axonemes(isolated and demembranated flagella) of the mutants did not display beating in
the presence of 1 mM ATP, but they displayed beating under the pressure of 40
to 60 MPa. This suggests that the application of pressure modulates thefunctional activities of some axonemal components. The motility analysis of the
double mutants lacking both of CP (RS) and various species of dyneins showedthat outer-arm dynein is indispensable for the movements. Previous studies
showed that the microtubule-sliding movements powered by dynein werereduced in the axonemes of the CP/RS-deficient mutants. We suggest that theapplication of pressure directly or indirectly activates the outer arm dynein andincreases the sliding movements, which induces bending movements in theparalyzed mutants.
3H0936

走化性誘因物質の進行パルス刺激によって誘発された一方向細胞運動

Directed cell migration induced by travelling waves ofchemoattractant
Akihiko Nakajima1, Satoshi Sawai1,2 (1Grad. Sch. Arts & Sci., Univ.
Tokyo, 2Res. Cent. for Complex Systems Biology, Univ. Tokyo)
Mirgration of Dictyostelium cells is mediated by chemotaxis toward thechemoattractant cAMP. It is believed that cells determine their direction of
motion by comparing the concentration difference between the head and rear of
the cell. However in aggregating Dictyostelium, concentration profile ofchemoattractant is not static, but takes the form of traveling waves. Because
reversal of spatial gradient occurs repeatedly after each passing wave, cells
could potentially move in the reverse direction in the wake of a passing wave.
This problem is referred to as ‘chemotactic wave paradox’. To address thisproblem, we designed and constructed a microfluidics system to generate
traveling cAMP pulses of user-specified wave-length and propagation speed.
Using this system, we studied chemotactic responses towards the travelingcAMP pulses and found that the migratory direction depends on the speed ofpulse propagation. Cells migrate toward the in-coming cAMP pulse for waves
with passage time of 3 ~ 10 min. In contrast, when the stimulus duration is lessthan 2 min, cells come almost to a halt. When stimulus duration is longer than
10 min, cells reversed their direction of motion at the back of the wave. These
results indicate that cells are able to move towards the correct orientation only
when stimulus duration time matches the cell response time. We will show how
cytosolic signaling such as phosphatidyl inositol and actin localization is related

to directed cell migration under the traveling cAMP pulses.
3H0948極性を持つアメーバ細胞の走化性運動に関する理論

Theory on the chemotaxis of an amoeboid cell with the cell
polarity
Tetsuya Hiraiwa, Tatsuo Shibata (Kobe, Riken)
Chemotactic amoeboid migration is an ubiquitous kind of cell motility, which isimportant for the immunological response, wound healing and developmental
processes. In this presentation, we would like to explain our recent theory on the
chemotactic migration of an amoeboid cell with the cell polarity, and share withyou our results for the observable statistical quantities about the migrating
direction based on the theoretical model in which we assume the following
facts; 1. The cell has the polarity which is spontaneously organized. 2. The
polarity is reoriented to the gradient direction the cell estimates from the binding
states of chemoattractants to receptors. 3. The cell migrates to the direction of
the polarity with a constant speed. 4. Chemotaxis is realized by the bias of thedirection in which more chemoattractants bind to the receptors according to thechemical gradient. Using this model, firstly, we confirme our theory bycomparison with the experimental data for the chemoattractant-concentration
dependence of the correlation time of the spontaneous migrating direction. Itshould be noted that the spontaneous migration can also be discussed by ourmodel as the case that the magnitude of the chemical gradient is zero. The
results agree with the experimental data for Dictyostelium discoideum.
Secondly, we derive the probability distribution of migrating directions andchemotaxis index. We find that the chemotaxis index is incremented byintroducing the cell polarity. The details will be discussed on the day.
3H1010Controlled cell migration with ultrasoundShinya Murakami1, Yo Otsuka1, Manabu Sugimoto1, Toshiyuki Mitsui2(1Grad. Sci. Eng., Aogaku Univ., 2Assoc Prof., Aogaku Univ.)

Ultrasound is not only used for ultrasound imaging but also used for ultrasoundhealing therapy to recover from bone fracture recently while the mechanism forhealing is not known surprisingly. Since it is reported that aggregation of cells isenhanced by ultrasound on a microscopic scale, it may be possible thatultrasound can control cell behavior closely related to the cell aggregation which
could be one of main causes for enhancing bone fracture healing. To clarify cellaggregation by ultrasound irradiations, we have observed cell migration under
optical microscopy with or without the irradiations directly. Ultrasound isgenerated from a homemade ultrasound needle made of tungsten wire with thediameter at 1.0 mm. The frequency of the ultrasound is near 1.5 MHz. The
power intensity of the irradiations is near 24 mW/cm2. In this conference, wewill show the cell migration under ultrasound irradiation and discuss the result,converging cells on ultrasound exposure area. Finally, we will discuss theinfluence of ultrasound on cell migration.
3H1022

筋肉と異なるストレスファイバ−の収縮特性

Contractile properties of stress fibers are distinct from those of
muscles
Shinji Deguchi, Tsubasa Matsui, Daiki Komatsu, Masaaki Sato (Tohoku
University)
Actin stress fibers play essential roles in cell adhesion regulation and tensional
homeostasis. Here we measured the contractile properties of single stress fibers
isolated from vascular smooth muscle cells. Cells were deroofed with low ionicstrength buffers to extract basal stress fibers. Western blotting analysis andnegative staining electron microscopy indicated that the extracted stress fibers
were intact with regard to structure and function. Individual stress fibers wereisolated from the substrate using two glass needles. Mg-ATP solutions wereadded to induce contractile activity of the stress fibers, and forces were
measured from the deflection of one of the glass needles with a known spring
constant. We obtained a convex form of contractile forces as a function of
sarcomere distance, which was similar to that of myofibrils. Interestingly, theshortening rate of the stress fibers drastically decreased when an external load
was applied. Thus, the shortening rate-loading relationship was sharply different
from that of muscles typically having a parabolic curve. We speculate that a
recently reported load-dependent decrease in ADP release rate of nonmuscle
myosin II may be responsible for this unique contractile property of stressfibers.

− S70 −

3H1034

Smooth muscle differentiation related transcription factor
CRP2 directly regulates physical properties of actin filaments
Takanori Kihara1, Satoko Shinohara1, Yasunobu Sugimoto2, Jun Miyake1(1Grad. Sch. Eng. Sci., Osaka Univ., 2Nagoya Univ. Synchrotron Radiat. Res.
Center)Actin filaments form many types of structures and dynamically regulate the cellmorphology and movement, and furthermore, they play a mechanosensory rolefor extracellular stimuli. Thus, actin filaments are the most important physicalcomponents of a cell and their modulation factors have the potential to directly
regulate the cell fate. In this study, we clarified that smooth muscle relatedtranscription factor, CRP2 (cysteine-rich protein 2), directly bound to actin
filaments and regulated their physical properties. The CRP2 molecule has adumbbell-like structure with 3 tandem clots as indicated by SAXS (small angle
X-ray scattering) analysis. CRP2 is directly associated with F-actin through its
N-terminal LIM domain and Gly-rich region. CRP2-bound actin filaments
showed higher persistent length as compared with unmodified actin filaments.
By induction of smooth muscle cells differentiation, intracellular CRP2
localizes to actin stress fibers as they are formed. Furthermore, smooth muscle
cells with active CRP2 expression showed higher mechanical stiffness. Now we
speculate that actin-bound CRP2 plays direct roles in the stabilization of smoothmuscle cells differentiation by regulation of the physical properties of actin
filaments. This study was supported by KAKENHI (24700454).
3H1046

細胞接着班における p130Cas と Src の動的相互作用を介した遊走制御機構の解明A distinct role for the dynamics of p130Cas at focal adhesion
via interaction with Src in the regulation of cell migration
Hiroaki Machiyama1,2, Hiroaki Hirata1, Yasuhiro Sawada1,2 (1Mechanobiology
Inst., Nat. Univ. Singapore, 2Dept. Biol. Sci., Nat. Univ. Singapore)
Cell migration is involved by the orchestrated regulation of remodeling of actin
cytoskeleton, actomyosin contraction and continuous focal adhesion turnover.
The phosphorylation of a focal adhesion protein p130Cas (Cas) has beenreported to regulate cell migration through the modulation of focal adhesion
turnover. However, it is still unclear how individual Cas molecules contribute to
the regulation of cell migration. The fluorescence recovery after photobleaching(FRAP) method was used to spatiotemporally analyze the dynamics of various
forms Cas mutants at focal adhesion in migrating cells. We find that
displacement of Cas molecules from adhesion sites is accelerated by theirphosphorylation but hampered by association with Src, although the Srcbinding of Cas at focal adhesion promotes Cas phosphorylation. Geneticallyengineered stabilization of Cas association with Src using the bimolecularfluorescence complementation (BiFC) method, which sustain Cas
phosphorylation but mitigate Cas displacement from adhesion complexes, result
in retarded cell migration. Myosin II inhibition that impedes displacement of
Cas molecules from adhesion sites also decreases cell migration, despitesustained Cas phosphorylation. Our findings indicate that both association with
Src and dissociation from Src are the key elements for Cas to activate Casphosphorylation signaling and thereby to drive cell migration.
3H1058Spatiotemporal regulation of signaling by active cytoskeletal
remodeling
Bhaswati Bhattacharyya1, Abhishek Chaudhuri3, Kripa Gowrishankar4,
Satyajit Mayor5, Madan Rao4 (1iCeMS, Kyoto University, 2Rudolf Peierls
Centre for Theoretical Physics, University of Oxford, UK, 3Raman ResearchInstitute, Bangalore, India, 4NCBS-TIFR, Bangalore, India)
Biochemical reactions performed by signaling and sorting complexes, both on
the surface and within the living cell, is often subject to the local regulation by
the active cytoskeleton. We have recently proposed that in many cases, thisspatiotemporal regulation is due to the the interplay between active contractility
and remodeling of the cytoskeleton which results in transient `asters', focusingof passive molecules to form clusters inside its core. This mechanism ofclustering gives rise to a significant increase in the local concentration of
signaling proteins, which enhances reaction efficiency and output signal. The
dynamic cytoskeletal elements that drive focusing behave as quasi-enzymes
catalyzing the chemical reaction. These ideas are applicable to the cortical actin
dependent clustering of cell surface proteins such as lipid-tethered GPIanchored proteins, Ras-proteins as well as many proteins that have domains tointeract with the actin-cytoskeleton. We discuss the implications of actin

dynamics by introducing an `activity temperature', which can work as aregulatory parameter for signaling replacing the details of cytoskeletal
dynamics. We show this using time-temperature-transformation (TTT) andArrhenius plots while `activity temperature' facilitate and/or inhibit(slow down)
chemical reactions.
3H1110Arp2/3 とアクチンを内包した油中液滴のフィロポディア様変形

Filopodia-like protrusions in water-in-oil droplets induced by
Arp2/3 and actin polymerization
Masataka Chiba1, Makito Miyazaki1, Takashi Ohki1, Shin'ichi Ishiwata1,2(1Dept. of Phys., Waseda Univ., 2WABIOS, Waseda Univ.)
When cell moves, cytoplasmic protrusions called filopodia spread out in the
direction of movement. Filopodia consist mainly of bundled actin filaments andare elongating by the actin polymerization. In the initial growth, the following
mechanism has been proposed. First, Arp2/3 complex is anchored to the cell
membrane via the VCA domain of WASP, which activates Arp2/3 andnucleates actin polymerization. Arp2/3 transmits the force of actin
polymerization to the membrane, protruding it. To verify this hypothesis, many
experiments have been carried out using cells, however, the minimalcomponents and physical conditions indispensable for the membrane protrusion
are still unclear. In this study, we used purified proteins and cell-sized water
droplets, surrounded by a phospholipid monolayer, in water-in-oil emulsions.
Inside the droplets we encapsulated fluorescently labeled G-actin, Arp2/3, andVCA, which was designed to bind to the inner surface. After the promotion ofactin polymerization, distribution of actin became inhomogeneous, andfilopodia-like protrusions were occasionally observed. In the absence of Arp2/3,
water droplets maintained a spherical shape. To understand the mechanism ofsuch deformations, we plan to visualize Arp2/3 and VCA in addition to actin
and clarify the relationship between the deformation and the localization ofthese three proteins. From these results, we will discuss the mechanism offilopodia formation and regulation.
3I0912

ヘモグロビンのアロステリック平衡のリアルな描像

A realistic picture of allosteric equilibrium of hemoglobin
Naoya Shibayama1, Sam-Yong Park2 (1Biophysics, Jichi Med. Univ., 2Protein
Design Lab., Yokohama City Univ.)
In general, the molecular mechanism of protein allostery has been explained byassuming an equilibrium between the T (tense) and R (relaxed) states within theframework of the two-state allosteric model. However, it has long beenrecognized by hemoglobin (Hb) experts that such a simple two-state model fails
to represent the relationship between structure and function in Hb in quantitative
terms. To overcome this situation, some researchers including us continue topursue the elusive “intermediate allosteric states” for decades. Here, we present
crystallographic evidence that the Hb molecule assumes many kinds of distinct
quaternary conformations that exist in equilibrium. In this study, we have
utilized a novel Hb crystal form, which contains three independent Hb
molecules with different quaternary structures. Significantly, this crystal allows
large-scale conformational changes of the containing three molecules in
response to the changes in ligation state and solution conditions. By using aseries of three isomorphous crystals obtained from different conditions, ninedistinct Hb conformations can be characterized by X-ray crystallography andoxygen equilibrium measurements. Notably, the nine conformations are
distributed continuously over the whole conformational space of Hb, ranging
from T to R2 through R, with various intermediate forms also present. These
results suggest a mechanism for the allosteric control of a protein’s function bychanging the delicate balance among the multiple states on its energy landscape.
3I0924

ヘムをヘム垂線のまわりに 90°回転した時の共鳴ラマンスペクトル変化：ヘムオキシゲネース

Effects of Heme Rotation around the Heme Normal by 90°
on Resonance Raman Spectra of Heme Proteins; Observationfor Heme Oxygenase
Sachiko Yanagisawa1, Hiroshi Fujii2, Saburo Neya3, Takashi Ogura1, Teizo
Kitagawa1 (1University of Hyogo, 2Okazaki Inst. Integrative Biosci., 3Chiba
University)
To understand the protein effects on resonance Raman spectra of heme proteins
further, we report here the resonance Raman spectra of two proteins in which
their heme orientations are different with regard to the rotation around the heme
normal by 90° in the same protein pocket. We employed heme oxygenase (HO)− S71 −

as a model protein; one is wild type (WT HO) and the other is a mutant (KDMY
HO) which involves replacements of four residues (K16N, D27A, M31K,
Y112F). Their X-ray structures indicated that the 4-vinyl of WT HO seems tohave the same environments as that of 2-vinyl of KDMY in crystal. The NMR
spectra confirmed the 90° rotation of the heme in solution, and their catalytic
activities demonstrated the corresponding differences in the α and δ cleavages of
the heme. The resonance Raman spectra of WT HO and KDMY HO are alikeregarding major heme skeletal modes, whereas some modes involving heme
side chains vary in frequencies and behaviors. To characterize such bands, wereconstituted the two proteins with vinyl- or propionate-deuterated hemes andcompared their resonance Raman spectra in their oxidized, reduced, and CObound forms. The vinyl associated modes exhibited frequency shifts between
the two proteins.
3I0936ヘムオキシゲナーゼにおける CO 光解離後の構造変化および COの放出経路

Protein motions and CO migration following CO photolysis inheme oxygenase
Masakazu Sugishima1,2, Keith Moffat2,3, Masato Noguchi1 (1Dept. Med.
Biochem., Kurume Univ. Sch. Med., 2Dept. Biochem. & Mol. Biol., Univ.
Chicago, 3BioCARS, Univ. Chicago)
Heme oxygenase (HO) catalyzes the physiological degradation of heme using
dioxygen and reducing equivalents to produce biliverdin, ferrous ion, and CO.
The HO reaction proceeds without severe product inhibition by CO generated in
the second step of the HO reaction, even though CO is known to be a potent
inhibitor of HO and other heme proteins. Therefore HO must have an efficientmechanism for discriminating between dioxygen and CO.
To explore protein motions and CO migration in HO following CO photolysis
which mimics CO generation during the HO reaction, we collected X-ray
diffraction data on the CO-heme-HO complex before and after continuous
illumination by CW laser at temperatures below 160 K. The weighted difference
Fourier map shows that CO was partially photolyzed from the heme iron, andphotodissociated CO had migrated to a hydrophobic cavity, as observed inprevious results at 35 K. In contrast to previous results, motions of the distal
helix, water, and heme were observed which are the reverse of motions observedin CO binding. Notably, similar motions were absent in dioxygen binding andphotolysis. These results imply that the CO-bound form is severely constrainedby steric hindrance between the distal helix and CO bound to the heme iron, andthat steric hindrance contributes to discrimination between binding of CO anddioxygen. Further, another putative CO trapping site, more polar than previous
sites, was found. Combining these studies with previous results, we will discuss
CO migration pathways in HO.
3I0948多様な生物種によるヘムオキシゲナーゼ反応の微調整戦略：オキシヘムの安定化Strategy of a variety of organisms for the fine-tuning of heme
oxygenase reactions: Stabilization of the oxyheme intermediate
Sayuri Takada, Taiko Migita (Dep. Biol. Chem., Fac. Agr., Yamaguchi Univ.)
Heme oxygenase (HO) catabolizes regioselective heme-ring opening to produce
biliverdin, free iron, and carbon monoxide. In a variety of biological species, theHO reactions participate in recycling of iron, signaling, supplying resources ofphotoreceptive-molecule production, or generating anti-oxidants. To attain theirown requirements for HO, each organism seems to take unique fine-tuning
strategy. The HO reaction proceeds successively by consuming oxygen
molecules and electrons, so that their constant supply is essential. In addition,formation of the first reactive species, hydroperoxy hemin, requires proton as
well as electron transfer to the oxyheme. In different species, HO functions indifferent milieu, thereby regulated finely and differently by respective active
sites. Our HO study is focused on such fine-tuning modes taken in different HOs
from different species. In this study, stability of oxyheme, the first converted
form of hemin in HO, was compared among the five HO complexes: rat HO1,
puffer-fish HO1, cyanobacterial HO1, soybean HO1, and pathogenic-bacterial
HO. The auto-oxidation rates of the oxyheme complexes showed unexpectedly
big difference, which correlate with pKa of the heme-bound water as well aswith g-agisotropy of the hydroxide-coordinated heme. Based on the crystal
structures of three heme-HO complexes out of the five, very-well conserved Gly
residue in the distal helix, closest to the heme iron, is proposed as the HB donor
to stabilize the oxyheme in the heme pockets.

3I1010Ultraviolet Resonance Raman Study on Indoleamine 2, 3Dioxygenase
Masayuki Hara1, Sachiko Yanagisawa1, Hiroshi Sugimoto2, Yoshitsugu
Shiro2, Takashi Ogura1 (1Grad. Sch. Sci., Univ. Hyogo, 2Harima Inst., Riken)
Indoleamine 2, 3-Dioxygenase (IDO) catalyzes incorporation reaction of two
oxygen atoms into L-tryptophan (Trp), generating N-formylkynurenine, whichis in the major catabolic pathway of Trp in mammals. Visible resonance Raman(RR) studies identified a key intermediate of ferryl-oxo (Fe=O) species. Thissuggested that two oxygen atoms were transferred to substrate one by one. The
Fe=O species seemed to lie in the compound II oxidation level and a question
arose whether such an intermediate could perform oxo-transfer reaction, sinceno example had been reported. Such a reaction might be possible if the substrateintermediate is unusually reactive. In the present study, we have adopted
ultraviolet (UV) RR spectroscopy to characterize the substrate bound to IDO,
since the technique selectively enhances vibrational spectra of aromatic
molecules. UVRR scattering was excited at 229 nm for the ferrous state of IDO.
UVRR spectrum of substrate-free IDO was primarily contributed by Trp andTyr residues. In the spectrum of the substrate-bound IDO, intensity of the W3band of Trp was increased. This was ascribed either to bound-Trp or Trp residue
in IDO. In the latter case, binding of Trp induced structural change of intrinsic
Trp in IDO. In order to distinguish these possibilities, we utilized indole-2-13Cand indole-D5 -labeled Trp. The results showed that bound-Trp and not the
intrinsic Trp exhibited the intensity change. The present study provides a new
method to study the structure of the bound-substrate to IDO.
3I1022

An Intermediate Conformation of Cytochrome Oxidase in the
Ligand-Free State Identified by Time-Resolved ResonanceRaman Spectroscopy
Izumi Ishigami, Takeshi Nishigaki, Kyoko Shinzawa-Itoh, Shinya Yoshikawa,
Satoru Nakashima, Takashi Ogura (Grad. Schl. Life Sci., U. Hyogo)
Cytochrome c oxidase (CcO) reduces dioxygen to water by heme a3 and drives
proton pump by heme a. In the present study, we applied green-pump andviolet-probe time-resolved resonance Raman (TRRR) spectroscopy to monitor
the dynamics of hemes after CO-photolysis with a time resolution of 10 ns. The
green and violet light were used for CO-photolysis and resonance Raman
excitation, respectively. TRRR spectra after CO-photolysis were measured atdelay time = 10 ns - 5 ms. The Fe-His stretching mode was located at 221
wavenumbers at delay time between 10 ns and 100 ns, and then decreased
exponentially with time with a time constant of 600 ns and at 5 ms reached 215
wavenumbers, which was the same value for equilibrium ligand-free form. Thisbehavior was similar to that reported earlier. In this study, we found that
intensity of the same mode decreased to two thirds at 100 ns relative to that at 10
ns and remained constant up to 5 ms, but was significantly smaller than theintensity for equilibrium ligand-free form. Results indicate that an intermediate
conformation exists after ligand dissociation at delay time = 0.002 to 5 ms. This
conformation might have a significantly smaller affinity to dioxygen comparedwith the conformation seen at 10 - 100 ns as judged from the Fe-His stretchingfrequency. The affinity control should be important to adjust the timing of
dioxygen binding appropriately relative to the timing of proton loading for an
efficient proton pumping.
3I1034

ポンプ・プローブレーザーと同期したパルスフローシステムの開発とそれを用いたタンパク質の時間分解赤外分光解析

Time-Resolved IR Analyses of Proteins Using a Novel Pulse
Flow System Synchronized with Pump/Probe Lasers
Minoru Kubo1, Satoru Nakashima1, Masao Mochizuki1, Kyoko ShinzawaItoh2, Shinya Yoshikawa1,2, Takashi Ogura1,2 (1Picobiology Inst., Grad. Sch.
Life Sci., Univ. Hyogo, 2Dept. Life Sci., Grad. Sch. Life Sci., Univ. Hyogo)
IR spectroscopy is a method to analyze structures and reactivities of functionalgroups, and powerful to reveal protein reaction mechanisms. This spectroscopy,
however, has the crucial shortcoming that the application to aqueous solution ispractically impossible in most cases because of strong IR absorption of water,which has prevented time-resolved (TR) IR measurements using a flow cell.
Recently, we developed a new pump-probe TRIR spectrometer based on the
femtosecond IR technology and using a spinning cell. Our spectrometer can
detect a 30 OD protein signal even in the presence of ~2 OD water backgroundin 50 sec of data accumulation, and is promising for flow-flash TRIR
measurements. Here, we developed a novel flow system that can be coupled− S72 −

with the pump-probe TRIR spectrometer. A flow cell was composed of two
CaF2 windows and a 50-m-thick Teflon spacer that has a 500-m-wide
observation channel. A syringe pump delivered protein solution to the cell bytriggering with a pulse generator at 1 kHz (or less). The pulse generator was also
used to trigger the pump/probe lasers at 1 kHz with an appropriate delay. The
repetition rate of pump illumination can be prescaled with an optical chopper
when needed to see slow protein dynamics. The system was tested on CObound cytochrome c oxidase (0. 5 mM) at a flow rate of 1 ml/min. Highperformance of our flow system was shown through the observation ofnanosecond-TR Fea3-CO and CuB-CO signals upon CO photolysis. Details will
be discussed at the meeting.
3I1046チトクロム c 酸化酵素のヘム a 側鎖に由来する共鳴ラマン線の帰属

The assignment of the resonance Raman bands of heme aperipheral substituents in cytochrome c oxidase
Miyuki Sakaguchi1, Yukie Katayama2, Hiroshi Fujii3, Hideo Shimada2,
Takashi Ogura1,2 (1Grad. Sch. Life. Sci., Univ. Hyogo, 2Picobiol. Inst., Grad.
Sch. Life. Sci., Univ. Hyogo, 3Okazaki Inst. Integr. Biosci.)
Proton-pumping process coupled to the O2 reduction reaction in cytochrome coxidase (CcO) generates the electrochemical potential of proton across themembrane. The putative proton-pumping pathway in bovine heart CcO has Hbond with heme a substituents, namely, the formyl, the 7-propionate and thehydroxyfarnesylethyl (HFE) groups, and the proton-pumping mechanism driven
by heme a is proposed. The functions of heme peripheries are remained to beelucidated and the structural changes of them are needed to be observed.
Resonance Raman (RR) spectroscopy has a potential to do it and assignments ofthe vibrational modes are necessary.
RR band of the formyl group has already been assigned and those of the 7propionate and the HFE groups are investigated in this study using bacterial
mutants. Although another proton-pumping pathway is proposed in bacterial
CcO, the structure of the redox centers and the RR spectra are almost identicalto the bovine heart CcO. Y406 and T50 have hydrogen bonds with the 7propionate and the HFE groups, and Y406F and T50A mutants show frequency
shifts of the RR bands at 364 and 1251 cm-1, respectively. These mutants havenormal absorption spectra and cytochrome c oxidation activities, suggesting
minimal effects of the mutations. The former band is assigned to the bending
mode of the 7-propionate group and we propose an assignment of the vibrationalmode of the HFE group to the 1251 cm-1 band. The present study would enable
us to monitor the heme peripheral groups during proton-pumping process.
3I1058シアン結合型構造から推察される一酸化窒素還元酵素の反応機構

Functional Implications from Structural Characterization of
CN-bound Nitric Oxide Reductase
Takehiko Tosha, Nozomi Sato, Norihiro Okada, Hiroshi Sugimoto,
Yoshitsugu Shiro (Harima Inst., RIKEN)
Denitrification, conversion of nitrate to N2, is an example of anaerobic
respiration. Nitric oxide reductase (NOR) catalyzes reduction of NO to N2 O;
2NO + 2H+ + 2e- => N2 O + H2 O, a key step for denitrification. NOR is
supposed to share the same progenitor with aerobic cytochrome c oxidase(COX) catalyzing four electron reduction of O2, based on the structuralsimilarity. While we know the structures of NOR and COX, structural factors
determining their catalytic properties are still unknown. Here, we characterized
the detailed structure of NOR, and compared the results with that of COX, toelucidate the structural elements controlling the function of NOR. The X-ray
structure of oxidized NOR indicated that the active site structures of NOR and
COX are similarly composed of heme iron and non-heme metal, while the nonheme metal is different between NOR and COX; Fe in NOR is replaced with Cu
in COX. To get insights into the structural property of NOR, we utilized CNbound form as a model for the substrate, NO, bound form. Spectroscopic data
showed that CN-bound to heme did not interact with non-heme iron in oxidized
NOR, contrasted with CN bridging heme and Cu in COX. Since binding of two
NO to the active site is required for the NOR catalysis, contrary to one O2
binding in COX, the CN ligand for heme in NOR avoid to interact with nonheme Fe to keep second substrate binding site. We will present the X-ray
structure of CN-bound NOR and further discuss the determinants for thefunctional difference between NOR and COX.

− S73 −

1PS001

アクチンの Tyr143 変異による生化学的性質の変化

Changes of biochemical properties of Dictyostelium actin
induced by mutation of tyrosine-143
Yuki Gomibuchi1, Taro Uyeda3, Takeyuki Wakabayashi2 (1Teikyo Uni.Grad.
Scienceand Engineering, 2Teikyo Uni. Dept. Judo Therapy, 3AIST)The frontal region of actin around Tyr143 has been proposed to be involved in
the binding of myosin. It also changes its conformation during thepolymerization (Murakami et al., 2010). Thus we introduced mutation to
Tyr143 and examined the biochemical properties, such as polymerizability andthe activation of myosin ATPase. In polymerization assay, it turned out that
actin with Tyr143Phe mutation showed poorer polymerizability in the presenceof NaCl compared with the wild-type actin. Tyr143Ile mutation showed,
however, only small decrease in polymerizability. In the presence of phalloidin
Tyr143Ile-actin showed almost normal polymerizability irrespective of theadded monovalent cations, whereas Tyr143Phe showed the somewhat decreased
polymerizability. The interaction with myosin moiety was compared with thatof the wild-type actin. The rate of hydrolysis of ATP by rabbit skeletal heavy
meromyosin (HMM) was determined by measuring the release of inorganic
phosphate in the presence of phalloidin and low concentration (4 uM) of actin.
Both of these mutant actins showed weaker ATPase activation. At higher actin
concentrations (6-10 uM), Tyr143Ile-actin showed also poorer activation,
whereas Tyr143Phe-actin showed the comparable activation. These data
indicate the importance of Tyr143 for the activation of myosin ATPase and forthe polymerization.
Reference
Murakami et al. (2010) Cell 143, 275-287.
1PS002

誘電緩和分光法によるアクチンフィラメントの水和特性の温度依存性

Temperature dependence of hydration properties of F-actin bydielectric relaxation spectroscopy
Yuichiro Okazaki, Asato Imao, Noriyoshi Ishida, George Mogami, Tetsuichi
Wazawa, Makoto Suzuki (Grad. Sch. Eng., Univ. Tohoku)
Polymerization of G-actin into F-actin is known to be endothermic reaction asshown by Oosawa et al., and thus it thought to be entropy driven. Our previous
studies by microwave dielectric relaxation spectroscopy (DRS) revealed that Factin has a dual hydration shell composed of constrained water (CW, dielectricrelaxation (DR) frequency fc <fcw (bulk water)) layer and hyper-mobile water(HMW, fc >fcw) layer which may be formed to a greater extent in an F-actin
solution than in a G-actin solution. In this study we examined a temperaturedependence of hydration properties of F-actin to understand the mechanism of
HMW formation around F-actin. DR spectra of F-actin aqueous solutions were
measured at 5, 10, 15 and 20 ° C. At every temperature the spectrum was
successfully fitted with two DR components, i.e. CW and HMW, in addition to
the bulk water component around actin. Assuming that the DR amplitude of theeach DR component is proportional to the number of hydration water, theamount of HMW decreased and that of CW increased, with increasing
temperature. We estimated the van’t Hoff enthalpy of transformation from bulk
water to HMW to be +13 kJ/mol, and from bulk water to CW -24 kJ/mol. These
results are consistent to the endothermic nature of actin polymerization, andsuggest that this is due to the change in the water structure around actin.
1PS003アクチン重合における水和変化の誘電緩和分光解析

Dielectric analysis of hydration change of actin bypolymerization
Asato Imao, Yuichiro Okazaki, Noriyoshi Ishida, Tetsuichi Wazawa, George
Mogami, Makoto Suzuki (Grad. Sch. Eng., Tohoku. Univ)
Actin is a major protein in cell and plays crucial roles in cellular functions. At
low salt concentrations (e. g., less than several mM KCl), actin adopts amonomeric form, G-actin, whereas at higher salt concentrations (e.g., 50 mMKCl) and in the presence of adenosine triphosphate (ATP), actin polymerizes toa filamentous form, F-actin. This polymerization reaction is endothermic andoccurs above a critical concentration. These endothermic reactions are believedto involve structural and dynamical changes in the surrounding water.
This paper introduces recent results on the rotational mobility of water aroundactin filaments (F-actin, a polymerized form of G-actin). Microwave dielectricrelaxation spectroscopy (DRS) was employed. The DRS measurements showedthat F-actin exhibits dual hydration properties as a structure-maker and astructure-breaker. Thus, in addition to the water molecules with loweredrotational mobility that form a constrained hydration shell around the F-actin,
there exist water molecules with rotational relaxation frequencies higher thanthose of bulk water, which are here referred to as hyper-mobile water. Thus, thepresent study revealed a significant increase of hyper-mobile water around Factin. This could be a clue to understanding the endothermic reactions of actin
polymerization and constructing the energetics of adenosine-triphosphatedriven protein functions.
1PS004アクチンの水和状態に及ぼすハライドイオンの効果

Effects of halide ions on the hydration properties of F-actin
Noriyoshi Ishida, Asato Imao, Yuichiro Okazaki, Tetsuichi Wazawa, George
Mogami, Makoto Suzuki (Grad. Sch. Eng., Tohoku. Univ.)
Actin is a cytoskeletal protein and polymerizes into filaments (F-actin) in highsalt condition. We investigated the effects of potassium halides (KCl, KBr, KI)on the hydration properties of F-actin. Actin was taken from chicken pectoral
muscle, and purified by a method of Spudich & Watt (1971). The dielectricrelaxation (DR) spectra of F-actin solutions (c = 6~7 mg/mL, 50mM KX, 2mM
MgCl2, 10mM HEPES (pH7.8), 1mM DTT, 0.1mM ATP ; X=Cl, Br and I) by
using high-resolution DR spectroscopy in the frequency range from 0.2 GHz to
26 GHz at 20.0 ± 0.01 °C. With a fixed volume fraction analysis at φ/c = 0.005
mL/mg, the extracted spectrum for the fixed volume including an actin filamentand a surrounding water layer was obtained by the Wagner mixture theory. The
DR spectrum of the fixed volume around an actin filament for every KX wassuccessfully fitted with two Debye components with DR frequencies, 20 GHz
and ~5 GHz. The total DR amplitude for each salt was similar and around 73
and composed of three parts assigned to be constrained water (fc ~5GHz),
hyper-mobile water (HMW, fc ~20 GHz), and bulk water (fc = 17 GHz). As aresult, the amount of constrained water appeared to be not sensitive to halideions, and the amount of HMW was found to increase slightly from Cl to I,
suggesting some absorption of large halide ions at the surface of actin.
1PS005

ミオシン S1 の水和に及ぼす共溶媒の影響

Cosolvent effects on the hydration state and the enzymatic
activity of skeletal myosin
Takuya Nakagawa, Shin-ichiro Yasui, Tetsuichi Wazawa, George Mogami,
Makoto Suzuki (Grad. Sch. Eng., Tohoku. Univ.)
Cosolvents such as urea and potassium halides are known to affect the hydration
state of protein, and in turn cause changes in the conformation and enzymatic
activity of protein. Myosin interacts with F-actin and generates mechanicalwork by ATP hydrolysis reaction. In this study, we have investigated effects ofthe cosolvents on the hydration state and enzymatic activity of myosin.
Myosin subfragment-1 (S1) and heavy meromyosin (HMM) were prepared fromchicken skeletal myosin by papain and chymotrypsin, respectively. By our highprecision dielectric relaxation spectroscopy (DRS), dielectric spectra of aqueous
solutions of myosin were measured to derive the hydration properties of myosin.
In this study, cosolvents of urea, 1,3-diethylurea (DEU), KBr, or KI were usedwith a reference aqueous solvent containing 30 ‒50 mM KCl, 10 mM HEPES(pH 7.8) and 2 mM MgCl2.
The effects of urea at 0.3 M on the sliding speed of F-actin were modest. DEU
decreased the sliding speed of F-actin to 1/20 in the gliding assay using myosin,
but had little effect on the Vmax of acto-HMM ATPase, indicating that DEU
uncouples the linkage between the ATPase and the mechanical work. DRS of S1solution showed that the hydration shell of S1 consists of constrained water of adielectric frequency of 3.8 ‒5.0 GHz. The content of the constrained wateraround S1 decreased by ~10%, when DEU was added or KCl was substituted
for KI in the solvent.
1PS006蛍光性カルモジュリンを用いた血管平滑筋ミオシン I および足場タ
ンパク質によるアクチン繊維の組織化メカニズム

Actin filament organization of smooth muscle and possibleroles of myosin I and scaffolding protein visualized byfluorescent calmodulin
Yoh Okamoto, Kai Sunada, Syougo Tokuda (Muroran Institute of Technology,
Division of Applied Science)
In contrast to the architecture of contractile apparatus of striated muscle, smoothmuscle has dynamic nature with respect to both of actin and myosin filaments. It
should be noticed that a significant decrease of G-actin pool was observed upon
the contractile stimulation of vascular smooth muscle according to Zang, W. etal.(2005). We have identified the presence of myosin I, a single headed myosin− S74 −

with lipid binding short tail in both porcine aorta smooth muscle and A10 cell.
An appreciable proportion of the myosin I existed in the cytosol of the A10.
(Hasegawa,Y. et al; 1996,1998) We have attempted to find out the role of thecytosolic myosin I with respect to the actin filament organization in vascularsmooth muscle cell. During the study of binding between the aorta myosin I and
F-actin, an appreciable proportions of F-actin has been sedimented with small
amounts of myosin I under physiological ionic strength. This phenomena was
attributed to the occurrence of myosin I induced F-actin bundling. During thestudies on myosin I, we have also found IQGAP1 in aorta smooth muscle.
IQGAP1 has been known as a scaffolding protein of F-actin along with small
GTPase, calmodulin. Both myosin I and IQGAP1 in aorta smooth muscle can be
key players for actin reorganization during contraction and relaxation cycles ofvascular smooth muscle. We have made a fluorescently labeled calmodulin forfurther characterization of F-actin bundling with myosin I or F-actin gel formingwith IQGAP1.
1PS007

ホ ッ キ 牽 引 筋 NAM Mg-ATPase 活 性 に 対 す る 2 種 類 の TM
（TM1,TM2) の影響

The effects of two kinds TM(TM1,TM2) to Mg-ATPase activityof hokki retractor muscle NAM
Yoichi Yazawa
We have studied Hokki clam, spisula sachalinensis, protein components ofmuscle natural actomyosin (NAM) and Mg-ATPase activities (= Casenditivities).
In autumn(october and november), Ca sensitivity of hokki clam retractor muscle
NAM ATPase activites showed low values (15~50%). Them of NAM preparedin other three seasons were higher than autumn(88~97%).
SDS-PAGE patterns of two kinds of NAM.
We was different from TM(TM1 and TM2). The difference between TM1 andTM2 is the first report about two types of TM function to Mg-ATPase activity.
1PS008

クライオ電子顕微鏡法を用いたアクチンーミオシン硬直複合体の高分解能構造への試み

Approach to higher-resolution structure of actin-myosin rigor
complex by electron cryo-microscopy
Norihiro Shimizu1, Yoshihiro Tsukada1, Yakuo Yasunaga1,2 (1Kyushu Inst. of
Tech., 2JST)
Actin-myosin interaction is involved in muscle contraction, cell motion, andintercellular transport, and the interaction mechanism in detail is not figured out
yet. We convince it necessary to obtain high-resolution structure of the rigor
complexes to elucidate the mechanism to produce strong binding between actin
and myosin. The previous studies have reported their 13 Å resolution density
maps but their resolution isn’t good enough to discuss the rigor structure at theatomic level. We also have been challenging to obtain high resolution maps
beyond 4 Å by cryo-electron microscopy and helical particle analysis. The
obtained filaments formed arrowhead every 36 nm. It is due to the behavior of
actin-myosin rigor complex: actin filaments take double-helical architecturewith a half pitch of 36 nm, and actin molecule bind to myosin molecule in pairs.
Therefore, those filaments were obtained as full-decoration. This time, we could
get to about 30 filaments (~2000 units) in addition to existing 5500 units fromearlier study (Tsukada, 2010), but need 10000 units or more number of units inorder to get high-resolution structure beyond 4 Å. We calculated that it isimportant to increase the number of better quality complex filament.
1PS009In vivo マウス心臓におけるサルコメア長のリアルタイム計測
Real-time measurement of sarcomere length in the mouseheart in vivo by using α-actinin-GFP

Akari Mizuno1, Fuyu Kobirumaki-Shimozawa2, Kotaro Oyama1, Takako Terui2,
Erisa Hirokawa2, Togo Simozawa3, Shin’ichi Ishiwata1, Norio Fukuda2(1Department of Physics, Waseda University, 2Department of Cell Physiology, The
Jikei University School of Medicine, 3Department of Physics, Gakushuin University)
The heart is a unique organ in which cardiomyocytes repeat contraction andrelaxation in response to electrical stimulation, thereby ejecting blood. Despite
numerous studies conducted thus far under various experimental settings, themolecular mechanisms of contraction and relaxation of cardiomyocytes tillremain elusive in vivo. In the present study, we expressed GFP at sarcomeric Zdiscs (α -actinin) by using the adenovirus vector system in adult mice, andconducted real-time imaging of the movement of single sarcomeres in

cardiomyocytes in the left ventricle under fluorescence microscopy at 10 nm
precision (at 100 fps). First, we found that sarcomere length (SL) was ~2.0 μm
in the isolated heart when perfused with 30 mM BDM (hence during diastole).
This value is close to what was obtained previously by other investigators in rats
using various experimental techniques. When perfused with Tyrode’s solution
containing 1 mM Ca2+, the heart started to exhibit beating with the diastolicand systolic SLs ~2.2 and ~1.7 μm in the left ventricle, respectively. Finally, we
attempted to visualize single sarcomeres in vivo in open chest mice underanesthesia. We found that SL was ~2.0 and ~1.7 μm during diastole and systole,
respectively, but varied by ~300 nm even in the same left ventricular cell. We
also found that sarcomere contraction occurred at the T-wave end point on theelectrocardiogram, followed by an in crease in the left ventricular pressure. At
the meeting, we discuss these findings at the molecular level.
1PS010チャネルロドプシンを発現した骨格筋管細胞における筋収縮と成熟の光操作

Optogenetic manipulation of myogenic contraction andmaturation in channelrhodopsin-expressing skeletal muscle
myotubes
Toshifumi Asano1,2, Toru Ishizuka1,3, Hiromu Yawo1,3,4 (1Grad. Sch. of Life
Sci., Tohoku Univ., 2JSPS, 3JST, CREST, 4Center for Neurosci., Tohoku Univ.
Grad. Sch. of Med.)
Traditionally, neuron and muscle have been electrically stimulated to be
induced the artificial activity and examined the specific functions. Although,this technique is simple and convenient, it is often nonuniform, nonspecific andinvasive with the electrical field stimulation using metal electrodes placed in theextracellular space. In this study, to improve the spatiotemporal resolution andto reduce the invasiveness, mouse muscle cell lines, C2C12 myoblasts were
genetically introduced a variant of light-gated ion channel, channelrhodopsingreen receiver (ChRGR), and subsequently induced into multi-nucleatedmyotubes according to the standard differentiation conditions. The LED lightpulses depolarized the membrane potential of a ChRGR-expressing myotube
and eventually evoked action potentials dependent on the intensity and durationof the illumination. The light-activated contraction of the photosensitive
myotube was obvious in synchronous to the light pulses of the given frequency
and the pattern (1-10 Hz, 20-100 ms). We also found that the optical stimulation
using pulsed flash light promoted the sarcomere assembly and the contractile
ability of the ChRGR-expressing myotubes. It is suggested that the myogenic
contraction and the maturation of skeletal muscle cells can be manipulated bylight using optogenetics. This technique could facilitate the studies such as thefundamental biological processes during myogenic development, the stem celltherapy for muscular disorder, and the bioengineering using the musclepowered actuator.
1PS011軟体動物横紋筋のコネクチン様タンパク質

Connectin-like protein in molluscan striated muscle
Yulong Bao1, Akira Hanashima1, Fumiaki Sodeyama2, Sumiko Kimura1(1Department of Biology, Graduate School of Science, Chiba University,2
Department of Biology, Graduate School of Science, University of Tokyo)
Connectin is an elastic muscle protein in vertebrate striated muscle. Invertebrate
striated muscle is also known to possess several connectin-like proteins.
Because the results of our SDS PAGE and immunoblot analyses have suggested
the presence of a connectin-like protein in molluscan muscle, we examined its
primary structure and compared it with those of some known connectin andconnectin-like proteins.
Using the genome information of Lottia gigantea, we searched for a connectinlike protein and determined its sequence by RT-PCR. As a result, the connectinlike protein of the limpet was estimated to be 54 kbp and have a molecularweight of 2,020,000. The domain structure of this protein was similar to that ofvertebrate connectin in that it possessed tandem Ig domains, a PEVK region at
the N-terminus and a kinase domain at the C-terminus. However, the protein had
characteristics substantially different from those of the already known connectinand connectin-like proteins. In particular, it was found to possess a tandem Fnregion consisting of 45 repeated Fn domains at the C-terminus and to lack asuper-repeat region.
Immunoblot analysis using a polyclonal antibody for the recombinant protein
showed that the antibody against the PEVK region reacted with a 3000-kDa
band of the limpet heart muscle. Furthermore, the observation with fluorescencemicroscopy revealed that the PEVK region of limpet connectin-like protein is
localized in the I-band of the striated muscle sarcomere.

− S75 −

1PS012環形動物斜紋筋のコネクチン様 4000K タンパク質の 47 アミノ酸
リピート

47 amino acids repeat of connectin-like 4000K-protein in
obliquely striated muscle of Annelida
Yui Nomiya1, Shuji Kanamaru2, Fumio Arisaka2, Sumiko Kimura1(1Department of Biology, Graduate School of Science, Chiba
University, 2Department of Life Science, Graduate School of Bioscience andBiotechnology, Tokyo Institute of Technology)
Connectin that exists in the striated muscle of vertebrates is an elastic proteinwith a molecular mass of approximately 3000 kDa. Invertebrates are also known
to possess various connectin-like proteins, one of which is a connectin-like 4000
kDa protein that is present in the obliquely striated muscle of Annelida. We
have determined the nucleotide sequence of the gene which encodes aconnectin-like 4000 kDa protein using a cDNA library of the body wall muscleof Neanthes sp. In the deduced a.a. sequence from the nucleotide sequence, we
discovered a sequence in which a unit of 47 amino acids was repeated 11 times.
This 47-amino acid sequence repeat contained a high proportion of E and Q, andno significantly homologous protein was found in the database. After preparing
a recombinant protein corresponding to this repeat sequence, we performed
analytical ultracentrifugation and measured far-UV CD spectrum to estimate its
secondary structure content. The sedimentation velocity experiment revealed
that the protein had an extremely asymmetric shape, and 48% of the whole
structure assumed beta-structure. It was thus demonstrated that this protein
could cover a longer distance than other ordinary proteins with the same size.
The fact that the connectin-like 4000 kDa protein of the obliquely striatedmuscle of Annelida has a distinct structure with the 47-amino acid repeat
sequence may be related to the resting length of a sarcomere in the Annelida
obliquely striated muscle, which is about twice as long as that in the vertebrate
striated muscle.
1PS013

プラナリアのコネクチン様タンパク質の配列

Sequential analysis of Planarian connectin-like protein
Kouhei Sasano1, Masayoshi Kurasawa1, Akira Hanashima1, Yulong Bao1,
Yuni Nakauchi2, Sumiko Kimura1 (1Grad. Sch. Sci., Chiba Univ., 2Fac. Sci.,
Yamagata Univ.)
The striated muscle of vertebrates contains connectin, an elastic protein with amolecular weight of approximately 3,000,000 that functions to maintain the
structure of sarcomeres. In addition to Ig, Fn3 and kinase domains, connectin
has a PEVK elasticity-relating region. On the other hand, invertebrates possessconnectin-like proteins some of whose domain structure are similar to those ofvertebrate connectin.
Because connectin antibodies has reacted with 550 kDa band in planarian(Platyhelminth) electrophoresis sample, the planarian is suspected to possessconnectin-like protein. Therefore, we searched for a possible candidate based on
the genome information, thereby revealing a tentative sequence of the planarian(Platyhelminth) connectin-like protein. The total length of the tentative
sequence was about 48 kb, and its molecular mass was estimated to beapproximately 1820 kDa if the sequence was fully expressed. Based on theabundance of Ig and Fn3 domains and the presence of a kinase domain, thistentative sequence is considered to be that of connectin-like protein. In thistentative sequence, we observed a repeat sequence of 39-amino acids was
repeated 34 times and a tandem Fn3 domain.
Based on this tentative sequence, we performed RT-PCR and determined thepartial sequence of the 8.4 kbp region, including the tandem Fn3 domain. The
results of the analyses revealed that the tentative sequence is expressed as
mRNA in planarian muscle.
1PS014in vitro 運動におけるアクチン束の自発的形成

Spontaneous formation of moving actin bundles in vitro
Masayuki Hoshida, Yuuto Maruko, Hajime Honda (Nagaoka University ofTechnology)
Actin filaments are forming both static and dynamic structures fulfilling theircellular functions in vitro. Some ones are supported by many kinds of actin
binding proteins but some others are spontaneously formed according just to
their physical properties. Understanding the formation mechanism of dynamic
structures of actin filament with respect to their dynamic property might beimportant for biophysical researches.
Frequently used assay for actin filament gliding under fluorescent microscope

are usually composed of myosin-coated glass slide and buffer mediums
sandwiched with another glass slide. The concentration of actin filament should
be reduced to below 1.0 mcg/mL in order to observe single filaments separately.
We introduced un-labeled actin filament at the concentration of 0.1 mg/mL
mixed with labeled filaments. After several ten minutes, bundles of moving
actin filaments are spontaneously emerged. The width of bundle is about 0.1
mm winding to form a meshwork structures with the size of about 1.0 mm.
Within the bundle of actin, filaments were moving bi-directionally with about
10 nm spacing. This mesh-like structure was formed when the actin
concentration was over 0.1 mg/mL. At lower concentration, the structure wasformed in the limited area but the shapes and the sizes are essentially the same inall cases. Therefore, these geometrical parameters of this structure should bedetermined by the physical properties of actin and myosin molecules on theglass side.
1PS015異なる滑り運動を持つ 2 種類のアクチン分子は独立したフィラメン
トを形成する

Independent formation of actin filament with two actin species
with different sliding velocities
Syunsuke Kinoshita, Kouhei Iwase, Hajime Honda (Nagaoka University ofTechnology)
G145V mutant of actin from dictyosterium was reported to slide about half to its
wild type along skeletal muscle myosin molecules on the glass slide. To
understand why the velocity was slow even driven by the same myosin, we tried
to co-polymerize these two actin molecules in the various ratios. Surprisingly,
polymerized portion of filaments were composed of same actin species of either
mutant or intact actin molecules. In addition, some filaments were tandemlyattached to each other to form a block copolymer. The block copolymers did not
attached to skeletal myosin molecules on the glass slide in the presence of ATP
in spite of the filament composed entirely from the same apiece did bind andslide along the glass surface at the same conditions.
The reason why these actin species polymerize independently might be caused
from the difference in the velocity of polymerization. However, the difference inthe binding constants between two molecules could not completely explain thereason why the block copolymers did not bind to myosin molecules. We areinvestigating the reason with respect to the difference of sliding velocity of eachspecies of actin molecules. Sliding property of the block copolymers will be
discussed in the meeting.
1PS016ヒト心筋 SPOC に対する疾患と加齢の影響

The effects of disease and aging on human myocardial SPOC
Tatsuya Kagemoto1, Mitsunori Yamane1, Cristobal G. Dos Remedios2, Norio
Fukuda3, Shin’ichi Ishiwata1 (1Department of Physics, Faculty of Science andEngineering, Waseda University, 2Bosch Institute, The University ofSydney, 3Department of Cell Physiology, The Jikei University School of
Medicine)
SPOC (SPontaneous Oscillatory Contraction) is a phenomenon observed in thecontractile system of striated (skeletal and cardiac) muscle at an intermediatelevel of activation between contraction and relaxation. We previously reported
that the period of the resting heart rate in various animal species is wellcorrelated with that of sarcomere length oscillation under SPOC conditions (forreview, see Ishiwata et al., Prog. Biophys. Mol. Biol. (2011) 105: 187-198).
Recently, we reported the SPOC properties of human cardiac muscle fibers,
such as SPOC period and propagation velocity of SPOC wave, in various
samples prepared from human hearts (47th Ann. Meeting of Biophys. Soc.
Japan: S122). In the present study, we examined the SPOC properties by using
single human cardiac myofibrils prepared from non-failing hearts aged 19 to 65
years and failing hearts (dilated cardiomyopathy (DCM)) aged 15 to 63 years.
The use of single myofibrils allowed us to observe the movements of individual
sarcomeres with no influence of connective tissues or intercalated discs. Our
preliminary results show that the SPOC period in human cardiac myofibrils issimilar to that observed in myocardial fibers. At the meeting, we will discuss theeffects of DCM and aging on human myocardial SPOC. This research has been
approved by Human Ethics Committee at Waseda University.
1PS017− S76 −

弛緩状態との境界領域における骨格筋筋原線維の自励振動状態（SPOC）

Characteristics of auto-oscillation (SPOC) for skeletal myofibrils
observed near the boundary region with relaxation conditions

Kaori Sato1, Shin’ichi Ishiwata2 (1Science and Engineering, Waseda
Univ., 2Waseda Bioscience Research Institute in Singapore (WABIOS))

Jun Ohnuki, Takato Sato, Koji Umezawa, Mitsunori Takano (Grad. Scl. ofAdv. Sci. & Eng., Waseda Univ.)

The contractile system of striated (skeletal and cardiac) muscle has the autooscillation state called SPOC (SPontaneous Oscillatory Contraction) state inaddition to contraction and relaxation states (Ishiwata, S., Shimamoto, Y.,
Fukuda, N. (2011) Prog. Biophys. Mol. Biol.. 105, 187-198.). This is observedat the intermediate levels of activation between contraction and relaxation. In
the SPOC state, the sarcomere length (SL) shows oscillation with a saw-tooth
waveform consisting of slow shortening and rapid lengthening. On the other
hand, the SPOC theory (Sato, K., Ohtaki, M., Shimamoto, Y., Ishiwata, S.
(2011) Prog. Biophys. Mol. Biol. 105, 199-207.) predicted that, as the activationlevel is lowered approaching to the relaxation, the amplitude of SL oscillation
decreases and its waveform tends to become a triangular one. In this study, we
aimed to experimentally examine this theory. We analyzed the oscillation
properties of skeletal myofibrils in the boundary conditions between relaxationand SPOC (ADP-SPOC; SPOC induced at the coexistence of MgATP, MgADP
and Pi in the absence of Ca2+). As a result, we observed that myofibrils showeda transition from a general SPOC state to a weak oscillation state by lowering
the ADP concentration (lowering the activation level), and the amplitude, theperiod and the waveform of SL oscillation became similar to those predicted by
the theory. Therefore, it is concluded that the theoretical prediction has been
experimentally confirmed. We will also report the other properties of weak
oscillation.

Myosin utilizes the free energy obtained from ATP hydrolysis for generating theforce which drives the unidirectional motion on an actin filament. Lever-arm
model, in which the swinging motion of the lever-arm region of myosin (called
the power-stroke) is the driving force, gains widespread support as this forcegeneration mechanism. However, the model is deduced mainly from structural
studies, and the energetic aspect in aqueous solution remains unclear. Therefore
it is critical to evaluate the free energy change associated with the lever-arm
swing. To do this, we calculated the free energy change accompanying thelever-arm swing by using the explicit-water all-atom model and the enhanced
sampling method. In the single myosin molecule system, we found that thepower stroke is accompanied by the unfavorable free energy change (to which
the electrostatic energy dominantly contributed). This result, however,apparently conflicts with the lever-arm model. One possible reason is that thefree-energy change is favorable but is not so large that the statistical error due tothe sampling insufficiency might lead us to the conflicting result. Therefore, we
improve the sampling efficiency by using better starting structures and reaction
coordinate. The effect of the amino-acid sequences should also be examined.
Another possible reason is the effect of the actin filament, so we recalculate thefree energy change accompanying the power-stroke in the presence of the actinfilament.
1PS021

1PS018Force transmission and anisotropic stiffening of reconstituted
cytoskeletonsDavid Head1, Emi Ikebe2, Akiko Nakamasu2, Peijuan Zhang2, Shoji Ando3,
Daisuke Mizuno2 (1University of Leeds, 2Kyushu University, 3Sojo University)
Stresses transmit from force generators in living cells induce a non-linear
cytoskeletal response which affects range of cell behaviors. Here we employ
microrheology to apply physiological-strength point forces to intermediatefilament (vimentin) networks and measure the transmission of 2D response at ananterior point. We observe both pronounced stiffening and a marked anisotropy,
with a largerstiffness parallel to the force. This is intuitively and quantitatively
explained in terms of the properties of individual filaments by an affine
continuum model. We argue that deviations from the theoretical model at low
frequencies is due to non-affine network deformation modes, rather thandistributed inter-crosslink separations.
1PS019高圧下におけるαアクチン構造安定性の理論的解析

Theoretical Analysis of α-Actin Stability at High Pressure
Nobuhiko Wakai1, Kazuhiro Takemura2, Takami Morita3, Akio Kitao2,4 (1Grad.
Sch. of Fron. Sci., Univ. of Tokyo, 2IMCB, Univ. of Tokyo, 3NRIFS, 4JST, CREST)
One of the main components of muscle fiber is α -actin, which plays many
important roles in cellular functions. Monomeric actin proteins can polymerize
into a filament with ATP hydrolysis. The amino acid sequences of actins arehighly conserved across species. Deep-sea fish actins have specific amino acidsubstitutions, Q137K/V54A or Q137K/L67P. Since only Q137K substitution is
located near the active site, this residue is expected to contribute to the pressure
tolerance. Although some experiments showed the effects of substitutions athigh pressure, the detailed mechanism of the pressure tolerance is still unclear.
Therefore, we analyzed high pressure effects on monomeric actins using
molecular dynamics simulations. Notable differences of excluded volume andsolvent-accessible surface area were not observed at high pressure. However,the deep-sea fish actins form a salt bridge between K137 and ATP at the activesite. This salt bridge may stabilize ATP at the active site even under highpressure. The orientations of the 137th residue are different between the deepsea fish actins and other species, which shows a possibility of different reaction
mechanism of ATP hydrolysis. In addition, the total number of the salt bridges
in the deep-sea fish actins increased due to the formation of several salt bridges
connecting pairs of the secondary structures and subdomains. These salt bridges
are suggested to be a key for the pressure tolerance.
1PS020ミオシンのレバーアーム・スイングの自由エネルギー計算とアクチ
ンフィラメントの影響Free energy calculation of lever-arm swing of myosin and theeffect of actin filament

ATP 合成酵素の結晶化

Crystallization of ATPsynthaseYasuo Shirakihara1, Hiromi Tanikawa1, Satoshi Murakami2 (1National
Institute of Genetics, 2Tokyo Institute of Technology)
ATP synthase is responsible for ATP production in living cells, and is amembrane protein located in the energy conversion membrane. We crystallized
ATP synthase from a thermophilic bacterium PS3, got diffraction patterns
extending to 7 A resolution but with a very high mosaicity. In the course of
improvement of those crystals, we suddenly got unable to reproduce them.
The problem was cured only partially, in spite of lots of efforts. Then we
isolated 27 strains living in unusual environments like hot springs as ATP
synthase source. We hoped, from a consideration of limited temperature range
where those bacteria are viable, that our new strains carefully isolated may besuited to structural study. We purified ATP synthase from 27 such strains and tothem applied crystallization screening test using conditions including the best
ones for PS3 ATP synthase. However, the results were discouraging.
We are now back to PS3, and are examining conditions for getting back goodold crystals. Those efforts are mainly on the PS3 ATP synthase preparation.
Examined points are: test of available different bacterial stocks, culture
conditions(temperature, airation, nutrition, and so on), membrane preparation
methods (lysozyme lysis, French press, sonication), preparation temperature andMg concentration in the buffer. Though definite answers are yet to come, wehave a feeling that we have to use a good strain stock, and that culture
conditions are very important. Details will be presented on why we think so.
1PS022

TF1 β E190D 変異体の外部トルクに対する影響

Effect of external torque on the rotation of TF1 βE190D
mutantTomohiro Kawakami1, Shoici Toyabe2, Hiroshi Ueno1, Seishi Kudo3, Eiro
Muneyuki1 (1Dept. Phys., Faculty of Science and Engineering, Chuo Univ.,
2
Faculty of Physics, LMU Munich, 3Dept. Appl. Phys., Sch. Eng., Tohoku
Univ.)The F0F1 ATP synthesizes ATP from ADP and inorganic phosphate (Pi) byproton motive force across the membrane. When isolated, F1 portion works as a
motor, hydrolyzing ATP to ADP and Pi. Furthermore, F1-motor converts freeenergy change derived from ATP hydrolysis into the kinetic energy of therotation of γ subunit. Recently, Toyabe examined the external torquedependency of the rotation rate of wild type F1-ATPase. He found that the
maximum work estimated from the stall torque coincided well with the freeenergy change of ATP hydrolysis, reached the conclusion of high energy
transduction efficiency. So, the next stage of the study is to elucidate themechanism of this high efficiency. For this purpose, comparison of WT tomutant is a potent approach and we used a mutant βE190D which rotates slower
than WT due to low catalytic activity. For applying torque, we adopt
electrorotation method. We found different external torque dependence of therotation of βE190D from that of WT. In case of WT, rotation rate changes
sharply around stall torque under our experimental condition. But the torque− S77 −

dependence of βE190D around stall point was rather small. Distant from thestall point, the rotation rate showed dramatic external torque dependency. We
will discuss torque generation source of F1 motor based on the observation.
1PS023

Effect of nucleotide structure on nucleotide binding androtation of F1-ATPase
Yohsuke Kikuchi1, Masaike Tomoko2, Shoichi Toyabe3, Hiroshi Ueno1, Eiroh
Muneyuki1 (1Dept. of Phys., Univ. Chuo, 2Dept. of Phys., Univ. Gakushuin, 3Fac. ofPhys., LMU Munich)
F1-ATPase is a molecular motor in which the central γ subunit rotates 120°
against the α3β3 ring while hydrolyzing an ATP. This 120° step rotation consistsof 80° substep and 40° substep. The 80° substep is triggered by the nucleotide
binding to catalytic site which is located on each β subunit. The 40° substep istriggered by the phosphate release from the β subunit. Thus nucleotide binding
plays an essential role in the rotation of the γ subunit. To study the couplingmechanism of the rotation and nucleotide binding to catalytic site, we compared
the affinity of nucleotide binding to catalytic site of F1 and a single β subunit byfluorescence quenching of genetically engineered βY341W with or without β
E190Q mutation. The single β subunit, separated from F1 complex, has ATP
binding activity but does not hydrolyze ATP. For complex, we measured the Kd3
and thermodynamic parameters such as enthalpy of the nucleotide binding. The
Kd3 of ATP binding to F1 was bigger than ADP at all temperature range (4-50
degrees) and ΔH0 of ATP (-28 kJ/mol) was smaller than ADP (-18 kJ/mol). But
Kd and ΔH0 of ATP or ADP binding to single β subunit were almost the same. A
comparison between (G, I)TP and (G, I)DP showed a similar tendency. The
results indicate the effect of complex formation on the nucleotide binding. To
discuss the mechanism of the rotation and the nucleotide binding, we estimatedtorque of F1 utilizing ATP, GTP or ITP hydrolysis. We will present the resultsof nucleotide binding and rotation of F1.
1PS024

F 1-ATPase の P-loop 変異体に対するリン酸の阻害効果

Inhibitory effect of Pi on F1-ATPase P-loop mutant
Hikaru Yoshida1, Ayumi Ito1, Jotaro Ito1,3, Syoichi Toyabe1,2, Hiroshi Ueno1,
Eiro Muneyuki1 (1Dept, of Physics. Chuo Univ., 2Faculty of Physics, LMU
Munich, 3School of Engineering, The University of Tokyo)
F1 -ATPase is an ATP-driven rotary molecular motor. Single molecularobservation revealed γ subunit rotates 120° against α3 β3 ring. This 120° step
consists of 80° substep and 40° substep. The former substep is triggered by ATPbinding and ADP release. The latter substep is triggered by ATP hydrolysis andPi release. In a previous study, two schemes of F1 rotation were proposed(Adachi et al. Cell, 130 (2007) 309-321). The difference between these schemes
was whether Pi releases before or after ADP. Because we cannot observe Pi
release, the experiment on its Pi release has been difficult. Recently, Watanabe
et al proposed that F1 released Pi after ADP (Nat Chem Biol. 2010 Nov; 6 (11)794-5). In order to obtain another clue as to this problem, a mutant which
releases Pi slowly will be useful. In the present study, we made a P-loop mutant(βG158S) based on (Sanjay et al. Biochemistry 2002, 41.14421-14429). We
measured inhibitory effect of Pi on ATPase activity and single molecule
rotation. 1 mM KPi decreased ATPase activity of the mutant about 20% at 200
uM ATP. On the other hand, 1 mM KPi did not affect turnover of wild type. In
rotation assay, 1 mM KPi decreased the rotation rates of the mutant about 40%
at 200 uM ATP. These results suggest that the P-loop mutant has strong affinity
for Pi and releases Pi slowly.
1PS025

Conformational changes in the β subunits of F1-ATPase
revealed by FRET measurements during the rotation of the γ
subunit

Mitsuhiro Sugawa, Masaru Kobayashi, Takashi Matsui, Tomoko Masaike,
Takayuki Nishizaka (Department of Physics, Gakushuin University)
F1 -ATPase is a rotary molecular motor in which a central γ subunit rotatesagainst hexagonally arranged subunits α3 β3, hydrolyzing ATP sequentially inthree β subunits. It remains challenging to uncover the conformational changesof the F1 complex during the rotation. Previous study has proposed sets of the βsubunit conformations during the rotation (Masaike et al., Nat. Struct. Mol.
Biol. 2008). Here we performed single-pair FRET measurement to detect
distance changes between two β subunits having a FRET pair (Cy3 and Cy5).
Transition between high (0.8) and low (0.5) FRET efficiencies was observed,
using F1(βE190D). Given the crystal structures, low FRET efficiency indicates
one β subunit in the open form but another in the closed form. On the other

hand, high FRET efficiency indicates both of the β subunits in the closed form.
Next, we performed a simultaneous measurement of FRET between two
fluorescently labeled β subunits and the rotation of the γ subunit. Although theresults were preliminarily, relationship between FRET efficiency and therotation angle was revealed. High FRET efficiency was occurred in one of thethree catalytic dwells. In other dwells, other two catalytic dwells and three ATPwaiting dwells, FRET efficiency was lower. These results suggest that in the
ATP-waiting dwell two of three β subunits would not take the closed form as in
the catalytic dwell. We are performing further experiments.
1PS026

F1-ATPase におけるトルクの入出力

Torque input/output profiles of F1-ATPase
Eiichiro Saita1,2, Kazuhiko Kinosita3, Masasuke Yoshida1,2 (1Dept. Mol. Bio.,
Kyoto Sangyo Univ., 2ICORP, JST, 3Dept. Phys., Waseda Univ.)
F1 -ATPase is an ATP-driven rotary motor, which generates torque by
consuming the chemical energy of ATP hydrolysis. Inversely, it can synthesize
ATP from ADP and Pi by using reverse torque applied externally. Average
output of the torque during ATP hydrolysis reaction is reported to be 40-50
pNnm. However, the detailed torque profile as a function of rotor angle remains
unknown. To know it, we have been measuring the torque by using magnetic
tweezers. A magnetic bead under a magnetic field can be approximated as aspring in the rotational motion, which allows us to measure the torque on amagnetic bead attached to the motor in a conservative manner. We note that thismethod can be applied to the torque measurement during ATP synthesis as wellas during ATP hydrolysis.
One of primary findings is that the torque output during ATP hydrolysis is notconstant but varies with the bead angle. A gradual decrease followed by a steep
jump in torque repeats at least three times per 120 degree rotation, indicatingthat F1-ATPase rotates with the transitions between at least three states. This isalso the case for the torque input profile during ATP synthesis, and each of thetwo profiles of both reactions is almost identical under various substrates
conditions. In addition, the amount of mechanical work calculated from thetorque profiles is almost equal to that of calculated free energy change of ATPhydrolysis. The detailed torque profiles obtained possibly explain how F1 ATPase attains very efficient energy conversion.
1PS027

H+/ATP ratio of FoF1-ATP synthase from the thermophilic
Bacillus PS3

Naoki Soga1, Kazuya Kimura1, Yuzo Kasuya1, Toshiharu Suzuki2, Masasuke
Yoshida2, Kazuhiko Kinosita1 (1Dept. of Phys., Waseda Univ., 2Molecular
Bioscience, Kyotosangyo Univ.)
FoF1-ATP synthase converts the energy of transmembrane proton flow into thehigh-energy bond between ADP and phosphate. The proton motive force thatdrives this reaction consists of two components, the pH difference (ΔpH) acrossthe membrane and transmembrane electrical potential (Δψ). The H+ /ATP ratio
and the standard Gibbs free energy of ATP synthase are important parameters ofcoupling between proton translocation and ATP synthesis. Here, to determine the
H+/ATP ratio and the free energy of ATP synthesis for the thermophilic Bacillus
PS3 ATP synthase, we examined how the proton motive force is balanced bycounteracting ATP hydrolysis. We measured the rates of ATP synthesis andhydrolysis with luciferin/luciferase system as a function of the proton motive
force, which was formed by acid-base transition and valinomycin-mediated K+
diffusion potential. For various nucleotide conditions we determined the proton
motive force at which FoF1 neither synthesized nor hydrolyzed ATP. The shift ofthe equilibrium proton motive force as a function of [ATP]/[ADP][Pi] indicatedthe H + /ATP ratio of 3. 0 ± 0. 4 and the standard Gibbs free energy of ATP
synthesis of 36±4 kJ/mol for this thermophilic enzyme.
1PS028

新ステップ解析アルゴリズムの F1− ATPase への応用

Application of New step finding algorithm to F1-ATpase
Akihiko Seino, Takeshi Nakagawa, Kazuo Sasaki, Kumiko Hayashi (Dept.
Appl. Phys., Sch. Eng., Tohoku Univ.)
Kerssemakers and coworkers proposed the step finding algorithm and theiralgorithm has been used in single-molecule experiments, because the
distribution of step size and dwell time makes it possible to obtain important
information about the molecule [J.W.J. Kerssemakers et al., Nature 442, 709(2006)]. In order to improve the performance of the algorithm in the presence ofnoise, we proposed a new step finding algorithm [Nakagawa et al. The 49th
Annual Meeting of the Biophysical Society of Japan, 3M1036]. In this

− S78 −

algorithm, the candidate steps, which are found by the first stage of the
Kerssemakers algorithm, are tested by Student’s t-test. Thus, we came to be able
to detect steps which could not be found in Kerssemakers algorithm. Besides, its
performance was almost equal to that of Kalafut and coworkers algorithm,
which developed recently [Kalafut & Visscher, Compt. Phys. Comm. 179, 716(2008)]. We implemented the algorithm by using LabVIEW and applied it tosingle-molecule experiments on F1-ATPase (F1). Note that F1 is a rotary protein
which rotates hydrolyzing ATP and the time course of its rotary angle is a
stepwise manner with the step size 120º. As an additional improvement of thealgorithm, we did a tilt step fitting analysis to 120º rotation of F1. This result
enabled us to get real-time measurement of rotary torque of F1.
1PS029

回転電場を用いた F1 -ATPase の一分子計測による拡散の Giant

Acceleration の観察
Giant Acceleration of diffusion in single-molecule experiments
Ryunosuke Hayashi1, Syuichi Nakamura1, Seishi Kudo1, Kazuo Sasaki1,
Hiroyuki Noji2, Kumiko Hayashi1 (1Dept. Appl. Phys., Sch. Eng., Tohoku
Univ., 2Dept. Appl. Chem., Sch. Eng., Univ. Tokyo)
The diffusion coefficient is one of parameters that are often measured in small
biological system to characterize their fluctuation. Giant acceleration ofdiffusion by use of tilted periodic potentials is one of the many theories on
diffusion in the field of non-equilibrium statistical mechanics. According to thetheory [1], when we apply a constant force to the colloidal particle in a periodic
potential, the diffusion may be greatly enhanced, as compared to free thermal
diffusion with an enhancement of up to 14 orders of magnitude for a realistic
experimental set up [1]. The diffusion coefficient as a function of the force
exhibits the resonance peak at the force value near the critical tilt, which gets
more and more pronounced as the environmental temperature decreases [1].
Because the structure of F1 has three-fold symmetry, the rotary potential isperiodic with a period of 120°. It has been found that a constant torque can beapplied to F1 by using an electric rotating field [2]. When we measured the
diffusion coefficient as a function of the external torque, we observed the giant
acceleration of diffusion in F1. We show the torque value at the resonance peakcorresponds to the critical tilt in the rotary potential of F1, which is estimated by
the fluctuation theorem.
[1] P. Reinmann et al., Phys. Rev. Lett. 87, 010602 (2001).
[2] T. Watanabe-Nakayama et al., Biochem. Biophys. Res. Commun. 366, 951957 (2008).
[3] K. Hayashi et al., Phys. Rev. Lett. 104, 218103 (2010).
1PS030

バネでつながれたモーター分子による ATP 合成

ATP synthesis by elastically coupled nanomotors
Yasuhiro Imafuku1, Nils Gustafsson2, Neil Thomas2 (1Department of Biology,
Kyushu University, 2School of Physics and Astronomy, University ofBirmingham)
We have studied a simplified analogue of ATP synthase, consisting of twoelastically coupled nanomotors pulling against each other in a molecular ‘tug of
war’. When one of the nanomotors is driven strongly enough by an external
free-energy source, it pulls the other ATP-driven nanomotor backwards so thatit synthesizes ATP. Monte-Carlo simulations of the molecular tug of war allow
us to study the dependence of the ATP synthesis rate on the free-energy drive Δ
G and on the stiffness λ of the spring coupling the two motors. The model
reproduces the experimentally observed dependence of ATP synthesis rate on
free energy at two crucial points, the maximum ATPase velocity and thethermodynamic equilibrium where the velocity of the ATPase is zero. We havealso investigated the variation of ATPase velocity with both ΔG and λ. There arethree distinct phases of operation: (i) a forward ATP-synthesis phase, (ii) a
backward ATP- hydrolysis phase, and (iii) a locked phase where the ATPase
velocity is close to zero. The locked phase becomes more extensive withincreasing stiffness λ. Its presence demonstrates the key role that elastic energy
plays in the operation of ATP synthase.
1PS031回転モーター F1-ATPase の強制回転における長短ヌクレオチド結合の解釈

The interpretation of long and short bindings of nucleotides torotary motor of F1-ATPase during forced rotation
Kengo Adachi1, Kazuhiro Oiwa2, Masasuke Yoshida3, Kazuhiko Kinosita, Jr.1
(1Waseda Univ., 2Adv. ICT Res. Inst., NICT, 3Kyoto Sangyo Univ.)

F1 -ATPase is a rotary molecular motor in which a central γ subunit rotatesagainst hexagonally arranged subunits α3 β3. Three β-subunits, each hosting acatalytic site, bind ATP sequentially and hydrolyze it to power the rotation of γ,
whereas they synthesize ATP from ADP and inorganic phosphate (Pi) when γ
rotates in reverse under an external force. The chemical reactions that occur inthree catalytic sites are tightly coupled to the mechanical rotation. Recently wehave succeeded in determining the angle dependence of nucleotide affinity bydirectly observing binding/release of fluorescently (Cy3) labeled nucleotides
during the forced rotation, revealing the entire reaction pathway of ATP
synthesis/hydrolysis.
In that study under the condition of uni-site catalysis (at most one of the threecatalytic sites is occupied), there are two types of nucleotide binding: shortbinding for <120° and long binding for ~240°. We interpreted the result as
follows: the long binding is regular pathway and when a site that is toaccommodate a nucleotide for ~240° happens to be vacant, due to failure in theinitial binding (during open to closed conformational changes) or to early
release the bound nucleotide, the site allows another chance of binding toward
the end of the ~240° period (in re-opening process). To confirm theinterpretation, here we attempt to identify the sites that are binding a Cy3nucleotide by employing defocus imaging technique which resolves the threedimensional orientation of a single fluorophore from the diffraction pattern.
1PS032

プラズモン増強電場を利用した蛍光 ATP と回転分子モーターの回
転の同時観察Simultaneous observation of fluorescent-nucleotides androtation of rotary molecular motor on plasmonic metalnanostructures
Hiroshi Ueno1, Shoichi Toyabe1,2, Eiro Muneyuki1 (1Dept. of Phys., Fac. ofSci. & Eng., Univ. Chuo, 2Sys. Biophys., Fac. of Phys. Munich Univ.)
We have previously developed the simultaneous observation system of darkfield and fluorescence images using gold nanoparticle as both an optical probewith a low frictional load and a plasmonic nanoparticle for fluorescenceenhancement. Metal nanostructures interact strongly with specific wavelengthof light, which causes a localized surface plasmon resonance. The plasmonicnanostructures generate large electric field near its surface which enhances thefluorescence of dyes. Actually, we have succeeded in a simultaneous
observation of Cy3-ATP binding/release and rotation of F1 -ATPase at 10ms
temporal resolution and found an enhancement of Cy3-ATP fluorescence
without increasing background noise. In addition, we found a change influorescence with 40° substep from 200° to 240°. Our results suggest that
conformational change of nucleotide-binding region of β subunit occurs with
40° substep at 200°. However, we cannot rule out the possibility that this change
is an artifact resulting from the inhomogeneity of the enhanced-electric field
caused by rotation. So, we redesigned the layout of the nanostructures in which
the plasmonic nanostructures were fabricated on the glass surface by direct
immobilization of nanoparticles or nanosphere lithography to form nanostructure array. Our improved system may afford a versatile experimental platform
for sensitive measurements of single molecule fluorescence and surface
enhanced Raman scattering in the future.
1PS033

FoF1-ATP 合成酵素のプロトン駆動力による回転運動の直接観察

Direct observation of H+-driven rotation of FoF1-ATP
synthase
Rikiya Watanabe, Kazuhito V. Tabata, Ryota Iino, Hiroyuki Noji (School ofEngineering, The University of Tokyo)
1YS1115The synthesis of ATP, the key reaction of biological energy metabolism, isaccomplished by the rotary motor protein; FoF1-ATP synthase (FoF1). In vivo,
FoF1, located on the cell membrane, carries out ATP synthesis by coupling to
the rotary motion driven by the proton motive force (pmf) across the membrane.
Fo F1 consists of two molecular motors; ATP-driven motor (F1) and protondriven motor (Fo). In 1997, we for the first time observed the ATP-driven
rotation of F1, and have thoroughly elucidated its operating principle as a rotary
motor protein in this decade. In contrast, despite more than 10 years waselapsed, the proton-driven rotation of Fo or whole enzyme; FoF1, has not been
observed yet. This is mainly due to the defects of the experimental system; i.e.
the damage on the membrane, the instability of pmf generation system, and its
interference with the detection system for rotary motions. In this year, to resolvethese technical issues, we fixed these defects one by one, and eventuallydeveloped the experimental system which enabled to observe the proton-driven
rotation of Fo F1 under the optical microscope. By using this experimental− S79 −

system, we directly observed the proton-driven rotation of FoF1 in clockwise,
which is the reverse direction of ATP-driven rotation, and also obtained various
information related to the “dynamics”, such as the stepwise rotary motion
coupled with the proton transport and ATP synthesis, which provided a clue forunderstanding the rotary catalysis mechanism of Fo F1 in the physiologicalcondition.
1PS034

分子動力学計算を用いて明らかにするγ 16°回転前後の Yeast F1-

ATPase の構造的特徴
Molecular Dynamics Simulation on Structural Characteristics
of Yeast F1-ATPase before and after 16-degree Rotation ofGamma Subunit
Yuko Ito1, Takashi Yoshidome1,2, Nobuyuki Matsubayashi3, Masahiro Kinoshita2,
Mitsunori Ikeguchi1 (1Yokohama-city univ. Nanobioscience, 2Kyoto univ. Instituteof Advanced Energy, 3Kyoto univ. Institute of Chemical Research)
We performed molecular dynamics simulations for crystal structures of yeast
F1-ATPase whose crystallographic unit contains two different states. One
complex binds Pi in the βE subunit, and the other liberated it. In the latterstructure, the position of central stalk is also rotated + 16 degree in thehydrolysis direction. Because single molecule experiments have proven that Pi
release occurs at the β E subunit, and the γ subunit is rotated after ATPhydrolysis and Pi release, these structures are supposed to represent snapshots
before and after Pi releases for the 40 degree substep, respectively. From theirmotional trajectories, we describe how the F1-ATPase complex changes the
structure after Pi release. Then, we propose a possible mechanism of the 40
degree rotation of the γ subunit.
1PS035Single-molecule analyses of the rotation and regulation ofhuman F1-ATPaseToshiharu Suzuki1,2,3, Tanaka Kazumi1,2, Chiaki Wakabayashi1, Eiichiro
Saita1,2, Shou Furuike4, Kazuhiko Kinosita Jr5, Masasuke Yoshida1,2 (1ATPsynthesis regulation project, ICORP, JST, 2Dept of Mol Bioscience, Kyoto
Sangyo Univ, 3Chemical Resources Laboratories, Tokyo Inst of Tech, 4Dept ofphysics, Osaka Med College, 5Dept of Physics, Faculty of Science andEngineering, Waseda Univ)
F1-ATPase (F1) is the smallest rotary motor in life. The rotation and regulation
mechanisms have been deeply investigated by using bacterial enzymes,
especially Bacillus PS3 F1(TF1). One hydrolysis reaction of ATP makes 120°rotation and during the 120°, two kinds of dwelling events, named ATP-binding
dwell and catalytic dwell, are observable by single molecule microscopic
analyses. Although researchers have desired to precisely compare theexperimental results to structural information from X-ray crystal study tounderstand the rotary catalysis mechanism, available structural information is
only from eukaryotic enzymes. Additionally, bacterial F1s have no sensitivity
toward most of chemical and protein factors affecting eukaryotic F1, especiallyhuman F1. To overcome the difficulties, we have established systems forrecombinant human F1.
Single molecule analyses showed counterclockwise rotation of the human F1
by the energy of ATP as seen in bacterial F1, but its rotation was remarkably
fast, 705±75 rps at 26°C and over 1,366±43 rps at human body temperature,
which was roughly 3-folds faster than TF1. Slow hydrolysable ATP-analogue,
ATP-γS, clarified the presence of catalytic dwell in it, although the angle wasdifferent from that of TF1. Rotation analyses using several factors, such as
inhibitory factor-1, revealed that another novel 3rd dwelling position is present
in human F1. These results suggest that rotation in mammalian F1 is notnecessarily same to that of bacterial enzymes.
1PS036

一分子 FRET 法によるキネシン頭部のヌクレオチド依存的な構造
変化の観察

Single molecule FRET observation of the nucleotidedependent conformational changes of the kinesin motor
domainRyosuke Komiyama, Michio Tomishige (Department of Applied Physics,
School of Engineering, The University of Tokyo)
Kinesin-1 is a motor protein that moves along microtubules by alternately
exchanging two motor heads. Our lab recently showed that ATP hydrolysis
cycles of trailing and leading heads are differently regulated depending on the
direction of the neck linker extension, although it is still unknown how thetension posed to the neck linker affects the nucleotide-binding site distal fromthe neck linker. Based on the recently solved crystal structure which revealed
nucleotide-dependent rotational motion within the motor domain, we
hypothesized that ATP binding/hydrolysis can be regulated by the neck linker
tension through restricting the subdomain rotation. To test this hypothesis, wedeveloped a single-molecule FRET probe to detect the rotational motion in thekinesin head. We labeled a residue in the rotating subdomain and a residue inthe microtubule-interface in kinesin monomer with donor and acceptor dyes.
The FRET efficiency distribution in nucleotide-free and AMPPNP states
showed distinct peaks separated by about 10%, indicating that the conformational difference before and after rotation can be distinguishable. Then, welabeled with this probe the leading head of the heterodimeric kinesin in which
trailing head is a immotile mutant head, and showed that the leading headmainly takes pre-rotating conformation even in the presence of ATP. These
results are consistent with the idea that ATP binding in the leading head isprohibited because the backward tension on the neck linker suppresses therotation in the head.
1PS037

キネシンのヘッド間張力がヌクレオチド結合部位に与える影響 (分子シミュレーションによる調査)

The effect of the internal tension between two heads on the
nucleotide binding site of kinesin-1 studied by All-atom MD
Ryo Kanada1, Tsukasa Makino2,3, Michio Tomishige3, Shoji Takada1 (1Grad.
Sch. Sci., Univ. Kyoto, 2BSI, Riken, 3Dept. of Appl. Phys., Univ. Tokyo)
The dimeric motor protein kinesin-1 walks along microtubules (MT) in a handover-hand manner by alternately hydrolyzing ATP and moving two heads. Theinternal tension between two heads may be related to the regulation for thecoordinated walking of kinesin, although the detail mechanism remains
unknown. In this study, we focus on the mechanism how the internal tension
imposed on the neck-linker region affects the nucleotide binding site in motor
domain allosterically. To address this problem, we constructed the dimerickinesin models bound on MT at on-path way " Tatp-Lapo" state (where thetrailing head is ATP bounded state and the leading head is in nucleotide-free)
and the monomeric model on MT at apo-state by using the new resolved x-ray
structures. We performed all-atom MD and compared the structures andfluctuations at nucleotide binding sites between the dimeric (with internaltension) and the monomeric model (without tension). Based on the comparison,
we will discuss the validity of “front-head gating” mechanism which proposes
ATP binding to the leading head is prohibited due to the internal-strain.
1PS038

スピンラベル ESR によるキネシンのネックリンカードッキング、
リンカー間歪み、ヌクレオチド−リンカーサイト間伝達の動的構造
解析

Structural dynamics of necklinker docking, interlinker strain
and nucleotide-to-linker transmission in kinesin probed byspin-label ESR
Satoshi Yasuda1, Shinji Takai1, Masafumi D. Yamada2, Shinsaku Maruta2,
Toshiaki Arata1 (1Grad. Sch. Sci., Osaka Univ., 2Grad. Sch. Eng., Soka Univ.)
We have used site-directed spin label (SDSL) ESR to explore 3 structural eventsin kinesin monomer (KM) and dimer (KD) at atomic resolution; neck linker(NL) motion, the NL strain by 2 motor heads (MHs) and intersite transmission
or communication. 1) We directly measured the distance between 2 SLs at
motor domain (MD) (at C223) and NL (at C336), by availing spectral
broadening. The NL-MD distance of KM on MTs was 1-2 nm (dock) in
AMPPNP state whereas >2 nm (undock) in the no nucleotide (NN) or ADP
state. 2) The distance between the C336s of NLs close to an NL-stalk junction of
KD on MT was 1-2 nm (unstrained) in NN state whereas >2 nm (strained) in
AMPPNP or ADP state. Using ESR, we for the first time detected the stress
imposed by 2 docked NLs of front and rear MHs in AMPPNP state or by the
dissociated and mobile MH in ADP state. 3) α-1 helix spans between nucleotide(nuc) and NL sites. We estimated steric hindrance, from SL mobility, aroundSL-cys residue 61, 62, 66, 67, 70, or 72 of α-1 in KM on MT. In NN state, themobility of C66 and C67 was changed reversely; the mobility of C66 decreased,
whereas that of C67 increased. Therefore, it is likely that nuc and NL sites
communicate with each other through “rotation” of this helix that induces
structural changes in both sites to regulate nuc affinity or NL docking to MD.
We would discuss the structural transmission mechanism in KD by combining
these data with the previous data on undock-dock transition and the length of
NLs in the KD (Sugata et al. BBRC ’04, JMB ’09).

− S80 −

1PS039

The role of electrostatic interaction in binding-site selectivity of
KIF1A on microtubule
Yukinobu Mizuhara, Kyohei Yamamoto, Jun Narita, Mitsunori Takano(Grad. Scl. of Adv. Sci. & Eng., Waseda Univ.)
KIF1A, a well-studied kinesin superfamily motor protein, functions as amonomer, where the biased binding to MT, which leads to force-generation, hasbeen observed both in vitro and in silico experiments. Cryo-electron microscopy
indicated that KIF1A binds to the α-β contact region within the dimmer (intradimer binding site), although there is another almost equivalent potential
binding site between the adjacent dimmers (inter-dimer binding site). It has been
indicated that the binding-site selectivity is achieved by the difference in the
KIF1A-MT electrostatic interaction between the intra- and inter-dimer bindingsites. On the other hands, binding of KIF1A to the inter-dimer binding site wasalso observed in our previous simulation study using a residue-based coarsegrained. However, it is possible that the binding-site selectivity was smeared inour simulation due to the coarse-graining of the protein model. Therefore, in thisstudy, we examined this possibility by using the all-atom model, and calculated
the binding energy between KIF1A and the αβ tubulin dimmer. We found thatthe binding energy for KIF1A binding to the intra-dimer binding site is lower
than that to the inter-dimer binding site. We then analyzed the binding energy at
the residue level, and examined whether the observed binding-energy differenceis originated from the electrostatic interactions (including the critical role ofloop 7 of KIF1A as has been suggested).

1PS040Regulation of motor activity of kinesin-6 by phosphorylation of
the N-terminal extension domain
Akihiko Sato1, Tim Davis2, Shin Yamaguchi1, Masanori Mishima2, Junichiro
Yajima1 (1Graduate School of Arts and Sciences, Univ. Tokyo, 2Centre for
Mechanochemical Cell Biology Warwick Medical School University of
Warwick)
Kinesin-6 is a microtubule-based motor protein localized at centralspindle inanaphase. It has been demonstrated that phosphorylation of the motor domain ofkinesin-6 on a conserved site within a N-terminal extension destabilised theinteraction between microtubules and kinesin-6. Furthermore, deletion of the Nterminal extension also reduced its affinity for microtubules. The molecularmechanism in which the N-terminal extension domain controls the interplay
between microtubule and kinesin-6 is unknown. Here, we investigate theinfluence of the N-terminal extension domain on motor activity. To do this, wehave engineered C.elegans kinesin-6 motor domain construct with the mutationof one residue to asparatic acid (T9D) in its N-terminal region predicted to
mimic phosphorylation of the conserved threonin. Using a microtubule sliding
assay in which a quantum dot is attached to the microtubule (Yajima J. et al.
Nat. Struct. Mol. Biol. 2008), we found that microtubules sliding over wildtypekinesin-6 underwent short-pitch rotation around their longitudinal axes, as
revealed by the tracking of the quantum dot in 3D, which has not beenpreviously seen for kinesin-6. In the case of T9D, sliding velocity decreased to
10% of wildtype kinesin-6 and rotations of sliding microtubules could not be
observed. This result suggests that phosphorylation in the N-terminal extension
domain will regulate torsional force as well as axial sliding force in kinesin-6.

motors and processibity of kinesin provides a good reference for the CNTs as anano heater at single molecular level for future applications of various proteinsin vitro and in vivo.
1PS042

重要である

Multiple kinesin molecules coordinate to ensure the longdistance walking: a DNA-kinesin hybrid nanomachine study
Yuya Miyazono1, Masayuki Endo2, Takuya Ueda3, Hiroshi Sugiyama2, Yoshie
Harada2, Hisashi Tadakuma3 (1Grad. Sch. Appl. Phys., Univ. Tokyo, 2iCeMS,
Univ. Kyoto, 3Grad. sch. of Frontier Sci., Univ. Tokyo)
Kinesin drives cellular transport using energy derived from ATP. However, howmultiple kinesin affect the vesicle transport remains elusive. Here we created a
hybrid nanomachine (DNA-tile-kinesin) using DNA-origami-tile as the skeletal
structure and kinesin as the functional module. Single molecule imaging of
DNA-tile-kinesin hybrid, on which 1 to 9 kinesin molecules were attached,
allowed us to evaluate the effects of number of kinesins participating in vesicletransport. Our results show that the number of kinesin involving movement
affects run length but not velocity of the DNA-tile-kinesin. Furthermore,disturbing the coordination of multiple kinesins, by replacing 1 molecule of 9molecules to defect kinesin, revealed that coordination is crucial for efficienttransport. Thus, the intermolecular coordination facilitates high processive
movement of transport complex.
1PS043Kinesin Eg5, a member of the kinesin-5 family, is essential for formation of
bipolar spindle during cell division. Eg5 is overexpressed in tumor cell andinduce significant cell multiplication. Monastrol and Strityl-L-cysteine (STLC),
potent inhibitor specific for kinesin Eg5 ATPase activity shut off mitotic
division and results in apoptosis. Therefore, these inhibitors are attracting
attention as an anti-cancer drug. In this study, we have tried to design andsynthesize Monastrol and STLC analogues composed of photochromic
molecules (azoboenzen and spiropyran derivatives) as kinesin Eg5 inhibitors,
and their photo-reversible inhibitory effects for Eg5 were examined upon ultraviolet (UV) and visible (VIS) light irradiations. STLC analogue, PAM-Cys was
synthesized by coupling reaction of 4-phenylazophenyl maleimide with SHgroup of cysteine. PAM-Cys showed reversible absorption spectral changes
upon UV-VIS light irradiations suggesting the cis-trans isomerization ofazobenzen moiety of PAM-cys. Preliminary experiments revealed that theMicrotubules dependent ATPase activity of EG5 was inhibited by PAM-Cys
reversibly upon UV-VIS light irradiation. We also examined inhibitory effectof spiropyran derivative, 1’, 3’, 3’ -Trimethylspiro [1 (2H) -benzopyran-2,2’-indoline] (TMSBI) for Eg5. TMSBI inhibited ATPase activity of Eg5 upon
UV light irradiation more effectively than VIS light irradiation.

We demonstrated that single carbon nanotubes (CNTs) can be used as a heat
conductor to induce local temperature change for skeletal muscle myosin(Annual meeting, 2011). However, adsorption of myosin on CNTs required very
high concentration of myosin as 30 mg/ml to observe continuous sliding of actin
filaments. To reduce number of motors on CNTs toward single molecular level,
here we used kinesin as a typical processive motor.
Multiwall CNTs (~170 nm diameter, ~10 μm length) were coated with kinesin(~0.1 mg/ml) purified from a bovine brain to observe sliding of fluorescentlylabeled microtubules in the presence of 1 mM ATP. During the sliding, a local
point of the CNT was temporarily heated with a laser irradiation (645 nm, φ ~2
μm, 2.7 mW) to supply external heat.
Microtubules moved on the kinesin-coated CNTs at a speed of ~0.80 μm/s. By
the local heating with laser, the speed increased to ~18. 0 μ m/s, indicating
temperature jump over 10°C as observed for the myosin-coated CNTs.
Our results suggest that the local heating using CNTs can be applied to kinesin

フォトクロミック阻害剤を利用したキネシン Kif18A の光制御

Photo-regulation of kinesin Kif18A using photochromic
inhibitor

カーボンナノチューブを用いたキネシン運動の局所加熱

Local heating of kinesin motors on carbon nanotubes
Yuichi Inoue, Mitsunori Nagata, Akihiko Ishijima (IMRAM, Tohoku Univ.)

フォトクロミック阻害剤を用いた有糸分裂キネシン Eg5 の光制御

Photo-regulation of mitotic kinesin Eg5 using photochromic
inhibitors
Kumiko Ishikawa1, Hideo Seo1, Kanako Touyama2, Shinsaku Matuta1 (1Div.
Bioinfo., Grad. Eng., Soka Univ., 2Dept. Bioinfo., Fac. Eng., Soka Univ.)

1PS044
1PS041

多分子キネシン間の協調性は運搬物を効率的に長距離輸送するのにHideo Seo1, Kumiko Ishikawa1, Kanako Touyama2 (1Div. Bioinfo., Grad.,Eng., Soka Univ., 2Dept. Bioinfo., Fac. Eng., Soka Univ.t)
The shape and function of the mitotic spindle depends on the cooperation actionof various kinesins. Recently, it was demonstrated that the kinesin Kif18A is acentral component for the correct alignment of chromosomes at the spindle
equator. BTB-1 is a small molecule potent inhibitor for Kif18A. BTB-1 inhibits
ATPase activity of Kif18A but not of other mitotic kinesins. BTB-1 also inhibits
the motility of Kif18A in a reversible manner. BTB-1 inhibits Kif18A in an
ATP competitive but microtubule -uncompetitive manner and slows down the
progression of cells through mitosis. Azobenzen is one of the photochromic
molecules that change their shapes and properties drastically upon ultra-violet(UV) and visible (VIS) light irradiation. Previously, we have incorporated
azobenzen derivative into functional region of ATP driven motor protein andsucceeded to control microtubule dependent ATPase activity reversibly by UVVIS light irradiation. Interestingly, BTP-1 shows structural similarity toazobenzen derivatives. In this study, we examined photo-reversible inhibitory
effects of various azobenzen derivatives for ATPase activity of Kif18A. Cis− S81 −

isomer of 4-(phenylazo)-phenol (4-PAP) generated by UV irradiation inhibited
ATPase activity of Kif18A more effectively than trans isomer of 4-PAP. Otherazobenzen derivatives were also examined.
1PS045

RAD54, A CHROMATIN REMODELER, TRANSLOCATES
ALONG DNA BY TRACKING THE DNA HELIX

Ichiro Amitani, Christopher Dombrowski, Ronald Baskin, Stephen
Kowalczykowski (Department of Microbiology, University of California,
Davis)
Rad54 is a member of the Snf2/Swi2 family of chromatin remodeling proteins.
The members of this family belong to the superfamily2 helicase. Nevertheless,they do not show helicase activity. Instead they translocate on DNA. Rad54
plays various roles during repair of DNA double strand breaks with Rad51. In
the initial phase of recombination, Rad54 binds to Rad51-ssDNA complex,
stabilizes the nucleoprotein filament and stimulates the homology search by
Rad51-ssDNA. Interestingly, in the late phase of recombination, Rad54 disrupts
the Rad51 nucleoprotein filament formed on dsDNA, which is inhibitory in thefollowing steps. Alteration of protein-DNA interaction is one of the importantroles of Rad54. In eukaryotes, the access to the target DNA is limited since the
DNA is wrapped around a histone octamer. Therefore the DNA has to be
exposed to allow the DNA repair machinery to gain the access to the DNA. It
has been reported that Rad54 has the ability to slide nucleosomes along DNA.
However, the molecular mechanism is not yet understood. Here, we report that
Rad54 generates 6-8 pN force and translocates along DNA by tracking the DNA
helix. Our observations suggest that Rad54 conforms to the twist diffusion
model when it slides nucleosomes. In this model, when Rad54 encounters anucleosome, Rad54 pulls and twists the nucleosomal DNA. The twist defectgenerated by the action of Rad54 propagates along the histone surface,disrupting the DNA-histone contacts and re-establishing new contacts. As aresult, the nucleosome’s position is changed.
1PS046

DNA を分解中の RecBCD の高速 AFM 観察

Structural dynamics of RecBCD enzyme during DNA
degradation processes studied by high-speed atomic force
microscopy
Yusuke Moriguchi1, Naofumi Handa2, Noriyuki Kodera3, Toshio Ando1,3(1Sch. Math. & Phys., Int. Sci. & Eng., Kanazawa Univ.,, 2Dept. Microbiology,
UC Davis, 3Bio-AFM Frontier Research Center, Inst Sci. & Eng., Kanazawa
Univ.)RecBCD, a heterotrimeric protein complex of the E. coli, is a highly processive
DNA helicase and nuclease that is involved in the seemingly conflicting
functions of recombinational DNA repair and the degradation of DNA. These
biological activities are regulated by a recombination-activating Chi-sequence
coded on DNA, in which RecBCD digests away DNA until the Chi-sequence isrecognized. At the Chi-sequence, RecBCD pauses, reduces the nucleaseactivity, and then switches its polarity to initiate the recombination process.
Although these properties have been extensively studied by biochemical andbiophysical methods, little is known about the detailed mechanism at thenanometer level. In this study, we applied high-speed atomic force microscopy(HS-AFM) to directly observe structural dynamics of RecBCD at work. Last
year, we reported successful direct visualization of DNA degradation processes
by RecBCD, from which we obtained kinetic parameters of the ATP hydrolysis
similar to those reported previously. However, the detail structure of RecBCD
was not seen in the AFM movies due to the positional fluctuations of themolecules. This time, we developed a new experimental setup in which NiNTA-containing lipid bilayers were used as a substrate on which the His-tagged
RecBCD was specifically immobilized. This setup enabled us to distinguish thedomain structures of RecBCD. In the presentation, we will show the obtained
results together with the HS-AFM movies.
1PS047高密度昆虫細胞によるアクチン発現

Expression of recombinant beta-actin in the high-density insect
cell culture
Takashi Ohki1, Shin'ichi Ishiwata1,2 (1Dept. of Phys., Waseda Univ., 2WABIOS,
Waseda Univ.)
Actin is the most abundant protein in the cytoplasm of eukaryotic cells, whichplays important roles in such processes as muscle contraction, cell division,
morphogenesis, organelle positioning, and vesicle trafficking. In our previous
study, we reported the expression method of recombinant beta-actin at high celldensity in a shaking Erlenmeyer flask. In the insect cell culture at stationary
phase (10-12 x 106 cells per ml), additional nutrients or medium exchange attime of infection restored the expression level of beta-actin per cell equivalent tothat in the cell culture at early exponential phase (1 x 106 cells per ml).
Therefore, the volumetric yield of recombinant beta-actin, which was
determined by the maximal cell density and the yield of recombinant protein percell, was at least ten-fold higher than compared to the manufacturer’s protocol.
The present study further developed the expression method of recombinant betaactin at the density of 30 x 106 cells per ml, which is ~30-fold higher than thecell density at stationary phase. This method requires medium exchange plus
additional nutrients and air ventilation after infection. The volumetric yield ofrecombinant beta-actin reached 350 mg per liter of insect cell culture. Here we
report in detail the effective method for the production of recombinant betaactin and the biological and the biochemical properties of recombinant betaactin.
1PS048

ATP、ADP と無機リン酸存在下での運動するアクチン繊維の変形

Deformations of moving actin filaments on myosin moleculesin the presence of ATP, ADP, and inorganic phosphate
Satoru Kikuchi, Kuniyuki Hatori (Grad. Sch. Sci. Eng., Yamagata Univ)
Deformations of actin filaments were evaluated by an analysis of fluorescence
images of short filaments, in which displacement (DP), peak intensity (PI), andspreading (SD) of fluorescence of dyes bound to actin filaments were measured
and the correlations between them were calculated. Using this approach withlabeling of rhodamine-phalloidin (Rh-ph), we have previously shown that actin
filaments perform the sliding movement with filamental deformations along
heavy meromyosin molecules and an increase of velocity occurs after
protractions of actin filaments (Kikuchi, S. and Hatori, K., BSJ annual meeting,2011). In order to examine this further, we tested whether changes offluorescence are distinct in single actin filaments that were directly labeled with
IC3-PE-maleimide (Cy3 analog supplied by Dojido). Fluorescence intensity ofactin filaments in labeling of IC3 was greater than that in Rh-ph. However, nodifference between IC3 and Rh-ph was observed in correlation values between
PI, SD, and DS. The presence of 4 mM ADP induced a decrease of DP, but not
PI and SD. On the other hand, DP and SD for IC3-labeled actin filaments wereincreased by 50% when 10 mM inorganic phosphate was added to the solution
with 1 mM ATP. This result shows that the presence of inorganic phosphatefacilitates the protraction of the filaments during sliding movement and theincrease of their velocity.
1PS049
1YS0900

生体分子モーターのストレインセンサー機構の発見とエネルギー変換への寄与の定量化

Discovery of strain-sensor mechanism in motor protein and thequantification for the energy conversion
Mitsuhiro Iwaki1,2,3, Keisuke Fujita2, Atsuko Iwane2, Lorenzo Marcucci2, Toshio
Yanagida1,2 (1QBiC, RIKEN, 2Grad. Sch. Biosci., Osaka Univ., 3Harvard Med.
Sch.)
It is believed that biological linear molecular motors (myosin, kinesin, dynein,etc.) generate force by dominantly structural changes in their composition.
Myosin’s swinging lever-arm model is a typical hypothesis, where the tilting of
the lever-arm domain is strongly coupled to the force generation. On the other
hand, random diffusion and forward catch of myosin head to actin is also
directly visualized during the directional motion. Myosin-V and -VI areappropriate target in that both (1) lever-arm swing and (2) Brownian search-andforward catch can be directly visualized during the directional motion.
Remaining mystery is which process is critical for the force generation? And
how is the mechanism for rectifying random Brownian motion? Here, we
answered the questions by improving single molecule measurement techniques(Iwaki et al., Nat. Chem. Biol. , 2009; Fujita and Iwaki et al., Nat. Commun. ,2012).
We report the strain-sensor mechanism to realize the rectification of random
Brownian motion and the quantification for the force generation. Also, we’ll
discuss the physiological meaning of the dual function (lever-arm swing and theBrownian seach-and-catch) in myosin transporters.
1PS050

統計処理による素過程推定手法の開発（分子マシンを例として）

Construction of statistical method for estimating elementary
processes and their characteristics
Hiroto Tanaka, Hiroaki Kojima (NICT)− S82 −

Bio molecular machines generally express macroscopic outputs (MOs) via
cyclical and/or multiple microscopic elementary events (MEEs). For example,
some bio molecular motors develop sliding or rotating movement (as MO) via
cyclical unitary stepping events (as MEE). It is important to reveal such MEEs
for understanding working mechanism of bio machines. However, such MEEs
are often difficult to observe directly, while expressed MOs are often easy toobserve. Therefore, a method that enables us to model MEEs and to estimate
their characteristics (size of unitary step, stepping ratio, rate constants, etc. formolecular motors) from “easily observable” MOs, would be a powerful tool toovercome difficulties of direct observation and help understanding working
mechanism of bio nano machines. We propose widely suitable method andprocedure to model elementary events, and to estimate their characteristic in thisstudy. We theoretically formulated statistical relation between MEEs and MOs
for general motion and propose a estimation method (procedure) of
characteristics of MEEs with non-linear fitting of “observable” MOs .
As a result of our tests for application, our method showed its potential todetermine model of MEEs and their characteristics successfully. Our method
and procedure proposed in this study can be a powerful tool to tackle more detailcharacteristics of bio molecular machines, and to improve apparent spatial andtime resolutions of measurement system by using statistical analysis.
1PS051

Photo-control of microtubule flexibility using photochromic
nucleotide analogue entrapped within GTP binding site of
tubulinNozomi Furutani-Umezu1, Takeshi Itaba2, Shinsaku Maruta1,2 (1Dep.
Bioinfo., Fac. Eng., Soka Univ., 2Div. Bioinfo., Grad. Sch. Eng., Soka Univ.)
Azobenzene is a photochromic molecule that undergoes rapid and reversible
isomerization between the cis- and trans-form in response to ultraviolet (UV)and visible (VIS) light irradiation, respectively. Previously, we haveincorporated azobenzen derivative into functional region of ATP driven motor
protein and succeeded to control microtubule dependent ATPase activity
reversibly by photo-irradiation. The results suggested that the photo-control
system using photochromic molecules might be applicable to other functional
biomolecules. It is known that tubulin utilize GTP to polymerize and the GTP isentrapped within the site of tubulin. Interestingly, it was demonstrated thestructure of the microtubules composed of GTP analogue GMP-PCP is more
flexible than that composed regular GTP. In the present study, we have
employed photochromic nucleotide analogue, Phenylazobenzoyl-iminoethylTri-Phosphate (PABITP) in order to photo-control flexibility of microtubules.
The microtubules composed of PABITP showed much more flexible shapechange during gliding on in-vitro motility assay than that composed of regular
GTP. The effect of photoisomerization of photochromic PABITP entrapped
within the site of microtubules induced by UV and VIS light irradiation for
flexibility of microtubules was examined.
1PS052ヌクレオチドを利用する生体分子機械へのフォトクロミックヌクレ
オチドアナログの応用

Possible application of photochromic nucleotide analogue tothe nucleotide required bio-molecular machines
Takeshi Itaba, Shinsaku Maruta (Div. of Bioinfo., Grad. Sch. of Eng., Soka Univ.)
Previously, we have synthesized photochromic nucleotide analogues
Phenylazobenzoyl-iminoethyl-Tri-Phosphate (PABITP) that change its structure
reversibly by light irradiation in order to photo-regulate the function of myosin
II. PABITP was hydrolyzed by skeletal muscle myosin and induced dissociationof acto-myosin. X-ray small angle solution scattering analysis of myosininteracting with PABITP showed that the radius of gyration values of S1·
PABITP(trans) and S1·PABITP(cis) are identical to those of S1·ATP and S1·
ADP respectively. The results suggested that the photo-isomerization of the
PABITP induce conformational change of myosin head, which may reflect
energy transduction. In this study, we examined the possible application of
PABITP to other nucleotide required bio-molecular machines, smooth muscle
myosin, myosin V, conventional kinesin, Kif18A, Eg5, tubulin and small Gprotein Ras. PABITP was hydrolyzed by these kinesin and myosin ATPase.
PABITP induced microtubule gliding on conventional kinesin and polymerization of tubulin to microtubules. We also examined the effect of reversible photoisomerization of PABITP for the nucleotide required bio-molecular machines.
1PT001タンパク質ドッキング過程における溶媒和自由エネルギー変化

Solvation free energy in protein dochking process

Taku Mizukami1, Hiroaki Siato2, Hidemi Nagao2 (1Materials Science,
JAIST, 2Natural Science and Technology, Kanazawa Univ.)
Hydration water is believed to play an important role in the process of proteinfunctional expression, for example in protein structural change, or in folding, orin the ligand binding process. The series of intense studies on the water
hydration has been done through last several decades. It showed thecharacteristic chemical/physical parameters, that is, low diffusion constant,vectorial or anisotropic motion, and protein dynamics controlled by water
motion. These results suggest a strong correlation mechanisms between waterand protein dynamics.
Recently, we have established a methodology to estimate the excess chemical
potential or the solvation free energy in the use of trajectory data from MD
simulation. In the case of a single globular protein, the protein structural changes
caused ~10 3 kcal/mol solvation free energy change which strongly correlatedwith the density of the first hydration and the second hydration shell water.
In this study, under the motivation to investigate the solvation effect in thecourse of protein docking process, we calculate the solvation free energy ofBarnase-barstar complex. From MD trajectory performed on each configuration,a solvation free energy was estimated by means of the classical MD simulationand the energy representation method. We investigated the correlation between
the solvation free energy depending on the distance and the configuration
among two protein, the hydration water density. These results suggest that thehydration water mediates the protein docking process.
1PT002

ミオシン周囲の水の密度揺らぎ

Density fluctuation of water around myosin
Takato Sato, Jun Ohnuki, Koji Umezawa, Mitsunori Takano (Grad. Scl. ofAdv. Sci. & Eng., Waseda Univ.)
Association and dissociation between myosin and actin are essential for force
generation of the actomyosin molecular motor. However, the origin of theintermolecular interaction involved in these processes is complicated due to thecoexistence of the electrostatic and hydrophobic interactions. In the previous
report, by conducting an extensive explicit-water all-atom molecular dynamics(MD) simulation, we showed that the ATP-induced dissociation of myosin fromthe actin filament is likely to be caused by the allosteric response of myosin in
the actin-binding loops (such as loop 2) and in the residues on the actin-binding
surface, which are electrostatically-controlled by the ATP charge. We also
found that the water density around the actin-binding surface slightly increases
upon ATP-binding (the orientation of water is also affected by the ATPbinding). In this study, we further investigated the hydration state of myosin
from the viewpoint of water density fluctuation around the myosin surface,because the water fluctuation is closely related to the hydrophobic interaction(Acharya, et al. 2010). We found that some regions with large water-density
fluctuation coincide with the actin-binding hydrophobic regions, and mobile
regions including surface loops generally exhibit large water-density fluctuations. By placing hydrophobic small probes in the system, we directly examine
whether those regions with large water-density fluctuation actually causeintermolecular attraction.
1PT003

中性子散乱による F-アクチン周辺水分子のダイナミクスの研究

Dynamics of Water around F-actin in Solution Studied by
Neutron Scattering
Satoru Fujiwara1, Marie Plazanet2, Toshiro Oda3 (1QuBS, JAEA, 2Univ.
Fourier, 3RIKEN SPring8 Center)
Proteins are constantly fluctuating under the influence of surrounding aqueous
environment. These thermal fluctuations, or dynamics, of the protein are
indispensable for the structural changes that make the protein function. Ultimate
understanding of the protein functions thus requires understanding of the
dynamics of the proteins including the hydration water. We have been
investigating the dynamics of F-actin. How the dynamics of water around Factin affects the internal dynamics of F-actin is particularly interesting because
the water around F-actin shows unusual behavior such that it has higher mobility
than bulk water (the hyper-mobile water). Here we employed quasi-elastic
neutron scattering (QENS) to characterize the dynamics of the hydration water
as well as the internal dynamics of F-actin. The QENS spectra of theconcentrated solutions of F-actin in D2 O and H2 O were measured with the
instrument IN5 in the Institut Laue-Langevin, Grenoble, France. The spectra
arising from the dynamics of the hydration water and those from the internal
dynamics of F-actin were extracted from the obtained spectra. The dynamics ofthe hydration water around the protein in the "solution" samples is characterized− S83 −

for the first time here, and it is shown that translational diffusion constant ofwater around F-actin has a similar value to that of the bulk water (2.8 × 10-5
cm2/s) but longer residence time (3.3 ps compared to 0.6 ps of the bulk water).
The results of the detailed analysis will be presented.
1PT004

Mean Exit Time Analysis about Water Molecules around
Membrane Surfaces
Eiji Yamamoto1, Takuma Akimoto1, Yoshinori Hirano2, Masato Yasui3, Kenji
Yasuoka4 (1Department of Mechanical Engineering, Keio University, 2The
Institute of Physical and Chemical Research (RIKEN), 3Department ofPharmacology, School of Medicine, Keio University, 4Department of
Mechanical Engineering, Keio University)
The structures and dynamics of water molecules in the hydration layer ofmembranes are known to be different from those in bulk. In order to investigate
the diffusivity around the surface of the membrane, we performed molecular
dynamics simulations of a 1-palmitoyl-2-oleoyl-phosphatidylethanolamine(POPE) bilayer and water molecules at temperatures ranging from 250-350 K.
The diffusion coefficient cannot properly characterize the diffusivity of watermolecules on membrane surfaces because water molecules do not remain
attached to the surface for all time. To characterize translational motion of thewater molecules near membrane surfaces, we introduce the concept of mean exit
time and show that translational motions of water molecules around the surfaceof the membrane are slower than those in bulk. Furthermore, we find that the
distribution of trapping times of water molecules on membrane surfaces obeys apower law distribution, and the power-law exponent depends on temperature.
These results imply that power-law trappings enhance the viscosity of watermolecules around the surface of membranes.
1PT005アルギニンによるカフェ酸の可溶化とそのダイナミクス

Solubilization and Its Dynamics of Caffeic Acid Induced by
Arginine
Daisuke Shinozaki1, Atsushi Hirano2, Tomoshi Kameda3, Tsutomu Arakawa4,
Kentaro Shiraki1 (1Facul. of Pure and Appl. Sci., Univ. of Tsukuba, 2NRI,AIST, 3CBRC, AIST, 4Alliance Protein Lab.)
Poor aqueous solubility of low molecular weight drug substances hampers theirbroader applications. We have shown that arginine increases the solubility ofsmall organic compounds as well as proteins. Here we examined the molecularmechanism of arginine on the solubility of caffeic acid that also has a low
aqueous solubility. The solubility of caffeic acid increased with increasing pH in
the aqueous solutions. At a given pH, arginine further enhanced its solubility.
Ratio of the solubility of caffeic acid in arginine solution to that in water was
almost constant over the wide range of pH. The interaction between two caffeic
acid molecules in the presence or absence of arginine was investigated bymolecular dynamic simulation. Our simulation showed arginine decreases
binding free energy between the caffeic acid molecules, which suggested theincreased solubility of caffeic acid by arginine was attributable to stabilizationof the dissociated state.

Calculation of dynamics and charges of water around small
solute molecules: MD and QM calculations
Takuya Takahashi (College of Life Sciences, Ritsumeikan Univ.)
The physico-chemical properties during enzymatic reaction such as ATPhydrolysis are affected by solvent conditions, where the solvent ions and wateraround biomolecules seem to play important roles. To elucidate the properties,classical MD simulations of water molecules around several types of solutes(poly-phosphates, AMP, and monovalent ions) were done with several watermodels. The hydrating water molecules around mono-phosphate and diphosphate molecules with a moderate negative charge showed increased
mobility of water compared with bulk water and the results were consistent withthe experimental results of the dielectric relaxation spectroscopy although no
HMW was observed around tri-phosphate molecules where both the SPCE and
TIP5P models were tried. As well as the classical MD, the QM calculations ofwater around phosphate molecules and other polar and non-polar small
molecules were done with Gaussian software. In case of the tri-phosphate
molecules (H5P3O10 ~ P3O10) with various protonated states (i. e., the net
charge of the molecule varies from neutral to -5 e), the calculated absolutecharge of water oxygen atom was smaller as the solute charge became negative
and the tendency was not changed in several calculation conditions such as
different system size, basis function and chemical model. The details of other
QM results will be shown in the meeting.
1PT008

力場パラメタが溶媒和ダイナミクスに及ぼす影響の探索

Investigation of force field parameter effect on solvation
dynamics
Yoshito Kondo, Takuya Takahashi (College of Life Sciences, Ritsumeikan
Univ)
Ions and water are important players in most of the biological phenomena. The
anomalous behavior of water around ions would indirectly affect dynamics ofbiomacromolecules. In addition, hyper-mobile water (HMW) which has higher
dielectric relaxation frequency than bulk water is observed around most of thesolutes called water structure breakers. NMR experiments also show theexistence of water molecules whose translational self-diffusion coefficient islarger than that of pure water around the structure breaker solutes. In ourprevious studies, molecular dynamics (MD) simulations of water molecules
were done around monatomic ion solutes by using conventional water models(TIP3P, TIP4P, TIP5P, SCP etc.). Then, the self-diffusion coefficients androtational relaxation times in all simulations were calculated to examine themotility change of water molecules due to the solute molecule. As the HMW
was not reproduced with the conventional water models, we developed themodified TIP5P water model which force field parameters were optimized withquantum mechanical calculations. After that, we could partly reproduce the
HMW. In this study, we tried a variety of parameters of force field to find themost suitable one that reproduces the dynamics of water more quantitatively.
The mobility change of water molecules in ionic solutions by the conditions of
the appropriate force field parameters from the analysis results will be shown.
1PT009

蛋白質水和の物理的特性．IV. 水和サイト動力学の定量解析

1PT006

Single molecule analysis for the energy conversion ofactomyosin in the presence of osmolyte
Keisuke Fujita1, Koji Itoh2, Atsuko Iwane1, Toshio Yanagida1,3, Mitsuhiro
Iwaki1,3 (1Grad. Sch. Front. Biosci., Osaka Univ., 2Fac. Sci., Chiba
Univ., 3QBiC, RIKEN)

Physical Properties of Protein Hydaration. IV. Quantitative
Analysis of Hydration-Site Dynamics
Kunitsugu Soda1, Yudai Shimbo1, Yasutaka Seki2, Makoto Taiji1 (1Lab.
Comp. Molec. Des., QBiC, RIKEN, 2Lab. Struc. Biol., Sch. Pharmacy, Iwate
Med. Univ.)

Actomyosin is a biological molecular machine of nanoscopic size that converts
the chemical free energy of ATP into mechanical work. Recent single molecule
measurements enabled us to detect the mechanical motion at a phenomenological level. However, the energy transduction at the substantial level is still
unsolved. It is expected that the majority of free energy of ATP to be used isoriginated from hydration and dehydration of surrounding water molecules.
Here, we modulated the hydration of actomyosin during an energy transduction
using osmolyte, Sucrose. From a viewpoint of osmotic pressure, the pressure at
0.5- 1 M sucrose is comparable with that in cell. In a cell, various biologicalmolecules exist and is crowding. This is far from conventional in vitro
environment. Our experiment would teach us to predict the energy transduction
in a cell.

[Background and Objective] In a series of studies on protein hydration, we
have newly defined the hydration site (HS) of proteins to report on static anddynamic properties of HSs and detailed molecular michanism of the dynamics
of internal hydration water (HW). These studies showed that HSs of a protein
are classified into Single HSs (SHS) and Clustered HSs (CHS) composed of one
and more than one polar groups (PGs) respectively. They confirmed that, in anyof SHSs, external CHSs and internal CHSs, HWs are very dynamic entity. Quite
different from that given by X-ray crystallography, the real view of HWs is far
from static. The aim of this study is to characterize quantitatively dynamics ofthe HW of lysozyme (Lzm).
[Method and Results] Performing MD simulation on an aqueous solution ofLzm, we obtained data on the fluctuation of the hydration state of Lzm. Analysisof the data led to the conclusion as follows: (1) Fluctuations of the hydration
state of all the SHSs relax very rapidly within a few tens of ps. (2) In CHSs,
HWs H-bonding to their respective PGs fluctuate in mutually correlatedmanner. Temporal fluctuations of HWs in the largest internal CHS, 'Hg', can be

1PT007低分子周囲の水のダイナミクスおよび電荷計算：MD および QM
計算− S84 −

approximately divided into 11 independent relaxational modes, about half or
five of which with larger amplitudes contribute to 76% of the whole fluctuation.
(3) Each of these major modes originates in correlated fluctuations of thehydration states of several specific PGs. Their relaxation times range widelyfrom 10 ps to several tens of ns.
1PT010誘電緩和分光測定によるオリゴリン酸塩の水和解析

Hydration study of oligophosphates by dielectric relaxation
spectroscopy
Kazuki Ishimori, George Mogami, Nobuyuki Morimoto, Makoto Suzuki(Grad. Sch. Eng., Univ. Tohoku)
Adenosine triphosphate (ATP) is an energy mediator in biological systems.
Recently, the importance of the hydration effects on ATP hydrolysis free energy
is being realized widely. In this study, we performed high-resolution dielectricrelaxation spectroscopy (DRS) for oligophosphates aqueous solution(orthophosphate, pyrophosphate and triphosphate; ≤ 20mM) as a model ofadenosine oligophosphates. The present DRS analysis can divide dielectric
properties of the solution into multiple relaxation components. Experiments
were performed by microwave network analyzer on 0. 2-26 GHz frequency
range with the sample solution controlled at 20.0±0.01 °C.
Firstly, we found two types of water that have different relaxation frequency (5GHz, 20 GHz) from bulk water (17 GHz) around solute ions. The 5-GHz
component was attributed to the constrained water, and the 20-GHz component
to the hyper-mobile water; HMW.
Secondly, with increasing valence of solute ions, the dispersion intensity of
HMW increased significantly. It suggested that the hydration state ofoligophosphates was strongly influenced by the valence rather than thepolymerized structure of phosphate unit.
1PT011

ATP，ADP，Pi の水溶液から脂肪酸アミン / オクタノール溶媒への移行：その熱力学特性とメカニズム

Thermodynamics and mechanism of transfer of ATP, ADP and
Pi from aqueous solution to fatty acid amine/octanol solvent
Hideyuki Komatsu (Dept. Bioscience & Bioinformatics, Kyushu Inst. Tech.)
ATP hydrolysis is an important exergonic reaction driving the thermodynamically unfavorable reactions in living systems. Although hydration is considered
to contribute significantly to energy profiles of chemical reactions in aqueouscondition, the extent of its contribution to ATP hydrolysis remains to beexperimentally assessed. In order to evaluate the effect of hydration on thethermodynamics of ATP hydrolysis, ATP hydrolysis in non aqueous condition(i. e., organic solvent) has been studied. For this purpose, fatty acid aminecontaining octanol was used as a model solvent, in which ATP, ADP and Pi arehighly dissolved. Approximate hydration energies were estimated from thepartition coefficients as Gibbs energy changes (ΔG) for transfer of ATP, ADP
and Pi from aqueous solution to the fatty acid amine-containing octanol, andcompared to ΔG for hydrolysis in the solvent. ΔG for the transfer was smaller
than that for hydrolysis in the solvent. Thus, the hydration energy may modestly
modulate the Gibbs energy of ATP hydrolysis in aqueous solution.
This energy profile is going to be refined by consideration of the enthalpy
changes for the transfers, which can be estimated from van’ t Hoff plots oftemperature-dependencies of the partition coefficients. In addition, in order toelucidate how ATP, ADP and Pi are stabilized in the solvent, ionizations ofATP, ADP and Pi and reversed micelle formation will be investigated by pH
titration and dynamic light scattering, respectively.
1PT101

リガンド結合状態及び非結合状態のケナガマンモスヘモグロビンの
結晶構造

The crystal structures of hemoglobin from the woolly
mammoth in liganded and unliganded states
Hiroki Noguchi1, Satoru Unzai1, Kevin L. Campbell2, Chien Ho3, Sam-Yong
Park1, Jeremy R. H. Tame1 (1Protein Design Laboratory, Yokohama City
University, Japan, 2Department of Biological Science, University of Manitoba,
Canada, 3Department of Biological Sciences, Carnegie Mellon University,
USA)
We have recreated the hemoglobin (Hb) of the extinct woolly mammoth using
artificial genes expressed in Escherichia coli. The globin sequences werepreviously determined using DNA recovered from frozen cadavers. Althoughhighly similar to the Hb of existing elephants, the woolly mammoth protein

shows rather different responses to chloride ions and temperature. In particular,
the heat of oxygenation is found to be much lower in the mammoth than in theelephant protein, which appears to be an adaptation to the harsh Siberian winter.
Many Arctic animals such as reindeer show a similarly low temperaturedependence of oxygen affinity in order to ensure oxygen transport to the limbs,
which may experience much lower temperatures than the body core, even inlarge, active and fur-covered mammals. In order to discover the structural basis
for the altered heterotropic effect we have determined the crystal structures ofthe Hb in the deoxy, carbonmonoxy and aquo-met forms. These models, the firstprotein structures from any extinct species, show many features reminiscent of
human Hb, but underline how delicate control of oxygen affinity relies on much
more than overall quaternary structure changes.
1PT102HIV-1 プロテアーゼによるダルナビル耐性の分子機構の解明

Structural change at the flap region of HIV-1 proteaseassociated with darunavir resistance
Hirotaka Ode1, Koji Suzuki1,2, Masayuki Fujino3, Masami Maejima1, Yuki
Kimura1,2, Takashi Masaoka1, Junko Hattori1, Yoshiyuki Yokomaku1, Atsuo
Suzuki2, Nobuhisa Watanabe2, Yasumasa Iwatani1,4, Wataru Sugiura1,4 (1Clin.
Res. Center, Nagoya Med. Center, 2Grad. Sch. Eng., Nagoya Univ., 3AIDS
Res. Center, NIID, 4Grad. Sch. Med., Nagoya Univ.)
Darunavir (DRV) is the most potent protease (PR) inhibitor (PI) currently
available for anti-HIV therapy. It has a higher genetic barrier to resistancedevelopment than the other PIs. However, it has been reported that DRV isvulnerable to the resistance acquisition, especially for the patients who havepreviously failed the PI-containing treatments. Here, in order to understand thebasis of DRV-resistance, we analyzed structures of the DRV-resistant PR.
We first induced DRV-resistant virus in the culture system with the DRV
concentration increased over 154 days. On day 154, we isolated the DRVresistant virus with 55.5 times higher IC50 against DRV than the wild-type(WT) clone. The analysis of the viral sequences revealed that the escape mutant
with five mutations in PR was dominantly induced during the viral replications.
Next, we determined a high-resolution (1.8 Å) crystal structure of the DRVresistant PR. It turns out that the DRV-resistant PR has an open configuration at
its flap region, which has not been observed in WT PR. Furthermore, themotional properties and DRV-binding modes of the PR were analyzed bymolecular dynamics (MD) simulations. MD simulations showed that the flap
region of DRV-resistant PR is more flexible than WT, and that the DRVinteraction modes are distinct. The results suggest that structural change at theflap region is involved in the resistance development of HIV-1 PR. Our findings
will provide useful information to develop effective PIs even to the DRVresistant HIV-1.
1PT103

P2X 受容体の ATP 認識機構およびチャネル活性化機構

1YS1030

Mechanism of ATP binding and channel activation in P2X
receptors

Motoyuki Hattori1, Eric Gouaux1,2 (1Vollum Institute, Oregon Health &Science University, 2Howard Hughes Medical Institute)
ATP is known as the vital energy source involved in energy metabolism,
biosynthetic reactions and active transport. It is also an essential extracellularsignaling molecule that activates two distinct families of ATP receptors:
ionotropic P2X and G-protein-coupled P2Y receptors.
P2X receptors are trimeric ATP-activated ion channels permeable to Na+ , K+
and Ca2+ , expressed throughout the human body including the nervous,
cardiovascular and immune systems and are implicated in various physiologicalprocesses such as synaptic transmission, smooth muscle contraction, taste andpain sensing and inflammation.
A recent crystal structure of zebrafish P2X4 receptor in an apo, closed state,
revealed the chalice-shaped trimeric architecture of P2X receptors. However,
due to the lack of a crystal structure in complex with ATP, the ATP binding site
and the mechanism of channel activation remained unclear.
Here we report the crystal structures of zebrafish P2X4 receptor in the presence
and absence of ATP at 2.8 and 2.9 Å resolution, respectively. The agonistbound structure reveals a novel ATP binding motif and an open channel pore
conformation. A structural comparison of two states suggests that ATP binding
induces cleft closure in an intersubunit agonist binding site, which expands theregion of the receptor proximal to the transmembrane domain, causing an irislike opening of the channel pore. This work provides the first structural insights
into how ATP binding activates P2X receptors based on atomic resolution
structures.

− S85 −

1PT104

20S プロテアソームの機能阻害と X 線結晶構造解析
Crystal structure of the yeast 20S proteasome in complex withnew inhibitor

Takuma Maekawa1, Kazuya Nishio2, Bahrudin Udin3, Ichiro Hisatome3,
Yasushi Saeki4, Keiji Tanaka4, Hiroshi Yamaguchi1, Yukio Morimoto2 (1Grad.
Sch. Sci&Tec, Univ. Kangaku, 2Grad. Sch. Physi, Univ. Kyoto, 3Grad. Sch.
Med., Univ. Tottori, 4Tokyo Inst., Rinshoken)
Proteasome is a multi-catalytic compound and has function for degrades unfold
or nonfunctional proteins. The function of proteasome involves ubiqutin
proteasome system that plays important role in living organism. We found a
new proteasome inhibitor that has no side effect in normal cell, but effect for thetumor cell degradation. To reveal the precise structure of the inhibitor-bindingsite and to obtain the fundamental date for development of more effective
inhibitor for anticancer reagent, we have determined the crystal structure of theyeast 20S proteasome in complex with the inhibitor.
Yeast 20S proteasome was prepared by over expression in yeast (Saccharomyces cerevisiae), and was purified by each chromatographies. Isolated 20S
proteasome was co-crystallized with small molecule inhibitor by using sitting
drop vapor diffusion method. The solution of 0.1 M MES-NaOH (pH 6.5), 15%
MPD, 50 mM magnesium acetate was used as reservoir solution, and protein
solution of 10 mg/ml proteasome was used as droplet. Diffraction images wererecorded at BL44XU, SPring-8, Japan, and analysed by using HKL2000 and
Refmac.
The inhibitor was bond to the S1 pocket at subunit β 5. Three-dimensional
orientation of amino acid residues around S1 pocket are available for occupationof one molecule of the inhibitor; TYR(135) and GLY(128) interact with -OH
and -C=O groups by hydrogen bond. It is considered that the inhibitor rather
than a bortezomib reagent has slightly weak binding-abilities in the S1 pocket
and hence induces no side effect for a human being.
1PT105Ⅳ型分泌系構成蛋白質、DotI の構造

Structure of DotI, a core component of the Type IVB secretion
system
Takuya Kuroda1, Yumiko Uchida1, Tomoko Kubori2, Hiroki Nagai2, Katsumi
Imada1 (1Grad. Sch. Sci. Osaka Univ., 2RIMD Osaka Univ.)Type IV secretion systems (T4SSs) are bacterial molecular machines that
directly translocate virulence effecter proteins or DNA into the target cells.
Many pathogenic Gram-negative bacteria utilize the T4SSs for infection of theirhosts. The T4SSs are evolutionarily related to bacterial conjugation systems,
and are divided into two subgroups, type IVA (T4ASS) and type IVB (T4BSS).
The Dot/Icm system of Legionella pneumophila is classified as a T4BSS andclosely related to conjugation systems of IncI plasmids. T4BSS is a multiprotein complex that form a channel spanning the inner and the outer membraneof bacteria. The core complex consists of, at least, six distinct proteins. DotI is
one of the core components. DotI is an inner membrane protein composed of asmall N-terminal region, a single transmembrane helix and a C-terminal
periplasmic domain. Here, we report the crystal structure of periplasmic
fragments of DotI and its homologue TraM in IncI plsmid conjugation system.
DotI and TraM share a same folding topology and their structures are similar tothat of VirB8, which is an inner membrane protein located most inside of thecore complex of VirB/D4 T4ASS of Agrobacterium tumefaciens, suggestingthat DotI and TraM are present at the inside of the core complex of T4BSS. DotI
forms an octamer ring with an internal diameter of 30Å and an external diameterof 120Å in the crystal. This ring structure is detected under certain conditions insolution, implying that the ring is also formed in the core complex in vivo.
1PT106枯草菌細胞壁溶解酵素阻害タンパク質 IseA の立体構造解析：特徴的な阻害ループを持つ弓鋸型新規構造の解明

Solution structure of an autolysin inhibitor IseA from Bacillus
subtilis: a novel hacksaw-like fold with a characteristic
inhibitory loop
Sadaharu Fukui1, Ryoichi Arai1,2, Naoya Kobayashi1, Hua Li3, Satoru
Watanabe3, Junichi Sekiguchi1 (1Appl. Biol., Fac. Tex. Sci. Tech., Shinshu
Univ., 2YREC, Shinshu Univ., 3SSBC, RIKEN)In Bacillus subtilis, LytE, LytF, CwlS and CwlO are vegetative peptidoglycanhydrolases, DL-endopeptidases, which cleave the D-glutamic acid-mesodiaminopimelic acid linkage, and they play important roles in cell separationand growth. IseA is a proteinaceous inhibitor against the DL-endopeptidases.

Here we report the solution structure of IseA determined by NMR spectroscopy.
(All NMR data were measured and analyzed at RIKEN NMR Facility.) The
structure of IseA comprises a single domain consisting of three α-helices, one
310 -helix and eight β -strands, which is a novel “hacksaw-like” fold. A
noteworthy region in the IseA structure is a flexible loop between β4 and 310helix, which looks like a blade of a hacksaw. Electrostatic potential distributionof IseA shows that most surface charges positively but the surface around theloop region charges negatively. In contrast, an active-site cleft of DLendopeptidase, LytF, is expected to charge positively. NMR chemical shift
perturbation of IseA, interacting with LytF, indicates that the potential
interaction sites are located around the loop region, the end region of α-helix,
and C-terminal region. These results suggest that IseA inhibits DLendopeptidase through direct interaction of the loop and around the region withthe active-site cleft of the enzyme.
1PT1074.1 蛋白質 FERM domian と calmodulin の結合解析

Unique structural changes in calcium-bound calmodulin uponinteraction with a peptide of protein 4.1R
Wataru Nunomura1,2, Noriyoshi Isozumi3, Shigeyoshi Nakamura4, Shinya
Ohki3, Shun-Ichi Kidokoro4, Yuichi Takakuwa5 (1Dept. of Life Sci., Grad. Sch.
Eng. & Resource Sci., Akita Univ., 2Cent. for Geo-Environment. Sci., Grad.
Sch. Eng. & Resource Sci., Akita Univ., 3Cent. for Nano Mat. & Tech.,
JAIST, 4Dept. of Bioeng., Nagoka Univ. of Tech., 5Dept. of Biochem., TWMU)
Calmodulin (CaM) binds to the FERM domain of 80kDa erythrocyte protein
4.1R (R30) independently of Ca2+ but, paradoxically, regulates R30 binding
to transmembrane proteins in a Ca2+ -dependent manner. We havepreviously mapped a Ca2+ -independent CaM binding site, pep11 (A264
KKLWKVCVEHHTFFRL), in R30 and demonstrated that, when saturated by
Ca2+, CaM (Ca2+/CaM) interacts simultaneously with pep11 and with S185, thebinding affinity of Ca2+/CaM for S185 increasing dramatically in the presence of
pep11. Based on these findings, we hypothesized that pep11 induced key
conformational changes in the Ca2+ /CaM. Using nuclear magnetic resonancespectroscopy (NMR) and differential scanning calorimetry (DSC) analyses, weestablished that apo-CaM was more stable when bound to pep 11 and that pep11
bound to apo-CaM through amino acid residues V108, M145 and A147 in the Clobe of CaM. Surprisingly, pep11 interacted with both N- and C-lobes of Ca2+
/CaM. FTIR analysis revealed that these changes in binding features uponinteraction of Ca2+/CaM with pep11 were accompanied by a dramatic increasein CaM β-sheet structures, but not α-helix structures, and by conformationalchanges of the carboxyl side chain of E141 in CaM, a key residue in CaM
interaction with Ca2+. We propose that these unique properties may account inpart for the previously described Ca2+ /CaM-dependent regulation of R30
binding to membrane proteins.
1PT108真空紫外円二色性分光により予測されたβ2-Microglobulin フラグ
メントの Steric Zipper 構造

Steric-Zipper Structures of β2-Microglobulin Fragment inAmyloid Fibrils Revealed by Vacuum-Ultraviolet Circular
Dichroism Spectroscopy
Koichi Matsuo1,2, Hirotsugu Hiramatsu3, Kunihiko Gekko4, Robert W. Woody2(1Synch. Radi., Hiroshima Univ., 2Biochem. Mol. Biol., Colorado State
Univ., 3Grad. Sch. Pharm. Sci., Tohoku Univ., 4Inst. Sust. Sci. Devel., Hiroshima
Univ.)The 21-29 region of β 2 -microglobulin (β2 -m21-29) was suggested as the corefragment of β2-m amyloid fibrils, but its detailed structure in solution remains
unclear. Vacuum-ultraviolet (VUV) circular dichroism (CD) spectroscopy usingsynchrotron radiation has a great advantage for the structure analysis of amyloid
fibrils and recently the experimental protein CD was successfully calculated
from the 3D coordinates by CD theory with the matrix method. In this study, we
combined the theoretical method with a VUVCD spectroscopy to reveal thesteric-zipper structures of β2-m21-29 [21NFLNCYVSG29] fibrils. The VUVCD
spectrum of β2-m21-29 fibrils exhibited negative peaks at 285, 250, 222, and 185
nm, and a positive peak at 202 nm. Two steric-zipper models, in which twoparallel β-sheets are linked with disulfide bonds and face in parallel or antiparallel directions, were constructed and simulated by molecular dynamics for
20 ns. We calculated the VUVCD spectra of the simulated model structures by
CD theory and found that the spectra are significantly affected by theorientations of aromatic side-chain of Phe and Tyr residues. Four steric-zipper
models composed of six β -sheets were constructed by varying the aromatic
stacking. One simulated model, in which all six β-sheets face in the anti-parallel− S86 −

direction, reproduced well the experimentally observed spectrum, suggestingthat β2-m21-29 fibrils form three types of steric-zipper structures with different
aromatic stacking between Phe and Tyr residues.
1PT109

どのようにβ-strnad はβ-sheet 中でその位置を決めるのか？

How do β-strands determine their positions in β-sheets ?
Hiromi Suzuki (Sch. Agri., Meiji Univ.)
We analyzed the role of amino acid pairing propensity for β-sheet formation.
Statistical scores were computed from the frequency of the inter-strand aminoacid pairs based on experimental protein structure data, taking the existence ofhydrogen bond formation into account. These scores suggested that residues ona strand tend to avoid inconvenient residues on the other strand as partners(negative selection) rather than to select convenient residues (positive
selection). Prediction accuracies for alignment order of adjacent strands in the
sheets from one edge to the other (Order) in the β-sheets were 77, 57, 39 and
22% for 3-, 4-, 5- and 6-strand sheets, respectively, by using these pairing scoreswith two restrictions: i) Strands form anti-parallel pairs if a loop region isshorter than 12 residues long, ii) All residues on β-strands have at least one
pairing partner(s). H-bond formation was almost completely predicted when
Order, direction and register were correctly predicted. Analysis of prediction
results showed that in more than half mis-predicted cases for 5-strand sheets,
one of edge strands was predicted as a middle strand. If two edge strands werecorrectly selected among β-strands, accuracies of Order prediction can be over
70 and 40 % for 5- and 6-strand sheets, respectively. Such increase of accuracies
may reflect important factors, convenient strand pairing and discrimination
between edge and middle strands, for β-sheet formation.
1PT110X 線小角散乱法による酵母 26S プロテアソームの構造研究

Structural investigation of the yeast 26S proteasome in an
aqueous solution by a small-angle X-ray scattering
Yuya Morita1,2, Takashi Matsumoto3, Akihito Yamano3, Yukio Morimoto1,2(1Graduate School of Science, Kyoto University, 2Research Reactor Institute,
Kyoto University, 3TX-ray Research Laboratory, Rigaku Corporation)
The 26S Proteasome is a large multiprotein complex involved in a regulateddegradation of ubiquitinated protein in the cell. The 26S proteasome iscomposed of two kinds of three sub-complexes, one core particle (CP, 20S) andtwo regulatory particles (RP, 19S). CP is formed by axial stacking of four
heptameric rings: two inner β-rings and two outer α-rings. Capping at each endof the CP is the RP that regulates the proteolytic function of the protease core.
The RP can be further divided into base and lid subcomplexes.
It has not been the way itself how the 26S proteasome degrades ubiquitinatedproteins. We have experimented that degradation process and/or particle
forming by a small angle X-ray scattering (SAXS). Scattering intensities werecollected by use of a BioSAXS-1000 installed FR-E X-ray generator with aprotein concentration of 1mg/mL. We have calculated a liberal structure of the
26S component by SAXS without any constraints on shapes of components.
In the session, we will compare our 26S SAXS model with the 26S electron
microscopy image, and present characteristic structure of our model.
1PT111intensity of those signals are stronger for liquid silk than regenerated silkaqueous solution with same concentration as liquid silk. From theseexperiments, it was confirmed that repeated type II Β-turn like conformation
was generated in Bombyx mori liquid silk.

Bombyx mori 液状絹の溶液 NMR 構造解析

NMR Study of Bombyx mori liquid silk
Yu Suzuki1, Toshimasa Yamazaki2, Akihiro Aoki1, Tetsuo Asakura1 (1Tokyo
Univ. of Agri. and Tech., 2NIAS)
A Bombyx mori Silkworm larve keeps silk in silkglnad at high concentrated(~20% w/w) aqueous solution state before spinning. Liquid silk is expected toform some ordered structure since liquid silk transforms to Β-sheet riched silk
fiber with high strength in a moment under normal temperature and pressure. In
fact, observation of α-helix like pattern in the CD spectra of liquid silk at theconcentrated state was reported. On the other hand, the 13C chemical shift ofliquid silk indicate random coil although liquid silk extracted from a different
kind of silkworm, S.c.ricini, which has polyalanine sequences (Ala)12 clearlyshows α-helix in the three carbons of Ala residues. In this study, atomic-leveled
conformation of Bombyx mori liquid silk was analyzed with several solution
NMR techniques.
1
H and 13 C resonance assignments were achieved with 1 H- 1 H NOESY,TOCSY, 13C HSQC and 13C HSQC-NOESY. The assignments revealed that the
Gly and Ala residues have several chemical shifts mainly due to forward andbackward amino acids. NOE signals between Ha-HN(i, i+2) which indicates
repeated type II Β-turn conformation observed in 1H- 1H NOESY spectra. The

1PT112
Solution stucture of the alternatively folded state of ubiquitin
Soichiro Kitazawa1, Tomoshi Kameda2, Maho Yagi3,4, Kenji Sugase5, Nicky
Baxter6, Koichi Kato3,4, Michael P. Williamson6, Ryo Kitahara1 (1Ritsumeikan,
univ, 2Computational Biology Research Center, AIST, 3Okazaki Institute forIntegrative Bioscience and Institute for Molecular Science, 4Nagoya City
univ., 5Suntory Foundation for Life Sciences, 6University of Sheffield)

Although significant progress has been made in understanding the basic folded
state of proteins, atomic coordinates of high-energy states, which are marginally
populated high free-energy states, remain largely unexplored by traditional
structural experiments. Here, We demonstrate a rational design for the highenergy conformer N2 of ubiquitin based on the high-pressure NMR study andstructure determination of the N2 by solution NMR spectroscopy. Using a singleamino acid replacement method, we created the ubiquitin mutant Q41N thatfavors the population of N2. High pressure NMR spectroscopy showed that theN2 is significantly populated in the mutant sample (~80%) at 1 bar than in thewild-type sample (~15%). The atomic details of the N2 showed that the α1helix, the following loop, the β 3-strand, and the β 5-strand change theirorientations relative to the remaining regions. Conformational fluctuation on the
microsecond to millisecond time scale, characterized by 15 N spin relaxation
NMR analysis, is remarkably increased for these regions of the mutant. These
structure and dynamic characteristics of Q41N are similar to those of the N2 of
the wild-type protein under high pressure. The rational mutation based on the
structure and dynamic characteristics of proteins derived by pressure
experiments is crucial for amplifying the particular fluctuations in proteins.
1PT113加圧による IPMDH への水分子の侵入と深海微生物由来タンパク質の圧力適応High-pressure-induced water penetration into IPMDH and pressureadaptation mechanism of the proteins from deep-sea bacteria
Takayuki Nagae1, Yuki Hamajima2, Takashi Kawamura3, Ken Niwa1,
Masashi Hasegawa1, Chiaki Kato4, Nobuhisa Watanabe1,3 (1Grad. Sch. ofEng., Nagoya Univ., 2Grad. Sch. of Life Sci., Rikkyo Univ., 3Synchrotron Res.
center, Nagoya Univ., 4Inst. of Biogeosci., JAMSTEC)
Several organisms have been found in deep-sea environments such as theMariana Trench, and their proteins are known to be adapted to high-pressure
environments. However, the pressure-adaptation mechanism of proteins fromdeep-sea bacterium is not well understood. For example, 3-isopropylmalate
dehydrogenase (IPMDH) from a deep-sea bacterium Shewanella benthica
DB21MT-2 (SbIPMDH) is more tolerant towards high-pressure stress than thesame enzyme from a land bacterium S. oneidensis MR-1 (SoIPMDH). To
elucidate the pressure-adaptation mechanism of IPMDHs, we have initiatedstructural studies on these proteins by a high-pressure protein crystallographymethod using a diamond-anvil cell. Crystal structures of SoIPMDH were
determined at 2 Å resolution at room temperature under pressures up to about
600 MPa. In the high-pressure structure of SoIPMDH, a new cleft is formed
together with water penetration, which is not observed in the structure underatmospheric-pressure [1]. Interestingly, several residues at the cleft vary
between SoIPMDH and SbIPMDH. Those residues could be the regions
responsible for pressure-adaptation of SbIPMDH. Results of high-pressure
structure analysis of IPMDHs will be reported. [1] T. Nagae, T. Kawamura,
L. Chavas, K. Niwa, M. Hasegawa, C. Kato & N. Watanabe. (2012), Acta Cryst.
D68, 300-309.
1PT114Amyloid-Fibrillation of Lysozyme Is Enhanced by C64-C80
and C76-C94 Disulfide Bonds

Ryohei Kono1,3, Kaoru Shinohara2, Yutaro Fujita2, Hideki Tachibana2,3 (1Med.
Univ. Wakayama, 2Dep. Biotech., School of BOST, Univ. Kinki, 3HPPRC, Instituteof Advanced Technology, Univ. Kinki)
Effect of presence or absence of specific disulfide bonds on the amyloidfibrillation reaction of hen lysozyme was investigated by using a set of
disulfide-variant proteins: 0SS, an all-disulfide-deficient variant; 1SS, singledisulfide-retaining variants (four molecular species); 2SS, double-disulfideretaining variants (two species employed); 3SS, triple-disulfide-retaining− S87 −

variants (four molecular species); and 4SS (WT). The extent of monomolecularfolding covered by this set of proteins ranges from global unfolding, throughlocal folding/unfolding, to global folding. Fibrillation reaction was carried out
with the addition of sonicated WT lysozyme fibril fragments as seeds, at various
incubation temperatures and periods, and was monitored with thioflavine T
fluorescence, CD spectroscopy, and scanning probe microscopy. All the data
showed that the presence of the disulfide bonds C64-C80 and/or C76-C94
enhanced amyloid-fibrillation when compared with the all-disulfide-deficientvariant, 0SS, indicating that these native disulfide bonds have a positive role inthe misfolding into regular intermolecular beta-structures. The presence of theother native disulfide bond, C6-C127 or C30-C115, had no direct effectcompared with 0SS under strongly unfolding conditions, but had an indirect
effect against fibrillation through protein stabilization under folding conditions.
1PT115Structural analysis of hNck2 SH3 domain at various pH: nonnative α-helix-rich monomer and native dimer
Masaji Shinjio1, Yoshitaka Matsumura1, Kaoru Ichimura1, Jianxing Song2,
Kihara Hiroshi3 (1Kansai Medical Univ., 2Department of Biochemistry, Yong
Loo Lin School of Medicine and Department of Biological Sciences, 3SR
Center, Ritsumeikan University)
hNck2 SH3 is a protein which takes β-structure at the native state. On the otherside, Liu and Song reported1) that the protein forms non-native α-helix-rich
structure below pH2 based on NMR spectroscopy. We have done X-ray solution
scattering study of hNck2 SH3 at various pH. hNck2 SH3 lost its tertiary
structure below pH2, judging from Kratky plots. That is, the non-native α-helixrich intermediate, found by Liu and Song1), is not compact. We also found thatmonomer- dimer transition occurred from pH2 to pH4 by X-ray solution
scattering. Below pH2, the protein is monomer and above pH4, the protein
forms dimer, resulting from I(0) analyses. Structural calculations were done byusing DAMMIN2) program. Elongated structure was obtained at pH2 andellipsoidal structure was obtained at pH8. The volume of the structure at pH8 isfound to be almost twice as large as that at pH2, which also supports the
structure below pH2 is monomer and the structure above pH4 is dimer. 1) Liu &Song (2008) Biophys. J., 95, 4803-4812 2) D.Svergun (1999) Biophys. J., 76,
2879-2886
1PT116

Crystal structure of the fatty acid binding protein 3 (FABP3)
with or without fatty acid
Mika Hirose1,2, Shigeru Sugiyama1,2, Mayumi Niiyama1,2, Hanako Ishida2,3,
Tsuyoshi Inoue2,3, Shigeru Matsuoka1,2, Michio Murata1,2 (1Grad Sch. of Sci.,
Osaka Univ., 2JST, ERATO, Lipid Active Structure Project, 3Appl. Chem.,
Grad Sch. of Eng., Osaka Univ.)
Recently, in biological membrane, it revealed that lipids were not only thecomponent of the membrane but also influenced the nature of membrane, andlipids were involved in various intracellular signaling affecting differentiation,
regulation, of growth, and gene expression. Thereby, lipids have been proposed
as a potential drug target. The structural studies of lipids are helpful toinvestigate the precise functions of lipids.
However, so far as we know, they show no clear and well separated electron
densities corresponding to individual atoms of a lipid molecule. To obtain welldefined lipids, we focused on the structure of fatty acid binding protein 3(FABP3) previously determined at 1.4 Å resolution.
In cell, long-chain fatty acids (FA) are insoluble molecules, therefore, binding
with FABP is essential for intracellular trafficking. Fatty acid binding proteins(FABPs) are 14-15 kDa cytosolic proteins found in different cell types and theiramino acid sequences exhibit considerable tissue specific variation.
We tried to purify and crystallize apo-FABP3 by conventional methods forstructure comparison of FABP3 with or without FA. However, it was difficult toremove endogenous fatty acids from FABP3 and impossible to obtain clear
electron densities of a lipid molecule. It was assumed that the binding affinity of
FABP3 to fatty acid was higher than other FABPs.
We will report a removal of endogenous fatty acids from FABP3 and structure
analyses of FABP3 with or without FA at high resolution.
1PT117結晶構造に基づくグラム陰性菌多剤排出ポンプ阻害剤開発に向けた研究

Structural study on inhibitor specifity of bacterial multidrug
efflux pumps

Katsuhiko Hayashi1,2, Ryosuke Nakashima1, Keisuke Sakurai1, Seiji
Yamasaki1,2, Chikahiro Nagata3, Kazuki Hoshino3, Yoshikuni Onodera3,
Kunihiko Nishino1, Akihito Yamaguchi1,2 (1ISIR, Osaka Univ., 2Grad. Sch.
Pharm. Sci., Osaka Univ., 3DAIICHI SANKYO COMPANY, LIMITED)
Pseudomonas aeruginosa highly expresses intrinsic resistance against various
antibiotics. Resistance in P. aeruginosa is mainly caused by two RND typemultidrug efflux pumps, MexB and MexY. Compound A (CA) inhibits both
MexB and AcrB of the major RND pump in Escherichia coli, but CA can’t
obstruct MexY. There is no useful inhibitor of MexY, which is impediment forovercoming of P. aeruginosa infection. CA derivative has great potential to be auniversal inhibitor for RND pumps.
First, in order to clarify the interaction manner between CA and AcrB/MexB,
we made AcrB-CA and MexB-CA co-crystals. With X-ray analyses, we find theinteraction between CA and F178 residue in AcrB and in MexB. MexY
homology model was constructed from AcrB and MexB crystal structure. The
corresponding position of F178 in AcrB/MexB is W177 in MexY, whichsuggests that the voluminous side chain of Trp may have the steric hindrance
rather than interaction with CA.
To verify this hypothesis, we exchanged F178 in AcrB and the W177 in MexY
each other, and checked whether CA inhibits or not. The result is remarkable,
and MexY W177F is inhibited but AcrB F178W isn’ t obstructed by CA.
Furthermore, the crystal structure of AcrB F178W certifies that AcrB mutant
has normal folding and that there is no space for interaction around the W178
residue.
This result can be not only the indicator of the way to make a universal inhibitorof AcrB, MexB and MexY but also the good example for structure based drug
design.
1PT118STEM トモグラフィー法を用いた細菌および真核細胞内超分子複合体の可視化

Structural visualization of supramolecules in bacterial andeukaryotic cells using STEM tomography
Masafumi J. Koike1,3, Shiho Minakata2, Akihiro Narita3, Michio Homma4,
Jiro Usukura2,5, Yuichiro Maeda3 (1RIMD, Osaka Univ., 2Grad. Sch. Eng.,
Nagoya Univ., 3Structural Biology Research Center, Nagoya Univ., 4Grad.
Sch. Sci., Nagoya Univ., 5EcoTopia Sci. Inst., Nagoya Univ.)
In order to form hypotheses on the functions on protein complexes in living
cells, it is essential to visualize the structures of these complexes. Electron
tomography is a powerful tool for structural analyses of supramolecules inside
cells. However, this technique does not work well for thick specimens, which
limits the area that can be visualized. To overcome this problem, we used STEM
(scanning transmission electron microscopy) for tomography analysis. Although
this technique is not commonly used in the field of biology, it provides highcontrast images of thick specimen. Using STEM we obtained high-contrast
structural images of supramolecules in both bacterial and eukaryotic cells. We
imaged the bacterial flagellar motor complex in Vibrio alginoliticus. The
flagellar basal body and ring structures were clearly recognized in the STEM
images we obtained. However, the cytoplasmic part of this motor structure was
not visualized due to the method of sample processing. In eukaryotic cells weutilized STEM to show for the first time images of the thin actin network
structure juxtaposed with the nucleus. Due to the increased thickness of the cellin this area, imaging was not possible using electron microscopy. By image
processing filaments in silico, the spatial location of the actin network was
visualized three dimensionally. We show that STEM tomography is useful toolfor imaging of supramolecular structures in the cell, and is particularly usefulfor imaging of thick specimens not amenable to conventional techniques.
1PT119EM Data Bank と Protein Data Bank の低分解能 3D 電子顕
微鏡構造データの類似構造検索システム

Similarity search system for low-resolutin 3D-EM strcutredata in EM Data Bank and Protein Data Bank
Hirofumi Suzuki1,2, Haruki Nakamura1,2 (1IPR, Osaka-univ., 2PDBj)
More than 1800 structure data by 3D electron microscopy (3D-EM) aredeposited on the Protein Data Bank (PDB) and the EM Data Bank (EMDB).
Extensive and indiscriminate comparison of such 3D-EM data was difficult forsome reasons: 1) EMDB data are not atomic coordinates but 3D maps which are
not able to be compared by existing tools for coordinates. 2) Various quality
values of the 3D maps such as resolutions and CTF-correction accuracies must
be unified for each comparison. We have been developing a structurecomparison system for 3D-EM data, in which dependencies of data types and− S88 −

resolutions were limited by comparing distance profiles of coarse-grainedmodels made by vector quantization of the structures instead of the structure
data themselves. Therefore, 1707 structures from 3D-EM data in PDB and
EMDB were compared mutually by very low computational costs. In thecomparison, most of 70S ribosome structures were distinguished from 80S
ribosomes. And also, distinguishment of 30S ribosome subunits from 50S
subunits and of group I chaperonins from group II were achieved. Using thismethod, we will provide a web service for similarity search of low-resolution
3D-EM structure data.
1PT120Residues contributing to the stability of bovine β lactoglobulin
elucidated by comparing with a chimera protein, Gyuba
Ryo Imaizumi, Hideaki Ohtomo, Masamichi Ikeguchi (Soka Univ.)

A chimeric β lactoglobulin, Gyuba is composed of the amino acid sequencesfrom bovine β lactoglobulin (BLG) in the secondary-structured regions andthose from equine β lactoglobulin (ELG) in the loop regions. Previously, thecrystal structure of Gyuba was determined and clarified that the threedimensional structure of Gyuba was very similar to that of BLG. However,thermal denaturation experiments revealed that the stability of Gyuba was
dramatically decreased as compared with that of BLG. Additionally, at pH 1.5,
BLG maintain its native structure, whereas Gyuba was denatured. The
comparison of the crystal structures between BLG and Gyuba suggested that theresidue 81 and 126 were responsible for the destabilization of Gyuba.
In this study, we characterized two mutants of Gyuba (E81V and Q126P), in
which the residues 81 or 126 of Gyuba were replaced with those of BLG,
respectively. At pH 7.0, near- and far-UV circular dichroism spectra of the twomutants were similar to those of Gyuba, indicating that the substitutions did notaffect with the structure. Thermal denaturation experiments at pH 7.0 showed
Tm of mutants were 5-6ºC higher than that of Gyuba. Additionally, E81V and
Q126P maintained their native structure at pH 1.5. These results suggest that theinteraction of Cγ1 of V81 with Cδ of K91 and Cδ1 of L93 and the stacking
between side chains of P126 and Y20 stabilize BLG structure.
1PT121

X-ray crystallographic analysis of a chemoreceptor protein ofVibrio cholerae, Mlp24
Kazumasa Sumita1, Yumiko Utida1, So-ichiro Nishiyama2, Ikuro Kawagishi2,
Katsumi Imada1 (1Grad. Sch. Sci. Osaka Univ., 2Dept. Front. Biosci. Sci.,
Hosei Univ.)
Vibrio cholerae is a pathogenic Gram-negative bacterium that causes the humandiarrhoeal disease cholera. V. cholerae actively swims in liquid environment
using a single polar flagellum. In nutrient-poor aquatic environments, thebacteria do not produce toxin. However, in nutrient-rich environment, such as
the lumen of the human small intestine, they begin to form colonies and express
pathogenic proteins. Thus, sensing of nutrient is the crucial for pathogenicity of
V. cholerae. Mlp24 is a chemoreceptor for multiple amino acids, including
serine, arginine and asparagine, which stimulate the expression of several
virulence factors. Interestingly, Mlp24 is required for the production of cholera
toxin upon mouse infection. These facts suggest that chemotaxis toward a set ofamino acids mediated by Mlp24 plays a crucial role in pathogenicity. To
elucidate the molecular mechanism of multiple amino acid recognition andpathogenicity of V. cholerae, we expressed, purified and crystallized the ligand
binding domain of Mlp24. Static light scattering measurement indicated that the
purified Mlp24 domain is in equilibrium between monomer and dimmer. Afterseveral days of incubation, rod-shaped crystals were grown from a solution
containing PEG400 as precipitant. Structure analysis of the crystal is underway.
1PT122

時計タンパク質 KaiB の KaiABC 複合体形成における構造変化

Structural change of the clock protein KaiB on the formationof the KaiABC clock oscillator complex
Ryosuke Tajika1, Risa Mutoh3, Hiroyuki Mino1, Masahiro Ishiura1,2 (1Grad.
Sch. Sci., Nagoya Univ., 2Center for Gene Research, Nagoya Univ., 3Inst. Prot.
Res., Osaka Univ.)
Circadian rhythms, biological oscillations with a period of 24 h in various
metabolic and behavioral activities, are observed ubiquitously from procaryotes
to eucaryotes. In cyanobacteria, three clock proteins, KaiA, KaiB and KaiC,
form the KaiABC clock oscillator complex, which plays essential roles ingenerating circadian oscillations. KaiA enhances KaiC autophosphorylation
whereas KaiB suppresses it. We believe that those functions are associated withstructural change of the KaiABC complex. We have previously demonstrated

that the structural changes of KaiB occur on the formation of a complex with
KaiA and KaiC. However, it is not clear how and when the protein complexes
are formed.
Pulsed electron-electron double resonance (PELDOR) is a well-establishedmethod to determine the distance between radicals with high accuracy. In order
to measure the distance between proteins, the site-directed spin labeling method
was used. We prepared a KaiB mutant, which has a Cys residue substitution at
the 22nd Val residue (KaiBV22C), attached maleimide spin label (MSL) to thecysteine residue (MSL-KaiBV22C). PELDOR result showed that the interdistance between the spin labels in MSL-KaiBV22C was calculated to be about
30 Å. In addition, the longer distance was obtained in the co-presence of KaiA
and KaiC. We are now investigating the time course of the formation andstructural modification of the KaiABC complex.
1PT123分裂酵母由来 SpHsp16.0 のシャペロン機能における N 末領域のフェニルアラニン残基の重要な役割

Phenylalanine residues of the N-terminus region play a crucialrole in regulating chaperone activity of SpHsp16.0 from fission
yeast
Yuya Hanazono1, Yoshiki Aikawa1, Nobuhiko Akiyama1, Tetsuya Abe2,
Kazuki Takeda1, Toshihiko Oka3, Masafumi Yohda2, Kunio Miki1 (1Grad.
Sch. Sci., Kyoto Univ., 2Dept. Biotech. Life Sci., Tokyo Univ. Agricult.
Tech., 3Grad. Sch. Sci. Tech., Shizuoka Univ.)
Small heat shock proteins (sHsps), which are widely found in all domains oflife, bind and stabilize denatured proteins to prevent aggregation. The sHsps
exist as large oligomers that are composed of 9-40 subunits and control theirchaperone activity by the transition of the oligomeric state. The sHsps have a
globular α -crystallin domain from which the peripheral N-terminal and Cterminal regions protrude. In order to elucidate how sHsps recognize substrates
and transit the oligomeric state, we performed crystallographic and biochemical
studies by use of SpHsp16.0 from fisson yeast, Schizosaccharomyces pombe.
We performed mutational analysis at well-conserved phenylalanine residues of
the N-terminal region. Size exclusion chromatography at elevated temperatures
and thermal aggregation measurements indicate that Phe6, Phe7 and Phe9 areprobably involved in oligomeric stability and interaction with substrates.
Moreover, we have determined the crystal structure of SpHsp16. 0 at 2. 4
angstrom. SpHsp16. 0 forms a 16-meric oligomer with 422 symmetry. The
SAXS analysis supports this oligomeric assembly. The crystal structure
indicates that the three phenylalanine residues (Phe6, Phe7 and Phe9) form ahydrophobic cluster at the center of the oligomer.
1PT124

Staphylococcal nuclease の触媒活性におけるループの役割

The role of the flexible loop in Staphylococcal nuclease on its
catalytic activity
Rumi Shiba, Hironari Kamikubo, Yoichi Yamazaki, Mariko Yamaguchi,
Mikio Kataoka (Graduate School of Materials Science, Nara Institute of
Science and Technology)
The molecular control mechanism of enzymatic catalysis is a fundamental
problem in protein science.
We have generated the mutant of Staphylococcal nuclease that takes a stablenative structure but loses enzymatic activity. The mutant maintains all of active
site residues but lacks a flexible loop (Δ44-49). The deletion mutant also keeps
substrate binding ability. In order to clarify how enzymatic activity is controlled,
we carried out X-ray crystal structure analysis of the mutant with and without an
inhibitor, prAp, as well as the WT. While the flexible loop (44-49) of WT takes
an open form in the absence of prAp, the loop shows large movement in thepresence of prAp to bring a closed form. Accompanying the motion, the residue
E43 which acts as a general base catalyst moves to point to the phosphate groupof prAp. The mutant Δ44-49 does not show such a large movement because it
lacks the flexible loop. E43 of Δ44-49 slightly moves to the similar position tothat of WT. However, the flexibility of E43 is apparently lost. Furthermore themovement upon binding prAp of the loop (76-90) which contains anothercatalytic residue R87 is larger for Δ 44-49 than WT, although the final
configuration of R87 is identical. The motions of the loops are closely coupledwith each other. We assume that the loop flexibility is an important factor of
catalytic activity and that the 44-49 loop controls the flexibility.
1PT125− S89 −

Athermal Fluctuations in Active Cytoskeletal Networks Follow
Truncated Levy Distribution

Heev Ayade, Syou Yamamoto (kyushu University)
A reconstituted active cytoskeletal networks consisting of an actin filament
network coupled to myosins (motor proteins) have been shown to display rich in
dynamical and mechanical behaviors that is often in contrast to passive,
equilibrium system. The motor proteins, which spontaneously generate forces,
kept the active cytoskeletal network out of equilibrium. The athermal
fluctuations observed in the network are linked to the active force generation by
motor proteins which give more relevant information including the interaction
with the surrounding materials. In prior studies, only the second moment of theathermal fluctuations has been investigated. There is no reason a priori to expect
Gaussian statistics in nonequilibrium systems. Indeed, the full displacement
distribution of the athermal fluctuations in active cytoskeleton recently probed
using video microrheology is found to be far from Gauss. In this study, weinvestigated the nonequilibrium statistics and dynamics of the active network byanalyzing the athermal fluctuations. The model developed here is based on Levy
statistics which is generally observed for the effectors whose impacts decays as
1/r2. It was found that the displacement distribution is obtained as a convolutionof truncated Levy distribution due to the finite-size of probe particles. The
truncation of Levy becomes less and power-law tail is enhanced as the probe
particle gets smaller.
1PT126

Atoms in molecules 法によるタンパク質αへリックス、βシートの水素結合相互作用の理論解析

Atoms in molecules analysis of hydrogen-bond interactions in
α- helix, and β-sheets of protein

1PT128

光駆動性陽イオンチャネルであるチャネルロドプシンの結晶構造解析

1YS0930

Crystal Structure of a light-gated cation channel,
channelrhodopsin

Hideaki E. Kato1, Feng Zhang2, Ofer Yizhar2, Charu Ramakrishnan2,
Tomohiro Nishizawa1, Kunio Hirata3, Jumpei Ito4, Yusuke Aita4, Tomoya
Tsukazaki1, Shigehiko Hayashi5, Peter Hegemann6, Andres D. Maturana4,
Ryuichiro Ishitani1, Karl Deisseroth2, Osamu Nureki1 (1Dept. of Biophys. andBiochem., Grad. Sch. of Sci., Univ. of Tokyo, 2Dept. of Bioengineering,
Stanford Univ., 3Harima Inst., Riken, 4Bioengineering Dept., Nagaoka Univ, ofTech., 5Dept. of Chem., Grad. Sch. of Sci., Kyoto Univ., 6Inst. of Biol.,Experimental Biophys., Humboldt-Univ.)
Channelrhodopsin (ChR) is a light-gated cation channel derived from algae that
conducts cations, including sodium ions, in a light-dependent manner. Because
the inward flow of sodium ions changes the electrochemical gradient andtriggers neuron firing, neurons expressing ChRs can be optically controlled withhigh temporal precision within systems as complex as freely moving mammals.
Although ChR has been broadly applied to neuroscience research, little is
known about its molecular mechanism. Here, we present the crystal structure of
ChR at 2.3 angstrom resolution, showing its basic architecture and molecularmechanism, and give the long-awaited answer to the question, “Where is thecation conducting pathway?” The integration of structural and electrophysiological analyses provides insight into the molecular basis for the remarkablefunction of ChR, and paves the way for the precise and principled design of ChRvariants with novel properties.

Hideki Yamasaki, Haruki Nakamura (IPR, Osaka Univ.)
Hydrogen-bond interactions (HBIs) between biomolecular systems play animportant role in physics, chemistry, and biology, and affect many biologicalprocesses, such as molecular recognition. In this study, HBIs of α-helix (AH),and parallel and antiparallel β-sheets (PBS and APBS) were investigated interms of both energetics and topological points of view, using the electron
density analyses of atoms in molecules (AIM) (Yamasaki & Nakamura, 2012)
in conjunction with quantum chemical calculations. The secondary structures
were built such that the numbers of peptide bonds of AH, and PBS and APBS
were increased from 2 to 12 or 16, respectively, in addition to extended models,followed by refining and analyzing with DFT-M062X/6-31+G* calculations.
For BS, the interaction energies (Ecp) were estimated with difference in totalenergy between dimer and the sum of each monomer. The basis sets
superposition error was corrected by counterpoise-correction method. For AH,
the correlation between total energy differences between AH and extended
peptide and the sums of electron density over the bond critical points (Σinterρb)were found with r2 of 0.99. For PBS and APBS, the correlations between Ecpsand Σinterρb of HBIs were also found with r2 of 0.99 and 0.97, respectively,
implying that Ecp were well reproduced with Σinterρb. Interestingly, while Ecps
were monotonically increased for PBS as the numbers of peptide bonds
increased, the stepwise increase of Ecps was found for APBS, implying thecooperative HBI effect in APBS.
1PT127

Go モデルによるフォールディングシミュレーションに対する天然構造の差異の影響について

Analysis of the influence of differences in native structures of aprotein on its folding simulations with a Go model
Shuji Abe, Masatake Sugita, Takeshi Kikuchi (Dept. Bioinf., Coll. Life Sci.,
Ritsumeikan Univ.)
Go model is a simple model with inter-atomic potentials between only native
contacts in a given protein. The native structure of a protein is determined by Xray crystallography or NMR spectroscopy. Although the 3D structures of aprotein determined by these methods show the same structures at the level of the
backbone fold, it has been reported that differences in the folding mechanisms
predicted by a Go model are observed depending on whether its 3D structure has
been determined by X-ray of NMR, or which structure in the NMR models has
been used as a native structure. That is, we have to clarify the following points;
1) the extent of the influence of the definition of the native structure on the
results of folding simulations by a Go model, 2) How can fruitful information on
the folding mechanisms of a protein be extracted using NMR structuralensemble. In this study, this problem is treated by performing simulations using
a coarse grained Go model which we have developed. Protein G, Protein L andα-spectrin SH3 domain are used for tests. Our results show that the differences
appeared in the folding simulations in Protein L is especially large and specific.
We will show the detailed results.

1PT129DNA ポリメラーゼηによるホスホジエステル結合形成を観察する

1YS1000

Watching DNA polymerase η make a phosphodiester bondTeruya Nakamura1, Ye Zhao2,3, Yuriko Yamagata1, Yue-jin Hua3, Wei Yang2(1Grad. Sch. of Pharmaceut. Sci., Kumamoto Univ., 2NIDDK, NIH, 3INAS,
Zhejiang Univ.)
A DNA polymerase catalyzes a nucleotidyl-transfer reaction with Mg2+, and a
new bond is formed between the 3'-OH of the primer strand and the α phosphate of dNTP and the phosphodiester bond between α and β-phosphates ofdNTP is broken. Until now, a number of structural and kinetic studies of DNApolymerases proposed the catalytic mechanism of the nucleotidyl-transfer
reaction. However, the actual reaction mechanism remains unknown. In thisstudy, we have visualized the nucleotidyl-transfer reaction process catalyzed byhuman DNA polymerase η (Polη) using X-ray crystallography. Polη, DNA anddATP were co-crystallized at pH 6.0 without Mg2+. The reaction was initiatedby exposing crystals to 1 mM Mg2+ at pH 7.0 and stopped at various time points
by freezing. The substrates and two Mg2+ are aligned for reaction within 40 sec,
but the new bond begins to form at 80 sec. Structures at reaction times from 80
to 300 sec reveal a mixture of decreasing substrate and increasing product of thenucleotidyl-transfer reaction. In sequence, the nucleophile 3'-OH is deprotonated, the deoxyribose at the primer end converts from C2'-endo to C3'-endo, andthe nucleophile and the α-phosphate of dATP approach each other to form the
new bond. A third Mg2+ ion, which arrives with the new bond and stabilizes theintermediate state, may be an unappreciated feature of the two-metal-ion
mechanism. Here we show a chemical bond formation at atomic resolution andin real time.
1PT130Endo-1,3-β-glucanase・オリゴ糖複合体の MD シミュレーション

Molecular dynamics simulations of endo-1,3-β-glucanase andoligosaccharide complex
Nobutaka Komichi1, Narutoshi Kamiya2, Haruki Nakamura2, Masayuki Oda1
(1Grad. Sch. of Life and Environ. Sci., Kyoto Pref. Univ., 2Inst. for ProteinRes., Osaka Univ.)
Endo-1,3-β-glucanase from Cellulosimicrobium cellulans DK-1, is composed of
catalytic domain and carbohydrate-binding module. The catalytic domain
belongs to glycoside hydrolase family 16, and catalyzes the hydrolysis of β-1,3glucans such as laminarin and laminarioligosaccharides. We have determinedthe crystal structure of catalytic domain, and analyzed its interactions with β-1,3-glucans using isothermal titration calorimetry. The catalytic domain would
have six subsites for the binding of glucose units, four subsites (-4 to -1) being
on the non-reducing end from the scissile glucosidic bond and two subsites (+1
and +2) on the reducing end. In this study, we carried out molecular dynamics
simulations to clarify the complex structure with laminarioligosaccharides
whose degrees of polymerization are 2 to 7. Each oligosaccharide was modeled− S90 −

and docked into the crystal structure of catalytic domain. Then, a spherical box
of water molecules was placed around the subsites. Molecular dynamics
simulations in explicit water were carried out at 300 K, and stable structures
were chosen from the trajectories by using the cluster analysis. The key residues
for the oligosaccharide recognition based on the subsite theory were identified.
In the bound oligosaccharide, the glucose unit on the non-reducing end wasmore fluctuated than that on the reducing end. Taken together with theexperimental results of binding thermodynamics, the energetic contribution on
the respective subsites could be elucidated.
1PT131αへリックス・βストランドレプリカ交換分子動力学シミュレーショ
ン法の開発と応用

Development and application of the helix-strand replicaexchange molecular dynamics method
Hisashi Okumura1,2, Satoru G. Itoh1,2 (1Inst. Mol. Sci., 2SOKENDAI)
Biomolecules such as proteins have complicated free energy surfaces with many
local minima. Conventional molecular dynamics (MD) and Monte Carlo (MC)simulations tend to get trapped in these local-minimum states. One of thepowerful techniques to avoid this difficulty is the replica-exchange method. We
propose helix-strand replica-exchange molecular dynamics method. We apply
this method to a design peptide and show the results.
1PT132PDB データベースによるアミノ酸ごとに最適化した主鎖二面角力場

Development of a force field of backbone dihedral angles foreach amino acid using Protein Data Bank
Yoshitake Sakae1,2, Yuko Okamoto1,3,4 (1Dept. of Phys., Nagoya Univ.,
2
IMS, 3Structural Biology Research Center, Nagoya Univ., 4Center for
Computational Science, Nagoya Univ.)
A force field is widely used in the field of molecular simulations forbiomolecular systems and is determined by an atomic model based on classicalmechanics. Many commonly used force fields for protein systems such as
AMBER, CHARMM, GROMACS, OPLS, and ECEPP have amino-acidindependent force-field parameters of main-chain torsion-energy terms. Here,
we propose a new type of amino-acid-dependent torsion-energy terms in theforce fields. As an example, we applied this approach to AMBER ff03 force
field and determined new amino-acid-dependent parameters for φ and φ’ angles
for each amino acid by using our optimization method, which is based on theminimization of some score functions by something in the force-field parameter
space, where the score functions are derived for the protein coordinate data in
the Protein Data Bank (PDB) [1-3]. In order to test the validity of the new forcefield parameters, we then performed folding simulations of α-helical and βhairpin peptides, using the optimized force field. The results showed that the
new force-field parameters gave structures more consistent with theexperimental implications than the original AMBER ff03 force field.
[1] Y. Sakae and Y. Okamoto, Chem. Phys. Lett. 382, 626-636 (2003).
[2] Y. Sakae and Y. Okamoto, J. Theo. Comput. Chem. 3, 339 (2004).
[3] Y. Sakae and Y. Okamoto, J. Theo. Comput. Chem. 3, 359 (2004).
1PT133

コフィルアクチンフィラメントの分子動力学モデルの構築

Molecular dynamics model of cofilactin filament
Tetsuya Fujii1, Yasuhiro Inoue2, Taiji Adachi3 (1Grad. Sch. Eng., Dept. ofMicro Eng., Kyoto Univ., 2Dept. of Biomechanics, Inst. Frontier Med. Sci.,
Kyoto Univ., 3Dept. of Biomechanics, Inst. Frontier Med. Sci., Kyoto Univ.)
Cofilin is an actin regulatory protein which dissembles and severs actin
filaments. The binding affinity of cofilin to actin filament is regulated via
biochemical pathways. In addition, it has been pointed out that tension in actin
filament prevents cofilin from binding to the filament. Because cofilin binding
put a twist on the helix of the actin filament that might be incompatible filament
conformation caused by the tension, we suppose that structural change of actin
filament is related to the mechanism on tension-induced inhibition of cofilinbinding. In order to obtain better understanding the mechanism at molecularlevel, we need to investigate energetic and structural effects on binding domainsof actin and cofilin caused by the conformational change of the filament.
To achieve the research mentioned above, in this study, we construct molecular
dynamics (MD) model of cofilin-decorated actin (cofilactin) filament. First, weplaced cofilins in the vicinity of their binding domains on actin along thefilament based on the PDB data of actin-cofilin complex. Second, MD
simulation for 20ns was conducted to obtain an equilibrium structure ofcofilactin filament. Because this equilibrium structure has included thermally-

induced various twist angles, a cofilin-bound actin trimer with a moderate angle
has been extracted from the equilibrium structure as a sample of cofilactin
complex. In this way, we succeeded to construct MD models of cofilactin
complexes with distinct twist angles.
1PT134ロボット機構の運動学に基づくタンパク質の内部運動解析

Internal Motion Analysis of Proteins based on Robot
Kinematics
Keisuke Arikawa (Fcl. Eng., Kanagawa Inst. of Tech.)
From the perspective of robot kinematics, we investigated a method forpredicting the internal motions of proteins using data from PDB. The protein
model used in this method is simple; it comprises main chains and linear springs
between the alpha carbons. The main chains are considered to be robot
manipulators that control the positions of the alpha carbons or the distances
between them, where the dihedral angles on the main chains correspond to thejoint angles of robot manipulators. We apply forces in static equilibrium to themodel and evaluate the deformation caused by them. The deformation
magnitude differs depending on the direction of the force, despite the magnitudeof applied forces being the same. It can be concluded that deformations of alarge magnitude occur easily. Here, forces are applied in two different manners.
One is the application of forces at the alpha carbons in the residues interactingwith other molecules and the other is the application of forces at the centroids ofthe atomic groups (e. g., secondary structures, domains, and subunits). The
formulation of basic equations for this method is similar to that for kinematic
analysis of robot manipulators. We applied this method to data from PDB ofsome proteins (e.g., calmodulin, lactoferrin, and aspartate transcarbamoylase).
Despite being a simple model, the results are almost consistent with the actualinternal motions.
1PT135コンピュータシミュレーションによる 4-helix bundle タンパク質を基にしたタンパク質分子間相互作用面の設計

Simulation study for designing protein-protein interaction
interfaces based on a four-helix bundle protein
Masaki Fukuda1, Yu Komatsu2, Ryota Morikawa1, Takeshi Miyakawa1,
Masako Takasu1, Satoshi Akanuma1, Akihiko Yamagishi1 (1Dept. of Mol.
Biol., Tokyo Univ. of Pharm. Life Sci., 2Grad. Sch. of Pure Appl. Sci., Univ. of
Tsukuba)
Some proteins have the ability to self-assemble and form fibers. The purposes ofour study are: i) designing binding interfaces on two proteins which specificallyinteract to each other; ii) inducing self-assembly through the intermolecularinteraction between the two proteins; and iii) forming co-polymers. A fourhelix bundle structure is chosen as a model for designing the binding interface.
Using the four-helix bundle as the scaffold, we designed the binding interfaces
on the target protein YciF involving charged and hydrophobic amino acidresidues. To evaluate the designed binding interfaces, we performed molecular
dynamics (MD) simulation of the variants of YciF proteins with the designed
binding interface using GROMACS [1]. For the initial state, two protein
molecules with the designed interface were placed with appropriate distance,
and their binding interfaces faced each other. After 5ns simulation, we
calculated the distance between centroid of binding interface of the molecules,
and visualized the structures using Pymol[2]. The details will be presented at theconference.
[1] Berendsen, et al. Comput. Phys. Commun. 91 (1995) 43. [2] http://www.
pymol.org
1PT136

透明で柔軟性を持った実用性の高いタンパク質分子模型

A Soft and Transparent Handleable Protein Model
Masaru Kawakami (Sch. Mat. Sci.)
The field of molecular and structural biology currently relies on computergenerated graphical representations of three-dimensional (3D) structures toconceptualize biomolecules. As the size and complexity of the molecular
structure increases, model generation and peer discussions become moredifficult. It is even more problematic when discussing protein-protein
interactions wherein large surface area contact is considered. This reportdemonstrates the viability of a new handleable protein molecular model with asoft and transparent silicone body similar to the molecule's surface. A full-color
printed main chain structure embedded in the silicone body enables users tosimultaneously feel the molecular surface, view through the main chain
structure, and manually simulate molecular docking. The interactive, hands-on− S91 −

experience deepens the user's understanding of the complicated 3D protein
structure and elucidates ligand binding and protein-protein interactions. Thismodel would be an effective discussion tool for the classroom or laboratory that
stimulates inspired learning in this study field.
1PT137

蛋白質熱安定性に及ぼす糖の影響

Effects of sugars on the thermal stability of proteins
Hiraku Oshima1, Ken-ichi Amano2, Masahiro Kinoshita1 (1Inst. Adv. Energy,
Kyoto Univ., 2Dep. Chem., Kobe Univ.)
It is experimentally known that the heat-denaturation temperature of a protein
Tm is raised by the sugar addition. The magnitude of this effect stabilizing theprotein depends on the sugar species [1]. In earlier work, we proposed a measureof the thermal stability of a protein, which is defined as the solvent-entropy gain
at 298 K upon protein folding S normalized by the number of residues [2,3]. S
was calculated using a hybrid of the angle-dependent integral equation theory
combined with the mulipolar water model and the morphometric approach. Here
we show that S can be calculated using the hard-sphere solvent whose number
density and molecular diameter are set at those of water. We then investigate theeffects of sugar addition on the thermal stability by considering water-sugar
solution modeled as a binary mixture of hard spheres. The three-dimensional
integral equation theory is employed. The thermal stability is determined by acomplex interplay of the molecular size of the sugar D and the total packing
fraction of the solution η. D is estimated from the volume per molecule in thesugar crystal, and η is calculated using the experimental data of the solution
density. We find that the thermal stability is considerably enhanced by additionof sucrose. In the presentation, we will discuss the effects of addition of not only
sugars other than sucrose but also osmolytes.
[1] J. F. Back, Biochemistry 18 (1979) 5191.
[2] K. Amano et al., Chem. Phys. Lett. 474 (2009) 190.
[3] K. Oda et al., J. Chem. Phys. 134 (2011) 025101.
1PT138

ヒトカルシトニンアミロイド線維形成阻害機構の解析

Amyloid fibril inhibition mechanism of human calcitonin
Naoharu Kouduki1, Hironari Kamikubo1, Tomoyasu Aizawa2, Yoko Ogawa3,
Yoichi Yamazaki Yoichi Yamazaki1, Mariko Yamaguchi1, Mikio Kataoka1(1Grad. Sch. Mat. Sci., NAIST, 2Graduate School of Life Science, Hokkaido
University, 3Osaka University of Pharmaceutical Sciences)
Human calcitonin (hCT) is a 32-residue peptide hormone. hCT is used as a
medicine for osteoporosis, but it easily forms amyloid fibrils underphysiological condition, which is a problem on medication. Salmon calcitonin(sCT) is known to inhibit amyloid formation of hCT. Combinational use of hCTand sCT is expected to improve the pharmacological activity. While the Nterminal region (9-19) of sCT takes an α-helix, the C-terminal half of the helix isdisordered in hCT. Although an interaction between the disordered region and
the C-terminal tail of hCT is proposed to play a role on the amyloid formation,
the detailed mechanism of the inhibition of the amyloid formation by sCT has
not been revealed. In this study, we investigated amyloid fibril formation of hCTin the presence of each of two chimera peptides, which are comprised of the 115residues of hCT and the 16-32residues of sCT and, the reverse combination.
The former region (1-15) contains the stable α-helix and the latter the flexible
region in hCT. Amyloid fibril formation was monitored by fluorescenceintensity from Thioflavin T. While the fluorescence intensity raised about
30~40 hours after incubation of hCT alone, the amyloid fibril formation was
inhibited in the presence of sCT, or each of the two chimera peptides, indicatingthat each of the N-terminal and the C-terminal regions of sCT possesses theinhibitory ability. From these results, we conclude that both the C-terminal
region and the N-terminal stable α-helix are engaged in the inhibition of theamyloid formation.
1PT139ダブル pH ジャンプによるアミロイド線維のほぼ可逆的な構造変化の解析

Analysis of Almost Reversible Conformational Change of
Amyloid Fibrils by Double pH-jump
Keiichi Yamaguchi1, Yuji O. Kamatari2, Mayuko Fukuoka1, Reiji Miyaji3, Kazuo
Kuwata1 (1Unit. Grad. Sch. of Drug Dis. and Med. Inf. Sci., Gifu Univ., 2Life Sci. Res.
Center, Gifu Univ., 3Sup. and Dev. Center for Tech. Ed., Fac. of Eng., Gifu Univ.)

immensely large minimum at 207 nm on CD spectrum at pH 2.9 (named pH2.9
fibrils), but aggregate-like fibrils with a minimum at 220 nm were formed at pH
7.5 (named pH7.5 fibrils) near its isoelectric point. In this study, firstly single
pH-jump from 2.9 to 7.5 was performed. As a result, the CD spectrum showedthat the pH2.9 fibrils instantly changed to pH7.5-like fibrils with a minimumat 218 nm, but the ellipticities were certainly distinct between pH7.5-like andpH7.5 fibrils, implying that the conformation of pH2.9 fibrils partially remains
even at pH 7.5. Moreover, the pH7.5-like fibrils almost returned to the pH2.9
fibrils by restoring the solution pH from 7.5 to 2.9. FT-IR spectra indicated thatthese conformational changes were caused by the disruption of highly ordered β-sheet and β-turn conformations, and the subsequent their reconstructions. In
addition, kinetic conformational changes of the fibrils after the single anddouble pH-jumps were examined using ANS fluorescence stopped-flow. As aresult, these conversions of the fibrils composed of a few phases wereaccomplished within several seconds. Thus, amyloid fibrils can be changed
readily between the distinct conformations separated by a low energy barrier
almost reversibly.
1PT140金属結合によるヒトプリオンタンパク質のペプチド断片の構造変化

Conformation change in peptide fragments of human prion
protein caused by metal binding
Kazuya Iwama1, Takahito Imaki1, Masahiro Yagi2, Wakako Hiraoka2 (1Grad.
Sch.Sci,Univ.Meiji, 2Sch.Sci,Univ.Meiji)
Prion diseases are transmissible spongiform encephalopathies (TSE) and lethal
infectious diseases caused by misfolding of prion protein (PrP). Human prion
protein (huPrP) binds metal ion at the residues 60-91 (His61, His69, His77,
His85) and residues 90-126 (His96,His111) in N-terminal. Histidine residue are
known as high-affinity sites to metal binding. Metal binding to PrP is considered
as an important process for misfolding of PrP. This report aims to investigate
conformation change in huPrP caused by metal binding. huPrP Peptide
fragments conteining His96 and/or His111 were examined for binding metalsuch as Cu2+, Ni2+ and Co2+. pKa values were determined with electro
titrimetric analysis (Metrohm, 848 titrino plus). Metal binding to peptide
fragment was observed using visible absorption spectra (Hitachi, U-2800
Spectrophotometer) and Circular Dichroism (CD) (JASCO, J-820). Secondary
structure of peptide fragment was observed using Far-UV CD. Addition of Cu2+
and Ni2+ to huPrP (93-102:GGTHSQWN) showed the shift of pKa value. Vis
absorption and Vis CD indicated the characteristic spectra resulting from metalbinding. It was derived from imidazole N bound to metal ion and from d-dtransition of metal ion. Far-UV CD spectrum showed the conformational changeof secondary structure induced by metal binding. We will also investigate metalbinding to peptide fragments of huPrP containing His111 by using UV/Vis
spectroscopy, CD and electron spin resonance (ESR) to further understandabout metal binding to huPrP.
1PT141

ユビキチンの温度-圧力-自由エネルギー地形図

Temperature-pressure-free energy landscape of ubiquitin
Tsubasa Yamamoto1, Minoru Kato2 (1Graduate school of Life Science,
Ritsumeikan University, 2Department of Pharmacy, Ritsumeikan University)
To understand the change of packing and/or solvation of protein denaturationfrom quantitative point, volume change Δ V and isothermal compressibilitychange ΔβT, and thermal expansion coefficient change Δα are needed. Thesethermodynamic parameters at given temperature and pressure are determinedfrom temperature(T)- pressure(p)- free energy change(ΔG) landscape,which is
obtained fraom combined series of pressure/temperature variable denaturation
experiments.
In this study, we explore the (T, p, ΔG) landscape of ubiquitin. FTIR spectra ofubiquitin was reversibly changed by temperature/pressure perturbation at pD 2.0
and pD 5. 5. To determined Δ G at given temperature and pressure,temperature/pressure transition curves were obtained from change in FTIR peakintensity at 1673 cm-1 which is assigned to native β-strand structure. Threedimensional (T, p, ΔG) landscapes and thermodynamic parameters (ΔV, ΔβT, Δ
α, entropy change ΔS and heat capacity change ΔCp,) at ambient temperatureand pressure (298K, 0.1MPa) were obtained.
1PT142

To obtain insights into the mechanism of amyloid fibril conversion, pH-jumpexperiments were performed using a H2 peptide of mouse prion protein.
Previously, we reported that H2 peptide formed ordered amyloid fibrils with an− S92 −

X 線小角散乱によるフェリチンアセンブリ反応の追跡
Ferritin assembly kinetics followed by small-angle X-ray
scatteringDaisuke Sato1, Masayoshi Inoue1, Ayumi Sunato1, Yoshiteru Yamada2,
Hideaki Ohtomo1, Seiichi Tukamoto1, Kanako Nakagawa1, Masamichi
Ikeguchi1 (1Soka university, 2JASRI/SPring-8)
Non-heme ferritin (Ftn) from Escherichia coli forms a spherical shell-shape with
4/3/2 symmetry of 24 subunits. It stores Fe(III) as ferrihydrite mineral within theprotein shell. Ftn dissociates into 12 dimers at acidic pH, and it is able toreassemble into the native 24-mer when pH is returned to a neutral one. The
circular dichroism (CD) spectrum and small angle X-ray scattering (SAXS)
have shown that the acid-dissociated Ftn dimer maintains native-like secondary
and tertiary structures. To clarify the assembly mechanism of apo-Ftn thereassembly of acid-dissociated apo-Ftn dimers was initiated by stopped-flow
mixing with appropriate buffers and monitored by synchrotron radiation at
SPring-8 (Beamline 45XU) and a photon-counting two-dimensional detector
PILATUS. The reassembly rate depended on pH; it was too rapid to measure at
pH 6.4 and slowed down at higher pHs. At pH 8, we succeeded to observe an
overall change from the initial scattering identical to that of acid-dissociateddimer to the final scattering identical to the native apo-Ftn. The process
consisted of at least two kinetic phases. A native-like radius gyration was
attained in the faster phase and the zero-angle scattering intensity increased inboth the fast and slow phases. The rate of the fast phase depended on the protein
concentration, suggesting that this phase corresponds to the assembly of dimers.
1PT143GroEL-GroES 複合体形成の熱力学的解析
THERMODYNAMIC PARAMETERS ASSOCIATED WITH
THE GroEL-GroES BINDING

Toshio Takenaka, Takashi Nakamura, Jin Chen, Kunihiro Kuwajima (Okazaki
Institute for Integrative Bioscoence)
The tetradecameric chaperonin GroEL forms a complex with its co-chaperonin
GroES and mediates the refolding of substrate proteins in the cell. Despite alarge body of studies to elucidate the underlying mechanism of the chaperoneassisted refolding of substrate proteins, the thermodynamic parameters associatewith the GroEL-GroES binding have not yet been determined. We therefore
combined NMR spectroscopy and isothermal titration calorimetry (ITC) to
quantitatively estimate the GroEL-GroES binding constant in solution. In 2-D
HSQC NMR measurements, by monitoring amide hydrogen signal intensities ofcross peaks in a mobile loop region of 15N labeled GroES as a function ofconcentration of single-ring of GroEL (SR1), we obtained the equilibrium
binding constants between GroES and SR1 at different temperatures in 20 mM
Tris-HCl , 100 mM KCl, 10 mM MgCl2, and 3 mM ADP (pH 7.5), and thevalues were compared with those obtained by ITC under the conditions.
1PT144

Local and Global Structural Analysis of Denatured Protein by
CW and pulsed ESR

Jun Abe1, Munehito Arai2, Satoshi Takahashi1, Seigo Yamauchi1, Yasunori
Ohba1 (1IMRAM, 2Grad. Sch. Art and Sci, Univ. Tokyo)
Details of structural information of unfolded proteins are limited despite theactive investigations in the past. As a structural model, a random coil has been
assumed. However, recent researches have revealed that denatured protein has
residual structures similar to secondary structures: helices and β sheet[1]. In ourstudy, we analyzed the structure of unfolded protein based on electron spinresonance (ESR) methods (CW-ESR and pulsed ESR) with the site-directed
spin labeling. We introduced the spin labels to 22 and 55th residue to measure
the distance between the helices H2 and H3 in the B domain of protein A(BDPA). ESR signals were measured for the unfolding state by adding
guanidinium chloride (GdmCl) as denaturant in the folded and unfolded states.
CW-ESR reveals that the rotational correlation time in the folded state (GdmCl0 M) is faster than that in the unfolded state (GdmCl 5 M) for single labeled
BDPA. This result indicates that the motion of main chain in denatured protein
become faster. For doubly labeled BDPA, we found that the well-defined H2H3 distance for the folded state. In contrast, a broad distribution of the H2-H3
distance is observed for unfolded BDPA. We will compare the obtained distance
distribution with that obtained by numerical calculation which is based onassumption of the random coil behaviors. [1] Shortle, D. and Ackerman, M.S.,
Science, 293, 487-489 (2001)
1PT145β2ミクログロブリン部分ペプチドの運動性とアミロイド線維形成能との相関

Correlation of the dynamics of β2-microglobulin fragmentswith their amyloidogenicity

Mahoshi Takeda1, Kazumasa Sakurai1, Takahisa Ikegami1, Hironobu Naiki2,
Yuji Goto1 (1Inst. Protein Res., Osaka Univ., 2Med. Sci., Univ. Fukui.)
The tendency of the amyloid fibril formation of a peptide depends on its
sequence. Recently, several programs which can predict amyloidogenicity of agiven sequence have been presented. Although these programs predict well theamyloidogenicity of individual sequence, they do not provide information about
the dynamics of the corresponding peptide in solution. Thus, in our study, weinvestigated dynamics of various peptides and correlated them with theiramyloidogenicity. We previously reported that β2 -microglobulin (β 2m) isdigested by Achromobacter protease I into 9 fragments (denoted as K1-K9).
Among them, K3 and K6 were found to form amyloid fibrils, consistent with theprediction by Aggrescan, one of the prediction algorithms for amylodiogenic
sequences. We investigated the urea-concentration-dependent transverse
relaxation rates (R 2 ) of K2, K3, K5, K6, K7 and K9. It was found that nonamyloidogenic peptides showed only slight increase in R 2 as the urea
concentration increased, indicating expansion of the peptide chain with anenhanced solvation. K3 also showed the same dependency at high urea
concentrations. However, at lower urea concentrations, it showed an abrupt
increase in R 2 at the amyloidogenic portion of the sequence, indicating aformation of a hydrophobic cluster. From these results, we propose that an
amyloidogenicity is related to the ability of hydrophobic cluster formation. The
prediction algorithm is assumed to identify specifically such sequences.
1PT146

タウタンパク質の凝集の初期段階の解析

In vitro analysis of the early stage of aggregation of tau protein
Teikichi Ikura, Nobutoshi Ito (Medical Research Institute, Tokyo Medical and
Dental University)
Tau protein is essential to assembly of microtubules, which mainly consist of
two types of tubulins. Hyperphosphorylation of tau protein abolishes its ability
to bind tubulin and promote microtubule assembly. When it is released fromtubulin, phosphorylated tau protein aggregates into paired helical filaments,
which are characterized as the neuropathological hallmarks of Alzheimer's
disease. The mechanism of aggregation of tau protein is still unsolved, though
great efforts have been made to elucidate it. The early stage of aggregation isespecially poorly understood because of difficultly of its detection. Here we
synthesized various peptides including microtubule binding domains, whichshowed high aggregation propensity, and then investigated their in-vitro
aggregation under various conditions by fluorescence and dynamic lightscattering measurements. As a result, the tau peptides quickly aggregated into
the oligomeric forms even in the absence of inducers. Their criticalconcentration of unpolymerization was less than 1 micro-molar in the absenceof inducers. Increase in the concentration accelerated the velocity ofaggregation. Ionic strength and phosphorylation state also affected the velocity
of aggregation. The mechanism of aggregation of tau protein at the early stage
will be discussed on the basis of these results.
1PT147

MSES 法によるタンパク質間相互作用の解析

Multiscale enhanced sampling simulation of protein
interaction
Kei Moritsugu1, Tohru Terada1,2, Akinori Kidera1,3 (1CSRP, RIKEN, 2Grad.
Sch. of Agri. and Life Sci., Univ. of Tokyo, 3Grad. Sch. of Nanobiosci.,
Yokohama City Univ.)
Protein interaction plays a fundamental role for biological processes such astranscription, signal transduction and immune response. Simulating protein
association dynamics will be challenging beyond the conventional computational studies such as predicting protein complex structures and calculating freeenergy differences via residual mutations at the complex interface. Since protein
complex formation involves atomistic interaction and desolvation process, inthis study, we aim to achieve a conformational sampling of protein interaction
on atomistic resolution and under physiological condition including explicit
solvent using multiscale enhanced sampling (MSES) simulation.
The MSES simulation allows an enhanced sampling of the full-dimensional, allatom model in explicit solvent, by coupling with the accelerated dynamics of thecoarse-grained degrees of freedom. Here, this method has been applied tobarnase-barster complex which has been studied in extensive experiments andcomputations. The sampling of the relative configuration of the two proteins has
been enhanced via coupling to a Ca-atom coarse-grained model which moves
freely around the complex structure. The interaction process at the interface has
been characterized by formation of the hydrogen bonds and desolvation in
atomic detail. The MSES has also been applied to the same system with a site-− S93 −

directed mutation at the barster interface to demonstrate the role of the hydrogenbonds at the mutated residue during the complex formation.

1PT148原子間力顕微鏡を用いたβ-カテニンの力学的挙動解析

Analysis of the mechanical behavior of β-catenin using AFM
Koichiro Maki1,2, Sung-Woong Han1, Taiji Adachi1,2 (1Department of
Biomechanics, Institute for Frontier Medical Sciences, Kyoto University,
Japan, 2Department of Microengineering, Kyoto University, Japan)
In cellular activities, mechanical stimuli are functionally converted into
biochemical signals. This process called mechanotransduction has an essential
role in cell-cell communication through mechanical interaction. Cells
communicate with each other through mechanotransduction which is induced byadherens junctions (AJs) -mediated intercellular tension. According to recent
studies, AJs adapting modulate its intensity to the intercellular tension to which
the mechanical behaviors of its components contribute. However, themechanical behaviors of the components such as β-catenin and α-catenin are
unclear. In this study, we focused on β-catenin, which interestingly forms asuperhelical structure, and analyzed its mechanical behaviors using atomic force
microscope (AFM). AFM cantilever was modified using a NHS-PEGMaleimide for the glutathione modification. The N-terminus GST-tagged β catenin was then modified using a GST-glutathione affinity. The AFM-tip was
approached and made contact to the NHS-ester modified substrate to allow
outside amine of β-catenin and NHS-ester binding. Then we obtained forceextension curves for β-catenin and found a linear-elastic region in both loading
and unloading processes under small tension less than 120 pN without any
relaxation. Moreover, relaxation event was not observed under high tension
more than 1000 pN, that is, β-catenin was not unfolded. These results indicatethat β-catenin exhibits stiff mechanical behaviors and can be a molecule whichstably transmits the intercellular tension.
1PT149

PPC, DSC のコンビネーション解析により明らかになったシトク
ロム c の新規多状態熱転移

A combination analysis of PPC and DSC revealed novel multistate thermal transitions of cytochrome c
Shigeyoshi Nakamura, Shun-ichi Kidokoro (Nagaoka University ofTechnology)
Molten globule, MG, state is known as the equilibrium and/or kinetic
intremediate state which has a key role on the stability and folding mechanizumof proteins. The thermal transition of the MG state of cytochrome c at extreme
acidic pH and high salt condition showed the complicated transition rather than
the simple two-state transition. We performed differential scanning calorimetry(DSC) and pressure perturbation calorimetry (PPC) analysis of thermaltransition of the MG state at pH 2.5 with 500 mM KCl. By DSC measurements,
the protein concentration dependence of the thermal transition was clearlyobserved in the range of 0. 25~18 mg/ml cytochrome c. The transition
temperature of the MG state shifted to lower temperature by increasing
cytochrome c concentration. In all cases, the thermal transition of the MG state
showed full reversibility. It strongly suggests that the thermal transition of the
MG state at pH 2.5 with 500mM KCl is the complicated transition including thereversible oligomerization process of the disordered molecules. By PPC
measurements, the thermal expansion coefficient showed the complicated shapecomposed by the broad positive peak at lower temperature and a broad negative
peak at higher temperature. It indicates that the positive and negative volume
changes are occurred at lower and higher temperature in this transition. The
results of the multi-state analysis with self-dissociation/association process will
be discussed.
1PT150蛍光共鳴エネルギー移動を用いたスタフィロコッカルヌクレアーゼのフォールディングにおける特異的凝縮過程の研究

Site-specific collapse of staphylococcal nuclease during thefolding followed by Förster resonance energy transfer
Takuya Mizukami1, Ming Xu2, Heinrich Roder2,3, Kosuke Maki1 (1Grad.
Sch. Sci., Nagoya Univ., 2Fox Chase Cancer Center, 3Dep. Biochem. Biophys.,Univ. Penn.)
Many proteins form compact intermediates in the early stages of folding.
However, whether the condensation of the polypeptide chain is driven by nonspecific hydrophobic interactions or by specific local interactions remains

elusive. Staphylococcal nuclease consists of an N-terminal β-barrel and a Cterminal α-helical domains. Previous pulsed H/D exchange studies showed thatsome amide protons in the β-barrel domain are protected from exchange within
10 ms of the refolding. In addition, previous studies using a series of single
tryptophan variants showed that a hydrophobic core comprising a large fractionof the β-barrel domain is formed in 100 μs time range.
In the present study, the collapses within the β-barrel domain and between thetwo domains were monitored by the time-dependent change in the apparent
efficiency of Förster resonance energy transfer. Variants with single
donor/acceptor pairs within the β -barrel domain showed an increase in theefficiency in 100 μ s time range. In contrast, a variant with a single
donor/acceptor pair in the α -helical/ β -barrel domains, respectively, showedvirtually no change in the efficiency in the submillisecond time range but anincrease only after 100 ms time range. These results indicate that the collapse
within the β-barrel domain occurs in 100 μs time range, while the α-helicaldomain is recruited to the rest of the molecule in the rate-limiting step of thefolding. Thus the condensation is suggested occurring in the specific andcooperative manner.
1PT151蛍光相関分光法を用いた網羅的 in vivo 酵母タンパク質動態解析

Global analysis of protein dynamics in living yeast cells using
fluorescence correlation spectroscopy
Takafumi Fukuda1, Chan-Gi Pack2, Yasushi Sako2, Shigeko Kawai-Noma1,
Hideki Taguchi1 (1Grad. Sch. of Biotech., Tokyo Tech, 2RIKEN ASI)
In the cell, the majority of proteins does not work alone, but interacts with otherproteins or themselve, to maintain the cellular function, constituting a so-called“protein community” in the cell. Previous efforts on mass spectrometry-based
protein interaction networks, interactomes, have provided a picture on theprotein community. However, those were “static” information after cells were
disrupted. For a better understanding of the protein community, it is important toobtain “dynamic” information in living cells, such as how proteins behave in thecell, how large they are, how fast they diffuse, what portions of proteins interact
with others. Therefore, we have started a large-scale analysis on dynamic
aspects of proteins in living cells using fluorescence correlation spectroscopy(FCS), which is a technique to investigate the diffusion properties offluorescence molecules in cells. Using a budding yeast GFP clone collection,
which is a Saccharomyces cerevisiae strain collection expressing GFP tagged
open reading frames, we have evaluated i) diffusion constant, ii) the populationof fast components and slow components, iii) abundance and iv) tendency for
self-assembly, of ~200 kinds of yeast proteins in living yeast cells.
1PT152マイクロ秒分解一分子 FRET 測定による蛋白質折り畳み運動の追跡

Microsecond-resolved traces of protein folding by singlemolecule FRET measurements
Hiroyuki Oikawa1, Yuta Suzuki2, Kiyoto Kamagata1, Munehito Arai3, Satoshi
Takahashi1 (1IMRAM, Tohoku Univ., 2Grad. Sch. Sci., Tohoku Univ., 3Grad.
Sch. Arts. Sci., Univ. Tokyo)
High-speed measurements of fluorescence signals from single molecules are
expected to reveal the detailed information of protein folding. However, thetime resolution of single-molecule measurements is usually limited to a few
milliseconds. To improve the time resolution, we developed a line-confocal
microscope with two-color detection system that was combined with the sample
flow system. By using the developed device, we can trace a time evolution offluorescence intensity and FRET efficiency from single molecules with the time
resolution of 30 μs. We investigated the equilibrium unfolding transition of B
domain of protein A (BdpA). The two cysteine residues were introduced into
BdpA and labeled by a FRET pair of Alexa 633 and Alexa 488. The labeled
sample shows a typical two-state unfolding upon the guanidium chloride(GdmCl) titration. The microsecond-resolved FRET efficiency time series fromthe single BdpA molecules revealed a significant conformational heterogeneity
in the chemically unfolded state. The heterogeneity of the unfolded BdpA is
substantiated by calculating the FRET efficiency histograms at the different
GdmCl concentrations. The distribution obtained in the absence of denaturant
consists of single peak for the native protein. In contrast, the distributions
obtained in the presence of higher concentration of denaturants are broad. The
data demonstrate the presence of multiple substates, which are separated bysizable energy barriers.
1PT153− S94 −

エレメントを用いた人工酵素の設計と機能解析

Element-based molecular design can reproduce an artificial
protein with an expected enzymatic activity
Hironari Kamikubo, Eri Sakai, Yoichi Yamazaki, Mariko Yamaguchi, Mikio
Kataoka (NAIST/MS)
We have revealed that proteins are comprised of structure and function elements,and linkers connecting each of the elements [1]. We are proposing that theelements act as building blocks responsible for structure and function. Here, we
are focusing on the concept of the function element as a building block realizing afunction. Assuming that our hypothesis is correct, it can be expected that acomplete set of the function elements alone reproduces its defining function.
Using the function elements of staphylococcal nuclease (SNase) extracted before,
we designed an artificial enzyme, in which the complete set of the function
elements are connected with appropriate linkers extracted from the other protein,
TSN domain of human p100, which structurally resemble the linkers of SNase.
The resultant artificial enzyme showed the enzymatic activity for the hydrolysis
reaction seen in SNase. The enzymatic reaction by SNase is known to require a
Ca ion. The designed enzyme also clearly exhibited the Ca2+ requirement for its
hydrolytic activity, indicating that the enzymatic mechanism of the designed
enzyme remains to be that of SNase. The present results support our concept of
the element as a building block, and also suggests the usefulness of function
elements in designing functionally modified protein. [1] R. Shiba et al.
“Systematic alanine insertion reveals the essential regions that encode structureformation and activity of dihydrofolate reductase” Biophysics 7, 1-10 (2011)
1PT154

水及び非水環境におけるタンパク質の立体構造安定性

Structural stability of a protein in aqueous and nonaqueous
environments
Satoshi Yasuda1, Hiraku Oshima2, Masahiro Kinoshita2 (1Graduate School ofEnergy Science, Kyoto University, 2Institute of Advanced Energy, Kyoto
University)
A protein folds into its native structure having the secondary structures such as α-helices and β-sheets in aqueous solution. The relative content of them largely
differs from protein to protein. Such structural variability, however, is notalways exhibited in nonaqueous environment. For example, it is known that
alcohol induces proteins to form α-helices [1]. Many membrane proteins in thelipid bilayer possess only α -helices [2]. Here we investigate the structural
stability of a protein in aqueous and nonaqueous environments using our freeenergy function F and its energetic and entropic components, Λ and S. Λ is ameasure of intramolecular hydrogen bonds. -S represents the efficiency of the
backbone and side-chain packing brought by the entropic effect arising from thetranslational displacement of solvent molecules or nonpolar chains of lipid
molecules. The α-helices and β-sheets are very advantageous with respect to Λ
and -S [3]. In terms of Λ, the α-helices are even more stable. We find thefollowing. In aqueous solution, the entropic effect is so strong that the β-sheets
are often chosen especially for the close packing of side chains. In nonaqueous
environment, by contrast, the entropic effect is considerably weaker and Λ
becomes more imperative in the structural stability. As a consequence, there is astrong tendency that the α-helices are exclusively chosen. [1] M. Kinoshita etal., J. Am. Chem. Soc. 122 (2000) 2773. [2] J. U. Bowie, Nature 438 (2005)
581. [3] S. Yasuda et al., J. Chem. Phys. 132 (2010) 065105.
1PT155

抗プリオン化合物のプリオンタンパク質への結合特性による分類

Characterization of anti-prion compounds according to thebinding properties to the prion protein
Yuji O. Kamatari1, Yosuke Hayano2, Keiichi Yamaguchi2, Junji HosokawaMuto2, Kazuo Kuwata2 (1Life Sci. Res. Center, Gifu Univ., 2Unit. Grad. Sch.
Drug Dis. Med. Info. Sci., Gifu Univ.)
To date, a variety of anti-prion compounds have been reported that are effective
in ex vivo and also in vivo treatment experiments. However the molecularmechanisms of most of these compounds remain unknown. Here we classified
anti-prion mechanisms into four categories; I: conformational stabilization, II:interference with the interaction between the cellular form of prion protein(PrPC) and the scrapie form (PrPSc), III: precipitation of prion proteins, and IV:interaction with proteins other than PrPC. To characterize the anti-prion
compounds according to this classification, we determined their binding
affinities to PrPC and their binding sites in PrPC using the surface plasmon
resonance and NMR spectroscopy, respectively. GN8 and GJP49 bind
specifically to the hot spot in PrPC, and act as ‘medicinal chaperones’ tostabilize the native conformation and interfere with the interaction between
PrPC and PrPSc. Thus mechanism I & II are predominant. While quinacrine andepigallocathechin bind to PrPC rather non-specifically. They may mainlyinterfere with the intermolecular interaction, and mechanism II may be applied.
On the other hand Congo red and pentosan polysulfate bind to PrPC and cause
its precipitation reducing the effective concentration of prion protein. So,
mechanism III is appropriate. The proposed characterization of diverse antiprion compound would help understanding their anti-prion activities as well as
facilitating further effective anti-prion drug discovery.
1PT156糖タンパク質 A の変性温度、可逆性及び熱によって誘起される重合体の解析

Conformational stability, reversibility and heat-induced
aggregation of glycoprotein A
Takafumi Iwura, Fumio Arisaka (Graduate School of Bioscience andBiotechnology, Tokyo Institute of Technology)
In order to elucidate the relationship of conformational stability, reversibility ofheat-induced denaturation and aggregation of protein, the conformation, Tm andreversibility of denaturation of glycoprotein A were measured by circulardichroism (CD) and differential scanning micro-calorimetry (DSC) in aqueous
solutions at a number of pH values. Under the same pH conditions, solutions ofglycoprotein A were incubated at 37° C and analyzed by size exclusion
chromatography (SEC), SDS-PAGE, fluorescence from di-tyrosine, CD, and
SEC in the presence of 4,4’-dianilino-1,1’-binaphthyl-5,5’-disulfonic acid (BisANS) to quantitate and characterize heat-induced aggregation of glycoprotein
A. It was observed that the conformational stability of glycoprotein A waslowered at lower pH based on the lower Tm with endothermic ΔH, whereas thereversibility of the protein was decreased at higher pH. Glycoprotein A formedsome fractions of large non-covalent aggregates during incubation at lower pH,
whereas incubation at higher pH tended to cause formation of covalent dimers
including disulfide bond and di-tyrosine without forming larger aggregates.
These results indicated that lower conformational stability caused non-covalentaggregation, whereas the covalent aggregation resulted in lower reversibility.
Thus the evaluation of both stability and reversibility is necessary to develop
optimal formulations and to predict the kinds of aggregation which could be
induced during storage.
1PT157Structural properties and folding process of hNck2 SH3 domain

Yoshitaka Matsumura1, Masaji Shinjo1, Tsutomu Matsui2, Kaoru Ichimura1,
Jianxing Song3, Hiroshi Kihara4 (1Dept. Phys., Kansai Med. Univ., 2Bio-SAXS,SSRL, 3Dept. Biochem., Yong Loo Lin School of Medicine, Dept. Biol. Sci.,
Facul. Sci., National Univ. Singapore, 4SR center, Ritsumeikan Univ.)
Liu and Song (1) reported that hNck2 SH3 domain takes equilibrium non-nativealpha-helix intermediate at acidic pH, though the protein takes native beta
structure at neutral pH. To elucidate the structure in more depth, we did CD and
X-ray slolution scattering of this protein at various pH. First, we didconcentration dependence of the protein at neutral pH by X-ray scattering, andfound that this protein forms monomer at low concentration, but dimer at
relatively higher concentration at pH 6. We also did pH titration of the protein atc. a. 1 and 3 mg/mL, monitored by CD and X-ray solution scattering,
respectively. I(0) shows the protein takes monomer below pH 2, and dimer
above pH 4. Kratky plots show that the shape of hNck2 SH3 domain at acidic
pH is not so compact as molten globule state is. According to these findings, theprotein seems to take like C-state. We have also done kinetic refolding
experiments by cryo-stopped-flow monitored by CD. The results show that
there were no burst phases even at low temperatures. We will discuss therelationship of kinetic intermediate onf the refolding process and equilibrium
intermediate at acidic pH of hNck2 SH3 domain, and the relationship of thefolding process of hNck2 SH3 and other SH3 domains at the poster. (1) Liu andSong (2008) Biophys. J. 95, 4803-4812
1PT158天然変性蛋白質 p53 TAD による CBP TAZ2 への極めて速い結合反応

Extremely rapid binding of the intrinsically disordered p53transactivation subdomain with the TAZ2 domain of CBP
Munehito Arai1,2,3, Josephine C. Ferreon2, Peter E. Wright2 (1Dept. Life Sci.,
Univ. Tokyo, 2Dept. Mol. Biol., The Scripps Res. Inst., 3PRESTO, JST)The tumor suppressor activity of p53 is regulated by interactions with thegeneral transcriptional coactivator, CREB-binding protein (CBP). We previously showed that the AD2 region of the intrinsically disordered N-terminaltransactivation domain of p53 tightly interacts with the TAZ2 domain of CBP.

− S95 −

To study the detailed structure of the TAZ2:p53 AD2 complex, here we perform
NMR HSQC titrations in which the unlabeled p53 AD2 peptide was added
incrementally to 15 N-labeled TAZ2. The cross peaks in the spectrum of the
TAZ2 domain shift in fast exchange and exhibit curvature, indicating thepresence of two binding sites. Global fitting of the titration curves show that
dissociation constants for primary and secondary binding are 32 nM and10. 2 M, respectively. This small Kd in nM range is amazing, because the
HSQC cross peaks show fast-exchange shifts, which indicate rapid dissociation.
To estimate the association and dissociation rates, kon1 and koff1 , respectively,
we perform global fitting of lineshapes of a series of titrations. We find a kon1 of
1. 7 × 1010 M-1 s-1, which is extremely fast but not beyond the diffusioncontrolled limit. koff1 is estimated to be 540 s-1 , which is reasonably fast toshow fast-exchange shifts. We suggest complementary electrostatics between
AD2 (-8) and TAZ2 (+14) plays a major role in determining the very rapid
binding kinetics of the AD2 motif. It is plausible that a large net charge causes
both intrinsic disorder and rapid binding, suggesting that fast association is an
intrinsic nature of IDPs.
1PT159STABILITY OF FOLDING INTERMEDIATE AFFECTS
THE AMYLOIDOGENIC PROPENSITY OF
IMMUNOGLOBULIN LIGHT CHAIN VARIABLE
DOMAIN
Yuta Kobayashi1, Yuki Tashiro2, Kyohei Ikeda2, Tetsuyuki Abe2, Daizo
Hamada1 (1Grad. Sch. Med., Univ. Kobe, 2Sch. Med., Univ. Kobe)
Immunoglobulins (Ig) consist of a set of heavy (HCs) and light chains (LCs),
and both chains contain variable and constant domains belonging to Ig fold.
Intrinsic sequence variation in the variable domains provides Ig the variation inmolecular recognitions for these anti-pathogenic agents. However, this also
provides a risk to generate the aggregation-prone Ig domains to cause diseases
such as AL amyloidosis.
Here we analysed folding and unfolding kinetics of isolated variable domains(VL) from amyloidogenic BRE and nonamyloidogenic REI kLCs which wereoriginally isolated from patients of AL amyloidosis and multiple myeloma,
respectively. Although these proteins share 85% of sequence identity, the native
state of BRE VL is significantly decreased due to the increased unfolding andthe decreased refolding rates compared to REI VL, suggesting the destabilisationof the native state play important role in amyloid formation by BRE VL. A close
analysis on the folding/unfolding rates, thermal stability of the native states andaggregation propensity of several chimeric mutants between REI and BRE VL
suggested that the stability of folding intermediates is one of the most importantfactors to determine the aggregation propensity of VL, and the stability of
unfolded state ralative to the native state may be less relevent.
1PT160Recognition of a TAR nucleic acid by HIV-1 Tat,an intrinsically disordered protein
Kohei Sodeyama1, Yuuki Hayashi1, Takashi Nakamura2, Koki Makabe2,
Kunihiro Kuwajima2, Munehito Arai1 (1Dept. Life Sci., Univ. Tokyo, 2OkazakiInst. Integr. Biosci.)
Previously, native protein has been considered to have one rigid tertiary
structure and exerts its function associated with its structure. Therefore, proteins
which have no defined structures have been considered less important. Recently,
however, it has been discovered that intrinsically disordered proteins (IDPs) are
functional and structured when they recognize their target molecules. Human
immunodeficiency virus (HIV-1) transcriptional activator Tat is known as an
IDP, and binds to the trans-activating responsive element (TAR) of HIV-1
RNA. Because Tat-TAR interaction is essential for virus gene expression andreplication, Tat has been a target for anti-HIV drug development. However, themechanism of TAR recognition by Tat remains elusive. To address thisproblem, we study the Tat-TAR binding reaction by thermodynamic,
spectroscopic, and kinetic approaches. As a model protein, Tat(37-72)C37W
including the TAR recognition site is used. Fluorescence titration andisothermal titration calorimetry measurements reveal the dissociation constant at
4 nM. The association and dissociation rate are measured by stopped-flow
fluorescence experiments. We also measure the Tat-TAR binding by NMR
titration. Detailed results will be presented in the poster.
1PT161

NRSF/REST の競合誘起天然変性による mSin3 の構造安定性制御
Control of Structure Stability of mSin3 by FrustrationInduced Intrinsic Disorder of NRST/REST

Katsuyoshi Matsushita1,2, Macoto Kikuchi1,3,4 (1Cybermedia Center, Osaka
University, 2Institute for Protein Research, Osaka University, 3Graduate
School of Science, Osaka University, 4Graduate School of Frontier
Biosciences, Osaka University)
Protein intrinsic disorder has various biological roles [1]. In particular,hydrophobic intrinsic disorder region (HIDR) exhibits a peculiar coupledbinding and folding with a target protein. Much effort has been devoted to theinvestigation of HIDR in the last decade. However, understanding of biologicalroles of HIDR seems not to be sufficient. In order to gain deep insight into theroles, we have theoretically investigated NRSF/REST (neuron-restrictive
silencer factor / RE1-Silencing Transcription factor) [2]. Recently, on the basisof calculation results of a model of NRSF/REST binding mSin3, we suggested
the possibility that intrinsic disorder of HIDR is induced by structural frustration[3]. In the present work, we further investigated the stabilization of mSin3 in thebinding. Evaluating change in structural order parameter of mSin3 withincreasing chemical potential of the binding, we showed that the structure ofmSin3 is sharply stabilized at a specific value of the chemical potential. In
contrast, the stabilization of the structure of mSin3 is comparatively gradual in
the case without the frustration, namely without the intrinsic disorder. From
this result, we concluded that the frustration-induced intrinsic disorder enhances
response sensitivity of mSin3 to concentration change of NRSF/REST. [1] P.
E. Wright and R. J. Dyson J. Mol. Bio. 293 (1999) 321. [2] M. Nomura et al., J.
Mol. Bio. 354 (2005) 903; J. Higo et al., J. Am. Chem. Soc. 133 (2011) 10448.
[3] Seibutsu Butsuri Supplement 1 51 (2011) S133.
1PT162

リゾチームの乾燥誘導凝集に対する G3LEA タンパク質及びモデ
ルペプチドの抑制効果

Measurement of the association constant of a G3LEA protein
or its model peptide with lysozyme in aqueous solution
Kentaro Yamakawa1, Takao Furuki1, Rie Hatanaka2, Hiroyuki Furusawa3,
Takahiro Kikawada2, Yoshio Okahata3, Alan Tunnacliffe4, Minoru Sakurai1(1Center for Biological Resources and Informatics, Tokyo Inst. Tech., 2National
Institute of Agrobiological Sciences, 3Department of Biomolecular Engineering,
Graduate School of Tokyo Inst. Tech., 4University of Cambridge, Department ofChemical Engineering and Biotechnology)
Group3 Late Embryogenesis Abundant (G3LEA) proteins are responsible forthe occurrence of desiccation tolerance in many anhydrobiotic organisms, but
the functional mechanism remains unclear. The major part of the primary aminoacid sequence of a G3LEA protein is composed of several times repeats ofcharacteristic 11-mer motif. In our previous studies (T. Shimizu, et al.,Biochemistry 49 (2010) 1093, T. Furuki, et al., Biochemistry 50 (2011) 7093),
we synthesized several kinds of 22-mer peptides (LEA peptides), each of which
has two tandem repeats of the 11-mer motif, and studied their structural andthermodynamic properties in detail. Recently, we have investigated to what
extent these peptides reduce aggregation of proteins (e. g. lysozyme) during
dehydration and compared the result with that from the similar experiment usinga native LEA protein (A. Avenae G3LEA protein, AavLEA1) (T. Furuki, et al.,BBA 1824 (2012) 891). As a result, it was found that the LEA peptide reduced
the aggregation of lysozyme during dehydration, whereas AavLEA1 promoted
the aggregation. In this study, we tried to measure the association constant (Ka)between AavLEA1 and lysozyme using the Quartz Crystal Microbalance(QCM) method. The interaction between lysozyme and AavLEA1 wasmoderately strong with the Ka value of 3. 2 x 106 M-1. In the case of thelysozyme-LEA peptide system, a change of the QCM frequency was observed
but the Ka value was too small to be quantified. These results were interpreted
from the pI value differences between the proteins studied.
1PT163

表面力測定によるシグナル伝達タンパク質間相互作用の研究

Interactions between signal transduction proteins studied bysurface force measurement
Hitomi Fujiwara1, Taizo Umemura1, Masaya Fujita2, Kazue Kurihara1,3(1Institute of Multidisciplinary Research for Advanced Materials (IMRAM)
Tohoku, 2University of Houston, 3World Premier International-Advanced
Institute for Materials Research)
Spoluation in Bacillus subtilis is controlled by phosphorelay signal transduction.
KinA and Spo0F are involved in this sporulation initiation process. In the firststep of this series of phosphorylation reactions, KinA binds ATP and
autophosphorylates at a histidine residue. The phosphoryl moiety on KinA isthen transferred to a single-domain response regulator Spo0F. We have studiedinteractions between these signal transduction proteins (KinA and Spo0F) using− S96 −

colloidal probe atomic force microscopy (AFM). KinA and Spo0F were
immobilized on a glass substrate and a glass sphere using Langmuir Blodget
films of a chelate amphiphile. The conditions of adsorbing proteins were
determined using quartz crystal microbalance and AFM. Surface force profiles
for KinA and Spo0F showed only repulsive force in the buffer solution
with/without ATP. Adding cofactor Mg2+ and Ca2+ to buffer solution with
ATP, attractive forces and adhesive forces were observed on approach and in
retraction process. These forces were not observed without ATP . These results
demonstrated the specific interaction between KinA and Spo0F only in thepresence of a substrate ATP and cofactor divalent ions.
1PT164GroEL 円順列変異体 CP86 のストップトフロー機能解析
Functional and Stopped-flow analysis of circularly permutated
GroEL mutant CP86

Toshifumi Mizuta1, Tatsuya Uemura2, Takuma Hirayama1, Kunihiro
Hongo1,2, Yasushi Kawata1,2, Tomohiro Mizobata1,2 (1Dept. of Chem. andBiotech., Grad. Sch. of Eng., Tottori Univ, 2Dept. of Biomed. Sci., Grad. Sch.,
Tottori Univ)
Escherichia coli GroEL is a member of the chaperonins, a well-conserved
protein folding assistant necessary for cell growth. The GroEL subunit consistsof three different functional domains. The apical domain contains GroES andsubstrate protein binding sites, the equatorial domain contains the ATPasebinding site which controls the overall functional cycle, and these domains arelinked by the intermediate domain.
We have used random circular permutation to study the structural and functional
characteristics of the GroEL subunit. In a previous study, we constructed acircularly permuted mutant of GroEL (GroEL CP86) that relocated the N
terminus and C terminus from its original position to a new site in the vicinity of
the ATPase binding site. This mutant was characterized by a ~50% decrease inits basal ATPase activity and, interestingly, a change in specificity toward
substrate proteins in refolding assays.
To probe the effects of this circular permutation in more detail, we introduced afluorescent tryptophan residue to monitor the dynamic movements of the CP86
apical domain (GroEL CP86 R231W). Stopped-flow fluorescence experimentsthat probed movements of the apical domain showed that almost all of the large
domain movements that were detected in the wild-type protein were lost in the
mutant. In spite of this, CP86 was able to assist the folding of MDH efficiently.
Functional assays provided details regarding a possible functional mechanism
for this mutant.
1PT165

Design of functional miniature peptides acting like the lipase
Yoshihiro Iida, Atsuo Tamura (kobe university)
Obesity has become a huge problem in modern life. It is a condition that thelipid droplet enlarges in cell. To avoid obesity, we should hydrolyze triglyceride(neutral lipid) in lipocyte. As a candidate for an anti-obesity drug, we tried todesign peptides having enzyme activity like the lipase to expose and hydrolyze
triglyceride. As a strategy for the design, we make the peptide to have thecatalytic triad composed of histidine, asparagine acid and serine. When themolecule forms the dimer, the two serine residues coming from each molecule
hold the triglyceride together. We make use of alpha-helical peptides that form
coiled-coil when dimerized. Based on the strategy, we synthesized peptides
me1-4. CD (Circular Dichroism) measurements show that the conformations ofme2, me3, and me4 are alpha helical coiled-coil. DLS (Dynamic Light
Scattering) measurements indicate that the particle sizes of designed peptides
are around 4nm, suggesting that they are dimers. The calcein leakage
experiment showed that me3 and me4 hydrolyzed the membrane. The
hydrolysis of p-nitrophenyl acetate (pNPA) showed that me4 had been
conferred esterase activity. Based on these result, we conclude that the designed
peptide can be regarded as a lipase mimic which is capable of hydrolyzing thelipid droplets.

1PT166異なるリガンドと結合した DHFR の構造転移のカメレオンモデル
による比較

Comparison of the conformational transitions of DHFR boundto the different ligands using the chameleon model
Toru Kimura1, Masaki Sasai2, Tomoki P. Terada2 (1Dept. of Applied Physics,
Grad. Sch. of Engineering, Nagoya Univ., 2Dept. of Computational Scienceand Engineering, Grad. Sch. of Engineering, Nagoya Univ.)
A well-characterized protein, dihydrofolate reductase (DHFR), has the

enzymatic reaction rates that are correlated with the conformational changes ofthe protein (Boehr et al., Science 313, 1638 (2006)). Its reaction cycle consistsof five chemical steps, which can be described as the conformational switches
between roughly two structures, the occluded and closed structures. Among
those five steps, we here focus the two steps connecting the similar pair ofprotein structures in the opposite directions. Since different ligands are bound to
DHFR in these two reactions, the difference in the two reaction rates should be
intrinsically related to the difference in the ligand-protein interactions. To
calculate the free energy landscape with the different ligands, we have extended
the chameleon model to include the ligand-protein interactions, so that theinteractions between ligand and protein is dependent on their local
environments, as is the case of intraprotein interactions. Here, using thisextended model, we examine the conformational transitions of DHFR in both
directions. From the calculated free energy landscapes, we discuss the transition
state structure and changes in thermodynamic parameters accompanying thesetwo conformational transitions. Furthermore, we will examine the mutational
effect on the chemical reaction rate of DHFR, which is experimentally measured
recently (Bhabha et al., Science 332, 236 (2011)).
1PT167

DHFR のフォールディング過程の多次元自由エネルギー曲面

Multidimensional free energy landscape of folding of DHFR
Takashi Inanami, Tomoki Terada, Masaki Sasai (Dept. of Comp. Sci. Eng.,
Univ. of Nagoya)Free energy landscape is a useful theoretical tool to analyze pathways andintermediates of protein folding. Multiple folding pathways or multiple
intermediates, however, are overlapped with each other to become indistinguishable in the one-dimensional landscape drawn with a single order parameter.
In this study, in order to analyze the distributions of multiple intermediates, weconstruct the two- and three-dimensional landscapes by using multiple orderparameters of folding. As an example, we calculate the multidimensional freeenergy landscape of folding of dihydrofolate reductase (DHFR) and discuss thefolding intermediate recently observed by M. Arai et al. (J Mol Biol (2011) 410:
329-342). For this simulation, we develop the extended Wako-Saito-MunozEaton model which correctly describes the tendency that regions near the N- andC- termini have the ordered structures to some extent at the early phase offolding.
1PT168

Effect of flexibility and electrostatic interaction on rapid
binding and unbinding of an intrinsically disordered protein
Koji Umezawa1, Kohei Nishimura1, Junichi Higo2, Haruki Nakamura2,
Mitsunori Takano1 (1Grad. Scl. of Adv. Sci. & Eng., Waseda Univ., 2IPR,
Osaka Univ.)
It is now well known that there are a large number of proteins that are disordered
under a physiological condition. The intrinsically disordered proteins (IDPs),
which are generally highly charged, are thus more flexible than ordered proteins(OPs). One of the interesting features of IDP is as the so-called “coupled folding
and binding”, which implies that IDP may adopt different bound conformations
depending on its partner proteins, leading to the “hub” property of IDP. The
coupled folding and binding of IDP also implies the chain-entropy loss uponbinding is greater than OP. Furthermore, IDP can capture distant partners via its
extended conformation and long-range electrostatic interaction. To examine
these potential capabilities of IDP, in this study, we focused on the effect of
flexibility and electrostatic interaction upon binding of KID, a well-studied IDP,
to its binding partner KIX, by using a combination of coarse-grained model andan enhanced sampling technique. The potential of mean force (PMF) showedthat increased flexibility destabilizes the final complex but stabilizes theencounter complex, which is suitable for rapid binding and unbinding. The
electrostatic interaction also contributed to the stability of the encountercomplex. In addition, we address the possible role of the phosphorylation of
KID (pKID), which has stronger binding affinity for KIX, in terms of thecompetition of the intra- and intermolecular electrostatic interactions.
1PT169Calculation of local dielectric constants of proteins using
dipole-dipole correlations
Asahi Konno, Jun Narita, Kentaro Fukunaga, Koji Umezawa, Mitsunori
Takano (Grad. Scl. of Adv. Sci. & Eng., Waseda Univ.)
In a classical dielectric model of the protein systems, the dielectric constants areusually assumed to be homogeneous inside and outside the protein, respectively.
However, it is obvious that this assumption is the zero-th order approximation of− S97 −

reality. The dielectric constant is expected to vary depending on the local
electrostatic environments and the structural constraints imposed on the system.
In this study, we calculated the local dielectric environments of protein-water
systems by employing the theoretical framework proposed by Guest et al.
(PCCP, 2011) where the dipole-dipole correlations are properly taken into
account. We used long molecular dynamics simulation trajectories of the threesystems (solvated myosin, solvated actin, and Fo in the solvated lipid bilayer
systems) to evaluate the dipole-dipole correlations. From the local dielectric
constant map, we found that the regions around the flexible loops tend to exhibit
a large dielectric constant. The regions with a large dielectric constant aresupposed to respond well to the approaching charges of the binding partner andstabilize the electrostatic self-energy of the charges. Similarly, we will discuss
the ion translocation path in Fo portion of ATP synthase in terms of the local
dielectric constant map. We also point out a technical problem that calculated
dielectric constants sometimes become too large or even negative, and discuss apossible solution to this problem.
1PT170シグナル伝達に関与する PDZ ドメインの分子動力学データに対す
る時系列解析

The time series analysis of molecular dynamics data about
PDZ domain in synapse
Naoko Kishida1, Hiroshi Fujisaki2, Mikito Toda3 (1Graduate School of
Humanities and Science, Nara Women's University, 2Nippon Medical
School, 3Faculty of Science Nara ,Women's University)
The functions of a protein are supposed to have relation to its structures. We
study the relationship between its functions and movement from data ofmolecular dynamics(MD) simulation. We analyse the protein called the PDZ
domain. For human being,we can find them in a synapse of the nervous system.
In particular, these proteins exist mainly in postsynaptic cells as domains ofrecepters. They play an important role in signaling in the nervous systems[1].
We chose one of the PDZ, and its PDB code is 1BE9. It is the third domain of
PSD-95, where PSD-95 is a recepter which is in the center of the postsynaptic
cell. For the signaling,the clustering of domain is essential, and is caused byinteraction with other proteins. Here, we pay our attention to the mechanism of
signaling. It is shown that it has a path within the domain and signals aretransduced along this path[2]. We study its motion with wavelet transform andcanonical correlation analysis. First, by wavelet transform,we can find the timedependent spectra and find which parts of the secondary structure move withlarge amplitude. Second, in order to find whether we can see the flow of thermalenergy along this path at room temperature, we compare the correlations
between amino acids by canonical correlations analysis.
[1]E.Kim and M.Sheng.2004.Nature review neurosience,5,771-781[2]N.Ota and D.A.Agard.2005.J.Mol.Bio.351,345-354[3]A.Dyle,et al.1996.Cell,85,1067-1076
1PT171

粗視化シミュレーションによる 3 ドメインタンパク質 suf1 のコ・トランスレーショナルフォールディングの研究

Co-translation folding of a three-domain protein surf1 studied
by coarse-grained molecular simulations
Tomohiro Tanaka, Naoto Hori, Syoji Takada (Dept. of Biophys., Kyoto Univ.)
Proteins are synthesized linearly from N-terminus in ribosome and gradually
extruded from it. The native interactions of extruded polypeptide chain may beinitiated before the protein synthesis is completed. Protein folding which
proceeds during the synthesis is termed co-translational folding. Because total
conformation space of a polypeptide chain under translation is smaller than thatof the full-length polypeptide chain of the protein, co-translational folding could
be facilitated kinetically. In addition, the rate of translation is not uniform, but isaffected by tRNAs abundance. Recently, it was found that the correct folding of
suf1, a three-domain protein, is maintained by the particular use of rare codons.
When the rare condos were replaced with codons by synonymous mutations,
successful folding yield substantially decreased. However, the detailed
mechanism of this phenomenon is unclear. To understand the mechanism, wedeveloped a coarse-grained (CG) model and simulated suf1 co-translational
folding as well as post-translational folding as a control. The CG model is a
mixture of Go like atomic interaction based CG (AICG) model and generichydrophobic interactions which makes correct folding and misfolding possible.
Via comprehensive simulations, we address the difference between co- andpost- translational folding.

1PT172

粗視化シュミレーションによるサテライトタバコモザイクウィルスの安定性とダイナミクス

Stability and dynamics of satellite tobacco mosaic virusesstudied by coarse-grained simulations
Koji Ono, Naoto Hori, Shoji Takada (Dept of Biophys, Kyoto Univ)
Satellite tobacco mosaic virus (STMV) is one of the smallest (about 170
angstrom diameter) and icosahedral virus (T = 1). The virus consists of 60
copies of single proteins to make a icosahedral capsid and a 1058-base RNA
genome. The existence and importance of the viral genome for its stability havebeen acknowledged. Recently, STMV was analyzed by an all-atom MD
simulation. The research indicated that their RNA is important to stable theircapsid structure. All-atom simulation is, however, limited of the system size andthe time scale. To overcome them, we used coarse-grained simulation (CafeMol
developed by Our lab) to simulate STMV virus for longer time that all-atom MD
could do. In this simulation, each amino acid is represented as a CG bead andinteractions are modeled with structure-based potential. We first carried out
simulation of STMV without their RNA in various environments to investigate
the STMV particle stability and dynamics. For example at low ion strength orhigh temperature, how the virus is deformed has been studied. We discuss
importance of the RNA genome for the stability of this virus.
1PT173

エピレギュリンー抗体の分子動力学シミュレーション

Molecular dynamics simulation of epiregulin and its antibody
Keiko Shinoda, Hideaki Fujitani (Laboratory for Systems Biology andMedicine, Research Center for Advanced Science and Technology,University ofTokyo)
We present a molecular dynamics study of protein target for cancer, epiregulin
and its antibody. Epiregulin is a member of the epidermal growth factor family
and stimulates EGFR-driven cancer cell growth such as pancreatic cancer. To
investigate the structural stability and thermodynamic property of theepiregulin-antibody complex, we have performed 1-μ s molecular dynamics
simulations and free energy calculations. In this meeting, we discuss the resultsof the simulations and compare with experimental results.
1PT174

Molecular Dynamics Simulation of Protein Using Robot
Dynamics Algorithm

Yu Yamamori1,2, Kazuhiro Takemura1, Akio Kitao1,2 (1Institute of Molecular
and Cellular Biosciences The University of Tokyo, 2Department of computational
biology Graduate school of frontier science, University of Tokyo)
In the simulation study of biophysical phenomena, the gap between the timescale of the simulation and that of the target process is a major bottleneck ofCartesian Molecular Dynamics (CMD). High frequency motions in proteinsprohibit CMD from enlarging the simulation time step beyond 1.0-2.0 fs. To
overcome this limitation, we adopted a strategy to consider the torsion angles as
the only degrees of freedom (DOF) which enables us to employ a method
developed originally in robotics, namely Articulated-Body Algorithm (ABA),
which can solve the equations of motion of internal coordinates with the small
computational cost that linearly depends on the number of DOF. The programof Molecular Dynamics in Torsion Angle space (TAMD) is equipped with anextended version of ABA, so-called Freed-end ABA developed by the authors.
The program can treat the AMBER and ECEPP force field and with NVE and
NVT ensembles. To evaluate our program, we compared the spectra of 20
distinct dipeptides calculated by TAMD to the results of the normal mode
analysis in torsion angle space. From good agreements in this comparison, we
confirmed that the program can give reasonable results. We also searched the
maximum time step length to give reasonable energy conservation based on thesimulations of three proteins, villin head piece subdomain, BPTI and ubiquitin.
We concluded that time step 4.0 fs can be employed with the original force field,
and 6.0 fs can be employed if we scale the torsion energy terms of the terminal
chemical groups.
1PT1754 量体型サルコシン酸化酵素の酵素-基質複合体の分子動力学的解析
Analysis of enzyme-substrate complex of heterotetramericsarcosine oxidase by molecular dynamic simulation

Akinori Hiroshima, Haruo Suzuki, Shigetaka Yoneda (Grad. Sch. Sci.,
Kitasato Univ.)− S98 −

Heterotetramic sarcosine oxidase [SO, EC 1. 5. 3. 1] catalyses the oxidative
demethylation of sarcosine to generate glycine, hydrogen peroxide, andformaldehyde or 5,10-methylene-tetrahydrofolate, depending on the availabilityof tetrahydrofolate. SO was first purified from Corynebacterium sp. U-96 [SOU96], and then from various sources. SO-U96 comprises four non-identicalsubunits (α, 110 kDa; β, 44 kDa; γ, 21 kDa; δ, 10 kDa) and one FAD, one
FMN, and one NAD+. The 3D structure of the SO-U96-complex withdimethylglycine [DMG] has been determined from X-ray crystallography. The
structural and biochemical analyses gave new insight into the cavity,
conformational change, and channeling of oxygen, sarcosine, and products
through the cavity. The authors performed the free energy perturbation
molecular dynamics simulations in which the bound DMG was mutated by the
coupling parameter approach into sarcosine, and analysed the simulated
enzyme-substrate complex from the structural and energetic viewpoints. The
mobility of substrate was also analyzed to examine the possibility of realistic
simulations of channeling of substrate and products. The GROMACS program,the GROMOS 53a6 energy parameters, and the SPC water parameters, and theparticle mesh Ewald method were used. The numbers of the atoms were from
130 000 to 160 000, depending on the number of the water molecules includedin the simulations.
1PT176アデニル酸キナーゼ反応機構に関する ONIOM 法による研究

Study on the reaction mechanism of adenylate kinase with
ONIOM method
Kenshu Kamiya (Department of Physics, School of Science, Kitasato
university)
Adenylate kinase catalyzes the reaction: ATP + AMP + Mg2+ -> ADP + ADP+ Mg2+ . In order to elucidate the nature of the reaction, we have been studying
the theoretical model using MM/QM. We constructed the complex structure of
enzyme and substrates, ATP and AMP with Mg ion, based on the PDB data ofcomplex with inhibitor. After the full optimization of the whole molecule invacuo with MM, we put TIP3P water molecules arround catalytic center Mg ion
with spherical boudary condition, and harmonic constraint potentials on thesurrounding atoms in the boudary region. Some model structures were sampledfrom 900 K MD simulation followed by structural nergy minimizations. In orderto obtain the resonable initial model of the transition state, we performed themolecular mutation calculation from the reactant to the product. Further
truncated models were used for the calculations with ONIOM method, thereactant, product, transition structures were optimized. In these calculations,
small barrier (several kcal/mol) shown in the low level model (19/834 atoms in
QM/MM, HF/6-31G(d):AMBER) vanishes with higher level, and the present
highest model (89/764 atoms in QM/MM, B3LYP/6-31+G(d):AMBER, embedmodel) gives the reaction barrier of 19 kcal/mol, and 10 kcal/mol exothermicity.
For further discussion, however, more qualitative consideration as well as freeenergies on the conformational effects of the surrounding atoms is necessary.
1PT177

酵素の多機能性に関する解析

Analysis of enzyme promiscuity
Chioko Nagao, Kenji Mizuguchi (NIBIO)
For a long time, enzymes are believed to catalyze specific reactions withspecific substrates. Specificity is essential for enzymes to work in the crowded
environment of a cell. However, recent experimental studies have shown thatmany enzymes can catalyze more than one reaction or have broad substrates (i.
e., enzyme promiscuity). It is becoming recognized that enzyme promiscuity isrelated to functional evolution, drug metabolism and protein engineering [1].
Although molecular mechanisms of promiscuity of each enzyme have been
investigated, there have only been a few attempts to define the generalproperties that characterize promiscuous enzymes, and no comprehensive
analysis using reaction constants have been made [2]. Moreover, a goodmeasure for describing promiscuity has not been established.
In order to elucidate how widespread the substrates and reactions are that an
enzyme can bind and catalyze, we performed a systematic analysis of the
enzyme data in the BRENDA database [3]. The enzymes 1) for which UniProt
ids were obtained from BRENDA or through PubMed ids and NCBI Entrez
links, and 2) which have Michaellis constants or turnover numbers for at leastten different substrates were selected for the analysis. At the fourth level ofEnzyme Commission Number and within an orthologous enzyme group, therelationships among sequence identities, reaction constants and substrate
similarities are discussed.
1. Nat Biotechnol 2009;27:157. 2. Biochemistry 2008;47:157. Bioinformatics
2010;26:2012. 3. Nucleic Acids Res 2009;37:D588.

1PT178

Folding of topologically complex proteins by an atomic
interaction based coarse grained model

Wenfei Li1,2,3, Tsuyoshi Terakawa2, Wei Wang1, Shoji Takada2,3 (1National
Laboratory of Solid State Microstructure and Department of Physics, Nanjing
University, 2Graduate School of Science, Kyoto University, 3CREST, Japan
Science and Technology Agency)
While fast folding of small proteins has been relatively well characterized byexperiments and theories, much less is known for slow folding of larger
proteins. Recent progress of experimental works on the folding of large proteins
revealed a number of new features arising from the topological complexity,
which poses new challenges to the current protein folding theories. Reasonably
describing the folding of such topologically complex proteins needs moresophisticated theoretical models. In this presentation, we introduce a newmodel, i. e., AICG model, developed within the framework of the energy
landscape theory. The features of this new model include: i) contact interactions
are delicately weighted according to the atomic interactions via a multiscale
protocol; ii) a realistically flexible energy function is used for the local
interactions. We applied this model to a number of topologically complex
proteins, including multi-domain proteins and knotted proteins, and it
reproduced all the major features of folding revealed experimentally. Our results
suggest that the structure-based model can capture the essential features of thefolding of proteins with complex topologies after delicately weighting thecontact interactions, and including realistic local flexibilities.
1PT179多様な分子コンホメーションを取り得る溶質の小角 X 線散乱プロ
フィルの近似計算法

A new approximation method for estimating SAXS profiles ofsolutes with multiple molecular conformations
Yasutaka Seki1, Shigeyoshi Nakamura2, Shun-ichi Kidokoro2, Takamasa
Nonaka1, Kunitsugu Soda3 (1Sch. of Pharm., Iwate Med. Univ., 2Dept. of
Bioeng., Nagaoka Univ. of Technol., 3High Perform. Molec. Simula. Team,
ASI, RIKEN)
Small-angle X-ray scattering (SAXS) has long been used as an important meansfor determining global-structure parameters of biopolymers in solution. It can beapplied not only to natively globular proteins with a practically single global
structure but also to natively unfolded proteins with many available
conformations such as intrinsically disordered proteins (IDP). In the latter case,however, we need to evaluate a large number of SAXS profiles for averaging
them, because the IDP consists of an ensemble with diverse conformations. The
program CRYSOL (Svergun, D. et al., J Appl. Cryst., 1995) has been commonlyused to calculate SAXS profiles of IDPs. We found that the calculated profiles
contain a non-negligible amount of error caused by the approximation taken in
estimating the contribution of protein hydration-layer. Though it is possible inprinciple to use the method combined with MD simulation (Seki, Y. et al.,Biophys. Chem., 2002) for removing the error, the computational cost would go
impractically high for IDPs. We present here a new approximation method for
estimating SAXS profiles applicable to solutes with multiple molecularconformations. Though the accuracy of this method is lower than that of the
MD-combined method in the large-K region, it gives sufficient accuracy insmall-K region with low computational cost. In the meeting, we will report on
the detail of approximation and its applicable range of scattering angle.
1PT180極低温電子顕微鏡による 26S プロテアソームの単粒子解析

Single particle analysis of 26S proteasome by cryo-electron
microscopy
Kaoru Mitsuoka1, Daisuke Kasuya2, Yasushi Saeki3, Takuo Yasunaga4 (1BIRC,
AIST, 2BIRC, JBIC, 3Tokyo Metropolitan Institute of Medical Science, 4KITEC)
The eukaryotic 26S proteasome functions to degrade a large fraction of
intracellular proteins. The 26S proteasome is composed of at least 33 different
subunits in 20S core particle (CP) and one or two 19S regulatory particles (RPs).
Proteins destined for degradation are marked by poly-ubiquitin, a degradation
signal that is recognized by the RPs. The proteolytic sites are sequestered inside
the CP and are accessible only thorough a narrow channel. Thus the RP
deconjugates ubiquitin chains, unfolds substrate proteins, and translocates theminto the catalytic CP. The CP was amenable to structure determination by X-ray
crystallography but the 26S holocomplex with 1 or 2 RPs has so far resisted all
crystallization attempts. Here we optimized the single particle analysis methods
by cryo-electron crystallography for the structural analysis of the 26S proteasome.

− S99 −

In the EM preparation, the RPs tend to dissociate from the holoenzyme and so weapplied chemical fixation in density gradient centrifugation (GraFix) to thecomplex. For the optimal contrast of the images, we used energy-filter to estimate
the thickness of the ice layer. For the easier pickup of the particles, we used thedefocus pairs. By these improvements, now we can collect suitable images for the
single particle analysis, which can deduce the three-dimensional architecture ofmacro-molecules without crystallization. We would like to discuss the structural
implications by the single particle analysis, too.
1PT181

Free energy decomposition and functional sites of proteins
Mami Saito, Shoji Takada (Grad.Sch.Sci.,Kyito Uni.)

Prediction of functional sites of proteins has long been studied, but still has
room to improve. A major way of predicting functional sites of proteins relies
on sequence and/or structure alignments. When a target protein has a novel
function or motif or under 30% homology, however, alignment-based methods
do not work well. For overcoming this problem, someone have paid attention tofree energy analysis because it should mirrors sequence and structure. In
particular, it was reported that free energy of residues in functional sites tends tobe higher than that in non-functional sites. Here, based on these observation, we
extend free energy analysis of functional sites using some standard molecularmechanics force field. We choose MMPBSA.PY script in AMBER11, which
automates post-processing of MD trajectories, to analyze energetics using
continuum solvent models. It can be used to break energies into "pieces" arisingfrom different residues. It can also calculate contribution of energy component(electrostatic energy or so). Using them, we survey free energy of residues infunctional sites and non-functional sites for an ensemble of enzyme set.
1PT182

時間分解蛍光偏光法によるタンパク質周囲の水和層の局所粘性測定

Measurement of the local viscosity of the hydration shellaround protein by time-resolved fluorescence anisotropy
spectroscopy
Tetsuichi Wazawa, George Mogami, Nobuyuki Morimoto, Noriyoshi Ishida,
Makoto Suzuki (Dept of Material Processing, Grad Sch of Engin, Tohoku
Univ)
Hydration water is vital for the function, dynamics, and conformation of protein,
and analysis of how the hydration states of protein change during the molecular
processes would give better understandings of the molecular mechanisms. In
this study, we have investigated time-resolved fluorescence anisotropy of afluorophore tethered to a protein in order to apply the technique to the analysis
of hydration states of protein.
Cy3, a fluorophore, was tethered to Cys-374 of actin, as a model protein,through a flexible linker so that the dye underwent rotational Brownian motionin the hydration shell. The rotational motion was anticipated to be subject to the
local viscosity of the hydration shell. A time-correlated single photon counting
method was used to measure the time-resolved fluorescence anisotropy of Cy3
tethered to actin.
The fluorescence anisotropy of Cy3 tethered to G-actin was investigated at room
temperature. The anisotropy decay was decomposed into two components,
which showed the time constants of > 40 ns and ~200 ps with anisotropy
amplitudes of 0.29 and ~0.04, respectively. The slow component reflects the
tumbling motion of G-actin molecules. The fast decay component would be
attributed to the rotational Brownian motion of Cy3. Now, the response of thefast decay component to solvent viscosity is being investigated to establish thetechnique to detect changes in the hydration state of protein.
1PT183Highly accurate statistical pickup method for single particle
3D analysis using electron microscope
Masaaki Kawata1, Chikara Sato2 (1ITRI, AIST, 2BRI, AIST)

Single particle analysis is an information technology-based structure determination method of biological macromolecules. Among data processing in the threedimensional (3D) reconstruction, particle pickup from micrographs is of criticalimportance for the final resolution. Quality of the image library also seriously
affects the processing time using the following sub-algorithms, i.e., classification, alignment and 3D reconstruction. We found a new approach based onclassification of local similarity (LC method), and compares its performancewith other various pickup methods. We further developed an extension of the
LC method using statistical particle recognition techniques to increase reliabilityof particle recognition. The recognition- and computation-efficiency of thismethod was superior to those of other methods; the modified LC method wasespecially robust against noise and independent of parameter settings. We
demonstrate the performance both with synthetic micrographs and cryomicrographs. The new method is a key for fully automated cropping of highquality particle images from micrographs, and a step for a fully automatedsingle particle reconstruction algorism.
1PT184

中程度傾斜撮影した電子顕微鏡画像からの単粒子 3 次元再構築

Single Particle Reconstruction with Medium Tilt Images byElectron Microscopy
Yutaka Ueno, Emiko Kobayashi, Shouhei Mine, Kazunori Kawasaki (AIST
Kansai)In three dimensional reconstruction of single particle molecules taking tilted
pairs of the same specimen in different angles greatly contributes reliability of
the reconstruction of the volume data from projection images. While the random
conical tilt method has been widely employed for a specimen with the same face
up, a more general method for randomly oriented particles was studied. We haveintroduced medium tilt by plus-minus 20 degrees that comprises 40 degrees
tilted pairs. The method relays on the fact: the images in the same orientation arefound in both tilted images and they are grouped into unified clusters in terms ofviewing directions. Once a major group of images of the particle in a certain
viewing angles exist in images from different tilt angles, these tilted pair gives
additional tilted view of the particle beyond the major tilt observation. They
contribute to cover wider projection angles minimizing loss of projection
information known as the missing cone situation. The method was firstexamined by simulated reconstruction with a theoretical model phantom. As
with the case with traditional tilting observation data collection requires
adjusting the viewing scope to locate the same as another tilted pair. Introducing
recent electron microscope with CCD camera and the tilting stage controller
greatly facilitated data collection. Possible automation for this tedious task will
be discussed together with our preliminary results with our single particle
analysis of deacetylase from Archaea.
1PT185

拘束付き分子動力学法を適用した、電子顕微鏡法のための原子モデ
ル構築ツールの開発

Development of atomic modelling tool for Electron
Microscopy, applying steered Molecular Dynamics method
Risa Yamashita, Ryota Nakao, Hiroshi Sakamoto, Takuo Yasunaga (Kyushu
Institute of Technology)
In most of studies of proteins using electron microscopy (EM), it is difficult to
discuss detail at the atomic level because three-dimensional electron
microscopic maps (3D-EM maps) are mostly low or medium resolution and thetypical resolution of the 3D-EM map of proteins is about 1 to 4nm. Thus wehave developed an atomic modelling tool based on a 3D-EM map under animage processing package of Eos , which we called SRMD and planed atomicmodels constructed even from the typical resolutions of EM maps. The SRMD
method is one of the extensions of steered molecular dynamics (MD) simulation
techniques, in which EM maps are used as a potential for MD and constraineach atoms within a target protein. This SRMD method tool did not have GUI
yet: When we use SRMD method tool, we need to use an editor to set
temperatures for simulated annealing and conditions of constraints from EMmaps. Furthermore, we cannot watch atomic models during modelling in realtime. To construct an atomic model precisely, there are two planning points.
One is embedding of SRMD into a model viewer with GUI to monitor models
and change modeling conditions easily during calculation. The other is tochange molecular conditions by a pointing device like IMD. Thus, it will bepossible to make atoms stacked unnaturally by 3D-EM map potentials rescued.
Here, we will report our progress of the SRMD techniques and examples for acomplex map of heme oxygenase and cytochrome P450 redutase, which isreconstructed from EM images.
1PT186Comparative survey of image processing packages for electroncomputed tomography

Nan Shen, Mingyue Jin, Takuo Yasunaga (Dep. Biosci. Bioinf., Inst. Tech.
Kyushu)Electron computed tomography (ECT) is one of the most widely applicable
methods for obtaining the three-dimensional (3D) reconstruction of organelles
or cell components. First, tilt series of two-dimensional (2D) projection imagesof the specimen are recorded in electron microscope, and then 3D structure iscomputed from the tilt series using some softwares. A common problem− S100 −encountered in this ECT is alignment accuracy of tilted projection images. Here,three ET software packages (IMOD, Inspect3D and TEMography) are wellknown and applied to tilt series alignment in different ways. In our previous
work, we have acquired tilt series from axonemes by cryo-transmission electron
microscopy (cryo-TEM), and reconstructed the 3D structure using IMOD,
Inspect3D and Eos we have developed, mainly. IMOD can automaticallydetermine tilt axis by using fiducial marker, while Inspect3D needs to manually
determine tilt axis but gave us more precise alignment than that of IMOD,
judging from obtained 3D. Thus we decided to compare them systematically, i.
e., our present purpose is to evaluate the quality of 3D reconstruction and theusability of these software packages, show the pros and cons of themsystematically and then make our developed Eos more convenient with higher
analysis ability. We acquired tilt series of model structure (phantom data) andaxonemes by cryo-TEM, and computed the 3D structure using IMOD,
Inspect3D, TEMography and Eos from each of them, respectively. Comparison
between them will here be reported and discussed.
1PT187

Gibbs energies of secondary structures for α-Chymotrypsinogen
A investigated by FT-IR spectroscopy

Koichi Murayama (Grad. Sch. Med., Gifu Univ.)
FT-IR spectra were measured over a temperature range of 25-90 degree, toinvestigate difference in Gibbs energies of secondary structures for α Chymotrypsinogen A (a-ctg). The curve fitting method combined with Fourier
self-deconvolution technique allowed us to explore details of the secondary
structure. The spectra of a-ctg in the amide I’ region reveal eight bands for antiparallel β-sheet, turn, α-helix, β-sheet, intermolecular β-sheet and side chain
vibration below 70 degree. Additional band for disordered appears over 70
degree in the spectra. Then, Gibbs energies for secondary structures and theirtransition temperatures (Tm) were calculated. The Tm for the four bands due tothe α-helix, turn and disordered structures are found to be around 60 degree.
Corresponding values for anti-parallel β -sheet and side chain are higher by
about 67 degree, and those for β-sheet, inter-molecular β-sheet structures werearound 63-64 degree. The Gibbs energies were obtained about 11-16 kJmol-1 forα -helix, β -sheet and turn. The value for intermolecular β -sheets was muchhigher than others, suggesting that intermolecular β-sheets formation need moreenergy than other secondary structures. In this study, I will discuss Gibbs energy
and Tm values for secondary structures of a-ctg obtained from FT-IR
spectroscopy in details.
1PT188

TIP3P モデルおよび Generalized Born モデルを併用した分子動力学計算によるタンパク質の構造サンプリング

Improvement of sampling efficiency through combined use ofmolecular dynamics simulations with implicit and explicit
solvent models
Hiroko Kondo1,2, Noriaki Okimoto2, Makoto Taiji1,2 (1Department ofComputational Biology, the University of Tokyo, 2Laboratory for Computational
Molecular Design, Quantitative Biology Center (QBiC), RIKEN)
Various experimental and computational studies have indicated that the protein
conformation and its function are closely linked. Investigating their dynamical
properties is consequently important to understand their functional properties.
The molecular dynamics (MD) simulation is powerful tool for these analyses
because we can observe the kinetics of protein at the atomic revel. It is howeverthat the time scale of the simulation is not enough for examining the significant
event including protein folding, domain movement, and so on. Therefore theenhanced sampling methods to overcome this computational limitation are so
important.
In this study we aim at the improvement of sampling efficiency, reduction in
actual simulation time by combining the results from implicit solvent simulations
and those of explicit one; the former is used for reducing the sampling space andthe latter for correcting the conformational distribution sampled by the former
simulation. The replica exchange method was adopted for rough conformational
sampling with implicit solvent followed by the explicit solvent simulation. Here,
we performed conformational sampling of a mini protein, chignolin to examine
the efficiency of our method. The results show that our new method covered awider region of conformational space as compared with the conventional MD
simulation with the explicit solvation model.
1PT189生体分子の分子動力学に対する時系列解析ー集団運動の揺らぎと構造変化の関係を探るー

Time-series analysis of molecular dynamics: Conformationalchange and dynamics of collective behavior
Kana Fuji1, Masakazu Sekijima2, Hiroshi Fujisaki3, Mikito Toda4 (1Graduateof school Humanities and Sciences, Nara Women's Univ., 2GSIC, Tokyo
Tech, 3Phys., Nippon Medical School, 4Sci. , Nara Women's Univ.)
Our study aims to analyze time series data of molecular dynamics simulation forproteins to extract collective behavior involving side chains and watermolecules. Our methodology consists of the following two steps: (1)conformational analysis (dihedral principal conponent analysis) (2) dynamical
analysis (the wavelet transformation and singular value decomposition(SVD)).
The wavelet transformation enables us to investigate time-varying features ofoscillatory motions, and SVD extracts collective variables involving multipleamino residues[1].
Our system is chignolin. In our previous work, we analyzed time series datausing implicit solvent method for dynamics simulation. Here, we study datausing explicit solvent method and will conpare these two cases.
In particular, we aim to extract collective behavior involving not only the main
chain but also the side chain. By analyzing relationship of collective behavior
between the main chain and the side chains, we can understand to what extent
collective behavior involving the main chain provides us with information
concerning collective behavior with the side chains. Furthermore, we expect thatthe study of collective behavior involving the side chain will give us an
important clue to understand molecular functions for other proteins.
[1]M.Kamada,et. al,Chem.Phys.Lett.502(2011)241-7.
1PT190イオンモビリティ質量分析と分子動力学シミュレーションを用いた
気相中におけるヒストン多量体の構造解析

Characterization of histone multimers in the gas phaseby ion mobility mass spectrometry and molecular dynamics
simulation
Kazumi Saikusa1, Sotaro Fuchigami1, Kyohei Takahashi1, Yuuki Asano1,
Aritaka Nagadoi1, Hiroaki Tachiwana2, Hitoshi Kurumizaka2, Mitsunori
Ikeguchi1, Yoshifumi Nishimura1, Satoko Akashi1 (1Yokohama City Univ.,
2
Waseda Univ.)
Nucleosome core particle (NCP) is the basic structural unit of eukaryotic
chromatins. In order to understand the mechanism of the NCP assembly anddisassembly, structural characterization of the histone multimers, H2A/H2B
dimer and H3/H4 tetramer, should be of great help. However, these atomiclevel structures have not been determined. In the present study, human histone
multimers were prepared and characterized by electrospray ionization ion
mobility mass spectrometry (ESI-IM-MS) and molecular dynamics (MD)simulation. Experimentally obtained arrival times of these histone multimer ionsshowed rather wide distributions, which suggested that histone multimers hadvarious conformations. To probe the conformers’ structures, several MD
simulations of the histone multimers were carried out in solution and in vacuo,
and it was identified that the histone multimers had a variety of structure. In
addition, the simulation showed that the conformers with smaller CCS values
had more compact tail regions than those with larger CCS values. On the other
hand, the CCS values of the folded core structure were almost kept identical in
the analyzed conformers. This implies that the disordered regions brought about
the structural variety of histone multimers. This paper presents that the
combination of IM-MS and MD simulation enables comprehensive characterization of the gas-phase structures of biomolecular complexes containing
disordered regions.
1PT191

カーネル正準相関分析と離散ウェーブレット変換を用いたタンパク
質立体構造の時系列解析

Time-series analysis for protein dynamics using discrete
wavelet transform with kernel canonical correlation analysis
Mayumi Kamada1, Mikito Toda2, Tatsuya Akutsu1 (1Inst. Chem. Res., Kyoto
University, 2Phys. Dept., Nara Womens Univ.)
Structural dynamics of proteins play an important role in expression of proteinfunctions. In particular, slow collective motions of proteins make a large
contribution to performing its functions. To understand a correlation between
protein functions and structural dynamics, we need a method to extract slow
collective motions from structural dynamics without losing important
information, and correctly characterize them. For this purpose, we use thediscrete wavelet transformation (DWT) for the time-series data of protein
structures obtained from molecular dynamics (MD) simulation. DWT is one oftime-frequency analysis methods used for denoising and data composition, and− S101 −opens a door to extract compact datasets keeping the features of both time andfrequency. In other words, it enables us to perform trend analysis of time-series
data for protein structural dynamics. In this work, we apply DWT to trajectorydata obtained by all-atoms MD simulation of Myoglobin[1,2], and analyze themconcerning a ligand pathway problem of Myoglobin. For a correlation analysisof structural motions involving ligand pathway, we apply, to the datatransformed by DWT, the kernel canonical correlation analysis (kCCA), which
is useful to analyze an association between two variables even when they have
non-linear association. We discuss the result of correlation analysis andusefulness of our strategy.
[1] Scorciapino MA et al., J Am Chem Soc. 131(33):11825-32 (2009)[2] Scorciapino MA et al., J Am Chem Soc. 132(14):5156-63 (2010).
1PT192生体分子における原子レベルでのストレステンソル解析

Atomic stress tensor analysis in biomolecules
Takakazu Ishikura, Tatsurou Hatano, Takahisa Yamato (Grad. Sch. Sci.,
Nagoya Univ.)
In 1985, Frauenfelder and co-workers proposed the “protein quake” concept toillustrate the molecular function of myoglobin, which experiences an allostericconformational change triggered by the dissociation of CO from heme iron.
Although the concept is intuitively appealing, only a limited number oftheoretical studies have addressed the protein-quake-model [1].
One of the possible ways to characterize the model is to introduce atomic stresstensors. However, any of the conventional methods considered only two-bodyinteratomic potential terms for stress tensors, which is not convenient because
the force field functions for molecular dynamics simulation contain multi-bodypotentials such as angle, torsion and improper terms. To address the issue, we
derived complete pair-wise expression for interatomic forces in biomolecules.
Then we have developed a computer program, CURP, for the atomic stresstensor analysis of molecules and performed test calculations for small
polypeptides [2]. As a result, we calculated the contribution to the stress fromeach component, and found out that the contribution from multi-body potential
terms was larger than non-valence potential terms. This means that the analysis
of atomic stress tensor must be performed with all of potential terms. In thispresentation, we will introduce the results obtained by stress tensor analysis
method for some systems.
[1] K. Koike, K. Kawaguchi, and T. Yamato, Phys. Chem. Chem. Phys. 10,
1400 (2008).
[2] T. Ishikura, T. Hatano, T. Yamato, Chem. Phys. Lett. (in press)
1PT201

細胞環境における蛋白質の構造安定性とダイナミクス

1YS1100

Protein stability and dynamics under cellular environments
Ryuhei Harada1, Yuji Sugita1,2,3, Michael Feig3,4 (1RIKEN AICS, 2RIKEN
ASI, 3RIKEN QBiC, 4Michgan state university)
The effect of crowding on the stability and dynamics of protein was examinedfrom explicit solvent molecular dynamics simulations of a series of protein G
and protein G/villin systems at different protein concentrations.
In these simulations, the native state was found to be significantly destabilized
under crowded environments. Especially, for the villin systems, where induced
extensive unfolding to extended structures. This finding is entirely contrary towhat is predicted from excluded volume effects but can be explained again
through stabilizing interactions with other proteins. These simulations are theclearest evidence so far that enthalpic interactions due to protein-protein
interaction can in fact overcome the entropic cost of extension under crowded
environments.
Hydration structure was further analyzed through radial distribution functions,
three-dimensional hydration sites and preservation of tetrahedral coordination.
Analysis of hydration dynamics focused on self-diffusion rates and dielectric
constants as a function of crowding. The results show significant changes inboth structure and dynamics of water under crowded environments. The
structure of water is altered mostly beyond the first solvation shell. Diffusion
rates and dielectric constants are significantly reduced following linear trends asa function of crowding reflecting highly constrained water. The reduced
dynamics of diffusion is expected to be strongly related to hydrodynamic
properties of crowded cellular environments.
1PT202

マクロファージ細胞株における LPS 活性化時の遺伝子発現の細胞間ゆらぎに細胞間相互作用が与える影響

Effects of the cell-cell communication on the populationalvariability of mRNA levels among clonal macrophages after
LPS stimulation
Mai Yamagishi1, Yoshitaka Shirasaki1, Nanako Shimura1,2, Nobutake Suzuki1,
Osamu Ohara1,3 (1RCAI, RIKEN, 2Grad. Sch. Med. Pharm., Chiba Univ., 3KAZUSADNA Res. Inst.)
Macrophages are activated by lipopolysaccharide (LPS) to start expressing
various kinds of cytokines/chemokines. In the previous studies, single cell
analyses using the AmpliGrid system and the BioMark system revealed that a
population distributions of mRNA level varied quite widely from gene to geneand changed in a gene-specific manner; some genes were activated after LPS
stimulation with a narrow populational distribution, but some with a broad one.
To understand the mechanism to produce such a wide populational variation inmRNA levels in spite of the genetic homogeneity of a clonal macrophage-likecell line, RAW264.7. We here examine whether or not cell-cell communication
has some impacts on this cell-to-cell variation. In the first experiment, we
examined the dependence of cell density on the populational variations ofcytokine mRNA profiles. The results indicated that only CXCL10 gene showeda decrease in the standard deviation of populational distribution of mRNA levels
with increase of the cell density while the other responding genes seemed little
affected. Because the cell density employed was not so high that cell-cell
contact plays a major role in cell-cell interaction, it is very likely that the celldensity effect is exerted by humoral factor(s). Analysis and characterization of
the humoral factor(s) will be presented.
1PT203

小腸絨毛下線維芽細胞における各種刺激による ATP 放出

ATP-releases with various stimuli in subepithelial fibroblasts
in intestinal villi
Kishio Furuya1, Sonoko Furuya2, Masahiro Sokabe3 (1FIRST ResearchCenter for Innovative Nanobiodevice, Nagoya Univ, 2National Institute forPhysiological Science, 3Dept. Physiol., Grad. Sch. Med., Nagoya Univ)
Subepithelial fibroblasts of the intestinal villi, which form a contractile network
under the epithelium, are in close contact with epithelial cells, neurons,
capillaries, smooth muscles and immune cells. Developing a primary culture ofthese cells, we revealed unique characteristics such as: cAMP-dependent cellshape changes between flat and stellate, cell-shape dependent mechanosensitivity with ATP release as a paracrine mediator, and expression of various
receptors for endothelins (ETs) (ETA, ETB), ATP (P2Y1), substance-P (SP)(NK1), etc. From these features, we proposed that subepithelial fibroblasts work
as a mechano-sensor and signal transduction machinery in the intestinal villi.
Here, we show that, in addition to the mechanical stimulation, receptor
stimulations by ET1 and SP, and hypo-osmotic stimulation induce ATP-releasefrom the subepithelial fibroblasts, using a real time ATP-imaging system. ATP
is released sparsely from a limited number of stimulated cells and the ability of
ATP-release is cell shape dependent. Peak concentration of released ATPreaches to 1-10 microM, being enough to propagate the Ca2+ waves to neighborcells or whole colonies. Differences of the effects of various blockers for ATPrelease and difference of the ATP-releasing kinetics suggest the existence ofseveral mechanisms of ATP-release in subepithelial fibroblasts. Thesemechanisms may concomitantly regulate villous functions such as villous
movement, nutrient absorption and intestinal maturation.
1PT204An optimal heterogeneity in a cell population maximizes theeffects of growth factors directing stochastic PC12 cell fatedecisionsKazunari Mouri, Yasushi Sako (Adv. Sci. Inst., Riken)
Recently, the heterogeneity arising from the stochastic fate decisions of clonalcells in the same environmental conditions has been reported for several cell
types. Here we focused on the stochasticity in PC12 cell fate decisions. PC12 isa cell line derived from rat pheochromocytoma, and is a model system of thedifferentiation of sympathetic neurons. In general, PC12 cells are stimulated
proliferation in the presence of a growth factor EGF, while extension of neuritis
to differentiate into neuron-like phenotype in the presence of another growth
factor NGF. In our work, however, we found that cells spontaneously induced
cell fates without any external induction stimuli, which resulted in heterogeneity
in a population. Serum contained in the culture medium suppresses spontaneous
differentiation and cell death. Stochasticity in cell fate decisions affected by the
spontaneity makes it difficult to predict the responses of cells to external stimuli
of growth factors. In our experiments, for example, EGF (NGF) stimulus
stochastically went so far as to induce differentiation (proliferation) depending− S102 −on the environmental serum concentrations. Careful quantification of the
response rates for growth factors suggested that under an appropriate
environmental condition to bring optimal heterogeneity caused by thestochasticity of cell fate decisions, the effects of the growth factors become
maximum, i.e., suppressing cell death, NGF directs PC12 cell fates efficiently todifferentiation and EGF directs to proliferation.
1PT205蛍光相関分光法を用いた単一細胞内グルココルチコイド受容体のホモダイマー形成の解析

Quantification of Glucocorticoid Receptor of homo-dimer insingle cell by using Fluorescence Correlation Spectroscopy
Sho Oasa1, Akira Sasaki1, Shintaro Mikuni1,2, Masataka Kinjo1 (1Grad. Life
Sci., Hokkaido Univ., 2Grad. Med. Department of Advanced Optical Imaging
Reasarch., Hokkaido Univ.)
The dimerization of GR (Glucocorticoid Receptor) in single cell is analysed. It
is known that GR is dimerized by adding Dex (Dexamethasone) in living cell.
However, when and where the dimerization of GR is occurred is still
controversial. Moreover, many previous studies of GR processing have been
carried out by using trangent-expressed cell. When the heterogeneous amountsof target protein is estimated in each cell such as trangent-expressed cell, thesingle-cell measurement might be better than conventional biochemical methods
and required for better understanding of GR activity. In our laboratory, to assess
protein contents in single cell, the small cavity (MicroWell) on the PDMS
(polydimethylsiloxane) chip was constructed [1, 2]. Single cell was easily
isolated in MicroWell and target protein was extracted and kept into theMicroWell. FCS (Fluorescence Correlation Spectroscopy) was performed toobtain number and concentration of target protein in MicroWell and protein
complex information from CPM (count per molecule). EGFP-GR dimerized byadding Dex is analysed by using CPM and the ratio between monomeric anddimeric EGFP-GR is estimated with different concentraton of EGFP-GR intrangent-expressed cell.
[1] Sasaki, A, Kinjo, M, Curr Pharm Biotechnol., 2010, 11, 117-121[2] Oasa, S, Sasaki, A, Kinjo, M, Biophysic Society meething, 2011
1PT206マスト細胞と後根神経節初代培養細胞の相互作用における接着分子の研究

Cell adhesion molecule 1 (CADM1) on mast cells promotesinteraction with dorsal root ganglion neurites by heterophilicbinding to nectin-3
Tadahide, Furuno1, Miho, Sekimura2, Keisuke, Okamoto2, Man, Hagiyama3,
Akihiko, Ito3, Ryo, Suzuki2, Naohide, Hirashima2, Mamoru, Nakanishi1 (1Sch.
Pharm., Aichi Gakuin Univ., 2Grad. Sch. Pharm. Sci., Nagoya City Univ., 3Fac.
Med., Kinki Univ.)
Functional interaction between mast cells and sensory nerves has been shown tooccur in various physiological and pathological situations. We previously
showed that an immunoglobulin superfamily adhesion molecule, cell adhesion
molecule 1 (CADM1), on both mast cells and sympathetic superior cervical
ganglia (SCG) promoted their attachment and communication via thehomophilic binding using in vitro coculture approach. Here we have studied therole of CADM1 in the interaction between sensory dorsal root ganglia (DRG)
and mast cells. Though CADM1 was hardly expressed in DRG, CADM1 onbone marrow-derived mast cells (BMMCs) accumulated in contact region with
DRG neurites. CADM1-deficient IC-2 mast cell line was defective inattachment to DRG, while it was recovered in CADM1-transfected IC-2 cells(IC-2CADM1 cells) to the similar level to BMMCs. Searching adhesion
molecules known as heterophilic binding partner of CADM1, DRG expressed
nectin-3 but not Necl-1, Necl-5, and class-I-restricted T-cell-associatedmolecule. CADM1 on mast cells and nectin-3 on DRG neurites werecolocalized in their attached region in coculture. The efficiency in calcium
response of attached IC-2CADM1 cells after specific activation of DRG was
significantly higher than that of IC-2 cells and was as similar as BMMCs. A
neutralizing antibody to nectin-3 inhibited both mast cell attachment andsubsequent calcium responses. This suggests that heterophilic binding between
CADM1 and nectin-3 mediates functional DRG-mast cell interactions in vitro.

1PT207

Duration of CaMKII activation required for plasticity ofdendritic spines revealed by photo-activatable CaMKII
inhibitor

Hideji Murakoshi1,2, Ryohei Yasuda3 (1National Institute for Physiological
Sciences, 2PRESTO, JST, 3Dept of Neurobiology, Howard Hughes Medical
Institute, Duke University Medical Center)Ca2 + /Calmodulin-dependent kinase II (CaMKII) is required for long-term
potentiation (LTP) and associated spine enlargement, which underlies learningand memory in the mammalian forebrain. Previously, it has been demonstrated
that exogenous CaMKII activity lasts for ~1 min during induction of spinespecific LTP and spine enlargement. Here, to determine the duration ofendogenous CaMKII activation required for CaMKII-dependent synaptic
plasticity, we developed a genetically encoded light-inducible inhibitor of
CaMKII activation by fusing autocamtide inhibitory peptide 2 (AIP2) to LOV2Jα derived from phototropin1. We applied it to the study of structural plasticity
of single dendritic spines by using 2-photon glutamate uncaging, and found that
~10 s of CaMKII activation is sufficient for inducing transient spine
enlargement, while ~60 s is required for sustained spine enlargement. The
different temporal requirement between these two phases must be due todifferences in the integration properties of downstream signaling. The design ofthe light-inducible CaMKII inhibitor shown here should be applicable to thedesign of similar light-inducible inhibitors for many different proteins.
1PT208

TPO 受容体の二量体安定化によるシグナルの正のフィードバック
制御

Positive feedback control of thrombopoietin signaling bystabilization of its receptor dimers revealed through singlemolecule imaging
Akihiko Sakamoto1, Takashi Kato2, Takashi Funatsu1 (1Grad. Sch. of Pharm.
Sci., The Univ. of Tokyo, 2Fac. of Ed. and Int. Arts. and Sci., Waseda Univ.)
The thrombopoietin (TPO) receptor, Mpl, is a single transmembrane cytokine
receptor which stimulates differentiation of platelet progenitor cells andmaintains hematopoietic stem cells. Like other cytokines, TPO induces
phosphorylation of Mpl, which recruits various adaptor proteins regulating cellproliferation and differentiation. Although dimerization of cytokine receptors isnecessary for their phosphorylation, the regulatory mechanism is poorly
understood. Here, we describe the regulatory mechanism of Mpl dimerization
by single-molecule fluorescence imaging in living cells. Fluorescently labeled
Mpl molecules associated and dissociated repeatedly on the plasma membrane.
As a result, Mpl molecules resided in monomers, dimers, and oligomers. TPO
increased the ratio of Mpl dimers or oligomers, which was resulted fromstabilization of Mpl dimers. Surprisingly, stabilization of Mpl dimers was
resulted from their phosphorylation rather than TPO binding itself. Furthermore,
TPO-induced stabilization of Mpl dimers was dependent on an adaptor protein,Shc, suggesting that phosphorylated Mpl dimers were crosslinked by Shc. By
contrast, a monomeric Mpl mutant inhibited TPO-induced phosphorylation of
Mpl and downstream cell proliferation. In addition, cholesterol depletion
reduced the stability of Mpl dimers, resulting in inhibition of theirphosphorylation. Thus, we propose positive feedback control of TPO signalingby stabilization of Mpl dimers.
1PT209

Rac1 recruitment to the archipelago structure of focal
adhesion through the fluid membrane as revealed by singlemolecule analysis
Akihiro Shibata1, Chen Limin1, Nagai Rie1, Fumiyoshi Ishidate1, Yoshihiro
Miwa2, Keiji Naruse3, Takahiro Fujiwara1, Akihiro Kusumi1 (1Institute for
Frontier Medical Sciences, Kyoto University, 2Department of Pharmacology,
Institute of Basic Medical Sciences, University of Tsukuba, 3Cardiovascular
Physiology, Okayama University Graduate School of Medicine, Dentistry, andPharmaceutical Sciences)
The small G protein Rac1 has been shown to regulate actin reorganization andpolymerization at the focal adhesions (FA), an integrin-based architecture of theplasma membrane (PM), mechanically linking the extracellular matrix with thetermini of actin stress fibers, providing the scaffold for cell migration. Previously,
we found that the FA is not a single entity of massive protein assembly, but rather
consists of many protein islands sparsely scattered in the fluid PM (archipelagoarchitecture), and membrane molecules undergo rapid diffusion in the PM even
within the FA zone. Here, using single fluorescent-molecule imaging, we
examined how Rac1 and its upstream activator GEFs, alphaPIX and betaPIX, arerecruited to the FA. We found that almost all of the PIX molecules were directly
recruited to the FA zone from the cytoplasm. Meanwhile, 81% of Rac1, which has
a geranylgeranyl (+ possibly a palmitoyl) hydrophobic anchoring chains, firstlanded on the general inner surface of the PM from the cytoplasm and then

− S103 −entered the FA zone via lateral diffusion in-on the PM, continuing diffusion even
within the FA zone (diffusion coefficient is 1/3 of that outside the FA). 11% Of
Rac1 molecules that entered the FA zone exhibited temporary or long-term
immobilization in the FA zone, suggesting the binding to the FA island proteins.
19% Of Rac1 arrived at the FA zone directly from the cytoplasm andimmobilized, suggesting that Rac1 is recruited to the FA protein islands both
directly from the cytoplasm and via the general PM area.
1PT210

ERK リン酸化・脱リン酸化反応再構成系の ERK 濃度に依存した応答特性

Response characteristics of reconstructed ERK
phosphorylation and dephosphorylation depending on ERK
concentration
Masahiro Takahashi1, Toshio Yanagida2, Yasushi Sako1 (1RIKEN ASI, 2Graduate
School of Frontier Biosciences, Osaka University)
ERK, the mitogen-activated protein kinase (MAPK) in the Raf-MEK-ERK
cascade, has two phosphorylation sites which are phosphorylated by a kinese,
MEK (MAPKK) and dephosphorylated by a phosphatase, MKP. Some
theoretical studies have shown that two enzymes with opposing effect (e. g.
kinase and phosphatase) can lead to zeroth-order ultrasensitivity which is anultrasensitive response at the steady-states depending on the substrateconcentration (Goldbeter, A. and Koshland, D., PNAS: 78 1981).
To show this type of ultrasensitivity experimentally, we reconstructed the ERK
phosphorylations and dephosphorylations in E. coli cells, in which concentrations of ERK, MEK and MKP were independently controllable using
extracellular chemicals. The attained protein concentrations were estimated byfluorescence intensities of Cerulean (a cyan fluorescent protein variant), Venus(a yellow fluorescent protein variant), and mCherry (a red fluorescent protein
variant) each fused on ERK, MEK and MKP, respectively.
We investigated the dependence of ERK phosphorylation on MEK and MKP
expression level. The ERK phosphorylation increased by the increase of MEK
concentration and/or the decrease of MKP concentration. Thus, the response
curve for ERK phosphorylation depended on the ratio of MEK to MKP
concentration and could be described by the Hill equation. When the ERK
expression decreased by a factor of 0.29, the Hill coefficient decreased by afactor of 0.69. This response is consistent with the theoretical prediction by the
zeroth-order ultrasensitivity.
1PT211

マウス胚盤胞における細胞内カルシウム濃度とメカニカルストレス
負荷の同時計測

Simultaneous investigation of intracellular calcium
concentration and mechanical stimuli to mouse blastocysts
Koji Matsuura1, Yuka Asano1, Ikuyo Sugimoto1, Mieko Kodama1, Keiji Naruse2(1Research Core for Interdisciplinary Sciences, Okayama University, 2Cardiovascular
Physiology, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences,
Okayama University)
Mammalian embryos are transported to the uterine cavity through the fallopian
tube during cell cleavage and blastocyst development. Fertilized oocytes may besubjected to a mechanical influence. Dynamic embryo culture systems havebeen developed to generate and apply mechanical stimuli (MS) similar to thosegenerated within the oviduct affecting the mammalian embryo. To investigate
the responses of the mammalian embryos to MS, we developed an dynamic air
actuation system to apply MS in a microfluidic channel by deforming a 0.1-mm
membrane, and investigated changes of intracellular calcium ion concentration([Ca2+]i) in mouse blastocyst applied MS. Both MS and ([Ca2+]i) were
quantified in the microfluidic channel. ([Ca2+]i) was measured in a stainedmouse embryo with Fluo-4 AM using confocal fluorescence microscopy. We
captured a z-series stack of sections encompassing the entire embryo. Whenblastocysts were compressed, fluorescence intensity (FI) in the blastocyst also
increased in response to the applied MS. Compressive force estimated from theshape of the blastocysts was approximately 0.5-2.0 μN according to a force
deformation curve for the mouse embryo. The sum of FIs increased by a factorof 1.1-1.2 times compared with those observed before MS. The increase in thesum of FI indicated that enhancement of ([Ca2+]i) would be induced by theseMS. Molecular mechanosensing systems such as mechanosensitive ion channels(MSIs) would play an important role in responses to these MS.
1PT212

バイセクティング GlcNAc とフコースの導入が水中 N 型糖鎖の構
造多様性に及ぼす影響

Effect of Bisecting GlcNAc and Core Fucosylation on
Conformational Properties of Biantennary Complex-Type
N-Glycans in Solution
Wataru Nishima1, Naoyuki Miyashita2, Yoshiki Yamaguchi1, Yuji Sugita1,2,3,
Re Suyong1 (1ASI, Riken, 2QBiC, Riken, 3AICS, Riken)
The introduction of bisecting GlcNAc and core fucosylation in N-glycans isessential for fine functional regulation of glycoproteins. In this presentation, theeffect of these modifications on the conformational properties of N-glycans is
examined at the atomic level by performing replica-exchange molecular
dynamics (REMD) simulations. We simulate four biantennary complex-type Nglycans, namely, unmodified, two single-substituted with either bisecting
GlcNAc or core fucose, and disubstituted forms. By using REMD as anenhanced sampling technique, five distinct conformers in solution, each ofwhich is characterized by its local orientation of the Manα1-6Man glycosidic
linkage, are observed for all four N-glycans. The chemical modifications
significantly change their conformational equilibria. The number of major
conformers is reduced from five to two and from five to four upon the
introduction of bisecting GlcNAc and core fucosylation, respectively. The
population change is attributed to specific inter-residue hydrogen bonds,
including water-mediated ones. The experimental NMR data, including nuclear
Overhauser enhancement and scalar J-coupling constants, are well reproduced
taking the multiple conformers into account. Our structural model supports theconcept of “conformer selection”, which emphasizes the conformational
flexibility of N-glycans in protein-glycan interactions.
1PT213

新規フォトクロミック HDAC 阻害剤の設計・合成ならびに阻害作用

Design and synthesis of a novel photochromic HDAC inhibitor
and its inhibitory effectKeiko Tanaka, Shinsaku Maruta (Dept. of Bioinfo., Fac. Eng., Soka Univ.)
Histone acetylation-deacetylation is one of the epigenetic regulations of gene
expression in eukaryotes. Histone deacetylase (HDAC) catalyze deacetylationof lysine residues in N-terminal domain of core histones and regulates genetranscription and expression. Inhibition of them induces transcriptionally active
chromatin, causing arrest of growth, differentiation and apoptosis. HDAC
inhibitors are thought to be effective for neurodegenerative disorders, cardiac
hypertrophy, inflammation and cancer. In this study, we designed andsynthesized novel photochromic HDAC inhibitor, composed of photochromic
region (azobenzene), linker, and zinc-interacting thiol, for photo-regulation ofdifferentiation or apoptosis. Azobenzen moiety is placed in hydrophobic
interacting region of inhibitor, therefore, light-induced structural change were
thought to be affect affinity against HDACs. Synthesized photochromic HDAC
inhibitor showed light-induced isomerization. Photochromic HDAC inhibitor
showed micro-molar order HDAC inhibitory effect in vitro. The inhibitor
composed of trans isomer azobenzene generated by visible-light irradiation
inhibited deacetylase activity of HDAC with higher Ki than that composed of
cis isomer azobenzene induced by ultraviolet-light irradiation. Development ofphotochromic HDAC inhibitor is thought to be useful for site-directed inhibitionof HDACs.
1PT214

PC12 細胞シグナル伝達系における PLS 回帰を用いた解析

PLS regression analysis of Signal transduction network andphenotypes in PC12 cell
Yuki Akimoto, Katsuyuki Yugi, Shinsuke Uda, Shinya Kuroda (University ofTokyo)
PC12 cells, an adrenal chromaffin cell line, are a well-characterized model ofnerve cells. This cell line exhibit several characteristic phenotypes such as
differentiation, proliferation and apoptosis in response to different external
stimuli. Previous studies reported that these various stimuli are redundantly
mediated by a few common signal transduction factors such as Akt, CREB and
MAPKs, which increase expression levels of some proteins known as
Immediate Early Genes (IEGs). However, quantitative relationships between
these factors and cell phenotypes are yet to be well characterized. To elucidate
this underlying relationship between the temporal patterns of the signaling
factors, IEGs and phenotypes we focus a statistical analysis. Statistical models
are constructed to predict temporal patterns of IEGs/phenotypes from those of
signaling factors such as Akt, CREB and MAPKs using Partial Least Squares(PLS) regression analysis. The expression patterns of signal transduction factors
and IEGs were measured by QIC method previously developed by our groupand the cell phenotypes are measured by some molecular biologicalexperiments. Using these data a regression equation was calculated in terms of− S104 −PLS regression. Finally, we obtained four principal component dimensions
characterizing characterize some phenotypes. This data driven approach iseffective to identify undefined aspects of complex temporal signal transduction
networks.
1PT215幹細胞様のふるまいを示す人工遺伝子ネットワークの構築

Synthetic biology of stem cells: creating artificial gene networkof "differentiation"
Sumire Ono1, Reiko Okura1, Yuichi Wakamoto2,3 (1Grad. Sch. Arts and Sci.,
Univ. Tokyo, 2Research Center for Complex Systems Biology, Univ ofTokyo, 3JST PRESTO)
Stem cells can differentiate spontaneously and irreversibly, producing a variety
of differentiated cells in context-dependent manners. It remains to be resolved
what kind of fundamental characteristics of intracellular network enable suchremarkable attributes of stem cells. Recently, Goto and Kaneko have proposed a
dynamical systems model of stem cells that can explain spontaneous bifurcationof cellular states using two-gene regulatory network. In this model, a “diffusible
inducer” causes cellular interaction, leading to coexistence of two cellular states.
The simplicity of the regulation proposed in the model should allow us tosynthesize such regulatory network artificially and thereby test the hypothesis
regarding the stem cell gene networks.
Inspired by this theoretical study, we aim to construct an Escherichia coli strain
that possesses an artificial gene regulatory network equivalent to the scheme
presented in the model. To realize cellular interaction through diffusible inducer,
we utilized the lux operon of Vibrio fischeri. However, the original lux operon has
positive regulation whereas the inducer acts negatively in the proposed model.
For this reason, we designed the actual network with three genes instead of two ina manner that tet operon mediates the lux operon's function. T7 RNA Polymerase
system and the lac operon were also used for regulation. RFP and YFP were also
introduced as reporters for the activities of the sub-networks. The results and theperspective will be discussed in this meeting.
1PT216

1PT218細胞分化の力学系モデル：安定な発生過程と細胞リプログラミングの理解へ向けて

A dynamical system modeling of cell differentiation forunderstanding of robust development and cell reprogramming
Chikara Furusawa1, Kunihiko Kaneko2 (1QBiC, RIKEN, 2Dept. Basic Sci.,
Univ. Tokyo)
Two important problems in stem cell biology are how to characterize thedifference in cellular states between undifferentiated stem cells and terminallydifferentiated cells, and how to understand the dynamics of cell reprogrammingfrom terminally differentiated cells to undifferentiated cells. In this study, toanswer these questions, we have carried out a dynamical systems modeling ofinteracting cells with a gene regulatory network to reveal stem celldifferentiation triggered by the cell-cell interactions. We have simulated cellular
dynamics using gene regulatory networks with a small number of genes (e.g,less than 10), and screened regulatory networks that can generate cell-type
heterogeneity through cell-cell interaction by using genetic algorithm
simulations. The results revealed that expression dynamics of cells that havepotential to differentiate (i. e. stem cells) generally show irregular (chaotic)
oscillation, whereas such complex oscillation is lost for terminally differentiatedcell types. Furthermore, we found that, to reprogram terminally differentiatedcells to undifferentiated cells, a perturbation to genes which are positively autoregulated by its products is important. The emergence of stable cellular statesthat are not chosen by normal development and its relevance to cancer cells will
also be discussed.

カタユウレイボヤ初期胚におけるラマン顕微鏡による細胞内分子の検出

1PT219Detection of bio-molecules localized in specific cells of Ciona
intestinalis embryo by Raman spectroscopy
Mitsuru Nakamura, Kohji Hotta, Kotaro Oka (Graduate School of Scienceand Technology, Keio University)
Localization of bio-molecules plays central roles in the specification of cell fatesand morphogenesis in the embryogenesis. For this reason, to trace the
distribution of bio-molecules, especially, mRNAs and proteins, is one of the
major issues in developmental biology. In current study, genetic manipulation
can be used to observe molecular distribution through the use of fluoresceinlabeling molecules. This technique, however, can result in phenotypic changes,
need prior identification of target molecules, and the number of labels
applicable is limited. Raman spectroscopy, in contrast, is a low-invasive and“label-free” technique, allowing imaging of the distribution of the number ofbio-molecules, including molecules without prior identification. In this study on
the Ciona intestinalis embryogenesis, we explore the application of Raman
spectroscopy within the developmental process to investigate the distribution ofbio-molecules without labeling. We applied Raman spectroscopy to collect the
spectra scattered from Ciona embryo at single-cell resolution for early
developmental stages. After the spectral analysis, we succeeded to detect
vegetal hemisphere-specific localization of some bio-molecules and its
localization is similar to maternal factors. This result suggests that Raman
spectroscopic imaging in low-invasive and label-free manner on the spatial
distribution of bio-molecules is feasible to investigate for developmental studies
in Ciona intestinalis.
1PT217this model the network of three genes is assumed; the network is composed ofgene A which maintains the pluripotent state, gene B which induces thedifferentiation to one lineage, and gene C which induces the differentiation to theother lineage. Each TF synthesized from each of gene A, B, and C is assumed tomutually represses the expression of other genes. We analyze how the difference
between the rate of the TF binding/unbinding and the rate of the histone
methylation/demethylation affects the process of transitions among the cell states.

過程の組織大変形シミュレーション

Simulations of large deformation during tissue morphogenesis
using reversible network reconnection model
Satoru Okuda1, Yasuhiro Inoue1, Mototsugu Eiraku2, Yoshiki Sasai2, Taiji
Adachi1 (1Inst. Frontier Med. Sci., Kyoto Univ., 2Riken CDB)
Tissue morphogenesis is accompanied by large three-dimensional (3D)
deformations, in which mechanical interactions among multiple cells are
spatiotemporally regulated. To reveal the deformation mechanisms, in thisstudy, we newly propose the reversible network reconnection (RNR) model.
The RNR model is developed on the basis of 3D vertex models, which express amulticellular aggregate as a network composed of vertexes. 3D vertex models
have successfully simulated morphogenetic dynamics by expressing cellular
rearrangements as network reconnections. However, in cases simulating large
deformations during dynamic tissue morphogenesis, a 3D vertex model canpossibly induce unphysical results and failures. We found this is because ofgeometrical irreversibility, energetic inconsistency, and topological irreversibility in network reconnections. Mathematical and numerical analyses suggested
that these problems can be resolved in the RNR model. In addition, todemonstrate the applicability of the RNR model, we simulated tissue
deformation of growing cell sheets and showed that the proposed model cansimulate large tissue deformations, in which large changes occur in the local
curvatures and layer formations of tissues. Thus, the proposed RNR model
enables in silico recapitulation of complex tissue morphogenesis.
1PT220細胞分化のエピジェネティック動力学の平均場理論

Expression of the marker genes of cell differentiation is regulated by the
epigenetic modifications of histones, and such epigenetic modifications are
regulated by the expression of genes which encode transcription factors (TFs).
We analyzed this entangled dynamics of gene expression and epigenetic
modification by developing a mean field model of the simplified gene network. In

細胞体位置と神経突起の伸長方向を制御した 3 次元脳回路再構成技術の開発

Mean field theory of epigenetic dynamics of cell differentiation
Takaya Koshii1, Tomoki P. Terada2, Masaki Sasai2 (1Department of Applied
Physics, Nagoya University, 2Department of Computational Science andEngineering, Nagoya University)

リバーシブル・ネットワーク・リコネクションモデルによる形態形成

Development of Three-dimensional reconstruction brain
circuit controlling the direction of neurite elongation and cellbody position
Aoi Odawara, Ikuro Suzuki, Masao Gotoh (Tokyo University of Technology)
3D reconstruction techniques in biological tissues have been expected to bebeneficial in validation of drug candidates and an effective transplanttechnology in regeneration medicine. Recently, 3D reconstruction techniques
using cells combined with biocompatible materials have been developed. In− S105 −particular, cell sheet transplantation techniques applied to heart and retina.
However, the 3D reconstruction of biological tissues that has an orientation as
the brain nerve tissues is still not developed.The aim of this study is to develop a
3D reconstruction technique of brain circuit, controlling the spatial arrangementof neuronal cells. We have developed a 3D neuronal circuit culturing techniqueby controlling the position of nerve cell body and the direction of neurite
elongation using collagen fiber orientation techniques and poly dimethylsiloxane (PDMS) micro-processing. As a result, we observed that the neurite grow in
one direction along the collagen fiber with cell body position fixed. In addition,
by observing neurite configuration in Z axis using confocal microscope, we
confirmed that neurite in 3D gel matrix are elongated in one way. Furthermore,a multilayer structure that mimics the six-layer structure of the cerebral cortex
was successfully created. We were able to confirm that neurite are elongated in
one way among three layers. Finally, to confirm the function of the 3D
reconstructed neuronal circuit, we were able to obtain the propagation of action
potential between two cell body layers using multielectrode arrays (MEAs).
1PT2213 次元ネットワーク再構成のための神経組織の脱細胞化条件

Decellularized conditions of neuronal tissue for thereconstruction of 3D neuronal network
Shota Amano, Ikuro Suzuki, Masao Gotoh (Tokyo University of Technology)
Artificial heart and joints has been translated into the medical field. In recent
years, transplantation techniques used by these artificial tissues have been
expected. Cell transplantation using cell sheet such as in epidermal tissue,corneal epithelium, and the heart, has been successful. The discovery of iPS cellmade it an important tool in creating a variety of tissues, but in cell culture, theuse of decellularized cellular components such as scaffold has been of interest
because of its ability to keep the three-dimensional tissue structure andextracellular matrix (ECM) stable. Until now, changes in decellularization
conditions and ECM evaluation in liver, lung, heart tissues, and muscles has
been investigated, but not in nerve tissue. Therefore, the aim of this study is toinvestigate the changes in decellularization conditions and ECM evaluation of
nerve tissue. Using rat brain, nerve tissue decellularization was performed using
de-sodium dodecyl sulfate (SDS), Trypsin/EDTA, and TritonX-100, decellularized tissues were stained using hematoxylin and eosin (H&E). In addition, theprocessing of each decellularized nerve tissue’s ECM was evaluated. Together
all the results of nerve tissue decellularization, will be reported at theconference.
1PT222一分子蛍光ソーティングを目指したマイクロ流体システムの構築

Construction of a microfluidic system for fluorescence-based
single-molecule sorting
Sho Saitoh1, Hiroyuki Oikawa2, Kiyoto Kamagata2, Satoshi Takahashi2(1Grad. Sch. Life Sci., Tohoku Univ., 2IMRAM, Tohoku Univ.)
Techniques of sorting micro-particles such as cells have found wide applications
in biological sciences; however, sorting of biological molecules with relatively
small molecular weights including proteins and nucleic acids is still difficult.
This is mainly because of the lack of the method to discriminate molecules
sensitively at the single molecule level. We are currently constructing afluorescence-based single-molecule sorting device, which is composed of an
optical system for the detection of fluorescence from fluorophores labeled tosample molecules and the microfluidic system for the sorting of molecules after
the fluorescence detection. As the optical system, we utilized a light correction
system based on a spherical mirror, which achieves a large detection area andhigh numerical aperture at the same time. As the microfluidic system, weconstructed a silica-based chip, in which an inlet channel and two outlet
channels for waste and collection were carved. The sample was introduced tothe inlet channel, and its fluorescence intensity was detected at the observation
point. Based on the detected fluorescence intensity, the flow was controlled todirect sample either to the waste channel or the collection channel. We utilized
miniature solenoid valves to switch the sample flow. We confirmed that thesystem can be used to sort a mixture of two kinds of microbeads with different
colors. To further examine the performance of the developed system, we are
currently conducting sorting experiments of DNAs with different lengths.
1PT223電子顕微鏡画像分類の利便化のためのツール開発

Development of a convenient tool for making processes ofparticle clustering and averaging in 3D reconstruction fromelectron micrographs

Tatsuya Kihara, Takuo Yasunaga (Dept. of Biosc. and Bioinfo., Grad. ofComp. Sc. and System Eng., Kyushu Inst. of Tech.)
Eos (Extensible object-oriented system for macromolecular imaging), we havebeen developing, has many tools to process electron micrographs andenvironment for software development. Some tools provide GUI (Graphical
User Interface) -based one in Eos but others not. A group of program forclassification and averaging has been also implemented as only CUI (Character
User Interface)-based ones. Therefore, we tried to develop a convenient toolwith GUI. This was implemented by an integration of different programs under
Eos and so only a few simple steps using mouse were necessary for processing
to be executed. I succeeded in developing the tool with GUI and it wasevaluated in actual analyses by some researchers. At the last meeting, wereported the evaluations of the tool using model data sets, phantoms. We herereport evaluations for actual electron micrographs in these analyses. Actually,
the tests showed some problems from the researchers such as limits of image
number, no function for resetting zooming size and so on. At present we solved
some of the problems, added to new functions and modified the tool to be moreconvenient. However some still remain. Furthermore, we try to test the tools
iteratively and add functions and parameters for more precise image analysis
and refinement.
1PT224

高重力を利用した非球形構造を持つマイクロハイドロゲル粒子の作
製法

High-gravity-based synthesizing method for hydrogel
microparticles with non-spherical structures
Masayuki Hayakawa1, Hiroaki Onoe3, Ken H. Nagai4, Masahiro Takinoue1,2(1Interdisciplinary Grad. Sch. Sci. and Eng., Tokyo Tech., 2PRESTO, JST, 3IIS,
Univ. of Tokyo, 4Dept. Phys, Univ. of Tokyo)
In recent years, hydrogel-based anisotropic microparticles have attracted muchattention due to their various applications including micrometer-scaled selfassembly, tissue engineering, drug delivery, and artificial cell models. By threedimensionally organizing such anisotropic microparticles encapsulating somefunctional molecules, it will become possible to construct self-assembled
systems with higher-order functions as living cells and living tissues. However,constructing such three-dimensional structures from anisotropic microparticles
is now difficult because it is unable to synthesize three-dimensionally complex
anisotropic particles such as colorful beach balls, gears, etc. due to the use of
two-dimensional microfluidic channels in the synthesis of microparticles. Thus,
it is desired to develop a method to generate three-dimensionally anisotropic
microparticles.
In this presentation, we report a method for generating three-dimensionallyanisotropic microparticles such as hemispherical microparticles. This method
was based on Centrifuge-based Droplet Shooting Device (CDSD) [1]; it is a
device to synthesize monodisperese microparticles with multiple compartments.
Here, we constructed three-dimensionally anisotropic microparticles byremoving a part of the multiple compartments of the microparticles. We believethat our method will contribute to the study of more complex self-assembled
systems of microparticles.
[1] Maeda et al., Adv. Mater. (2012)
1PT225

ハイドロゲルマイクロファイバー内での血管内皮細胞の管構造形成

Tube Formation of Endothelial Cells in the Hydrogel
Microfiber
Hiroaki Onoe1,2, Shoji Takeuchi1,2 (1IIS, The University of Tokyo, 2ERATO
Takeuchi Biohybrid Innovation Project, JST)
Creating vascular network in a macroscopic artificial tissue has been stated as
an essential element for constructing long-life artificial tissues having nutrient/metabolite circulation. Various attempts have been made to create blood vessels
in an artificial tissue though, the arrangement of vascular network in a designed
3D configuration have still been a challenging issue.
Recently, we have proposed a cell-encapsulating core-shell hydrogel fiber,
named “cell fiber”. One important feature of our “cell fiber” is that we canfreely manipulate the fibers with keeping cellular activities and assemble theminto designed 3D configurations. Here we present the tube formation ofendothelial cells in the collagen/alginate core-shell hydrogel microfiber. We
found that encapsulated MS1 (mouse pancreatic endothelial cell line) and
HUVEC (human umbilical vein endothelial cell) spontaneously create tube-likemonolayer cell structures along the axial direction of the fiber at meter-lengthscale. Because the produced endothelial tubes are free-standing and can be
manipulated in a culture medium with the support of the alginate shell, our− S106 −endothelial cell fibers would be a fundamental tissue unit for constructing threedimensional (3D) macroscopic tissues with vascular network and for analyzing
cellular behaviors (ex. vasculogenesis or angiogenesis) under intercellular/
chemical/mechanical/fluidic interactions.
1PT226

分子ロボットによる人工パンスペルミア

Artificial Panspermia by Molecular Robots
Masahiro Endo1, Kei Fujiwara1,2, Syougo Hamada1,3, Satoshi Murata1,4,
Shin-ichiro. M Nomura1,5 (1Undergrad. Sch. Eng., Tohoku Univ., 2JSPS.
Postdoc., Tohoku Univ., 3Assi Prof. Tohoku Univ., 4Prof. Tohoku Univ., 5Assoc
Prof. Tohoku Univ.)
Molecular robotics is an emerging research field aiming at development of asystematic methodology to design complex molecular machines, which have apotential to overcome difficult problems that natural living system cannot solve.
Recently, owing to the rapid progress in DNA nanotechnology, many types ofmolecular devices have been proposed. Those devices, usually made of several
artificially synthesized DNA, have shapes and functions designed by humans.
However, these devices work only in test tubes. Extending their operation to thewider conditions, such as in vivo, in low temperature and in high/low pH, is oneof the important research targets of molecular robotics. An example of suchextreme environment is the space, where not only the zero pressure but intensive
radiations surround the molecules. Here, we think artificial pansperimia* is agood target for molecular robotics, and focus on environmental resistance ofmolecular devices against cosmic radiation and freeze-dry treatment. As a test
piece, we have designed a DNA nanostructure and evaluated its functionality (inthis case, the shape) after re-hydration. Atomic force microscopy observation
showed that the structure well maintains the designed shape throughout thetreatment. * “Panspermia” is a hypothesis about the origin of life, assuminginterstellar travel of living organisms.
1PT227細胞膜上の分子ロボットの作成に向けてPrototype molecular robots working on cellular membrane
Yusuke Sato1, Kei Fujiwara2, Shogo Hamada3, Satoshi Murata4, Sin-ichiro
Nomura5 (1Undergrad. Sch. Eng., Tohoku Univ, 2JSPS. Research Fellow.
Tohoku Univ, 3Assi Prof. Tohoku Univ, 4Prof. Tohoku Univ, 5Assoc Prof.
Tohoku Univ)
Recent progress of micro-nano technology enables us to design " molecularrobots" [1,2,3]. Molecular robot is a system of molecular devices in which DNA
nanostructures, DNA devices and modified proteins are combined as acoordinated system. They are in the scale of up to several tens nanometer, andhuge number of the same robot can be produced in a test tube by self-assembly.
We are investigating interaction between such molecular robots and living cells.
In the operations of the molecular robots, the following features are important:
1) target cell specificity and 2) launching/landing back to/from the target. Here,
we present a design of prototype robot working on cellular membrane. The
robot has hydrophobic " legs" that holds onto the membrane. Because ofdiffusion of lipid molecules in the membrane, the robot is also expected to show
lateral diffusive behavior. To verify this, single molecular tracking experimentwith fluorescent-tagged robot has been conducted. The data obtained by totalinternal reflection fluorescence (TIRF) microscopy clearly showed lateral
diffusion of the robot in several seconds. In the future applications, these robots
floating on the membrane will protect cells from virus infection, or will used tocontrol the specific receptors on the cell.
[1] G. M. Churchet al. Science 2012, 335, 831-834 [2]W. Winfree et al. PNAS
2012, vol.109, no.17, 6405-6410 [3]H. Sugiyama et al. JACS 2010, 132, 15921597
1PT228by external magnetic field. It was predicted that the accumulation pattern of themagnetic particles is strongly effect the whole droplet movement. The droplet
can easily transfer the structure into giant liposome by passing through thelipidic oil/water surface. Thus, we are now designing a "movable" molecularsystem on using this chassis. The result will be reported.
1PT229Bio-hybrid system has been attracting attention in recent years. The biologicalmacromolecule and living organism are worked as very small and highly
effective actuator and these bio-actuators are expected as next generation
motivity. Especially, several bio motors were reported. However, the research
field of bio-actuator is in advance, many unsolved problem are exist. For
example, biological reaction is not well known, evaluation of bio actuator is notenough, therefore developing control method for such kind of reaction isimportant. In this study, to realize the new bio motor, which can be applied tomicro-nano systems power source (built-in mechanical factor), we used Vibrio
alginolyticus, some kind of marine bacteria, specific motile mode “Surface
swarming” as actuator. At first, we tried to drive micro beads on surface
swarming of V. alginolyticus. Furthermore, we succeeded in controlling
movement of surface swarming by ratchet shape micro channel and then driving
the micro gear in the closed micro channel. As a next step, to transmit power ofrotational movement, we fabricated the open micro channel and micro object(floating pillar, micro gear etc.) and succeeded in driving the object in the open
micro channel. In addition, we revealed that rotational power of floating pillar
by deformation of nano probe. Finally, we assembled the micro gear with shaft
like crank. Therefore, it became possible transmitting the power from rotational
movement to outside. Thus, we achieved to construct base of bio-motor.
1PT230Evaluation of microtubule deformation by live cell imaging
and image analysis
Ryosuke Tanaka, Takeomi Mizutani, Hisashi Haga, Kazushige Kawabata(Grad. Sch. Life. Sci., Univ. Hokkaido)
Deformation of cytoskeleton is thought to be a converter of mechanical stimulus
into chemical activity in living cells. Although recent reports showed that
actin fiber, one of cytoskeleton, acts as a converter, contribution of othercytoskeletons is still unclear. As a first step to elucidate this question, we began
to evaluate deformation of other cytoskeletons.
We evaluated microtubule deformation by live observation of GFP-tagged
tubulin expressing cells and image analysis. We observed microtubules
dynamically bent in contrast to straight actin fiber. Therefore, longitudinal
deformation of microtubule is difficult to evaluate by using actin fiber
deformation analysis. To overcome this problem, we developed image analysis
software aimed to extract fluorescent intensity profile along curved microtubuleand identify the same points on the fiber from a set of time course images.
Microtubule deformation was evaluated by time change of distance between theidentical points. We successfully evaluated deformation of curving microtubule.
The next step of our study is to evaluate validity of our analysis. In this annual
meeting, we will also report microtubule deformation under cell deformation.
Besides, we will discuss roll of microtubule deformation as a cellular sensor.
1PT231

Molecular robotics is a new research field to develop an artificial molecularsystem. The major methodology, self-organization of biomacromolecules, isquite suitable for biological applications. Thus, designing smart drag delivery
system (DDS) is strongly expected in the future. Here we aim to create a" remote-controllable" molecular system. We constructed the prototype withwater-in-oil droplet (10 to 50 micrometer in diameter), incorporated magnetic
nano particles. The magneto-droplets showed three-dimensional motion induced

マイクロファイバーゲルマトリックスの弾性パターニングによる三次元細胞運動のメカニカル制御

Mechanical control of 3-D cell movement in elasticallymicropatterned matrix of micro-fiber gels

分子ロボティクス：リモコン分子クロラ・プロトタイプの構築

Molecular robotics: Construction of a remote control moleculecrawler prototype
Daiki Komatsu, Kei Fujiwara, Shin-ichiro M. Nomura (Tohoku University)

バクテリア駆動マイクロギアの構築とトルク計測

Building and torque measurement of bacteria driven micro gear
Masaru Kojima1,2, Tatsuya Miyamoto2, Masahiro Nakajima3, Michio
Homma4, Toshio Fukuda2,3 (1Present address: Grad. Sch. Eng. Sci., Osaka
Univ., 2Dept. Micro-Nano Sys. Eng., Nagoya Univ., 3Center for Micro-Nano
Mech., Nagoya Univ., 4Div.Bio. Sci., Nagoya Univ.)

Aya Ogata1, Satoru Kidoaki2 (1Grad. Sch. Eng., Univ. Kyushu, 2IMCE, Univ.
Kyushu)The cell movements are critically affected by the stiffness of extracellularsurroundings of 2-D substrate surfaces or 3-D matrices. To scrutinize themechanobiology of the cell motility, systematic design of the extracellularmechanical surroundings is quite essential. In this study, we have developped
the novel methodology of microelasticity-design of 3-D cell culture matrix
which consists of nonwoven mesh of swollen micro-fiberous gels with welltuned different elasticity, in order to clarify the effect of matrix stiffness on the
3-D cell movement. Nanofiber mesh of photocurable styrenated gelatin (StG)− S107 −including radical generator camphoquinone was prepared by electrospinning,cross-linked in ethanol media, and swollen in aqueous solution, which
successfully produced swollen micro-fiber gel matrix. By regulating thephotocuring conditions, elastic modulus of fiber gel sheet was well-tuned.
Assessment of movement of 3T3 fibroblasts on the prepared gel sheets withdifferent stiffness showed that cells extended well on the surface of the sheet
and did not enter the inside of soft matrix (1.4 kPa), whereas cells got into thehard sheet (15.8 kPa) and finally reached to the bottom of the sheet 6 hours after
seeding. This result suggested that 3-D cell mechanotaxis can be driven bymatrix-stiffness dependent manner as well as the 2-D mechanotaxis. Mechanismof such 3-D mechanotaxis and possible manipulation of 3-D cell movement by
the design of stiffness of gel sheet are discussed.
1PT232

微視的弾性境界の曲率設計による細胞メカノタクシスの高効率誘導

Efficient manipulation of cell mechanotaxis: effect of thecurvature of micro-elasticity boundary
Ayaka Utsumi1, Satoru Kidoaki2 (1Grad. Sch. Eng., Univ. Kyushu, 2IMCE,
Univ. Kyushu)
The directional cell movements induced by a mechanical gradient on a substratesurface, so-called mechanotaxis, are useful model phenomenon to investigate
the mechanobiology of cell motility. So far, we have established the elasticity
boundary conditions required to induce mechanotaxis of fibroblasts with certain
reproducibility; sharp linear boundary with enough narrow width and enoughhigh elasticity jump in the single-cell adhered interface on cell culture gels.
However, the induction efficiency of mechanotaxis was significantly affectedby the shape of moving cells, suggesting the importance of a shape interaction
between cells and elasticity boundary. In this study, to assess the effect of the
shape interactions on the induction efficiency of mechanotaxis, a curvature of
the elasticity boundary was systematically designed with a custom-built maskfree photolithography using elasticity-tunable photocurable styrenated gelatin.
Fixing the elastic gradient strength as 30-40 kPa/ 50 μm, the radius of curvatureof boundary was designed as 50, 100, 250, 500, 750, and 1000 μm in bothconvex and concave shapes. Migration of 3T3 fibroblasts was observed on theseboundaries and efficiency of induction of mechanotaxis was analyzed. As theresult, highly efficient mechanotaxis was induced on the convex boundary with
100 μ m in radius and on the concave boundary with 750 μ m in radius.
Interestingly, directional cell migration toward a softer region emerged on the
concave boundary with 50 μm in radius for the first time.
1PT233

線虫内部への蛍光ナノビーズの選択的導入

Selective Injection of Fluorescent Nanobeads into
Caenorhabditis elegans
Masahiro Nakajima1, Hirotaka Tajima2, Masaru Kojima2, Naoya Nakanishi2,
Naoki Hisamoto3, Michio Homma3, Toshio Fukuda1,2 (1Cent. For Micro-nanoMechatro., Grad. Sch. of Engineer., Nagoya Univ., 2Dept. of Micro-Nano Sys.
Engineer., Grad. Sch. of Engineer., Nagoya Univ., 3Div. of Biol. Sci., Grad.
Sch. of Sci., Nagoya Univ.)
We present a novel selective injection of fluorescent nanobeads into
Caenorhabditis elegans (C. elegans) based on bio-nanomanipulation techniquefor in-vivo single cell analysis.
The nanomanipulation system was constructed under hybrid microscope. The
hybrid microscope consists of the optical microscope (OM) and environmentalscanning electron microscope (E-SEM). It realizes detail analyses of biological
specimens by fluorescent imaging by the OM, and nano-scale manipulation by
the imaging of the E-SEM.
Based on the bio-nano manipulation system, we propose a nanoinjection methodby the nanoprobe insertion for embeddedness of nanobeads. The nanoprobe wasdesigned to have the four tips to fix single nanobead. It was fabricated byfocused ion beam (FIB) process. The nano-scale probe size is considered to beimportant for minimal damage to the biological specimens.
In this work, as a biological specimen, the C. elegans was used, because it is oneof the important model organisms for various biological analysis. The
nanoinjection technique was applied to embed a fluorescent nanobead into a
specific cell inside C. elegans for in-vivo analysis. We inserted the nanoprobe
into the body of C. elegans at a depth in approximately 6 μm. The size offluorescent nanobeads was 500 nm in diameter. The proposed system isconsidered to be important as future nano-surgery system for life innovation
using model organism.

1PT234W/O ドロップレット内での酵母を用いた G タンパク質共役受容体のリガンドアッセイシステム

Yeast-based ligand assay system for detecting G proteincoupled receptor activation in water-in-oil droplets
Takashi Sakurai1, Ryo Iizuka1, Yasuyuki Tanigaki2, Rui Sekine3, Dong H.
Yoon3, Tetsushi Sekiguchi4, Jun Ishii5, Akihiko Kondo6, Naoto Nemoto2,
Shuichi Shoji3, Takashi Funatsu1 (1Grad. Sch. of Pharm. Sci., The Univ. ofTokyo, 2Grad. Sch. of Sci. and Eng., Saitama Univ., 3Major in Nanosci. andNanoeng., Waseda Univ., 4Nanotech. Research Center, Waseda Univ., 5Org. of
Advanced Sci. and Tech., Kobe Univ., 6Grad. Sch. of Sci. and Tech., Kobe
Univ.)
G protein-coupled receptors (GPCRs) are involved in many different cellular
processes, and are targets of many therapeutic drugs. We developed a yeastbased ligand assay system for detecting GPCR activation in water-in-oil (W/O)
droplets. Our system is based on linking between the genotype and thephenotype using W/O droplets. DNA library encoding peptides, in vitrotranscription-translation system, and budding yeast cells expressing target
GPCR are co-encapsulated into droplets using a microchannel. Yeast cells are
genetically engineered to express GFP in response to ligand stimulation. The
generated droplets were collected into a microcentrifuge tube and incubated toallow the gene translation and ligand stimulation. The droplets would be
fluorescent when synthesized peptides stimulate GPCRs, enabling easy
identification of ligand candidates. Using this system, we firstly demonstrated
cell-free GFP synthesis in the presence of yeast cells in W/O droplets, indicating
the presence of yeast cells did not inhibit protein expression. Next, we
encapsulated yeast cells expressing Ste2p (an endogenous GPCR) and the geneof α-factor (ligand of Ste2p) in the droplets. After incubation, GFP-expressing
cells were clearly observed in response to α-factor stimulation. The α-factor
synthesized from the five-molecule DNA template in the droplets successfully
induced GFP expression. Our system will provide an effective approach for thehigh-throughput screening of peptide ligands targeted at mammalian GPCRs by
evolutionary molecular engineering.
1PT235フロー型乳酸バイオセンサの開発とその脳内乳酸測定への応用

Development of a flow-type lactate biosensor and its
application to the measurement of lactate in the mouse brain
Kaoru Yamazaki1, Mai Ichikawa1, Ryo Shimazaki1, Hideo Mukai2, Minoru Saito1(1Graduate School of Integrated Basic Sciences, Nihon University, 2Faculty of
Medicine, Saitama Medical University)
Lactate biosensors which enable us to measure lactate concentration in bloodand other body fluids are applicable to some medical fields such as sports
medicine. We fabricated a lactate biosensor by a flow-type sensing system, inwhich the injected sample flows into an immobilized enzyme (lactate oxidase;
LOD) column. The column was fabricated by packing an immobilized enzyme
support into a 30 mm long column. The support was obtained by immobilizing
LOD on silica particles (particle size: 30-60 mesh). The immobilization was
performed by loading LOD onto the aminoalkyl-bonded particles withglutaraldehyde. The column was kept at a constant temperature (37°C). The
generated H2 O2 or consumed O2 in the column was detected by the H2 O2electrode or O2 electrode. The fabricated biosensor could measure lactate
concentration up to 10 mM. By using it, we next measured lactate levels of themouse brain. The samples were prepared by homogenizing the brain slices in
ACSF. The results showed the differences of lactate levels among each part ofthe brain and among each age of the mouse.
1PT236マイクロチャンバー内でのβ -グルコシダーゼ 1 分子の酵素活性のモニタリング

Monitoring the single-molecule enzymatic activity of β-glucosidase
in a microchamber array chip
Kentaro Tahara1, Ryo Iizuka1, Takao Ono2, Kiyohiko Igarashi3, Masahiro
Samejima3, Takanori Ichiki2,4, Takashi Funatsu1,4 (1Grad. Sch. of Pharm. Sci.,
The Univ. of Tokyo, 2Grad. Sch. of Eng., The Univ. of Tokyo, 3Grad. Sch. ofAgri. and Life Sci., The Univ. of Tokyo, 4JST-CREST)
β-glucosidase (BGL), which catalyzes the hydrolysis of cellobiose to glucose, is
one of the important enzymes in bioethanol production. We aim to obtain BGL
mutants with improved hydrolytic activity by directed evolution. To screen
many potential mutants easily and precisely, we are trying to develop the single-− S108 −molecule assay for BGL activity in a microchamber array chip.
The microchamber array chips were fabricated by micromolding processes with
polydimethylsiloxane polymer. BGL1B from the basidiomycete Phanerochaetechrysosporium was confined with fluorogenic substrate [Fluorescein di-β-Dglucopyranoside (FD-Glu), TokyoGreen-β Glu (TG-Glu) or Resorufin β -Dglucopyranoside (R-Glu)] in microchambers with a volume of 50 fL. The
enzyme produced a large number of fluorescent molecules by hydrolyzing
fluorogenic substrates, leading to fluorescence increase in microchambers.
However, in the absence of BGL1B, TG-Glu was found to be hydrolyzed
spontaneously in microchambers, whereas the others not. We also proved that
R-Glu is more adequate for the enzymatic assay than FD-Glu. Unlike FD-Glu
that requires a two-step hydrolysis to generate maximum fluorescence, R-Glu
requires only a single-step hydrolysis reaction to attain full fluorescence,
thereby affording a higher rate of fluorescence increase and ensuring the linear
relationship between the fluorescence intensity and the BGL activity. Hence,using R-Glu, we are trying to monitor the single-molecule enzymatic activity of
BGL1B in microchambers.
1PT237フェリチン空洞内でのレアアース(Eu、Tb、Tm、Y)ナノ粒子合成

Synthesis of rear earth (Eu, Tb, Tm and Y) nanoparticles in
apoferritin cavity
Tomoaki Harada, Hideyuki Yoshimura (Meiji Univ.)
Eu, Tb, Tm and Y nanoparticles were fabricated in the cavity of a cage shaped
protein, recombinant apoferritin (fer0). Apoferritin is a spherical protein of 13
nm diameter with a cavity of 7 nm diameter. It is known to mineralize several
metal ions in the cavity. Eu nanoparticles were synthesized by mixing 0.1 mg/ml
apoferritin with 100 mM MOPS (pH 6.8) and 1 mM Eu(NO3)3 6H2O and kept
standing for 24 hours at room temperature. This solution was centrifuged for 3
minute at 12000 rpm to remove small amount of precipitation. The supernatant
was dialyzed with 20 mM NH3 acetate, whose pH was adjusted to 8.0 by adding
NH3, to stabilize core in the ferritin cavity. Tb, Tm and Y nanoparticles wereprepared as the same method, except 100 mM MOPS (pH 7.0) was used for Y
nanoparticles. The nanoparticles formation was confirmed with transmission
electron microscopy (TEM) with and without aurothioglucose negative staining.
The lattice structure of the cores was observed by high resolution TEM(HRTEM). Some of cores were showed lattice structure, however most of cores
were amorphous. These nanoparticles were analyzed with an EDX and thespecific peaks of expected elements were observed. The solution of Eu
nanoparticles was applied to gel-filteration (Sephadex G-25) with elution bufferof 5 mM NH3 acetate (pH 8.0) and was dried at 60℃. Subsequently, it was
sintered at 800℃ for 4 hours in the air. The sintered specimen showed electronic
diffraction pattern corresponding to the lattice spacing of Eu2O3.
1PT238

カゴ状蛋白質アポフェリチンを使用したガドリニウムナノ粒子の作成

Synthesis of gadolinium-based nanoparticles using the protein
cage of apoferritin
Hiroko Fukano, Hideyuki Yoshimura (Physics. Univ. Meiji)
Gadolinium-based nanoparticles are expected as a better contrast agent formagnetic resonance imaging (MRI), since their effect for the relaxation rate ismuch higher than those of chelated gadolinium (III) complexes used in the
medical field. Here we report the synthesis of gadolinium oxide nanoparticles
in the apoferritin cavity which is known to reserve metals as oxide or oxyhydroxide state. The apoferritin coated-gadolinium nanoparticles weresynthesized by the following simple procedure; the mixture solution of 0. 1
mg/mL recombinant apoferritin, 1 mM Gd(NO3)3 6H2O, and 0.1 M MOPS (pH7.0) was just incubated for overnight at 4 ºC. The synthesized nanoparticles
were characterized by TEM and EDS. Homogeneous and spherical nanoparticles coated with protein shell were confirmed by TEM. The average diameter ofthem was determined to be 5.8 ± 0.5 nm by evaluating 900 particles. According
to the high-resolution TEM images, the nanoparticles showed single-crystal,multi-crystal, or amorphous state. With EDS analysis, the peaks of gadolinium(L α; 6. 06 keV, L β; 6. 71 keV, M α; 1. 19 keV) were detected only at thesynthesized nanoparticles. The electron diffraction pattern suggested that thenanoparticles were gadolinium oxide. However further investigation of crystal
identification is needed because the electron diffraction image was broad andweak.

− S109 −2PS001

タンデム PomA を固定子とする Na+駆動型キメラべん毛モーターの回転計測

Rotation Measurement of Na+-driven Chimeric Flagellar
Motor with Tandem PomA
Yong-Suk Che, Yoshiyuki Sowa (Department of Frontier Bioscience, Hosei
University)
Four PomA and two PomB, homologs of MotA and MotB in the H+ -driven
flagellar motor of Escherichia coli, form a stator complex in the Na+ -driven
flagellar motor of Vibrio alginolyticus. The motor torque is generated by theinteraction between the cytoplasmic domain of PomA and the C-terminal
domain of FliG, a component of the rotor. It was shown that a tandem fused
PomA is functional as a torque generator in V. alginolyticus (Sato and Homma,
JBC 2000). Furthermore, a chimeric stator (which consists of PomA and thechimeric fusion protein PotB) works as a Na+-driven flagellar motor in E.coli(Asai et al., JMB 2003). In the last annual meeting, we demonstrated thattandem PomA dimer was expressed as a single polypeptide in E. coli andswimming speed of E.coli cells with tandem PomA was about half of that withthe monomeric PomA, suggesting that tandem PomA functions as a torquegenerator in E.coli.
Here, in order to characterize the details of tandem PomA, we carried out
rotation measurements of single flagellar motors in E. coli cells expressing
tandem PomA using 1 m beads attached to truncated flagellar filaments.
Maximum motor speed with tandem PomA was 80 Hz, similar to that withmonomeric PomA. However, the motor speed with tandem PomA distributed
broadly compared to that with monomeric PomA. We also measured motor
speeds with tandem PomA with several combinations of mutants in thecytoplasmic charged residues, which are thought to be crucial for torquegeneration. Details of these results will be discussed.
2PS002

枯草菌べん毛モーターの異なる 2 種類の固定子 MotA サブユニッ
トと MotP サブユニットの細胞質内ループの荷電アミノ酸残基の機
能的役割

The functional role of the charged residues of two different
stators: MotA subunit and MotP subunit in B. subtilis flagellar
motor
Yuka Takahashi, Masahiro Ito (Grad. Sch. Life., Univ. Toyo)
The flagellar motor is energized by either H+ or Na+ motive force. MotAB-type
stators use H+, while MotPS-type and PomAB-type stators use Na+ as couplingions. The MotAB-type stator flagellar motor torque in E. coli is considered to begenerated by electrostatic interactions at the interface between rotor and stator
as indicated by previous studies. There are conserved charged residues in thecytoplasmic loop of MotA, which probably interact with the conserved charged
residues of the C-terminal domain of rotor FliG. However, it is not clear whether
the electrostatic interaction between the rotor and the Na+-type stator PomAB isrequired. Here we studied a flagellar motor that consists of two different stators,
MotAB and MotPS, in B. subtilis and tried to identify critical charged residues
for torque generation in each MotA and MotP subunit. We identified theconserved charged residues in the cytoplasmic loop of MotA and MotP. B.
subtilis with mutations in conserved charged residues and several other charged
residues were measured for swarming and stator subunit protein expression
levels. These charged residues in the cytoplasmic loop of stators can be divided
into two types: those important to torque generation by interaction between rotor
and stators, and those involved in stabilization of the structure of stators. Thus,
important charged residues for motility in the H+ -driven MotAB and Na+ driven MotPS were identified in this study.
2PS003

べん毛モーターの回転と GFP 標識固定子の同時観察

Simultaneous observation of the rotation of flagellar motorand GFP-labeled stator unit in a functioning flagellar motor
Mizuki Nakajima1, Hajime Fukuoka2, Yuichi Inoue2, Hiroto Takahashi2,
Akihiko Ishijima2 (1Grad.Sch.Life.Sci.,Tohoku Univ., 2IMRAM.Tohoku Univ.)
The bacterial flagellar motor is driven by the influx of coupling ions, and theinteraction between stators and a rotor is thought to generate the motor rotation.
It have been estimated the number of stators in a motor from the stepwise
increment of rotational speed or the bleaching steps derived from GFP-labeledstator units. However, it is not quantified how many stator units are required foreach step of speed-increment, and the timing of stator assembly in the step ofspeed-increment is not measured. To clarify these questions, the rotational speedof motor and stator assembly should be measured simultaneously. To observe
stator assembly, we newly designed GFP-fused stator units based on
PomAPotB, which functions as the Na+ -driven stator units in an Escherichia
coli cell. When we observe the tethered cell expressing GFP-PomAPotB, thestepwise increment of rotational speed was detected when the motility medium
without Na+ was replaced to the medium containing 85 mM of Na+. Next, wedeveloped the microscopic system to simultaneously obtain the bright field
image (wavelength more than 600 nm, taking by high speed camera) for themeasurement of rotational speed and the fluorescence image (wavelength 500 560 nm, taking by EMCCD camera) for the observation of stator assembly. We
would like to discuss this simultaneous measuring result in the annual meeting.
2PS004

蛍光観察による細菌鞭毛モーターの解析

Imaging of fluorescently-tagged motor components of bacterial
flagella
Yoshiyuki Sowa, Yong-Suk Che (Department of Frontier Bioscience, Hosei
University)
Bacteria, such as Escherichia coli, can swim by rotating their helical flagella,
protruding from the cell body. Rapid rotation of each filament (up to several
hundreds hertz) is driven by a motor located in the cell envelope at the base of
the filament. This motor is powered by the flux of ions across the cytoplasmic
membrane. We have measured its rotation by a bead assay, in which we attach asmall s bead onto the flagellar filament or hook of the motor and detect its
motion. This assay is very useful for measuring the basic properties of the motorand detecting elementary steps in flagellar rotation. However, we can detectmotor rotation only via the filament or hook, although the rotational torque isgenerated at the cytoplasmic side of the motor components, FliG, FliM, FliN,
MotA and MotB. To understand how the motor works, direct observation of
motor movements at the site of torque generation is required. In this study, we
aim at developing the assay to detect motor dynamics by fluorescent microscopy
at single-molecule level. To track movements of single fluorescent dyes withnanometer accuracy, we have built total internal reflection fluorescence (TIRF)
microscope and are currently optimizing it. We also have succeeded in labeling
MotB with rhodamine dyes, which are more suitable for single-molecule
tracking than fluorescent proteins such as GFPs. We confirmed the rhodaminelabeled MotBs function in a motor and currently observe them by TIRF
microscopy.
2PS005

べん毛輸送装置構成タンパク質 FliP ペリプラズム領域の構造機能解析

Structural and functional analyses of a periplasm region of
FliP, a component of the flagellar protein export apparatus
Takuma Fukumura1, Yukio Furukawa1, Yumiko Saijo-Hamano1, Katsumi
Imada2, Keiichi Namba1,3, Tohru Minamino1 (1Grad. Sch, Frontier Biosci.,
Osaka Univ., 2Grad. Sch. Sci. Osaka Univ., 3QBiC, RIKEN)
Most of flagellar proteins are transported by a specific export apparatus from thecytoplasm to the growing distal end of the flagellum where they self-assemble.
The export apparatus consists of a water-soluble ATPase complex and a protondriven export gate made of six membrane proteins FlhA, FlhB, FliO, FliP, FliQ,
and FliR. The ATPase complex escorts flagellar proteins from the cytoplasm tothe export gate. It has been reported that FlhA plays a role in energy
transduction along with the ATPase complex. However, the structure andfunction of other gate proteins still remain unknown. FliP is the only protein thathas a signal peptide in its N terminus. FliP has a molecular weight of about 26
kDa and is predicted to have four transmembrane helices (TM). FliP has arelative large periplasmic domain (FliPp) with ca. 8.5 kDa between TM-2 andTM-3. A deletion of FliPp results in a loss-of-function phenotype. We focusedon FliPp to elucidate the role of FliP in flagellar protein export. Alaninesubstitution of highly conserved residues of FliPp indicated that Glu-178 is
critical for the export function. We constructed an E. coli expression system
of FliPp from Thermotoga maritima for structural analysis. The results ofsize exclusion chromatography, static light scattering and analyticalultracentrifugation indicated that purified FliPp forms homo-tetramer. Far-UV
CD spectra showed that the secondary structure is mostly -helix.
Crystallization for X-ray structural analysis is now in progress.
2PS006− S110 −2 種類の異なる固定子から構成されるべん毛モーターのトルク特性の解析

Torque-speed relationship of the flagellar motor consisting of
two distinct stators
Naoya Terahara1, Yukina Noguchi2, Syuichi Nakamura1, Nobunori Kamiike1, Masahiro Ito2, Keiichi Namba1 (1Graduate School of Frontier
Biosciences, Osaka University, 2Graduate School of Lifescience, Toyo
University)
The bacterial flagellum is an organelle for cell locomotion and consists of ahelical propeller and a rotary motor at its base. The rotary motor is powered byelectrochemical potential difference of ions across the cytoplasmic membrane.
Rotational energy is mediated by inward ion flux through the channel of thestator complexes. Most bacterial flagellar motors are driven by theelectrochemical gradient of H+, and their stator complexes consist of MotA and
MotB. It has been found recently that the Na+-dependent stator required for the
motility of alkaliphilic Bacillus is expressed from a pair of genes, motPS.
Furthermore, Bacillus subtilis, which belongs to neutrophile, has not only
motAB but also motPS on its genome. We have shown that B. subtilis MotPS
plays a major role at an elevated pH, Na+ concentration and viscosity, and thatboth MotAB and MotPS contribute in motility of B. subtilis. However, there has
been no direct evidence to date that these two stator complexes coexist in theflagellar motor. We therefore measured the rotation speed of single motors bybead assay under various conditions to see whether these two stators actuallywork together in a motor. A spherical bead was attached to the sheared flagellar
filament, and the rotation of the bead was followed by a photo-detector. Motortorque was calculated from the rotational speed, bead size and radius of the beadrotation. The details of the analyses will be reported at the conference.
2PS007

べん毛特異的分子シャペロン FlgN から紐解くⅢ型蛋白質輸送制御

Reading of the flagellar type III protein export regulation froma flagellar specific chaperone FlgN
Akira Hida (Graduate School of Frontier Biosciences, Osaka University)
The bacterial flagellum is a rotary motor for motility. It builds up with basal
body, hook and filament. Most of flagellar component proteins are transported
to outside the cell from inside by the flagellar type III protein export apparatus.
It consists of six integral membrane protein (FlhA, FlhB, FliO, FliP, FliQ, FliR)and three soluble proteins (FliH, FliI, FliJ). In addition, FlgN, FliS, FliT and
FliA act as flagellar specific chaperones. The export apparatus is considered toregulate the protein export.
FlgN binds to the hook-filament junction protein, FlgK and FlgL. FliS binds tothe flilament protein FliC. FliT binds to the flilament cap protein FliD. Thesechaperones act as bodyguard to prevent their premature aggregation. Not only
that, FlgN interacts with FliI, FliJ and FlhAC. Through FlgN interacting withFliI and FlhAC, FlgN facilitates the export of FlgK and FlgL. Thus, FlgN seems
to have many functions for the protein export regulation. In the same way, FliSand FliT too. However, most of the regulatory mechanism and chaperone’ sfunctions remains unknown in detail.
In previous time, to investigate the regulatory mechanism, we carried out
genetic analysis of a Salmonella ΔfliH-fliI flhB(P28T) ΔflgN mutant and its
suppressor mutants which have FlhA(D456V, T490M) and FliS(I7V, L114stop,
W122stop). We are continuing its analysis. As a result, We thought that FlgN is
the most important key protein to clarify the regulatory mechanism.
2PS008Controlling Bacterial Flagellar Motor Rotation by Optical
Spanner

Tsai-Shun Lin, Chien-Jung Lo (National Central University)
Bacterial Flagellar Motor (BFM) is a powerful molecular machine that canpropel bacterium to swim in low Reynolds number environment. Many studies
have focused on the mechanical properties of the BFM using electro-rotationand optical tweezers. However, due to the rotational nature of the motor motion,it’s not easy to control the rotation by a symmetry external potential such as
optical tweezer. The external torque can be applied to a micron size
birefringent particle through circular-polarized light. We synthesize uniform
vaterite micro particles with surface coating that can be attached to the bacterial
flagella. While the motor rotates, we can apply external torque toaccelerate/decelerate the motor. We will show our latest result of the torquespeed relationship of single bacterial flagellar motor.
2PS009Insights into myosin V stepping mechanism using point
mutations in the converter

Sergey V. Mikhailenko1, Takashi Ohki1, Jun-ichi Yoshimoto1, Shin'ichi
Ishiwata1,2,3 (1Sch. Adv. Sci. Eng., Waseda Univ., Japan, 2Org. Univ. Res.
Initiatives, Waseda Univ., Japan, 3WABIOS, Waseda Univ., Singapore)
Dimeric myosin V is an intracellular transporter, which moves cargoes towards
the barbed end of actin filaments in cells. Its ability to perform as individual
molecules implies that the ATPase cycles in two head domains must be
coordinated to ensure the efficient unidirectional motility. The kinetics ofnucleotide binding to myosin V is directionally modulated by load, suggestingthat the head-head communication may be achieved via intramolecular load
generated when both heads are bound to actin. Here we used point mutations in
the converter domain to directly test the effect of the intramolecular load on theprocessive stepping of myosin V. The converter is a compact structure, whichtransmits tiny conformational changes at the nucleotide-binding site to the leverarm. To disturb the transmission mechanism, we replaced with alanines, one
at a time, two phenylalanine residues that form a hydrophobic cluster with
the C-terminus of the relay helix. These mutations are inferred to reduce
intramolecular load but affect neither the nucleotide binding nor actin affinity.
We found that the F697A mutation, which completely eliminates intramolecularload, abolishes the motility of myosin V motors. At the same time, the F749A
mutation, which only partly reduces intramolecular load, significantly increases
the proportion of backward steps, providing strong experimental evidence thatthe efficient unidirectional processive stepping of myosin V is ensured by thehead-head communication based on the intramolecular load.
2PS010

負荷存在下におけるアクチン−ミオシン VI 相互作用の自由エネル
ギーランドスケープ解析

Free energy landscape analysis of the actin-myosin VI
interaction under the applied force
Masahito Ikeo1, Masaki Sasai2, Tomoki P. Terada2 (1Dept. of Applied Physics,
Grad. Sch. of Engineering, Nagoya Univ., 2Dept. of Computational Scienceand Engineering, Grad. Sch. of Engineering, Nagoya Univ.)
Myosin VI moves toward the minus end of an actin filament, oppositely to the
motions of myosin II and myosin V. Besides, the large step size (~36nm) of
myosin VI, which has the short lever arms, suggests that the Brownian motion in
the weakly bound state plays an important role in the search for the strong
binding position on the actin filament.
A single molecule measurement using the single headed myosin VI (Iwaki et al.,Nat. Chem. Biol. 5, 403 (2009)) has shown that the probability of strong bindingof myosin VI to the actin filament is dependent on the direction of the applied
force. We here simulate this experimental situation by extending the methods
which we previously used for simulating the Brownian movement of myosin II
in the weakly bound state along the actin filament (Takano et al., PNAS 107,7769 (2010)). Using the coarse-grained model of the actin-myosin VI system,
we performed Langevin molecular dynamics simulations to obtain the effective
free energy landscape in the presence of the different forces applied to myosin
VI. We examine the difference of actin-myosin interface between forward andbackward pulling conditions and correlate this difference with the obtained
effective free energy landscape. Finally, we also discuss on the importance of
this difference in the context of reaction cycle of the myosin VI.
2PS011

ミオシン 6 の双機能性とレバーアーム構造の関係

Relation between myosin VI’s dual functionality and its leverarm structures
Keigo Ikezaki1,2, Tomotaka Komori1, Toshio Yanagida1,2,3,4 (1Graduate
School of Frontier Biosciences, Osaka University, 2Quantitative Biology
Center (QBiC), RIKEN, 3Center for Information and Neural Networks(CiNet), 4WPI, Immunology Frontier Research Center)
Myosin VI is a molecular motor which walks along actin filaments using theenergy of ATP hydrolysis in a dimmer state. Myosin VI has unique aspects
differ from other myosin families. As one of the functional aspects, myosin VI
has two different rolls, a transporter and an anchor, in a cell. Recently, we
performed simultaneous observations of myosin VI during steps which revealed
that myosin VI has unique stepping mechanism taking both distant and adjacentbinding states to the actin filament. We assume that this stepping mechanism isimportant for enabling its dual function. We also assume that myosin VI’s leverarms, which has only two light chains, decrease a steric hindrance between themand enable to take the adjacent binding state for its dual function. Currently, toconfirm this point, we are investigating the stepping behaviors of recombinant
myosins which have modified lever arms.

− S111 −2PS012

アクトミオシン活性に対する尿素とトリメチル N オキシドの中和効果

2PS015Counteractive effect of urea and trimethylamine N-oxide on
the activity of the actomyosin motor

ミオシン首振り運動の分子動力学シミュレーション

Molecular dynamics simulation for the swinging lever-arm
motion of a myosin molecular motor

Kuniyuki Hatori, Ryusei Kumemoto (Grad. Sch. Sci. Eng. Yamagata Univ.)

Tadashi Masuda (Symbiotic Sys. Sci., Fukushima Univ.)

It is known that urea induces unfolding of proteins with solvation whereas
trimethylamine N-oxide (TMAO) stabilizes their structure due to peripheral
hydration. In addition, deleterious effect of urea on the structure of certain
proteins is completely counteracted by the presence of TMAO. We examined
whether their counteraction is effective in actomyosin functions. CD
spectroscopy showed that urea decreased unfolding transition temperature (Tm)of heavy meromyosin (HMM), on the other hand, TMAO increased its Tm. The
decrease of Tm by 0.6 M urea was recovered by 0.3 M TMAO. Both urea and
TMAO in the concentration range of 0-1.0 M considerably decreased the sliding
velocity of actin filaments on HMM molecules under in vitro motility assay.
Similarly, actin-activated ATPase activity was decreased by urea and TMAO
although the ATPase activity of HMM alone was not suppressed. The ureainduced decrease of ATPase activation was further reduced by the addition of
TMAO. Meanwhile, urea and TMAO were found to have counteractive effects
on motility at concentrations of 0.6 M and 0.2 M, respectively. The difference ofcounteractive effects on HMM structure and on the motility suggest that waterrelated action in addition to the structural dynamics of HMM structure
contributes to actomyosin activity.

I have proposed a theory called "Driven by Detachment (DbD)" mechanism forthe working principle of myosin molecular motors. In this theory, the powerstroke of myosin is not directly caused by the hydrolysis energy of ATP, but isproduced by distortion in a myosin molecule, which is generated during thedocking of myosin against an actin filament.
To verify the validity of this theory, a series of molecular dynamics (MD)simulation have been conducted. However, it was found that very longcalculation time is necessary to generate the distortion in a myosin moleculeduring the docking process. Therefore, instead of directly producing the prepower stroke configuration, external force was applied to the end of neckdomain of a myosin molecule.
The MD software used was Gromacs code with its default Gromos96-43a1 force
field and SPC water model. The initial structure of myosin was taken from PDB
file 1M8Q. The AFM pull function of Gromacs was used to apply external force
to the end of the neck domain toward the minus-end direction of an actin
filament. The residues assumed to be engaged in the docking were fixed to thespace.
When the applied force was 17 pN (10 kJ/(mol*nm^2)), the neck domain bent
about 60 degrees around the junction between the head and neck domains after
48 nm. If the applied force was removed, the neck domain returned to the initial
position. As shown in these simulations, the junction between the head and neckdomains works as an elastic element that can produce the power stroke ofmyosin molecular motors.

2PS013アクチン線維が滑走中のミオシン線維トラックからの ADP 遊離速度計測

Measurement of ADP release from bipolar myosin filament
track along which actin filament slides
Takahiro Maruta, Hiroyuki Okubo, Takahiro Kobatake, Shigeru Chaen(Department of Integrated Sciences in Physics and Biology, College of
Humanities and Sciences, Nihon University)
The motility assays have shown that along myosin thick filament, actin filaments
moved at a fast speed towards the central bare zone and at a slower speed awayfrom the bare zone. The force and displacement of myosin crossbridges
interacting with actin filament in the backward direction was smaller than that in
the forward direction. Sellers and Kachar speculated that myosin heads in the
backward movement might rotate 180 degrees so as to face the actin in the rightconfiguration, and might be constrained so that the detachment of the rotatedheads from actin would occur at slower rate. Recently, we have found that thethermal activation energy of the backward movement was significantly higher
than that of the forward movement, suggesting that the backward movement
causes the myosin heads to be constrained, and increases the energy required for
the ADP release step. However, there is no direct evidence for the slow ADP
release rate in the backward movement. Here, in order to examine whether rate of
ADP release step depends on the direction of the actin movement, we measured
nucleotide turnover rates of bipolar myosin thick filament along which actin
filament slides by monitoring the displacement of prebound fluorescent ATP
analog, Cy3-EDA-ATP on flash photolysis of caged ATP.
2PS014

細胞性粘菌ミオシン II の SH1 へリックス領域の変異がその運動特性に与える影響

Effect of mutation of the SH1 helix region of Dictyosterium
myosin II on the motile characteristics
Tsubasa Koyama1, Takahiro Maruta1, Sosuke Iwai2, Shigeru Chaen1(1Department of Integrated Sciences in Physics and Biology, College of
Humanities and Sciences, Nihon University, 2Department of Biology, Facultyof Education, Hirosaki university)
A human myopathy has been reported to be caused by a point mutation in a fast
myosin heavy chain gene. The mutation, glutamic acid to lysine at the 706th
amino acid, is located at the SH1 helix region of the myosin II motor domain.
The SH1 helix region acts as a linker for transmitting the structural changes ofATP-binding site in the catalysis domain to the lever arm. To investigate theeffect of the mutation on the actin-myosin motility, we have introduced acorresponding mutation into the SH1 helix of Dictyosterium myosin II (E683K).
The mutation resulted in a decrease in the actin-myosin sliding velocity (about
1/8 of the wild type), a decrease in thermal stability and the thermal aggregationof the myosin, which might be implicated in the disease process. This suggeststhat SH1 helix has an important function in the motor activity.

2PS016

Movement of cortical actin cables is driven by type V myosins
in fission yeast cells during interphase
Jun Kashiwazaki, Issei Mabuchi (Dept. of Life Sci., Faclt. of Sci., Gakushuin
Univ.)
Actin cytoskeleton is involved in various cellular activities such as cellmorphogenesis, membrane traffic, polarized growth and cytokinesis. In thefission yeast Schizosaccharomyces pombe, actin filaments form three major
structures; patches, cables and contractile rings. During interphase, actin cables
extend from the growing ends toward the cell equator. Although actin cables
have been shown to be important for establishment of cell polarity, their detailed
behavior has still not been clear. We found that thick actin bundles were formed
15 min after addition of the actin stabilizer, Jasplakinolide (Jasp), in interphasefission yeast cells. Interestingly, these actin bundles showed an oscillatory
movement along the long axis of the cell. We aimed to elucidate the mechanismof this movement. Most of the actin bundles moved along cell cortex at a
maximum speed of 0.14±0.04 m/sec. To identify factors which are involvedin the movement of the actin bundles, we treated various mutant cells with Jasp.
We found that type V myosins and the formin For3 are required for the rapid
movement of actin bundles. Especially, Myo51/Myo5 (Win et al., 2001; Motegi
et al., 2001), which is one of the two type V myosins in fission yeast,
colocalized with actin cables at the cell cortex. These results suggest that actin
cables elongating from ends of interphase cells are guided toward the midregion by cortical type V myosins.
2PS017

Contribution of the conformational change of myosin II in the
coupled sliding and binding process of the force-generation
Qing-Miao Nie1,2,3, Masaki Sasai1, Tomoki P. Tereda1 (1Dept. of Comp. Sci.
Eng., Nagoya Univ., Chikusa-Ku, Nagoya 464-8603, Japan, 2Institute for
Molecular Science, Okazaki 444-8585, Japan, 3Dept. of Applied Physics,
Zhejiang Univ. of Tech., Hangzhou 310023, China)
The mechanism of the actomyosin motor is still under debate. In the mostwidely believed lever-arm model, it has been hypothesized that the mechanicalforce is generated by the conformational change of the myosin head which isstrongly bound to the actin filament. In contrast, a single molecule observation
has shown that the unidirectional Brownian motion of the myosin head, while
keeping weakly bound to the actin filament (Kitamura et al., Nature (1999)).
Furthermore, a molecular dynamics simulation study has suggested that theunidirectional Brownian motion is indeed possible due to the asymmetric slopeof the energy landscape arising from the electrostatic interaction between actin
and myosin (Takano et al., PNAS (2010)). In that simulation, however, the− S112 −contribution of the large conformational change of the myosin head has not been
investigated in depth. In the present study, to resolve the controversy between
the lever-arm model and the unidirectional Brownian motion model, we study
the contribution of the conformational change of myosin head during thecoupled sliding/binding motion of myosin on the actin filament. The structuralensemble of actin-myosin complex is sampled with a coarse-grained model with
Go-like interactions, and the free energy landscapes of the three different
chemical states of myosin are calculated. The coupling of the conformationalchange of the myosin head and the process of the weak-to-strong binding to theactin filament is also investigated using a double-well structure-based model ofmyosin head.
2PS018コフィリンと HMM のアクチンフィラメントへの相互排他的な協同的結合はアクチンフィラメントの協同的構造変化に依存する

Cooperative conformational changes of actin filaments drive
mutually exclusive cooperative binding of cofilin and HMM to
the filaments
Nobuhisa Umeki, Taro Uyeda (Advanced Industrial Science and Technology)
Cofilin and HMM of myosin II both bind to actin filaments cooperatively. Thereis ample evidence that HMM or S1 causes cooperative conformational changes ofactin filaments, but corresponding biochemical evidence for cofilin-induced
cooperative conformational changes was lacking. Thus, we made copolymers ofcofilin-actin fusion protein and actin, and found that fluorescence of pyrene
attached to actin was quenched, and DNase loop of actin became more susceptible
to subtilisin cleavage, demonstrating cooperative conformational changes inducedby cofilin. Cosedimentation assay showed that copolymerization with cofilinactin increases affinity of actin to cofilin, indicating that conformational changes
induced by cofilin attracts further cofilin binding through a domino effect. In
contrast, copolymerization with acto-S1 fusion protein decreased the affinity ofactin for cofilin while increased the affinity for HMM in 1 mM ADP + 0.1 mM
ATP. These results indicate that cooperative conformational changes induced bycofilin or S1 is the basis for cooperative binding of cofilin and HMM to actin
filaments. Furthermore, these data as well as those of fluorescence microscopy(Nishikawa et al., this meeting) indicate that cooperative binding of cofilin and
HMM for actin filaments is mutually exclusive. We suggest that these positive
and negative effects that the binding of a specific actin binding protein exerts on
the binding of the same or other actin binding proteins is involved in functional
differentiation of actin filaments in vivo.
2PS019分子数を制御したキネシンとダイニンの綱引きによる積極的な両方向性運動の再構成

Reconstitution of tug-of-war between defined numbers ofkinesin and dynein as active bidirectional transport
Kazuyuki Yamamoto1, Ken'ya Furuta2, Hajime Honda1, Hiroaki Kojima2(1Department of Bioengineering, Nagaoka University of Technology, 2Bio ICTlab, NICT)
Microtubule-based intracellular transport is fundamental for many biologicalprocesses. The transport is typically achieved by the cooperation of oppositely
directed motor proteins, kinesins and dyneins that are engaged to the samecargo. Strikingly, many cargos move bidirectionally, alternating frequently
between plus end- and minus end-directed motion. Because this property
appears ineffective in delivering a cargo, many researchers are now asking how
such active fluctuations of the cargo contribute delivery to its destination via thecomplex microtubule network. In the cell, it is extremely difficult to identify the
number and class of engaging motor on moving cargos, much less the presenceof various regulatory factors. To circumvent this problem, we constructed
minimal assemblies containing defined numbers of kinesin and dynein on single
DNA scaffolds, so that we can clearly quantify the effect of linking oppositely
directed motor proteins together in vitro. To construct the motor assemblies, we
linked recombinant human kinesin-1 and cytoplasmic dynein 1 by DNA
scaffolds containing up to 5 binding sites. Using a total internal reflection
fluorescence microscope, we show that dynein-kinesin assemblies displayed
stochastic, bidirectional motion along microtubules, which was reminiscent of
typical intracellular transport. Further analysis to evaluate the performance of
these assemblies on an artificial microtubule network will be presented.
2PS020Ascaris 精子をもちいたアメーバ運動装置の in vitro 再構成

1YS0945

Simultaneous reconstitution of MSP-based protrusion andretraction in the amoeboid sperm of Ascaris

Katsuya Shimabukuro1,2, Naoki Noda3, Masasuke Yoshida2,4, Murray Stewart5,
Thomas M. Roberts6 (1Ube Nat. Colg. Tech., 2ICORP, JST, 3MBL, 4Kyoto Sangyo
Univ., 5MRC, 6Florida St. Univ.)
Crawling movement in eukaryotic cells requires coordination of the force forleading edge protrusion with that for cell body retraction. In Ascaris sperm,
protrusion and retraction are powered by a motility system comprised of major
sperm protein (MSP) in place of the actin apparatus typically associated withcell migration. By adjusting the concentration of cell-free sperm extracts, wereconstituted protrusion and retraction simultaneously in MSP filament
meshworks, called fibers, that assemble behind plasma membrane-derived
vesicles. As polymerization of filaments at the growing end of the fiber pushed
the vesicle ahead (protrusion), filament rearrangement and disassembly pulled
the rear of the fiber forward (retraction). Because nematode sperm do not use
motor proteins to power retraction, we used these fibers to explore themechanism of retraction by focusing on the cytoskeletal dynamics that occur
during the transition from protrusion to retraction. Using a combination offluorescent speckle microscopy, electron tomography, and advanced
polarization microscopy, we found that although fibers progressively lost
filaments during retraction the average length of the filaments did not change.
The surviving filaments were able to move and reorient so that their packing
density was conserved. To do so, fewer filaments packed into a smaller volume
and this rearrangement appears to provide the driving force for retraction.
2PS021

Identification of leg and crank proteins of Mycoplasma mobile
on gliding machinery by electron microscopy

Hiroki Yamamoto, Makoto Miyata (Graduate School of Science, Osaka City
University)
Mycoplasma mobile, a fish pathogen, glides by a unique mechanism based on
three huge proteins unrelated to conventional motor proteins. To date, the “leg”
structure on the surface of the gliding machinery and the molecular shapes ofisolated component proteins have been clarified, by freeze-fracture electron
microscopy and rotary-shadowing electron microscopy, respectively. However,
the whole structure of the machinery or the assignment of component proteins in
the machinery has been unknown. In the annual meeting of last year, we reduced
background by using volatile buffer and digested scaffold proteins, and thensucceeded in visualizing the machinery by negative-staining electron
microscopy. In this year, we achieved higher contrast images by (i) rotaryshadowing and (ii) negative-staining in the absence of scaffold proteins. The
observation of each mutant lacking one of Gli349, Gli521 or Gli123 suggested
that Gli349, a “leg” protein is sticking flexibly and irregularly from themachinery surface with 2-3 nm thickness and 30-80 nm length, and Gli521, acrank protein is aligned in 5 nm parallel to the cell membrane with 3 nm
diameter.
2PS022

ヒト肺炎 Mycoplasma pneumoniae の滑走運動装置と構成タン
パク質の構造解析

Structural analysis of gliding machinery and component
proteins of Mycoplasma pneumoniae
Yoshito Kawakita1, Daisuke Nakane1, Lisa Matsuo1, Tsuyoshi Kenri2, Makoto
Miyata1 (1Osaka City University, 2National Institute of Infectious Diseases)
Mycoplasma pneumonia, a human pathogenic bacterium glides with repeatedcatch and release of sialylated oligosaccharides on host cell surfaces. At a pole,
this organism forms a membrane protrusion, the attachment organelle which iscomposed of a surface receptor, named P1 adhesin, and the internal rod
structure. The rod structure can be devided into three parts, Terminal button,
Paired plates, and Wheel complex from the tip. In the present study, we
identified two novel component proteins of the rod structure, and assigned
eleven component proteins onto the organelle, based on the data of peptide massfingerprinting, YFP fusion, and electron microscopy. Next, we expressed andpurified four Wheel complex proteins coded by MPN311, MPN312, MPN387
and MPN526, a Terminal button protein coded by MPN309 and P1 adhesin
coded by MPN141, for crystallization. We used chemically synthesized genes tosolve the codon problems and induced these genes in Escherichia coli at a lowtemperature, and obtained pure proteins, of which the proteins of MPN311 and
MPN387 showed high solubility and yields.
2PS023− S113 −Mycoplasma mobile の滑走運動に方向性を持たせる“あし”の結合
Directed binding of Mycoplasma mobile may cause directeddisplacement in gliding

Akihiro Tanaka1, Daisuke Nakane1, Takayuki Nishizaka2, Makoto Miyata1(1Graduate School of Science, Osaka City University, 2Faculty of Science,
Gakushuin University)
Mycoplasma mobile, a pathogenic bacterium forms membrane protrusion, thegliding machinery, and glides on solid surface smoothly up to 4.5 m/s and 27
pN in the direction of protrusion. M. mobile is thought to glide by repeatedcatch-pull-release of sialylated oligosaccharide fixed on solid surface byhundreds of 50 nm flexible legs sticking out from the gliding machinery.
However, the directed displacement of cell is difficult to explain, because thelegs are flexible. To elucidate this mystery, we examined a starved cell binding
to sialylated oligosaccharide, by pulling forward and backward at a speed of 160
nm/s, using a bead manipulated by a laser trap. Tension was generated at similarrate for forward and backward, 0.09 ± 0.03 pN/nm. In these processes, thetension occasionally shifted down, suggesting temporal detachment of legs fromsialylated oligosaccharide fixed on solid surface, namely “slippage”. The
slippage occurred at tension of averages, 22.5 ± 13.9 pN and 48.4 ± 28.0 pN
for forward and backward, respectively, whereas no significant difference wasfound in the occasional decrease in tension between both directions. The tension
at slippage did not differ significantly between directions, when two types ofnon binding mutants lacking valid legs were artificially bound to glass. These
results suggest that M. mobile binds to solid surface in a direction dependent
manner, and this directed binding may cause the directed gliding.
2PS024

Leg movements suggested from inhibition of mycoplasma
gliding by free sialylated oligosaccharid
Taishi Kasai1, Daisuke Nakane1, Hideharu Ishida2, Hiromune Ando2,3,
Makoto Kiso2,3, Makoto Miyata1 (1Graduate School of Science, Osaka City
University, 2Faculty of Applied Biological Sciences, Gifu University, 3iCeMS,
Kyoto University)

2PS026

表面タンパク質のらせん流で動くバクテリア

Helical Flow of Surface Protein Required for Bacterial
Locomotion
Daisuke Nakane1, Keiko Sato1, Hirofumi Wada2, Mark J. McBride3, Koji
Nakayama1 (1Grad. Sch. Biomedical Sci., Univ. Nagasaki, 2Dept. Phys., Univ.
Ritsumeikan, 3Dept. Biological Sci., Univ. Wisconsin-Milwaukee)
A large group of bacteria belonging to the phylum Bacteroidetes show acharacteristic rapid surface movement known as gliding motility. Bacteroidetes
gliding motility is different from other motility systems, but its mechanism is
unknown. Here, we visualized the dynamics of cell adhesin SprB, and proposed“helical loop track” to explain the motility.
A soil bacterium, Flavobacterium johnsoniae has a 669 kDa cell-surface
adhesin, SprB, which is responsible for the gliding motility and cell adhesion.
Electron microscopic experiments revealed that SprB formed 150 nm long
filaments, and dynamic movements of SprB were observed by fluorescencemicroscopy. SprB was located dispersedly on a long pitch of helical loop along
the membrane, and moved on the cell surface along an apparent right-handed
helical closed loop. When the apparent velocities of SprB signals were
determined with respect to the glass substratum, about half of the SprB signals
moved with velocities of 3.4 m/s and the other half moved with velocities of-0. 5 m/s. Taking the velocity of cells (1. 9 m/s) into consideration, SprBsignals migrating in opposite directions appeared to move at the similar speedswith respect to the cell. The results suggest a model for Flavobacterium gliding,
supported by mathematical analyses, in which adhesins always moved on an
endless right-handed helical track at a constant speed, while some SprB tightly
bound to the solid surface, generating rotation and translation of the cell.
2PS027

Gene manipulation of gliding bacterium, Mycoplasma mobile
Isil Tulum, Atsuko Uenoyama, Makoto Miyata (Graduate School of Science,
Osaka City University)

Mycoplasmas, known as a pathogen of human pneumonia, bind to solid surfaces
and glide to one direction. In gliding, mycoplasmas catch sialylated
oligosaccharides, the common target of many pathogens. In previous studies, weanalyzed the inhibitory effects of synthesized sialylated oligosaccharide on
binding of gliding mycoplasmas and concluded the affinity, the cooperativity,
and the recognized structure. In the present study, we analyzed the effects of
those compounds to the gliding of M. mobile cells, to know the relationshipbetween sialylated oligosaccharide binding and the gliding mechanism. Gliding
speed was reduced by the addition of sialylated oligosaccharide with time, andthe decrease appeared accelerated with time after the decrease became obvious.
The decrease in gliding speed should be caused by the decrease in the working
legs in the addition of free sialylated compound. The acceleration in decrease
may suggest that the binding inhibition of a leg affect the affinity of neighboring
legs. The cells showed pivoting around the gliding machinery before the detach.
The shortage of propelling force caused by the free sialylated oligosaccharide
may induce remaining of legs after stroke on glass, some of which form thecenter of cell pivoting. All of these results support our assumption that the leg
after stroke should be removed by the continuous cell movement caused byother legs in the gliding mechanism.

Mycoplasma mobile, a pathogenic bacterium, glides on solid surfaces with aunique mechanism, not related to known bacterial motility systems orconventional motor proteins. The gliding machinery is composed of three hugesurface proteins and more than ten internal proteins. To elucidate the gliding
mechanism and the roles of component proteins, the genetic manipulation
including homologous recombination should be a powerful tool. However, thisorganism has not been transformed so far by standard procedures used for other
Mycoplasma species. In this study, we examined and improved the steps oftransformation as follows. (i) The electroporation conditions have been adjusted
to fit to the M. mobile cells which are much tougher than the other species. (ii)
The recovery time after electroporation was elongated from 2 h to 12 h. (iii) The
growth medium was modified to get clearer colony shapes. Then, we achieved
the transformation of M. mobile with efficiencies ranging 10-5 to 10-7 percompetent cells by 10 g of vector DNA, and also fluorescence labeling of cells
by using enhanced yellow fluorescent protein (EYFP). We are constructing
special vectors allowing us gene expression and homologous recombination.

2PS025

2PS028Swarm Dynamics of Vibrio alginolyticus
Szu-Ning Lin, Chien-Jung Lo (Department of Physics and Institute ofBiophysics, National Central University, Taoyuan County 32001, Taiwan)
Swarming is an important step for bacterial colonization and spreading. We
investigate the swarming dynamics of the dual flagellated bacterium, Vibrio
alginolyticus, in different growing stages and external conditions. While
growing on agar surface, Vibrio alginolyticus elongate and synthesize lateral
flagella that can move rapidly in the thin liquid layer above agar. The cellmotion is constrained by the cell to cell interaction and hydro-dynamicalinteraction with the liquid boundary conditions. We measured the colony
spreading speed and the single cell motility in different growing stages and agar
concentrations. The cells lengths are widely distributed. The cell density ishigher in the colony edge. Edge-cells show parallel alignments while interiorcells show swirling motion with different time and length scale. We found theedge-cells shows waving while the interior-cells shows turbulent like collective
motion. Far behind the spreading edge, cell density reduced down to single-celllayer. We exam the collective motion and cluster size distribution in thiscondition. Vibrio alginolyticus shows different motility dynamics in different
growing stage. We aim to build up a simply model to explain the collective
motion of Vibrio alginolyticus.

低温電子線トモグラフィーによって明らかとなった細菌べん毛蛋白質輸送装置の構造と機能

Structure and function of bacterial flagellar protein export
apparatus revealed by electron cryotomography of Salmonella
minicells
Akihiro Kawamoto1,2, Yusuke V. Morimoto1,2, Takayuki Kato2, Kelly T.
Hughes3, Toshio Yanagida1,2, Keiichi Namba1,2 (1QBiC., RIKEN, 2Grad. Sch.
of Frontier Biosci., Osaka Univ, 3Dept. Med. Sci., Univ. Fribourg)
The bacterial flagellum is a motility organelle composed of a basal body thatfunctions as a rotary motor and a long helical filament that works as a propeller.
Most of flagellar axial proteins are translocated into the central channel of thegrowing flagellum for self-assembly at the distal end by the flagellar protein
export apparatus. A structural study on many different types of intact flagellar
basal bodies by electron cryotomography (ECT) has suggested that the export
apparatus is highly conserved in their structures across species. However,soluble components of the export apparatus have not yet been clearly observedin the basal body of Salmonella entrica because the wild-type cell is about 1 min diameter, which is too thick to visualize the intracellular structures by ECT indetail. To visualize the structure of the export apparatus in the cell, weconstructed Salmonella minicell by overproducing FtsZ to make the cell smallenough for a high-resolution ECT analysis and carried out the structural analysis− S114 −of intact flagellar basal body. Comparison with the structure of a purified basal
body revealed the density of the export platform and the ring structure derived
from FliI within and below the C ring. The flagellar basal body of the ΔfliH-fliIbypass mutant, which maintaines flagellar protein export and motility in theabsence of FliH and FliI showed that the two densities within and below the C
ring were completely lost,suggesting that the platform structure is not essential
for flagellar protein export.

modified fliK constructs into C-ring mutants. A recombinant FliK with the sizeof 373 a.a. produced short hooks and the other recombinant FliK with the size of
570 a.a. produced long hooks, suggesting that FliK is dominant in hook length
control even in the C ring mutants. However, the hook length in C-ring mutantsvaried in wider range than that of the wild type. We will be discussing theinteraction between the C ring and FliK.
2PS032

2PS029

細菌べん毛モーターの固定子組込み機構に対する MotB(D33N)点
変異の影響

Effect of the D33N mutation in MotB on stator assembly of thebacterial flagellar motor
Shuichi Nakamura1, Tohru Minamino2, Nobunori Kami-ike2, Seishi Kudo1,
Keiichi Namba2,3 (1Department of Applied Physics, Tohoku University, 2Graduate
School of Frontier Bioscience, Osaka University, 3Riken Quantitative Biology
Center)
The bacterial flagellar motor converts the energy of proton translocation acrossthe membrane through the proton-channel stator complex into torque. The
MotA/B proton channel complex is firmly anchored to the peptidoglycan (PG)
layer through the PG-binding domain of MotB to act as the stator of the motor.
The stator units dynamically associate with and dissociate from the motor
during flagellar motor rotation, and an electrostatic interaction between MotA
and a rotor protein FliG is required for efficient stator assembly. However, theassociation and dissociation mechanism of the stator units still remains unclear.
In this study, we analyzed the speed fluctuation of the flagellar motor of
Salmonella enterica wild-type cells carrying a plasmid encoding a nonfunctional
stator unit, MotA/MotB(D33N), which lost the proton conductivity. The
wild-type motor rotated stably but the motor speed fluctuated considerably
when the expression level of the MotA/B(D33N) stator was increased. Rapid
accelerations and decelerations were also observed. These results suggest that
the MotA/B(D33N) stator retains the ability to associate with the motor but
rapidly dissociates from it as well. We propose that the stator dissociation
process depends on the proton translocation through the proton channel.
2PS030MS リングに突然変異が生じたサルモネラ菌べん毛モータの構造安定性と出力特性

Structural stability and rotational characteristics of theflagellar motor of Salmonella MS-ring mutant
Shun Taga1, Akira Asaumi1, Shuichi Nakamura1, Fumio Hayashi2, Kenji
Oosawa2, Seishi Kudo1 (1Department of Applied Physics Tohoku
University, 2Department of Chemistry and Chemical Biology Gunma
University)
The bacterial flagellar motor is a rotary nanomachine fueled by the proton
motive force. Two ring structures, MS ring and C ring, together act as a rotor,
and responsible for torque generation by interacting with a stator complex
consisting of MotA and MotB. It has been known that the flagella of a
Salmonella FliF mutant are fragile as compared with those of wild-type strain,suggesting that the MS ring plays an important role in keeping stability of the
motor structure. In this study, the mechanical strength and the rotational
characteristics of the FliF mutant motor were analyzed to understand how MS
ring is involved in the structural stability. Quantitative measurement of thestrength revealed that the mechanical strength of the FliF mutant motor
decreases up to a half of wild-type motor. However, both the swimming speed
and the motor speed of the FliF mutant were almost same with those of wildtype strain. These suggest that the defect of MS ring can decrease the structural
stability of flagellar motor without impairment of torque generation.
2PS031

The role of the flagellar C ring in hook-length control by FliK
Kaoru Uchida, Tatsuya Yamasaki, Shin-Ichi Aizawa (Grad. Sch. Pref. Univ.
Hiroshima)
The flagellar hook has an average length of 55 nm, which is controlled mainly
by FliK. We previously demonstrated that mutations of any one of the fliG,
fliM, and fliN genes gave rise to short hooks in Salmonella enterica serover
Typimurium. FliG, FliM, and FliN form the cytoplasmic ring (C ring) at theflagellar base, which is a multifunctional structure necessary for flagellar
protein secretion, torque generation, and switching of the motor rotation. We
also showed that hook length of the C-ring deficient mutants broadly distributes
around 55 nm, suggesting a tight length-control by the C ring. In order toelucidate the role of the C ring in hook-length control, we introduced two

べん毛モーターのトルク発生器における帯電残基の性質

Characterization of the electrically-charged residues in thetorque-generating unit of the flagellar motor
Satoshi Inaba, Shin-Ichi Aizawa (Prefectural University of Hiroshima)
FliG is one of the rotor components of the flagellar motor in Salmonella enterica
serovar Typhimurium. It is said that five conserved charged residues (K264,
R281, D288, D289, R297) in the C-terminal domain of FliG are directly
involved in torque generation. These five charged residues formed a ridge on thesurface of the domain. Among these residues, R281, D288 and D289 are
belonging to the same -helix. R281X and D288X/D289X gave rise to motilitydefective mutants by substitution with either Thr or Ala. In order to examine thefunctional roles in detail of the residues R281 and R288/R289, we substituted
these residues with all 20 amino acids.
Among variants of R281X, four (R281D, E, G, and P) were Mot-, and the rest
swam at various speeds. Distribution of their rotational speeds continuously
dispersed in a wide range, indicating that torque generated in the motor may
change according to changes in conformation near the R281 site. Among
D288X/D289X pair variants, three pairs (D288E/D289E, S/S, Y/Y) swam,
while the rest were Mot-. To fill the gap between R281 and D289, we chose
D284 and I285 and substituted them with 20 a.a. The variants I285D, R, T
swarmed. The variants D284E, F, R, and V swarmed. These results indicate that
D284 and I285 at the middle of the -helix are not directly involved in torquegeneration. In summary, a pair of D288/D289 residues might be the major
element of the torque generator, and that R281 residue might play a subsidiaryrole in the torque generator.
2PS033Meaning of HAP1 in the length measurement of the hook
Kohei Dono, Kaoru Uchida, Sin-Ichi Aizawa (Grad. Sch., Pref. Univ.
Hiroshima)

In the bacterial flagella, the hook occupies a region between the rod and HAP1
in an average length of 55 nm to connect the filament to the basal body. The
hook is composed of FlgE. We analyzed the hook in a temperature-sensitive
flgE mutant of Salmonella enterica seover Typhimurium. The mutant cells
produced wild-type flagella when grown at 30 ℃ but short hooks without
filaments at 37℃. We found by electron microscopy that the mutant hooks
produced at 30℃ had wild-type length (average length: 55 nm) in intact flagella,
whereas they were shorter (45 nm) in the hook basal body (HBB) obtained byacid treatment of intact flagella. SDS-PAGE showed that the HBB preparation
did not contain HAP1 that is supposedly attached on the distal end of wild-type
hook, indicating that interaction between the mutant hook and HAP1 might
becoma weaker by a mutation of the hook protein. As a consequence, welearned that the hook length that we have so far measured was length of the hookregion including HAP1, and that authentic hook length was about 45 nm and
HAP1 length was about 10 nm in the wild-type “hook”. The HAP region (HAP1and HAP3) in intact flagella is distinguishable from the hook and filament by its
appearance and thin width. However, surface appearance of the HAP region
resembles that of the hook than the filament, which led us to measure hooklength including HAP1. We will show length distribution of HAP region in wild
type and mutants.
2PS034コフィリンのアクチン線維への協同的結合の直接観察

Direct observation of cooperative binding of cofilin to actin
filaments
Kimihide Hayakawa1, Hitoshi Tatsumi2, Takafumi Goto2, Masahiro Sokabe1,2(1FIRST Research Center for Innovative Nanobiodevice, Nagoya Univ., 2Dept.
of Physiol., Nagoya Univ. Grad. Sch. of Med.)ADF/cofilin proteins are ubiquitously distributed actin severing proteins ineukaryotic cells. Several biochemical and electron microscopic studies havesuggested that cofilin binds to the actin filament in a cooperative manner. We
directly observed the binding of single Alexa-labeled cofilin molecules to actin
filaments in solution by a single molecule imaging technique. We analyzed thenon-contiguous (isolated, non-cooperative) cofilin binding to an actin filament− S115 −using low concentration of cofilin (10 nM-100 nM), in which measurement of
the binding was made on a short segment (600 nm in length) in the filament The
rate constant of cofilin binding to the actin filament was estimated at 0.06/ M^1s^-1, which agrees with the value obtained with biochemical techniques. The
rate constant elevated to 0.18/ M^-1s^-1 when the concentration of cofilin wasincreased to 10 M. This result indicates that our single molecule measurements
could detect the process of the cooperative binding of cofilin in solution. In low
cofilin concentrations (10 nM-100 nM), long lasting cofilin bindings followed
by a subsequent cofilin binding in the same segment (600 nm) of the filament
were observed. While the rate constant of the preceding cofilin binding was
0. 06/ M^-1s^-1, the rate constant of the subsequent cofilin binding waselevated to 0.18/ M^-1s^-1. This result suggests the binding of cofilin to theactin filament accelerates the binding of another cofilin to the neighboring (lessthan 600 nm) binding site on the actin filament.
2PS035微小管結合蛋白質 4 を介したアクチンフィラメントと微小管の相互作用の解析

Analysis of microtubule-associated protein 4-mediatedinteraction between actin filaments and microtubules
Shoma Saito1, Ayumu Kuramoto1, Tsuyoshi Yamasaki2, Taro Q.P. Noguchi2,
Yurika Hashi3, Susumu Kotani3, Kiyotaka Tokuraku1 (1Grad. Sch. Appl. sci.,
Muroran Inst., 2Miyakonojo Nation. Col. Tech., 3Kanagawa Univ.)
Microtubule-associated protein (MAP) 4 is a non-neural MAP that plays
important role in the intracellular organization of microtubules. Recent study
revealed that MAP4 also binds to actin filaments, but not actin monomer, at the
C-terminal part of the Proline-rich region in the microtubule-binding domain(Matsushima et al., J.Biochem., 2012). Since the binding affinity of MAP4 foractin filaments was the same order with that for microtubules, we believed thatthe interaction of MAP4 to actin filaments was physiologically significant. In
this study, we examined MAP4 functions when both actin filaments andmicrotubules (or unpolymerized tubulin) coexisted in a sample. To distinguishactin filaments and microtubules under fluorescence microscopy, thesefilaments were labeled with different fluorescent dyes. When microtubules weremixed with actin filaments that preincubated with MAP4, bundles consisted ofboth microtubules and actin filaments were observed. Moreover, when
unpolymerized tubulin instead of microtubules was mixed with actin filamentsthat preincubated with MAP4, elongation of microtubules along the actin
filaments was observed. These results suggest that MAP4 bound to actin
filaments retain microtubule binding and microtubule assembly-promoting
activities. On the other hand, when actin filaments were mixed withmicrotubules that preincubated with MAP4, bundles containing both actin
filaments and microtubules were not observed, suggesting that MAP4 bound to
microtubules lost actin filament-binding activity.
2PS036血管平滑筋細胞から単離したストレスファイバの収縮特性の計測

Measurement of contractile properties of stress fibers isolated
from vascular smooth muscle cells
Tsubasa S. Matsui1, Daiki Komatsu2, Masaaki Sato3, Shinji Deguchi2 (1Grad.
Sch. Life Sci., Tohoku Univ., 2Grad. Sch. Eng., Tohoku Univ., 3Grad. Sch.
Biomed. Eng., Tohoku Univ.)
Several lines of evidence have suggested a critical involvement of nonmuscle
myosin II in the fundamental cellular functions of proliferation, differentiation,
and apoptosis. In nonmuscle cells, nonmuscle myosin II forms stress fibers (SFs)along with actin filaments and exerts contractile forces on the extracellularmatrix. Compared to increasing reports studied at the whole cell level, thecontractile properties intrinsic to SFs themselves remain poorly understood. In thepresent study, we characterized the contractile properties of single SFs isolated
from cultured vascular smooth muscle cells. SFs were extracted from the cells
treated with hypotonic buffers and detergents. Western blotting showed that main
constituents of SFs were maintained in the isolated materials. We manipulated theisolated SFs by using two fine glass needles and measured their contractile forces
under physiological MgATP concentrations. The results showed that SFs could
generate forces of several tens of nano-Newtons. SFs suddenly relaxed at high
MgATP concentrations, and some fragments of F-actin were dissociated from theSFs. Interestingly, we observed rotational motions of SFs under passive stretch aswell as active contraction conditions. These results may be useful in theunderstanding the biophysical roles of SFs in cell behavior.
2PS037IQGAP confines myosin-II to the cytokinetic contractile ring

Masak Takaine, Osamu Numata, Kentaro Nakano (Grad. Sch. Life & Env.
Sci., Univ. Tsukuba)
During cytokinesis, myosin-II concentrates at the equatorial cortex with actin
filaments (F-actin) and is supposed to generate forces to divide the cell into two,
which is called the contractile ring (CR) hypothesis. Several lines of evidence
indicate F-actin-independent recruitment of myosin-II and its specificinteraction with the equatorial F-actin, although their details are still unknown.
We investigate the dual regulation of myosin-II localization using the fission
yeast S. pombe. IQGAP Rng2 is implicated both in the F-actin-independent
recruitment of myosin-II to cortical dots and in its sufficient concentration on
CR F-actin. Rng2 is required for the dot localization of Myo2 (the essential
myosin-II) via its tail. Rng2 shows a slower exchange rate and lower mobility
than Myo2. Myo2 is inhomogenous and unstable in CR of the rng2 mutant andpreferably associates with ectopic F-actin induced by overexpression of theactin-modulating moiety of Rng2, Rng2Ns. Biochemically, Rng2Ns slows
actin-activated Mg-ATPase of myosin-II. These results suggest that Rng2
tethers the Myo2 tail to cortical dots and accumulates myosin-II to CR F-actin
by modulating its ATPase-coupled turnover.
2PS038

キネシン Eg5 に対するフォトクロミック阻害剤を用いた細胞分裂の光制御

Photo-regulation of cell division using photochromic inhibitors
for kinesin Eg5
Kei Sadakane1, Kumiko Ishikawa1, Hideo Seo1, Banri Yamanoha2, Shinsaku
Maruta1 (1Division of Bioinfomatics, Graduate school of engneering, Soka
University, 2Department of development of environmental engineering ,Faculty of engineering, Soka University)
Previously it has been demonstrated that some ATP driven motor proteinskinesins are directly involved in regulation of the cell division cycle. Eg5 is oneof the kinesin and its biochemical properties and structure were well studied.
Interestingly Eg5 is activated in M phase of cell cycle and performs stabilizing
and positioning of spindle. It is also known that Eg5 is overexpressed in tumor
cell and induce significant. Monastrol and STLC, which are potent inhibitor
specific for kinesin Eg5 ATPase activity, shut off mitotic division and result inapotosis. Therefore, these inhibitors are attracting as anti-cancer drug.
Azobenzene and spiropyran, a widely studied photochromic compound, can bereversibly isomerized between the cis and trans forms by ultra-violet (UV) andvisible (VIS) light irradiation, respectively. We have recently demonstrated theMonastrol and STLC analogues composed of photochromic molecules inhibit
ATPase activity Eg5 reversibly upon UV and VIS light irradiation in vitro.
Therefore, it is strongly expected that the photochromic inhibitors are applicable
to reversible photo-regulation of cell mitosis. In this study, we have examinedthe effects of the photochromic inhibitors for mammalian cells. Effects of thephoto-chromic inhibitors were evaluated by survival number of mammaliancells, HeLa cells or A172 cells. The experimental results suggested that theeffects of photo-chromic inhibitor for cell division were controlled by UV andVIS light reversibly.
2PS039蛍光顕微鏡観察による細胞性粘菌由来のコフィリン、HMM と F-

actin の協同的相互作用の解析
Analysis of cooperative interaction of Dictyostelium-derived
cofilin and HMM to F-actin using fluorescence microscopy
Yusuke Nishikawa1, Ayumi Satoh1, Taro Q. P. Ueda2, Kiyotaka Tokuraku1(1Grad. Sch. Appl. Sci., Muroran Inst., 2Nation. Inst. Adv. Indust. Sci. Tech.)
In crawling cells, cofilin and myosin II are localized in the anterior and posterior
regions, respectively. It has not been understood the molecular mechanism of
the differential localization so far. In the last meeting, we reported thatcooperative binding of cofilin and HMM for F-actin was mutually exclusive,and concluded that the mutually exclusive binding might contribute to thedifferential localization of cofilin and myosin II. However, since the cooperative
binding was analyzed by use of proteins derived from different origins, such as
human cofilin, Dictyostelium HMM, and rabbit actin, it has been remained aconcern that the cooperative interaction containing the mutually exclusive
binding is not a physiological phenomenon. In this study, therefore, we used
proteins derived from only Dictyostelium to analyze the cooperative interaction.
To visualize cofilin and HMM, we prepared recombinant Dictyostelium cofilin(D-cofilin) and HMM (D-HMM) fused with mCherry and GFP, respectively.
Actin was purified from Dictyostelium (D-actin) or rabbit (R-actin), and waslabeled with fluorescent-phalloidins. The cooperative binding for 40 nM F-Dactin was observed in the range of 100-150 nM D-cofilin. The cooperative− S116 −binding of D-cofilin was also observed for F-R-actin, suggesting that thecooperative binding of cofilin was not origin-dependent phenomenon. We arenow examining the cooperative binding of D-HMM to F-D-actin. We will
present detailed data of the cooperative interactions containing mutually
exclusive binding at this meeting.
2PS040棘皮動物コラーゲン性のキャッチ結合組織における可逆的な硬さ変化の分子機構Molecular mechanisms of reversible changes in stiffness ofechinoderm collagenous 'catch' connective tissues
Akira Yamada1, Yasuhiro Takehana2, Masaki Tamori2, Tatsuo Motokawa2(1Adv. ICT Res. Inst., NICT, 2Grad. Sch. Biosci. Biotech., Tokyo Inst. Tech.)'Catch' connective tissues shown in echinoderm animals such as sea urchins, sea
stars, and sea cucumbers can extensively and reversibly change their stiffness ina few minutes under the regulation of the nervous system. Neurotransmitterssuch as acetylcholine, adrenaline, and some oligopeptides cause changes in the
stiffness of the living pieces dissected from these tissues. They are no more
effective, however, after the treatment with detergent solubilizing cell
membranes. There are some granule-containing cells in the tissues, which areassumed to secret some factors under the regulation of nerves causing changes
in the tissue stiffness. The tissues contain a large amount of the extracellularmatrix mainly consisting of collagen fibrils. It seems that cross-linking between
the fibrils are formed or broken during the change of the stiffness of the tissues.
Its molecular mechanisms are, however, not yet fully understood. We isolated aprotein factor called 'tensilin' from an extract of sea cucumber body wall dermis,
one of the known 'catch' connective tissues. It stiffens the detergent-treateddermal pieces and induces aggregation of collagen fibrils isolated from thetissue. We also isolated a crude extract solution which softens the detergenttreated dermal pieces. It is possible that there are other factors affecting on
interactions among dermal fibrils and the stiffness of the tissues. Molecularmechanisms of the stiffness changes of the 'catch' connective tissues should beclarified by purifying and characterizing these factors.
2PS041

Cooperative function of raft domains and actin skeleton
utilized for anthrax toxin assembly and internalization: asingle-molecule study
Yukihiko Kudo1,2, An-An Liu1,2,3, Shihui Liu4, Kenichi Suzuki1,5, Takahiro
Fujiwara1, Dai-Wen Pang3, Stephen Leppla4, Akihiro Kusumi1,2 (1Institute forIntegrated Cell-Material Sciences, Kyoto University, Japan, 2Institute for
Frontier Medical Sciences, Kyoto University, Japan, 3College of Chemistry
and Molecular Sciences,Wuhan University, 4Division of Intramural Research,
NIAID, NIH, USA, 5The National Centre for Biological Science/The Institutefor Stem Cell Biology and Regenerative Medicine (inStem), Bangalore, India)
How raft domains and cortical actin filaments (actin-based membraneskeleton) collaboratively work in the plasma membrane (PM) is one of the
key issues in understanding the functions of the raft domains andinternalization of raft-associable receptors. To address this question, we
examined the mechanism by which a component of anthrax toxin, called
protective antigen (PA), bound to the cell surface receptor (PA-receptor
complex, called PARC) form heptamers in the PM and internalized. The
lethal and edema protein components of the toxin are known to only bind to
PARC heptamers, and after internalization, cross the endosomal membrane toenter the cytoplasm. The process for the PARC heptamer formation has notbeen totally understood, because, previously, only PARC monomers andheptamers were detectable due to methodological limitations. Here, by using
CHO cells stably expressing the PA receptor, CMG2, and the PA specificallylabeled with a fluorescent dye precisely at a 1:1 mole ratio, we succeeded inobserving intermediate PARC clusters and the recruitment of a specific lipid
to those clusters by simultaneous dual-color single-molecule tracking. Withan increase of the cluster size, the residency time of the raft lipid at theclusters was prolonged. Furthermore, the PARC intermediate clusters induced
actin polymerization, which is enhanced with an increase of the cluster size.
How these events are correlated and lead to heptamer formation and thesubsequent internalization is under investigation.
2PS042

核内を運動する粒子の分子同定と、可視化、トラッキング解析

Imaging, tracking and analysis on the intranulclear moving
particles

Soyomi Uchibori1, Maiko Kuramochi1,2, Saori Mimatsu1,2, Emiko Kobayashi1,2,
Kaoru Katoh1,2 (1Biomed. Res. Inst, AIST, 2Grad. Sch. Live & Env. Sci., Univ.
Tsukuba)
Cell nucleus is a site of gene expression. The expression process should require
dynamic change in architectural framework of nuclear structures and active
transport of many kinds of molecules and subcellular components. Many
researchers microinjected fluorescently labeled molecules into the nucleus andshowed high mobility of the molecules in nucleoplasm. Moreover, advaned influorescently tagged protein leads to more precise analysis on dynamics ofnulceoplasm. However not so many studies were reported in the living
unstained nucleus. We, therefore, used pupil projection apodized phase contrast(PPAPC) microscopy, (external phase contrast with special frequency filtering).
The PPAPC revealed many moving particles in living cell nucleus, which wereunable to observe with conventional phase contrast microscopy. We reported
cell cycle dependent movement of the intranuclear particles. The particles
were identified by interactive observation of optical (PPAPC) and electron
microscopy. Here, we identified the molecules involved in the moving particles,with immunofluorescence method. Moreover, we tracked the moving particles
and analyzed the trajectory. Motile mechanisms of the particle and biologicalmeaning of the movements were discussed in the presentation.
2PS043

クライオトモグラフィーによる動的生細胞内イメージングの開発

Development for dynamic Live Cell imaging by cryo-electron
tomography
Atsuko H. Iwane1,2, Ruriko Ogawa2, Rina Nagai1, Akihiro Kawamoto1,2,Kazuhiro Aoyama1,3 (1Osaka Univ. Grad. Sch. of Front. Biosci. Speci. Res.
Promotion Group, 2RIKEN Quantitative Biol. Center Cell Dynamics Res., 3FEI
JAPAN Application Lab.)
Cryo-electron tomography of intact cells is an emerging technology thatcompliments crystallography, NMR and single molecule imaging techniques. Its
strength is in that it reveals the spatial arrangements of key proteins andcomplexes during intracellular signaling and mechanical events like motility
and division. In this meeting, we describe dynamical Live Cell imaging by
cryo-electron tomography using the Titan Krios microscope. Observed living
cells were grown directly on Holey Carbon Support Film, 200 meshmolybdenum EM specimen grids (Quantifoil R2/2). We analysed normal cell
movement and cell division on Quantifoil using Image J and compared theseproperties with those of cells grown on a normal cell culture plastic plate. After
phosphate-buffered saline washing and rapid freezing, the specimen grids weretransferred onto the microscope immediately while keeping the environment
under liquid N2. Cells were imaged over an angular range from -70 degrees to
70 degrees at 2 degrees x cosθ tilt increments automatically and analyzed with
Amira software to provide 3D images. We will present several 3D images inseveral types of Live Cell and would like to discuss them with everyone.
2PS044

Live cell imaging of the growth factor-induced nuclear
translocation of ERK in PC12 cells
Yuki Shindo1,2,3, Kazunari Mouri4, Kayo Hibino3, Masaru Tomita1,2, Yasushi
Sako4, Koichi Takahashi1,3 (1Inst. Adv. Bio. Sci., Keio Univ., 2Syst. Biol. Prog.
Grad. Sch. Media & Governance, Keio Univ., 3RIKEN QBiC., 4RIKEN ASI.)ERK (extracellular signal-regulated kinase) is one of the most major MAPKs
and its dynamic activation regulates cell fate such as the growth anddifferentiation in PC12 cells. Specifically, nuclear translocation of ERK after
the activation of Raf/MEK/ERK cascade leads to further activation of proteinsin the nucleus such as the transcription factor, resulting in cell fate decisions.
However dynamics of the ERK translocation within individual cells have notbeen well characterized. For further understanding and quantitative modeling of
this signal transduction system, dynamical behavior of ERK in response to
extracellular signals in single cells should be addressed. EGFP-tagged ERK wastransfected into PC12 cells, and visualized in living cells using confocal laser
microscopy, which allows quantitative measurement of the dynamics of ERK
nuclear translocation induced by growth factor stimuli. The results revealed thatthe amount and time course of the translocation were varied among individual
cells. To investigate the mechanism to induce such heterogeneity, translocationof Raf, the MAPKKK in the ERK cascade, to the cell membrane upon growth
factor stimuli are being investigated, using TagRFP-tagged Raf. Thus, we will
discuss the relation between dynamics of ERK and Raf, and further perspectives
toward single molecule measurement and modeling.

− S117 −2PS045

Live-cell imaging and analysis of cAMP-induced cAMP
response in Dictyostelium using microfluidics chambers
Fumihito Fukujin1, Keita Kamino1, Satoshi Sawai1,2,3 (1Graduate School ofArts and Sciences, University of Tokyo, 2Research Center for Complex Systems
Biology, University of Tokyo, 3PRESTO, Japan Science and Technology
Agency)Transient synthesis and secretion of cyclic AMP (cAMP) induced byextracellular cAMP mediates intercellular signaling during aggregation of
Dictyostelium. The response is characterized by a fast initial peak that appears
about 1min after exposure to an increase in extracellular cAMP followed bydose-dependent attenuation to the pre-stimulus level. There is also a fast
oscillatory component of about 3 min periodicity during this prolonged
response. However, biochemical pathways responsible for each of theseproperties are not revealed. Analyzing sensitivity of the response to variations in
activity of the biochemical components is helpful to this end. We developed
continuous flow microfluidics chambers that allow fast switching of thestimulus and drug application. We found that the overall dose dependence ofcAMP-induced cAMP response is similar to those observed in a larger
macroscopic chamber reported earlier. To take advantage of the current setup,
we studied cells continuously bathed with inhibitors of PI3Kinase and actin
polymerization. We will discuss implications of our observations to the
topology of underlying biochemical networks.
2PS046

高密度な集団中におけるサルモネラ菌の走化性応答の解析

Chemotactic response of Salmonella in high cell density
Takahiro Abe, Shuichi Nakamura, Seishi Kudo (Depertment of applied
physics, graduate school of engineering, Tohoku University)
Many bacteria can swim by rotating flagella, and migrate to favorable
environments for growth. When cell density is low, the chemotactic response inwhich smooth swimming (running) and change of direction (tumbling) arerepeated has been known as a biased random walk. However, it still remains
unknown how increase of cell density influences the chemotaxis. In this study,
we analyzed chemotactic responses of Salmonella by a capillary assay under an
optical microscope with changing cell density to characterize the chemotaxis
in high cell density. As a result, we found that chemotactic responses toconcentration gradient of attractant were significantly affected by cell density.
At least three distinct states were observed in the collecting process, which are a
gentle collecting state, a fast collecting state and a stationary state. Duration
time of the gentle state in high cell density were shorter than those in low celldensity, implying that hydrodynamic interactions among cells generate oriented
flows and improve the efficiency of chemotaxis.
2PS047円弧パターンを用いた Navicula pavillardii の運動解析

Trajectory analysis of Navicula pavillardii cells by using an arc
pattern
Kazuo Umemura1, Takahiro Haneda1, Yoshikazu Kumashiro2, Kazuyoshi
Itoga2, Teruo Okano2, Shigeki Mayama3 (1Fac. Sci, Tokyo Univ Sci, 2Tokyo
Women's Medical Univ, 3Fac. Edu, Tokyo Gakugei Univ)
Some types of diatom cells actively glide on a solid surface. The actin-myosin
system has been proposed to explain the movement of diatoms. Althoughmotility studies on diatom cells have been performed since 1950, single-cell
analysis using a microchamber has not been performed for this purpose. In
2011, we reported trajectory analysis of Navicula sp. diatom cells that were shutin a hole (diameter, 600 m). In that study, we found that the diatom cells
moved freely in the center of the hole and that movement was prohibited at theedge of the hole . In the current study, we fabricated a polydimethylsiloxane(PDMS) micro pattern involving arc grooves (width, 40 m; depth, 25 m) inorder to observe single Navicula pavillardii cells. The diatom cells glided along
the edge of the grooves while repeating inversion. Consequently, the velocity of
the cells was not affected by the curvature radius of the arc groove. Furthermore,
the velocities for the clockwise and counterclockwise directions were almost
the same as expected. In contrast, the velocity of each cell was obviously
accelerated when N,N-dimethyl-p-toluidine (DT) was injected into the culture
medium. The average velocities were 10.2 and 13.0 m/s in the absence andpresence of DT, respectively. However, the maximum velocity among 150 cells
was almost 18 m/s in both absence and presence of DT, which suggests that thevalue represents the mechanical limit of the cell motility.

2PS048

基質伸縮に対する運動性細胞毎の応答の違い

Difference of migrating directions among fast crawling cell
types on a substratum with cyclic stretch-shrinkage
Chika Okimura1, Takafumi Mizuno2, Katsuya Sato3, Yuta Nakashima4,
Kazuyuki Minami4, Hitomi Nakashima1, Yoshiaki Iwadate1 (1Grad. Sch. Med.,
Yamaguchi Univ., 2Biomedical Research Institute, AIST, 3Inst. Tech. Sci.,
Grad. Sch., The Univ. Tokushima, 4Grad. Sch. Sci. Eng., Yamaguchi Univ.)
Living cells are constantly subjected to various mechanical stimulations. They
must sense the mechanical aspects of their environment and respond
appropriately for proper cell function.
Cells adhering to substrata must receive and respond to mechanical stimuli fromthe substrata in order to decide their shape and/or migrating direction. In
response to cyclic stretch-shrinkage of the elastic substratum, intracellular stressfibers in slowly crawling cell types, such as fibroblasts and endothelial cells, arerearranged perpendicular to the stretch-shrinkage direction, and then the shapeof those cells becomes extended in the same direction.
On the other hand, in the case of Dictyostelium cells, a kind of fast crawling cell
types, not the shape but the direction of migration is regulated by the cyclic
stretch-shrinkage. They migrate perpendicular to the stretch-shrinkage. Fanshaped keratocytes, isolated from fish epidermis, are also known as fast
crawling cell types. It is well known that treatment with staurosporine induces
fragmentation of lamellipodia of keratocytes, which is followed by formation of
motile cytoplasts. We observed the migration in keratocytes and their motile
cytoplasts on the substratum with cyclic stretch-shrinkage. They showed the
directional migration different from Dictyostelium cells, i.e. migrated parallel tothe stretch-shrinkage direction. We will discuss how different the reaction incell migration in response to the cyclic stretch-shrinkage of substratum among
the fast crawling cell types.
2PS049

コレラ菌走化性因子ホモログの局在制御機構の解析

Localization control of chemotaxis-related signaling
components of Vibrio cholera
Hiroki Okabe1, Tomoyuki Nakamura2, Hiremath Geetha3, Ikuro
Kawagishi1,2,3 (1Dept. Frontier Biosci., Fac. Biosci. Appl. Chem., Hosei
Univ, 2Dept. Frontier Biosci., Grad. Sch. Eng., Hosei Univ, 3Res. Cen. MicroNano Tech., Hosei Univ)
Vibrio cholera, the causative agent of cholera, has three sets of chemotaxisrelated signaling proteins, but only System II is directly involved in chemotaxis.
System II components localize constitutively to a cell pole. By contrast, System
I and III components show clustering at a pole and lateral membrane in cells
cultured with standing (microaerobic conditions) but not with shaking (aerobic
conditions), suggesting that System I and III functions under oxygen-limiting
conditions. The addition of NaN3 upon shaking incubation induces localizationof these proteins, suggesting that it is not limitation of oxygen itself but somechange in energy metabolism that triggers the localization of these components.
In this study,we examined the effects of NaN3 on localization of System I, II and
III histidine kinases, CheA1, CheA2 and CheA3, under various conditions. At
30℃, CheA2-GFP localize to a cell pole with any concentration of NaN3 tested,
but at 37℃, it localized to a cell pole and lateral membrane regions with 0.2%
NaN3. Fractions of cells with polar and lateral localization of CheA1-GFP NaN3
and CheA3-GFP increased with increasing concentrations of NaN3 at 30℃ At
37℃, localization of CheA1-GFP was observed at lower concentrations of
NaN3. CheA3-GFP localized to the pole at 37℃ even in the absence of NaN3.
Immunoblotting demonstrated that NaN3 did not affect the expression levels of
the GFP fusions to CheA1, CheA2 and CheA3. These results suggest that Che
systems I and III of V. cholera may be active in the host.
2PS050クラミドモナスにおける走光性符号のレドックス調節

Redox-regulated switching of the phototactic sign inChlamydomonas
Shota Mochiji1, Norikazu Ohnishi2, Jun Minagawa2, Ritsu Kamiya3, Ken-ichi
Wakabayashi1 (1Dept. Biological Sciences, Grad. School of Science, Univ.
Tokyo, 2Div. Environmental Photobiology, National Institute for Basic
Biology, 3Dept. Life Science, Faculty of Science, Gakushuin Univ.)
The unicellular green alga Chlamydomonas shows positive or negative
phototaxis depending on the light intensities, circadian rhythms, etc. Switchingof direction (or “sign”) of phototaxis is important for the cells to stay underproper light conditions for photosynthesis. However, its mechanism has been

− S118 −unclear. Recently, we showed that the cellular redox (reduction-oxidation) poise
determines the phototactic sign: cells show positive phototaxis after treatment
with reactive oxygen species (ROS), whereas they show negative phototaxis
after treatment with ROS quenchers. This finding raised two questions of 1)
how cellular redox poise changes in vivo and 2) what is the molecular basis forthe redox-regulated switching of the phototactic sign. Regarding 1), we focusedon state transitions of photosynthesis, because the ROS generation has beensuggested to greatly change depending on the state of photosynthesis. Lowtemperature fluorescence spectroscopy revealed that cells showing positive
phototaxis are in State 1, whereas those showing negative phototaxis are in State
2. We propose a model that changes in the intracellular ROS amount, caused bystate transitions, function as a signal for the phototactic sign switching.
Regarding 2), we found that two previously isolated phototaxis-deficientmutants have defects in redox sensing. We also isolated a new mutant that
responds to the treatment with redox reagents in an opposite manner to that ofwild type. We will present the results of phenotypic analyses.
2PS051

細胞性粘菌で探る細胞運動ダイナミクスと走電性応答の関係性

Cell migration dynamics and its relevance to electrotaxis in
Dictyostelium cells
Hiroaki Takagi1,3, Masayuki J. Sato2,3, Masahiro Ueda2,3,4,5 (1Sch. Med., Nara
Med. Univ., 2Grad. Sch. Front. Biosci., Osaka Univ., 3JST, CREST, 4Grad. Sch.
Sci., Osaka Univ., 5QBiC, Riken)
Directional cell migration is a fundamental process associated with many
physiological phenomena. On the signaling process realizing directional cell
migration, the dominant view is that cells respond to external signals passively
and digitally. Recently, however, a complementary view in which cellular
spontaneous dynamics without external signals plays an important role toproduce cellular behaviors attracts considerable attention. In order to verify thelatter view, a relationship between spontaneous random cell migration anddirectional cell migration with external guidance cues (direct current electricfield) was extensively investigated in Dictyostelium discoideum. It was
confirmed that a generalized Langevin model with asymmetry for cellularpolarity could successfully reproduce the statistical characteristics of
spontaneous cell migration. Also, it was shown that the Langevin model
extended with additional velocity bias in accordance with the electric fieldstrength could describe directional cell migration under electrotaxis
quantitatively. Based on these results, the hypothesis that electrotaxis is realizedby biasing spontaneous cell migration properly, and velocity memory is relevant
to cellular flexible response for uncertain environment was proposed and tested
through a series of electric field reversal experiments and the model simulations.
2PS052細胞運動のための力と生化学の相互作用

Force-biochemical interaction for cell migration
Yuichi Sakumura1, Yoshiaki Iwadate2 (1Sch. Info. Sci., Aichi Pref. Univ., 2Grad.
Sch. Sci., Yamaguchi Univ.)
Motile cell generates traction force to show crawling migration. Althoughbiochemical signals such as Rho family small GTPases have been well studied,
it is still unclear how the migrating cell manages and integrates the traction force
and biochemical signals. Our experiment using force microscopy shows that
migrating Dictyostelium cell generates a small traction force at the front relative
to the other directions. Keratocyte also migrates by generating relatively small
traction force at the front direction. In addition, our experiment using GFPmyosin II revealed that the cell accumulates myosin II at the intracellular space
where force was added from extracellular side, suggesting that the celltransduces extracellular force to a biochemical signal through myosin II
accumulation. How the cell integrates intra- and extracellular interactions for
migration? To examine the mechanism of the cell migration based on theexperimental observations, we developed a simple computational model cellwhich has a hexagonal shape with a spring-dumper system and contains forcebiochemical interactions. The model cell migrates on the adhesive substrate
where the cell repeats adhesion and exfoliation. Using the model cell, we showthat the cell can migrate to the direction at which it generates small traction
force relative to the other directions. This suggests that exfoliation facilitated bymyosin II accumulation not at the front side plays key role for cell migration.
2PS053

歩行中ミオシン X の高速 AFM 観察

Direct observation of walking behavior of myosin X by
high-speed atomic force microscopy

Yusuke Sakiyama1, Noriyuki Kodera2, Osamu Satou3, Mitsuo Ikebe3, Toshio
Ando1,2 (1Sch. Math. & Phys., Int. Sci. & Eng., Kanazawa Univ. 2Bio-AFM
Frontier Research Center, Inst Sci. & Eng., Kanazawa Univ. 3Dept.
Physiology, Univ. Massachusetts Med. Sch.)
Double-headed myosin X, one of the actin-based motors, is involved infilopodia formation and intrafiropodial transport. Structurally, each head of
myosin X is comprised of a motor domain, an ~11 nm long neck domain (leverarm domain) consisting of three IQ motifs bound to calmodulins or calmodulinlike light chains, and a proximal tail of a stable alpha-helix which ishypothesized to extend the working stroke of myosin X. The head is followed
by a coiled-coil domain for possible dimerization and then by two uniqueglobular tail domains. Recently, single molecule fluorescent microscopy
revealed that the artificially dimerized tail-truncated myosin X can moveprocessively on single actin filaments with a large step size of ~34 nm [Y. Sun
et al., Nat. Struct. Mol. Biol. (2010)]. This large step size can be explained by
the hypothesis of the stable alpha-helix extension to the lever arm domain [i.e.,
total lever arm length is estimated to be ~26 nm (~11 nm + ~15 nm)]. However,this structural evidence on the functioning molecule is still missing. Thus, here
we have been performing high-speed atomic force microscopy imaging todirectly observe behaviors of myosin X moving along an actin filament. We
established an appropriate motility assay condition in which phospholipid
bilayers were used as a substrate. Using this assay condition, we successfully
visualized myosin X molecules that were moving with a large step size of ~36
nm. The details of the structural dynamics are now under investigation.
2PT001

独立してフォールドする構造ドメインデータベースの構築

I-StDom: A dataset of Independently foldable Structural
Domains automatically delineated from protein structures
Yuki Umezawa1,2, Teppei Ebina2, Yutaka Kuroda1 (1Dept of Biotech. & Life Sci.,
Tokyo University of Agriculture & Technology, 2Brain Science Inst., RIKEN)
Protein domains that can fold in isolation (structural domains) are significant
targets in diverse areas of proteomics research as they are often readily analyzed
by high-throughput methods. In this process, the ability to identify sequence
regions corresponding to structural domains in novel proteins with unknown
structures is an essential step. Computational methods for predicting domain
regions from sequence information only are thus becoming valuable. In
developing such predictors, reliable structural domain datasets are necessary aslearning data.
In this study, we constructed I-StDom, a dataset of Structural Domains that canfold Independently and in isolation (ISDs). We identified ISDs from multidomain proteins cataloged in SCOP and CATH by assuming that they form little
inter-domain interactions, such as H-bonds and hydrophobic cores, and which
we calculated from the protein’s atomic coordinates. We parameterizedinteractions, and optimized them so as to select the maximal number of protein
domains cataloged in PDB. 48089 ISDs were selected using the optimal
parameters (less than 11 MC-MC, 9 MC-SC, and 7 SC-SC H-bonds, and 7hydrophobic cores) with 29.7% of them having sequence similarity to a domain
cataloged in PDB. This is more than 10% higher than for the unfiltered SCOP
and CATH domains, which strongly suggested that domains in I-StDom canfold in isolation. I-StDom is available at http://domserv.lab.tuat.ac.jp/i-stdom.
html.
2PT002ADS 仮説に基づくタンパク質のデノボ立体構造予測法の開発
Development of a de novo protein structure prediction method
based on the antecedent domain segment(ADS) hyphothesis

Kota Nakamori, Shintaro Minami, Kengo Sawada, George Chikenji (Grad.
Sch. of Eng. Univ. of Nagoya)Protein structure prediction is one of the most important and fundamental
problems of biology. In particular, the prediction of novel protein structures, forwhich we cannot use template, is a very significant from the viewpoint ofbiophysics. Currently, one of the most successful method for predicting novel
protein structures is the fragment assembly (FA) method. The success of themethod is, however, limited to low contact order (CO) proteins, probably
because the FA method emphasizes local interaction too much. (R.Bonneau.
Protein Sci. 2002;11:1937-1944). In order to predict novel protein structureswith high CO, we developed a method for novel protein structures prediction.
The idea of the medhod is based on the antecedent domain segment (ADS)
hyphothesis, which was originally proposed by Lupas et al. In this hypothesis,
ancient protein structures were formed by self-assembling aggregates of shortpolypeptides. Our method mimick this aggregation processes for protein tertialy− S119 −structure assembly. Thus, the method enphasises non-local interactions rather
than local ones. One of the advantage of the method is that the method candirectly be applied to oligomer targets. We will report the detailed description of
the method and the results of benchmark tests.
2PT003

配列順序に依存しない構造関係性を用いた長い挿入配列の構造モデ

sites identified using an existing pocket detection algorithm, ghecom. We
applied our method to all-pair similarity searches for the 3.4 million known andpotential ligand-binding sites. Consequently, we discovered ca. 24 million
similar binding sites. Here we report those results including all the discoveredpairs. Our database is expected to be useful for predicting protein functions andrapid screening of target proteins in drug design. The PoSSuM database isavailable at http://possum.cbrc.jp/PoSSuM/.

リング

Modeling structure of large insersion regions in homology
modeling bu utilizing non-sequential structure relationship
Yuki Nakagawa, Shintaro Minami, Kengo Sawada, George Chikenji (Grad.
Sch. of Engineering, Nagoya Univ.)
Homology modeling is a powerful method for predicting protein structures byusing a template structure having a similar amino-acid sequence to the target
protein. However, in the region where the amino acid sequence is very different
between the target and the template or large insertions exit, the predictedstructure for such a region remains unreliable.If such an unreliable region is solarge that three or four secondary structures have to be constructed, de novo
modeling is needed. Currently, such de novo modeling, however, does notalways provide an accurate prediction. Here, we propose a new homology
modeling method that can effectively generate reasonable structure candidatesof such unreliable regions. The idea of the method is based on the observationthat if we search similar structures of the reliable region of the target protein in
PDB by a non-sequential structure alignment program, we almost always findgood structure candidates of such unreliable regions. In the presentation, we willreport the detailed description of method and results of benchmark tests.
2PT004

Common structural / sequence features of proteins that sharenon-sequential structural similarity

2PT006

Carbohydrate play an important roles in molecular recognition mechanism.
Especially, carbohydrate-protein interaction are associated with importantbiological process such as cell to cell communication, cellular immunity andcell recognition.Recently, the number of carbohydrate binding proteins wereachived over 4000 structures. It is desired to extract new biological findings
from these large amount of structual data. In this study, We have carried out
data mining of the Protein Data Bank (PDB) to collecting carbohydrate binding
proteins, and construct the prediction model of carbohydrate binding site inprotein sequence using support vector machine. We analyzed binding site
propensity for each types of carbohydrate. We also evaluated this prediction
model for 5-fold cross validation.The sensitivity and specificity are achieved an
81% specificity of prediction at 64% sensitivity. Area Under Curve (AUC) wereachieved 0.81.
2PT0072PT005

PoSSuM: タンパク質の既知及び潜在的基質結合部位類似性検索のタンパク質における部位間での同時的補償的置換に基づく隣接残基
対予測

Kengo Sawada, Shintaro Minami, Geroge Chijenji (Dept. of eng., Nagoya Univ.)
Recently, many researchers have reported interesting examples of nonsequential alignments such as circular permutations and domain or region
swaps. For example, Alexj et al. conducted a comprehensive large-scale studyof non-sequential alignments between available protein structures in Protein
Data Bank (PDB) and found that 35.2% of all significant alignments are nonsequential [1]. Furthermore, Aysam et al. identified significant non-sequential
structural similarity between some new folds and existing structures in PDB [2].
These observations suggest that appropriately utilizing non-sequential template
enables us to predict new fold targets.
The goal of this research is to identify the structural and sequence features ofproteins that share non-sequential structural similarity, in order to predict new
folds utilizing non-sequential templates. In this work, we collected structures
sharing non-topological similarity with one of the super-folds such as
Ferredoxin-like fold, and found common features among them. We will reportdetailed description of the method and the results in the presentation.
[1]Alexej Abyzov and Valentin A Ilyin. 2007. BMC Structural Biology 7:78
[2]Aysam Guerler and Ernst-walter Knapp. 2008. Protein Science. 17:13741382

サポートベクターマシンを用いた糖鎖結合部位予測手法の開発

Development of method for predicting carbohydrate bindingsite using support vector machine
Masaki Banno, Kentaro Shimizu (Grad Sch Agricul. life sci., Univ Tokyo)

Prediction of Contact Residue Sites Based on Concurrent andCompensatory Substitutions Between Sites in Protein Evolution
Sanzo Miyazawa (Gunma Univ., Grad. Sch. Eng.)
Structural and functional constraints originating in residue-residue interactions
act on each amino acid in a protein, because a protein must fold into a uniquethree-dimensional structure and play a specific function. Selective constrains on
amino acids in proteins are recorded in amino acid orders in homologous protein
sequences and also in the evolutionary trace of amino acid substitutions. Thus,
statistical analyses of amino acid orders and of the evolutionary trace of aminoacid substitutions can provide information on residue pair couplings. A
challenge is to extract direct dependencies between residue sites by excluding
indirect correlations that originate from indirect interactions through otherresidues within a protein or even through other molecules. Recent attempts ofdisentangling direct from indirect dependencies of amino acid types between
residue positions in multiple sequence alignments of proteins have revealed thatthe strength of inferred residue couplings is an excellent predictor of residueresidue proximity in folded structures. Here, we report an alternative attempt ofinferring site couplings from concurrent and compensatory substitutions
between sites at each branch of a phylogenetic tree. Accuracy of contact
prediction based on the present method is comparable to that of the maximum
entropy method.

ためのデータベース

PoSSuM: a database for searching similar pairs of known andpotential ligand-binding sites in proteins
Jun-ichi Ito1,2,4, Yasuo Tabei3, Kana Shimizu1, Koji Tsuda1,3, Kentaro Tomii1,2(1CBRC, AIST, 2Department of Computational Biology, Graduate School of
Frontier Sciences, The University of Tokyo, 3Minato Discrete Structure
Manipulation System Project, JST, 4National Institute of Biomedical Innovation)
We proposed a fast alignment-free method that can enumerate similar pairs frommillions of ligand-binding sites in the Protein Data Bank (PDB). In our method,
ligand-binding sites are first encoded as feature vectors based on their
physicochemical and geometric properties of amino acid residues. Once feature
vectors representing ligand-binding sites are converted to bit strings, called
structural sketches, which is obtained by random projections of the vectors, amultiple sorting method is applied to the enumeration of all similar pairs interms of the Hamming distance.
We have developed our new database, called Pocket Similarity Search using
Multiple-sketchsorts (PoSSuM) to compile all similar pairs detected using our
method as a relational database. In the latest version of our database, we used
the following two datasets: 226,630 small ligand-binding sites obtained fromprotein-ligand complexes in the PDB, and 3,134,413 potential ligand-binding

2PT008

3DMET 構築のためのキュレーション支援システム
Supporting system for manual curation of the 3DMET
database

Miki Maeda (National Institute of Agrobiological Sciences)
We have developed the 3DMET database, a three-dimensional structure
database of natural metabolites. Automatic conversion from 2D- to 3Dstructures causes conversion errors especially for chiral information. Thus, weapplied automatic confirmation and manual curation to each 3D-structure.
During the work, we need some supporting system and have developed.
"Structure-Checker" is a supporting system for self-judge of the conservation
between two structures. The user interface is web browser and two MOL files
are sent to the web server. In the server, the structures are converted to InChI
and each columns are compared. If a column does not correspond to one
structure, the color of the column is changed. Curators judged manually basedon the results. To understand the results, curators need basic knowledge for the
InChI description.
"Molecular Viewer" is a system for manual check of 3D-structures andliteratures, developed for chemists not familiar to computer. The user interfaceof this system is also web browser. Book name, pages, and chemical name are− S120 −selected in order; a 3D-structure of the molecule is shown. For this 3Dvisualization, J-mol is used as a plug-in program for web browsers.
The 3DMET is now available as Release 2.1. The Release 2.2 will be opened toaccess until this presentation. The recent development and changes of 3DMET
will be also reported.
2PT009

配列順序を無視したタンパク質構造アライメントの性質

Some characteristic features of non-sequential structuralalignment: difference between sequential and non-sequentalalignments
Tatsuo Mukai, Kengo Sawada, Shintaro Minami, George Chikenji (Grad. Sch.
of Eng. Univ. of Nagoya)
Recently, a number of interesting examples that show non-sequential structure
similarity have been reported, where non-sequential structural similarity is the
structural similarity in which structurally equivalent regions are aligned indifferent order in the sequence of the compared proteins. For example, Minami
et al. demonstrated that the usage of a non-sequential structure alignment
program yielded many structure alignments with significant similarities
between proteins of known three-dimensional structure and newly determined
protein structures that possess a new fold. This finding suggest that many new
folds can be modeled by permutating old folds.
In order to develop a new prediction method for new fold targets utilizing thenon-sequential structure relationship, we first must clarify characteristic featuresof non-sequenttial structural relationship. As a first step towards the goal, we
performed a large scale non-sequential alignment between protein pairs
deposited in PDB and compare their features with those of sequentialalignments. We found several characteristic features found only in nonsequential alignment. For example, protein size and solvent accessibility werelargely not conserved in non-sequentially similar pairs. These finding may be
usefull for developing a new De Novo prediction method. In the presentation,
we will report detailed description of the method and the results.
2PT010Prediction of the types of carbohydrate-binding proteins
Zhenyi Ge, Wei Cao, Kazuya Sumikoshi, Shugo Nakamura, Kentaro Shimizu(Department of Biotechnology, Graduate School of Agricultural and Life
Sciences, The University of Tokyo)
Carbohydrate-binding proteins or lectins, which are defined as carbohydratebinding complexes which are not directly modified by carbohydrate, areimportant complexes that participate in many biological processes asintercellular information transfer and cell recognition. Though there are avariety of methods to classify lectins, classifying lectins based on structural andevolutionary sequence similarities is more popular because of some highly
conserved amino acid sequence motifs in CRD (Carbohydrate Recognize
Domain). Depending on this way, lectins could be classified into C-type lectins(require calcium for recognition), S-type lectins (also known as Galectins,
which require free thiols for stability), L-type lectins (have a feature structure
called “jelly roll fold”) and so on. We developed a tool to detect and classify
lectins by HMM (Hidden Markov Models). Several corresponding profile
HMMs are composed and are prepared for scoring entry sequences. Sequencesin low score section are regarded as NOT lectins and sequences in thecorresponding range of scores are classified into fitting types of lectins.

2PT011Analysis of DNA-binding sites using novel amino acid index formolecular recognition (MR-index)
Katsutoshi Hibino1, Yasunori Yokoyama2, Shigeki Mitaku2 (1Dept. ofComputational Science and Engineering, Grad. Sch. of Engineering, Nagoya
Univ., 2Dept. of Applied Physics, Grad. Sch. of Engineering, Nagoya Univ.)
Protein-DNA interaction plays an important role in many biological processes.
However, the mechanism of protein-DNA interaction is still not clear.
Prediction of DNA-binding sites in DNA-binding proteins using their physicalproperties is very useful for understanding mechanism of protein-DNA
interaction. There are many experimentally determined structures in Protein
Data Bank database, but only a few protein-DNA complexes are available. In
addition, it is very expensive and time-consuming process to solve the structureof a protein-DNA complex through experimental methods. Therefore, asequence-based computational prediction of DNA-binding sites in DNAbinding proteins is required. In this work, we used a novel amino acid index formolecular recognition (MR-index) to develop a system for predicting DNAbinding sites in DNA-binding proteins. Although MR-index was originallydeveloped for predicting allergen-unique fragments (AUFs), it is found thatmost proteins have MR-index peaks at a constant rate. Also, MR-index peaks
locate at positions with large structural fluctuations such as loop regions, andthose sites tend to interact with other molecules. We took account of physicalproperties around MR-index peaks and aimed to develop the system forpredicting DNA-binding sites in DNA-binding proteins. The discrimination
analysis showed that the MR-index peaks around DNA-binding sites havecharacteristic distribution of physical properties.
2PT101

アミノ酸保存度と相互作用エネルギーを組み合わせたタンパク質ーリガンド結合部位予測手法の開発

Protein-Ligand Binding Site Prediction by Using Energybased Method with Amino Acid Conservation Scores
Hiroto Tsujikawa, Yusuke Umeda, Mizuki Morita, Shugo Nakamura, Kentaro
Shimizu (Grad. Sch. Agric. Life Sci., Univ. Tokyo)
Protein-ligand interaction is one of the most important steps for expression ofprotein functions. Identifying ligand binding sites helps protein function
analysis and structure-based drug design. Recently, a variety of computational
methods for predicting ligand binding sites are proposed. Among these methods,
the energy-based method which calculates protein-ligand interaction energy ona three-dimensional protein structure is shown to realize high precision. In thisstudy, we developed a new method which combines the energy-based methodwith amino acid sequence conservation scores. Our method locates methyl
probes around the protein surface and calculates van der Waals interactionenergy between each probe and the protein. In addition, we calculate amino acidconservation scores by constructing position-specific scoring matrix (PSSM)
and add their weighted scores to the interaction energy. It is considered thathighly conserved amino acid residues are expected to be closely related toprotein function and appear in functional sites. Probes with energeticallyfavorable for binding sites and within a short distance are clustered and they are
ranked according to their total interaction energies. As a result, in 80% proteins,at least one site in top three predicted sites predicts ligand binding sites
correctly. We will evaluate the performance of our method and compare it withother existing methods, and show the importance of amino acid sequenceconservation in ligand binding sites prediction.
2PT102

蛋白質・蛋白質相互作用ネットワークにおけるドメイン及び非ドメイン間相互作用の解析

Analysis of Domain and Non-Domain Interactions in ProteinProtein Interaction Network
Komori Satoshi, Sarai Akinori, Fujii Satoshi (Kyusyu Institute of Technology)
The analysis of protein-protein interactions and their network has been usuallybased on protein chains and domains. However, non-domain regions are often
involved in the interactions and serve function as well. Here, we haveconsidered non-domains as well as domains in the protein-protein interactions.
The information of domains within the individual chains was obtained from
Pfam, and sequence-based clustering was carried out. Non-domains were
defined as the regions outside the domains, within certain length threshold. We
have examined physical contacts among all pairs of domains and non-domains
within each complex structure of PDB, and identified 15,742 domain-domaininteractions, 15,218 domain-non-domain interactions and 12,804 non-domainnon-domain interactions. Then, we constructed the interaction network. These
pieces of information on domains and non-domains and their interactions were
implemented into PDBnet, which enables users to examine protein interaction
network in terms of domain and non-domain interactions. We have comparedcharacteristics of interactions due to domains and non-domains, such as aminoacid composition, interaction type and packing. We will also make systematic
analysis on the relationship among various properties at molecular structure,interaction and network levels, in order to get insight into the function ofproteins. Furthermore, we will apply the present analysis to the systematic
predictions of protein-protein interactions based on the catalog of interacting
domains and non-domains.
2PT103

進化的に保存されたタンパク質間相互作用の複合体立体構造の予測

3D complex structure assignment for evolutionary conserved
protein-protein interactions
Takeshi Kawabata, Haruki Nakamura, Akira Kinjo (Institute of Protein
Research, Osaka University)− S121 −A huge amount molecular interaction data are accumulated to elucidate entire
landscape of cellular activities. 3D complex structures of interacting proteinscan add their physical mechanisms, such as interacting residues and driving
binding forces. The goal of this study is to gather all the reported molecularinteractions, and to predict their 3D structures using homology modeling as
many as possible. Another goal is to propose biologically important interacting
protein sets to be solved their 3D complex structures in the future. However,current experimental molecular interaction data are not comprehensive andreliable. To overcome these problems, we compare interactions of several
different organisms, and consider conserved interactions to be highly reliable.
As the preliminary study, we analyzed yeast and human interactromes.
The INTACT database stored 70, 000 and 40, 000 two-body protein-protein
interactions for yeast and human, respectively. Among these interactions, 3D
complex structures for 1.5 % yeast and 6 % human pairs can be modeled using
homologies for known 3D complexes. To reduce false positive data, we
extracted conserved interactions (interologs) between the two species. Numberof the interologs are about 5,000, they may be more reliable and should bebiological important. 3D complex structures can be assigned for 11 % and 24 %of the interlogs for yeast and human, respectively. The remaining 4,000 protein
pairs are good candidates protein pairs to be solved their 3D complex structures.
2PT104相互作用プロファイルを使った正解相互作用部位の絞込みによる再ドッキング法の研究

Development of re-docking method by obtaining nativeinteraction pattern
Nobuyuki Uchikoga1, Yuri Matsuzaki2, Masahito Ohue2, Yutaka Akiyama2,
Takatsugu Hirokawa3 (1Dept. Phys., Sch. Sci. & Eng., Chuo Univ, 2Grad. Sch.
Comput. & Sci., Tokyo Inst. Tech., 3CBRC, AIST)In a study of protein-protein Interaction network, it is important to predict whichpairs of proteins interact in living cell and how these bind each other. We
approach to these problems using rigid-body docking algorithm, generating
many decoys including false positives. Although this docking method is popularand useful, there are some cases with no near-native decoys. In previous work,
we developed a method of generated Profile of Interaction FingerPrints (P-IFP)
rather than generating near-native decoys. Then, we applied a profile method to
obtaining region including native interacting residue pairs for re-docking
process.
We have been developed Interaction FingerPrints (IFP) for the post-docking
analysis of protein-protein rigid-body docking. IFP is composed of binary statesof interacting amino acid residues of each proteins, as a scale for measuring
unique similarities between the complex structures. Then, IFP can be used moreeasily to evaluate similarities between decoys than RMSD method, which
depends on methods of superposing their structures. Additionally, IFP can be
useful to assemble interaction profiles from various decoys.
In this work, we examined re-docking process after generating P-IFP for rigidbody docking decoys. As results, we could obtain a set of decoys with higher
similarities than that of decoys generated in the initial docking process. Redocking method using IFPs is expected to improve cases without near-native
decoys in an initial docking process.
2PT105タンパク質-低分子リガンド相互作用における結合様式の網羅的比較
解析

Comprehensive comparisons of interactions in small-ligand
recognitions by proteins at the atomistic level
Kota Kasahara1, Kengo Kinoshita1,2,3 (1Grad. Sch. Information Sci., Tohoku
Univ., 2ToMMo, Tohoku Univ., 3IDAC, Tohoku Univ.)
Illuminating molecular mechanisms of ligand recognitions by proteins is a longstanding problem in the biophysics. Toward this, the 3D structural data ofprotein-ligand complexes provide fruitful information and recent continuous
innovation of experimental techniques has been skyrocketing the structural data.
However, it is not straightforward to mine information and knowledge from thecomplicated data, consisting of the Cartesian coordinates of atoms.
Here, we developed a method to extract information about molecular interactions
form structural data of protein-ligand complexes and comprehensive comparisonsof their interactions were performed. In this study, the interacctions in a proteinligand complex was described as a network, nodes of which indicates amino acidresidues or ligand atoms, and edges of which indicates interactions. The similarity
between a pair of complex was calculated by alignment of these networks. We
performed all-against-all comparisons of binding modes on a dataset consisting of
35,338 protein-ligand complexes. Further, similarities in the SCOP classificationand ligand 2D structure were also calculated and consistency among these threesimilarity measures was evaluated.
As a result, basically, pairs with similar protein folds had similar ligands andinteractions. However, there were many exceptions, i.e., dissimilar interactions
with similar folds and ligands, and similar interactions with dissimilar folds andligands. Novel relationships among proteins and ligands were mined by ourstudy.
2PT106

分子ネットワークにおける構造情報と相互作用情報の統合

Integration of Structural and Biochemical Information intoProtein Interaction Network
Yuji Kawakami, Akinori Sarai, Satoshi Fujii (Kyushu Institute of Technology)
Interactions among proteins have been mostly studied by structural andbiochemical methods. Both kinds of interaction data have advantages anddisadvantages, and complement with each other. Thus, the integration of thesedata will be useful for research. On the other hand, the quality of theseexperimental data is not necessarily uniform, making it more difficult to analyze
them. Here, we try to integrate the structural and biochemical information ofprotein-protein interactions. The structural information is based on PDB. We
used DIP, BOND, HPRD, IntAct and Mint databases for biochemicalinteraction data. We first evaluated the quality of the experimental data, and add
the information along with the interaction data. We unified the biochemical data
by using Uniprotkb ID and removed the data redundancy among them. Then we
integrated the data into PDBnet by using Uniprotkb ID and PDB chain ID.
Owning to this integration, PDBnet now contains more comprehensive
interaction network. Many biochemical interactions are not present in the
structural interactions. This would help researchers working on structures to
identify missing potential interactions for structural analysis. On the other hand,some interactions are present only in the structural information. The integrationof structural information into the biochemical network also helps researchers tounderstand the molecular mechanism of protein interactions and to develop
potential drugs.
2PT107

酵素反応の分類と代謝パスウェイ検索への応用

A classification of enzymatic reactions and its application tometabolic pathway search
Susumu Goto, Masaaki Kotera, Toshiaki Tokimatsu, Yuki Moriya, Zen-ichi
Nakagawa, Ai Muto, Minoru Kanehisa (Inst. Chem. Res., Kyoto Univ.)
Enzymatic reactions are key components in the metabolic pathway and havebeen classified by the Enzyme Nomenclature Committee of the International
Union of Biochemistry and Molecular Biology. This classification (EC number
system) is based on six classes: oxidoreductases, transferases, hydrolases,
lyases, isomerases, and ligases, and each class is further subdivided according tomore detailed reaction mechanisms such as bond types and functional groups.
While quite useful, the system also has some limitations. For example, similar(resp. totally different) reactions in terms of mechanisms of chemicaltransformation are sometimes classified into different classes (resp. a sameclass). We, therefore, developed a new classification system for enzymatic
reactions based on chemical transformation mechanisms and functional groupsof chemical compounds.
First, we defined substrate-product pairs of enzymatic reactions. Each pair was
structurally aligned, from which reaction centers and different and matchedparts were extracted (RPAIR database). Reaction (chemical transformation)
patterns were then defined using the extracted parts and stored in RCLASS
database. This can be considered as a classification of substrate-product pairs
and their associated reactions by the reaction patterns. As of June 2012, 2,516
RCLASS entries have been registered and further classified into 384 groups
based on functional group conversions. This resource including some pathway
tools utilizing RCLASS is available on the GenomeNet (http://www.genome.
jp/reaction/).
2PT108

アポリポプロテインにおける疾患感受性のある点変異付近のアミノ酸配列断片の物性的特徴

Physical characteristics of amino acid fragments around point
mutation with disease susceptibility in apolipoproteins
Ryouta Masai, Yasunori Yokoyama, Shigeki Mitaku (Dept. Applied Physics,
Grad. Sch. of Engineering, Nagoya Univ)
Apolipoproteins are proteins that bind lipids to form lipoproteins. They serve as
enzyme cofactors, receptor ligands, and lipid transfer carriers that regulate the− S122 −metabolism of lipoproteins and their uptake in tissues. Some mutations in
apolipoproteins are related to inheritable disease. In this work, we studied
physical properties of amino acid mutations in two groups: mutations with andwithout disease susceptibility in apolipoproteins. The analysis was carried out
by two steps: First, we enumerated peaks of MR-index plots from amino acidsequences, and the mutations which changed the position of the MR-index
peak were used for further analysis. Second, we calculated the averagehydrophobicity and charge density of seven residues, and changes in themagnitude as well as the position of MR-index peaks. MR-index is an index that
estimates allergen-specific amino acid sequence and binding site. The result
indicated that the properties were significantly different between the mutations
with and without disease susceptibility. The results will be useful for theprediction of inheritable diseases of multi-factors.
2PT109L-リシン酸化酵素の X 線結晶構造解析

Structure of L-lysine α-oxidase from Trichoderma viride
Tadahisa Sano1, Tatsuya Kawaguchi1, Marie Amano2, Kengo Shinyashiki2,
Haruka Nakata2, Takashi Tamura2, Kenji Inagaki2, Hitoshi Kusakabe3,
Katsumi Imada1 (1Grad. Sch. Sci. Osaka Univ, 2Grad. Sch. Env. & Life. Sci.
Okayama Univ, 3Enzyme Sensor Co. Ltd.)
L-amino acid oxidase (LAO) is a dimeric flavoenzyme that catalyzes oxidative
deamination of an L-amino acid to produce a 2-oxo acid with ammonia andhydrogen peroxide. Most of known LAOs show wide substrate specificity, butalmost no activity for L-lysine oxidation. Unlike other LAOs, L-lysine oxidase (LysOX) from Trichoderma viride, which is found during the search for
antitumor agents, exhibits strict substrate specificity for L-lysine. Thus, LysOX
is an attractive candidate for an enzyme-based amino acid sensor. Moreover,
LysOX has broad antitumor activity. To understand the strict substrate
recognition mechanism of LysOX, structural information is essential. Here we
report the crystal structure of LysOX from T. viride at 1.9 Å resolution. Since noactive enzyme was expressed in E. coli, we purified LysOX from wheat branculture of T. viride, and used it for crystallization. The diffraction data werecollected at Spring-8 beamline BL41XU and the structure was determined bymolecular replacement using the structure of L-amino acid oxidase from Vipera
ammodytes as a search model. LysOX is composed of three domains and forms
a tetramer in the crystal. The structural refinement is now in progress.
2PT110Differences between key amino acid residues for the transitions Lto R- and R- to L-protofilaments of Salmonella flagellar filament

Fumio Hayashi, Hidetoshi Tomaru, Eiji Furukawa, Kanami Ikeda, Kenji
Oosawa (Department of Chemistry and Chemical Biology, Graduate School ofEngineering, Gunma University)
Salmonella cell swims by rotating its flagellar filaments. The filament (~ 10 m)
is composed of ~30,000 molecules of a single protein flagellin and forms lefthanded helical structure under physiological conditions. The cell repeats swimand-tumble and consequently move toward their favorite environments andaway from unfavorites. The swim-and-tumble is caused by the change ofthe filament shape introduced by reversal of flagellar rotation, and the change ofthe filament shape is called polymorphic transformation. To elucidate thetransformation mechanisms of flagellar filaments, we applied genetic
suppressor analysis within flagellin molecule against SJW1655 producing Rtype straight filament and SJW1660, HFG180, and HFG195 producing L-typestraight filament. We isolated about 100 revertants from every mutant,determined the second mutation sites, investigated the localization of the aminoacid residues on L- and R-type flagellin molecules, measured their swimmingand swarming speeds, and observed their flagellar filaments. From these results,
we propose that S106, A416, A427, and R431 are key residues involved intransitions from L- to R-type protofilaments, whereas D107 and G426, and S448
are keys from R- to L-type protofilaments. From the differences in the key
residues, we suggest that L to R (R to L) transition is not just the inverse
reaction of R to L (L to R) transition.
2PT111アミロイドベータペプチドのオリゴマー形成メカニズムに関する構
造学的解析

Structural analysis of the oligomerization mechanism ofamyloid beta peptide
Hitomi Yamauchi1, Shigeto Iwamoto1, Takashi Saito2, Toshiyuki Kohno3,
Takaomi Saido2, Hiroaki Terasawa1 (1Facul. of Life Sci., Kumamoto
Univ., 2RIKEN BSI, 3Sci. of Med., Kitasato Univ.)

Amyloid beta peptides (Aβ) form senile plaques following oligomerization in
the brain. Aβ oligomers are thought to be neurotoxic and to play a key role in
Alzheimer’s disease (AD). Aβ40, 42 and 43 are major species of Aβ. It is known
that Aβ42 and 43 are more prone to aggregation than 40. Thus, the length of the
C-terminus is assumed to promote Aβ oligomerization and neurotoxicity.
This study aims to investigate the structure of A oligomers and theoligomerization mechanism associated with AD.
To examine the influence of the C-terminus on A properties, we compared theoligomerization propensity of A by PICUP (Photo-Induced Cross-linking of
Unmodified Proteins). All species formed dimers, however, large oligomers(trimers and tetramers) were formed at higher rate by A42 and 43. Further,
NMR analyses suggested that the C-terminal structure of A42, 43 differs fromthat of 40. Besides, the flexibility of the C-terminus was higher in A40 than in
42 and 43 revealed by NMR. The results showed that the interaction between
the C-terminus of A42 and 43 is stronger than that of 40.
To investigate the cross-linking sites, we used ESI-TOF MS to analyze fortryptic decomposition product of A dimer stabilized by PICUP. It was foundthat H6-K16 fragments were cross-linked. In A dimers, this region may exist in
close proximity to the corresponding region on the other molecule.
In conclusion, our data suggested that A forms parallel dimers. Moreover, adifference in C-terminal structure is important in the formation of large
oligomers.
2PT112

Role of the Tyr residue stacked on the si-face of theisoalloxazine ring moiety in ferredoxin-NADP+ oxidoreductase
from Bacillus subtilis

Daisuke Seo, Hiroshi Naito, Erika Nishimura, Takeshi Sakurai (Division ofMaterial Science, Graduate School of Natural Science and Technology,
Kanazawa University)
Ferredoxin-NADP+ oxidoreductase (FNR, [EC 1.18.1.2], [EC 1.18.1.3]) fromthe low-GC gram-positive bacterium Bacillus subtilis (BsFNR) is similar tobacterial thioredoxin reductases (TdRs) in amino acid sequence. The crystal
structure analysis of BsFNR revealed the presence of the aromatic residue Tyr50
stacked on the si-face of the isoalloxazine ring of the FAD prosthetic groupwithin a distance of approximately 3.5 Å. This conformation has been often
found among the flavoprotein super family including FNR from plastid, but
TdRs lack such the residue. To investigate the role of Tyr50 in catalysis with
NADPH and ferredoxin (Fd), we performed site specific mutation analysis.
While Tyr50Trp mutant retained reactivities with NADPH and Fd of ~20 and~40%, against those of the wild type respectively, replacements to nonaromatic
Gly and Ser residues drastically decreased the reactivities with both NADPH
and Fd.
2PT113アミロイド構造の決定におけるタンパク質揺らぎの役割の解析

NMR study on the role of protein fluctuation in determining
the amyloid conformation
Yumiko Ohhashi, Motomasa Tanaka (Brain Science Institute, RIKEN)
Aggregation-prone proteins often misfold into multiple distinct amyloid
conformations. Although amyloid conformation is crucial factor to determine its
toxicity or infectivity, the formation mechanism of diverse conformation
remains unclear. Yeast prion Sup35, the [PSI +] ptotein determinant, forms
several types of amyloid with different conformations. Sup35 is translation
termination factor composed of three domains (N, M, and EF). An EF is
globular domain carrying translation termination function. N and M are
intrinsically disordered domains required for amyloid formation, however,biological function is unknown. In the present study, we elucidate the role ofprotein fluctuation in determining the amyloid conformation using NMR
spectroscopy. We found that the Sup35-NM mutant, S17R, dramatically altered
the amyloid core region. The S17R-type amyloid conformation can propagate towild-type Sup35-NM easily. These results raised an intriguing possibility that
there are (more than) two non-overlapping amyloid core regions in the Sup35NM sequence and one of the regions is selected depending on the condition
under which Sup35-NM amyloid is formed. NMR analysis with CLEANEXPM and STD revealed that the amino acid residues in both amyloid core regions
showed fast exchange rates and their transient interactions in the oligomers
might act as a nucleus for amyloid growth.
2PT114− S123 −抗真菌活性をもつ DUF26 タンパク質 Gnk2 の構造と機能

Structure and function of Gnk2, a DUF26 protein with
antifungal activity

Takuya Miyakawa1, Yoriko Sawano1, Ken-ichi Hatano2, Masaru Tanokura1(1Grad. Sch. Agric. Life Sci., Univ. Tokyo, 2Fac. Eng., Gunma Univ.)
Plants have a variety of potent defense mechanisms against pathogens,including the synthesis of secondary metabolites and antimicrobial proteins. We
found that the endosperm of Ginkgo biloba seeds contains ginkbilobin-2(Gnk2), a novel antifungal protein. Gnk2 consists of 108 amino acids andinhibits growth of plant and human pathogenic fungi. Gnk2 also belongs to
DUF26 protein family in plants and has a homology (28-31% identical) to theextracellular domain of cysteine-rich receptor-like kinases (CRKs) from Oryza
sativa and Arabidopsis thaliana. The CRKs are involved in the hypersensitive
reaction, which is cell death reaction caused by infection of plant pathogens.
Here, we present the structure and function of Gnk2 to provide the first insights
into the biochemical properties of DUF26 protein family. Crystal structure
showed that the conserved motif of DUF26 protein family considerably
contributed to maintain the overall structure of Gnk2 by forming three disulfide
bonds. In addition, NMR experiments showed that Gnk2 recognized Dmannose using a binding site surrounded by the residues, Asn11, Gly91, Ala92,
Arg93 and Glu104. Their hydrophilic side-chains probably contribute to bindingof D-mannose by hydrogen bonds with the free hydroxyl groups of themonosaccharide. The growth-inhibitory assay against a yeast variant lacking
mannose chain on cell surface showed that the interaction with mannose chain
was an essential event in antifungal mechanism of Gnk2.
2PT115The short α2-helix of the phospholipase C-δ1 pleckstrin
homology domain contributes to stable IP3 binding

Michikazu Tanio, Katsuyuki Nishimura (Institute for Molecular Science)
The phospholipase C (PLC) -1 pleckstrin homology (PH) domain has acharacteristic short -helix (2) from residues 82-87. The contributions of the2-helix toward the inositol 1, 4, 5-trisphosphate (IP3) binding activity andthermal stability of the PLC-1 PH domain were investigated. Native-PAGE
analyses indicated that disruption of the -helical conformation by replacementof Lys-86 with proline resulted in reduced affinity for IP3 and in thermal
destabilization of the IP3 -binding state. Although the mutant protein withreplacement of Lys-86 with alanine showed a slight reduction in thermal
stability, the IP3 -binding affinity was similar to that of the wild-type protein.
Replacement of Phe-87 with alanine, but not with tyrosine, also resulted in
reduced affinity for IP3 and in thermal instability. The 1 H-15 N HSQC NMR
study of the selectively [-15N]Lys-labeled PLC-1 PH domain indicated thatthe conformation of the 2-helix couples with the IP3 -binding activity of the
PLC-1 PH domain. These results indicated that the helical conformation of the2-helix and the phenyl ring of Phe-87 play important roles in the IP3-binding
activity and thermal stability of the PLC-1 PH domain. Based on these results,
the biological role of the 2-helix of the PLC-1 PH domain is discussed interms of membrane binding.
2PT116ヒト心臓脂肪酸結合蛋白質 (FABP3) の脂肪酸結合メカニズムに関する研究

Study of the Fatty Acid Binding Mechanism to Human Heart
Fatty Acid Binding Protein (FABP3)
Daisuke Matsuoka1,2, Shigeru Matsuoka1,2, Mika Hirose1,2, Mayumi
Niiyama1,2, Hanako Ishida1,3, Fuminori Sato1, Shigeru Sugiyama1,2, Michio
Murata1,2 (1JST ERATO, 2Grad. Sch. of Sci., Osaka Univ., 3Grad. Sch. of Eng.,
Osaka Univ.)
Understanding the interaction modes between a protein and a small molecule isessential to explain a ligand binding to a protein and to elucidate how watermolecules and lipid molecules contribute to the stabilization of proteins.
However, unlike water molecules and hydrophilic substrates, hydrophobic
molecules interact with proteins mainly through van der Waals interactions, and
it is hard to characterize clearly the ambiguous interaction mode.
We are now studying the binding modes of fatty acids to a human heart fatty
acid protein (FABP3) through molecular dynamics (MD) simulation, isothermal
titration calorimetry and cryogenic X-ray crystallography. We first performed
MD simulations of native and mutant FABP3 binding a stearic acid (C18:0 fattyacid). When the residues lining the binding cavity are mutated to smaller aminoacids in silico, the conformations of the fatty acids and positions of watermolecule in the binding cavity are different from ones observed in the native.
Then we performed MD simulations of native FABP3 binding lauric (C12:0),
myristic (C14:0), and palmitic (C16:0) acids. Within the simulation runs, thepalmitic and stearic acids keep the U-shape conformation in the binding cavity,
but the myristic and lauric acids show L-shape conformation.

In addition to the computational study, the calorimetric experiment alsosuggests the interaction modes are different between these fatty acids. Fromthese results and our high resolution crystal structure, we want to discuss thebinding modes of fatty acids to FABP3.
2PT117

Actinin-4 変異体の機能解析

The prediction of three dimensional structure for mutants ofactinin-4
Nami Miura1, Miho Banno2, Kazufumi Honda1, Akihiko Miyanaga1, Tesshi
Yamada1 (1Div. Chem. Clin., Natl. Cancer Ctr. Res. Ins., 2Mitsui Knowledge
Industry Co., Ltd.)
Actinin-4, an actin-bundling protein previously identified by our laboratory, isclosely associated with cell motility and cancer metastasis. Three mutations in
the ACTN4 gene encoding the actinin-4 protein are associated with dysfunctionof podocytes due to familial focal segmental glomerulosclerosis. We recently
identified a novel alternative splice-variant of ACTN4 (ACTN4-sp) that was
specifically expressed in high-grade neuroendocrine tumor of the lung (HGNT).
The expression of ACTN4-sp, which differs from ACTN4 by three amino acids,
was correlated with poor prognosis of patients with HGNT.
ACTN-4 consists of four spectrin repeats, a pair of C-terminal EF-hands, and an
actin-binding domain (ABD), which itself consists of a tandem pair of calponin
homology (CH) subdomains (CH1 and CH2). All of the mutations in threefamilial mutations and ACTN4-sp were restricted to the CH2 subdomain of the
ABD. Moreover, these mutations were located on the side of the helix, facing
the helix-helix interface between CH1 and CH2.
These findings suggest that the three-dimensional structures of the mutations
and ACTN4-sp differ from actinin-4, and that these changes were responsiblefor differences in the biological characteristics of the disease.
2PT118蛋白質濃度感受性蛍光蛋白質の開発

New fluorescent protein indicator for intracellular protein
concentration
Takamitsu Morikawa1, Katsumi Imada2, Keiko Yoshizawa3, Toshio
Yanagida1,3, Takeharu Nagai4, Tomonobu Watanabe1,3 (1Graduate School of
Frontier Biosciences, Osaka University, 2Department of Macromolecular
Science, Graduate School of Science, Osaka University, 3RIKEN Quantitative
Biology Center (QBiC), 4The Institute of Scientific and Industrial Research,
Osaka University)
Typical intracellular protein concentration of cell is about 350mg/mL, whichmakes the cell a very crowded environment. Recently, it was reported that suchmolecular crowding could alter enzyme activation. To observe the effects oflocal protein concentrations inside a cell, we developed a new protein
concentration indicator by using YFP.
YFP is sensitive to protein concentration such that an increasing protein
concentration causes its fluorescence intensity to decrease. To enhance thissensitivity, we inserted several glycine residues at beta-strand 7.
We next took advantage of CFP being insensitive protein concentration by
combining it and a YFP with one glycine insert (hereafter called YFP1G) via a
flexible linker. The ratio of the 480nm (CFP) and 525nm (YFP) fluorescentlight intensity depended on the protein concentration. Increasing the protein
concentration caused the fluorescence intensity of CFP to increase and that of
YFP1G to decrease. These results suggest our CFP-YFP1G chimera can act as aprotein concentration sensitive FRET probe.
2PT119Interactions of SicP with SptP subdomains in SicP-binding
domainYurie Kawashima, Fumio Hayashi, Kenji Oosawa (Department of Chemistry
and Chemical Biology, Graduate School of Engineering, Gunma University)
Many of Gram-negative bacteria cause infectious diseases. Some of the bacteria
construct nano-size of syringe-like structures and export virulence proteins“effector proteins” into host cells using the syringes. Each effector required themost appropriate chaperone for efficient export of the effector. Salmonella
effector protein SptP, which is involved in cytoskeletal remodeling of the host
cell, interacts with SptP-specific chaperone SicP at its N-terminal domain, andthe atomic structure of the complex (SicP-SptP1-158) is revealed. According
to the complex structure, SptP36-136 seems to be totally involved in SicPinteraction, and the two helices, SptP77-86 and SptP93-105, seem to interact with
SicP directly. Thus, the subdomains are responsible for SicP-SptP interactions.
To quantitatively reveal the effects of the subdomain on their interactions, we− S124 −prepared several truncated SptPs and SicP and examined whether the truncatedSptPs interact with SicP in vitro by Native-PAGE and western blotting as thefirst step.
We constructed pET3c-based plasmids expressing SptP1-136, SptP1-105, and
SptP1-86, and the three kinds of the truncated SptPs were individually prepared
under denaturing conditions. We also constructed pET3c-based SicP expression
plasmid and prepared SicP under native conditions. Thus, we prepared thetruncated SptPs and SicP individually, and then we examined whether thetruncated SptPs interact with SicP. In this meeting, we will show the results anddiscuss further efforts.
2PT120Purification and characterization of glycine-proline-fused typeof InvC involved in Salmonella type III secretion system
Aya Watanabe, Fumio Hayashi, Kenji Oosawa (Department of Chemistry andChemical Biology, Graduate School of Engineering, Gunma University)
Some of Gram-negative bacteria export virulence proteins “effector proteins”
into host cells using type III secretion systems (T3SS). Salmonella T3SS
consists of syringe-like structures, effector-specific chaperones, T3SS-specific
ATPase, and so on. InvC is the T3SS-specific ATPase in Salmonella, and theimportance of its ATPase activity is demonstrated by mutant analyses. To reveal
the detailed properties of InvC, InvC having a long extra amino acid sequence at
the N-terminal end had been examined. In this study, we aimed to prepare InvC
whose extra sequence was as short as possible. For this purpose, we used
pET50b as an expression vector, because a target protein is expressed as ahighly soluble His-Nus-tag fusion protein and the tag is removed by HRV3C
protease, which has highly sequence-specific endoprotease activity at 4 °C. We
cloned invC into the SmaI site by in-fusion method, and then transferred theplasmids into E.coli BL21(DE3) cells. The crude extract including His-NusInvC was prepared from the culture of the cells, and His-Nus-InvC was preparedusing Ni-NTA agarose column. After that, His-Nus-InvC was cleaved by
HRV3C protease. As the result of the sequential procedure, we prepared InvC
having two amino acid residues, glycine and proline, as the extra sequence.
Here, we termed the glycine-proline-fused type of InvC as GP-InvC. We applied
GP-InvC to several column chromatography steps for further purification andthen examined ATPase activities of GP-InvC under various conditions.

2PT121X 線 1 分子追跡法による II 型シャペロニン協同性評価

Cooperative Motion Analysis of group II chaperonin at Singlemolecule Level using Diffracted X-ray Tracking
Hiroshi Sekiguchi1,2, Ayumi Nakagawa4, Kazuki Moriya4, Mayuno Arita4,
Yohei Yamamoto4, Kouhei Ichiyanagi2,3, Masafumi Yohda4, Naoto Yagi1,
Yuji Sasaki3 (1Research Utilization Div., JASRI, 2JST CREST Sasaki
Team, 3Grad. School Frontier Sci., Univ. Tokyo, 4Dept. Biotech. Life Sci.,
Tokyo Univ. Agricult. Tech.)Group II chaperonin, found in archaea and in the eukaryotic cytosol, is anindispensable protein that captures an unfolded protein and refolds it to thecorrect conformation in an ATP dependent manner. ATP-induced structuralchanges are essential for chaperonin foldase activity; the closure and opening of
the chamber are the focus of group II chaperonin studies. We had previously
reported that the diffracted X-ray tracking method (DXT) could trace ATP
induced twisting motion of group II chaperonin ring at single molecule levelwith high accuracy. In DXT, gold nanocrystal immobilized on one side ofchaperonin-ring is used as tracer for structural change of chaperonin. The
timing, speed and direction of twising motion of chaperonin-ring after ATP
binding could be analyzed by DXT method in detail. In this study, we checked
how ATPase deficient mutant subunits modulate the speed or frequency of
twisting motion. We controlled the number of ATPase deficient mutant within
one chaperonin ring, constructed the ATP deficient hetero-ring using circularpermutated connected mutants, and evaluated the effects of those mutants tochaperoning-ring's twisting motion. We will discuss intra- and inter-ringcooperativity of group II chaperonin from such motion analysis view at single
molecule level.
2PT122

PAP の酵素活性制御因子としてのトリプトファン残基近傍のコンフォメーションとダイナミクス

The dynamics and conformation of Trp208 and Trp237 as
regulating factor of enzymatic activity of Pokeweed AntiviralProtein

Hiromichi Nakashima1, Yukihiro Fukunaga2, Keiichi Watanabe3, Shoji
Yamashita4, Etsuko Nishimoto4 (1Institute of Biophysics, Faculty ofAgriculture, Graduate School of Kyushu University, 2Interdisciplinary
Graduate School of Engineering Sciences Kyushu University, 3Department ofApplied Biological Sciences, Saga University, 4Molecular Biosciences,
Bioscience and Biotechnology, Kyushu University)
Pokeweed Antiviral Protein (PAP) belongs to a family of Ribosome Inactivating
Proteins (RIPs) and works as N-glycosidase to remove a specific adenine
residue from ribosomal RNA. We have focused on Trp208 andTrp237 uniquelyarranged in PAP as the activity regulating factor. Trp208 locates at the entrance
lid of active site and Trp237 is arranged at the C-terminal domain. We haveshown that enzymatic activity of PAP is dramatically affected by substitution of
Trp208 and Trp237. Additionally, changes of dynamics of Trp208 and the
distance between Trp237 and Tyr172 were observed when PAP bound with Nacetylglucosamine, and these changes induced the enhancement of enzymatic
activity of PAP. These results demonstrate that dynamics motilities andconformations of Trp208 and Trp237 play important roles in regulating PAP
activity. In the present study, we investigated in detail the correlation between
the conformation and dynamics near the active site, dynamics in the C-terminal
domain and the enzymatic activity of PAP based on the fluorescence
spectroscopic data. To analyze the conformational changes, we measured time
resolved anisotropy decay and FRET between Tyr and Trp by sub-pico second
laser based TCSPC method. Results suggest that Trp208 approached closer
nearest neighboring Tyr at the active site as temperature increasing. Then, theinternal rotational motion of Trp208 was not observed. Other properties on the
dynamics and conformation near the active site will be also discussed compared
the enzymatic activity of PAP.
2PT123

フォトンファクトリーの小角散乱ビームラインの現状

Current Status of Small-Angle X-ray Scattering Beamlines at
Photon Factory
Nobutaka Shimizu1, Noriyuki Igarashi1, Takeharu Mori1, Ohta Hiromasa2, Yasuko
Nagatani1, Takashi Kosuge1, Kenji Ito1 (1KEK, Photon Factory, 2Mitsubishi
Electric SC)
BL-6A and BL-10C at Photon Factory (PF) has been dedicated for small-angle
X-ray scattering (SAXS) experiment. BL-6A is a bending magnet beamline andwas moved from the previous BL-15A last summer. This beamline is optimized
for measuring samples of various forms, such as a solution, a fiber, and amembrane. CCD detectors (C7300, C4880, Hamamatsu photonics) have used
for the time-resolved measurement in a sub-second region. Moreover fast speed(200Hz framing rate) pixel-array detector PILATUS 300K (Dectris) will be
installed in this September. BL-10C is also bending magnet beamline and is
optimized for measuring a biological solution sample. The data in the broad
angle region can be measured with an imaging plate detector R-AXIS VII(Rigaku). We are upgrading BL-10C especially for high-throughput
measurement of Bio-SAXS experiment, and developed and installed an
automatic solution mixing machine which is a part of a solution-sample
changer. Construction of new BL-15A, which light source is a short-gap
undulator, is planned for SAXS filed, this will be opened from the autumn of2013. Moreover, the national project for structure life science research has been
started from this April for 5 years, and the SAXS beamlines, BL-10C and new
BL-15A will be opened to this project from April in 2013. In this presentation,
we will introduce not only the current status of these beamlines, the construction
plan of new BL-15A and the upgrade plan based on this project but our resultsof protein solution structure analysis.
2PT124

Legionella DotI and DotJ form a multimeric subcomplex
associated with the core complex of the Dot/Icm type IVB
secretion system
Tomoko Kubori1, Takuya Kuroda2, Katsumi Imada2, Hiroki Nagai1 (1RIMD,
Osaka Univ., 2Grad. Sch. Sci., Osaka Univ.)Type IV secretion systems (T4SSs) play a central role in the pathogenicity ofmany bacterial pathogens, including Agrobacterium tumefaciens, Helicobacter
pylori, and Legionella pneumophila. L. pneumophila utilizes a protein transportsystem categorized as a type IVB secretion system (T4BSS), which is asophisticated supramolecular complex composed of Dot/Icm proteins, totranslocate a large array of “effector proteins” into the cytosol of host cells. Due
to interact with host cellular systems, effector proteins play pivotal roles for theestablishment of infection. Composed of five component proteins, the corecomplex of the Legionella Dot/Icm T4BSS spans the bacterial inner and outer− S125 −membrane and is anticipated to form a protein-transport channel. However, themolecular structures of the core complex and potential peripheral subcomplexes
have not yet been revealed. We report here that two inner membrane spanning
proteins, DotI and DotJ, form a multimeric complex that associates with theinner-membrane components of the core complex. DotI and DotJ show a mutual
dependency to maintain their protein levels in bacteria. From these results andother evidence, we hypothesize that DotI and DotJ form a ring like structure thatassociates with the core complex to function as a transport conduit on the inner
membrane.
2PT125

基 質 及 び 阻 害 剤 に よ っ て 誘 起 さ れ る ABC ト ラ ン ス ポ ー タ ー

ABCB1 の構造・ダイナミクス変化
Dynamics and Structural Changes of ABCB1 transporter
induced by binding of substrates and inhibitors
Wei-Lin Hsu, Yurika Watanabe, Tadaomi Furuta, Minoru Sakurai (Center for
Biol. Res. & Inform., Tokyo Tech)
ATP-binding cassette (ABC) transporters form one of the largest protein
families. ABC transporters translocate a number of substrates across cellular
membrane by coupling with ATP hydrolysis. Several of these membraneproteins are clinically important in causing, as example, multidrug resistance tocytotoxic pharmaceuticals.
The crystal structures of some ABC transporters have recently been determined.
They reveal similar arrangements of the conserved ATP-hydrolyzing
nucleotide-binding domains (NBD), but various architectures of thetransmembrane domains (TMD), with the exception of common coupling
helices that are necessary for transmitting conformational changes. Despite thefact that a wealth of research has been conducted on these structures to resolve
the mechanistic problems about how ABC transporters couple with ATPhydrolysis in cytosolic domains to translocate substrates through thetransmembrane pore, the specific process about how substrates bind to theproteins and the resultant protein structural change still remains elusive.
Our present study focuses on the mechanism of protein structure changes
induced by binding of ATP, substrates or inhibitors in the mouse ABCB1
transporter with an inward-facing conformation. The structure fluctuation
simulated by the GROMACS package (version 4.0.7) shows the atomic-leveldescription of the protein structural changes. The result sheds light on themechanism about the drug translocation process of ABC transporter and themolecular design of inhibitors.
2PT126

核磁気共鳴法による DNA ミスマッチ修復蛋白質 MutL の解析

NMR study on DNA mismatch repair protein MutL
Ryota Mizushima1,2, Tomoyo Takai2, Young-Ho Lee2 (1Grad. Sch. Frontier
Biosciences, Osaka Univ., 2IPR, Osaka Univ.)
DNA mismatch repair (MMR) works in the last process of gene repair system,
enhancing the DNA replication fidelity up to 100 - 1000 folds. Familial nonpolyposis colon cancers called Lynch-Syndrome (LS) caused by mutations ofgenes coding MMR proteins occupies 2 - 5 % of total colon cancers. About 80% of those who hold such genetic mutations suffered from colon cancers in theirlife. I am currently engaged in studying one of the MMR proteins called MutLin terms of structural biology by the method of NMR and other biophysicaltechniques. MutL homologs can be divided into two groups: E coli. andEukaryotic MutL. The former does not possess endonuclease activity (MutH
does instead) and the latter does. In this research, I deal with the latter. MutL iscomprised of three parts: dimerized and endonuclease C-terminal domain(CTD), ATPase N-terminal domain transiently dimerized thorough ATPbinding and hydrolytic cleavage and the linker region connecting CTD and
NTD. Inter-domain configuration between CTD and NTD is thought to be
regulated thorough ATP hydrolysis cycle. Moreover, this configuration
dynamics regulates endonuclease activity of MutL and interaction with other
MMR proteins. No NMR study has been officially published on both E coli. andeukaryotic MutL so far. The purpose of this research is to elucidate theregulatory mechanisms of MtuL endonuclease activity through analyzing theinteraction between CTD and NTD at the residue level by means of NMR.
2PT127

選択的スプライシングによる新規タンパク質アイソフォームの機能・構造予測情報を得るためのツールの開発と公開

Development of a functional and structural annotation tool ofnovel protein isoforms produced by alternative splicing

Masafumi Shionyu1, Ken-ichi Takahashi1, Mitiko Go1,2 (1Fac. Bio-Sci.,
Nagahama Inst. Bio-Sci. Tech., 2ROIS)
Alternative splicing (AS) is thought to be a major mechanism for diversifying
transcriptome and proteome in metazoans. Especially, more than 90% of multiexon genes in human genome are shown to undergo AS using a next-generation
sequencing method. Although the accumulation of novel transcript data
produced by AS are accelerated by new experimental technologies, a large
portion of proteins encoded by the transcripts (termed "AS isoforms") is notknown in their 3D structures and functions. Therefore, information whether AS
events affect 3D structures and/or functional sites is expected to be useful forstudies of novel AS isoforms. For this purpose, we have developed a pipelinethat automatically annotates users' uploaded transcript sequences. Given atranscript sequence, the pipeline identifies regions altered by AS events(termed "AS regions") in a putative protein sequence encoded by the transcript,
and assigns functional information such as domains and inter-molecularinteraction sites obtained by the 3D structure information. Furthermore, thepipeline predicts the possible 3D structure of the AS isoform using structural
constraints of hydrophobic core residues mainly. Based on the pipeline, we havebeen developing a web tool, named AS-EAST (Alternative Splicing Effects
ASsessment Tools). Using our web tool, users can inquire whether their own
transcripts encode a novel AS isoform and infer the impact of AS on its
molecular function. AS-EAST is a freely available at http://as-alps.nagahama-ibio.ac.jp/ASEAST/.
2PT128EGFR 分子 C-末端の天然変性ドメインの 1 分子 FRET 時系列計測
Single-molecule FRET measurement of a intrinsicallydisordered C-tail domain of an epidermal growth factor receptor

Kenji Okamoto, Yasushi Sako (RIKEN)
Structural dynamics of biomolecules, especially proteins, has been known
to have indispensable roles in living systems. While conventional X-ray
crystallography or NMR is powerful tools to investigate a molecular structure,single-molecule FRET (smFRET) measurement has advantages in observing its
dynamics. smFRET can investigate structures of, for example, the intrinsicallydisordered proteins (IDP) and its time resolution enables realtime observation of
structural changes. Epidermal growth factor receptor (EGFR), which is amembrane protein and triggers cellular signal transduction pathways, has theintrinsically disordered (ID) C-tail domain in its intracellular part. The C-taildomain has several phosphorylation sites, one of which binds a protein Grb2
after phosphorylation. Years ago, Grb2-binding kinetics was found to be
nonlinear system and it has been suggested that structural dynamics of this ID
domain plays an important role. We introduce smFRET measurement of EGFR
C-tail domain using recently developed apparatus and statistical data analysis,
which is based on the hidden Markov model and the variational Bayes. Time
series of 2ch fluorescence signals are obtained from single EGFR C-tailmolecules immobilized in vitro. Obtained data are analyzed to reconstruct thestate transition trajectory (STT) while the number of states is also estimatedfrom data. Further analysis on those STTs gives us details of dynamics, such as
possible paths or rates of state transitions.
2PT129タンパク質の変性及びクロスベータ転移に対する浸透圧効果

Effect of osmotic pressure on protein unfolding and cross-betatransition
Kazuki Takeuchi, Mitsuhiro Hirai (Grad. Sch. Eng., Gunma Univ.)[Introduction]
Proteins are designed to function in crowded environments. However, how aprotein folds into its native structure under crowed macromolecular condition isstill ambiguous. To clarify the crowding effect on the thermal stability ofproteins, we carried out synchrotron radiation wide-angle and small-angle Xray scattering (WAXS, SAXS) experiments. We employed so-called "osmotic
stress" method by using a high molecular weight neutral polymer (osmolyte) insolutions.
[Experimental]
Polyvinylpyrrolidone (PVP) with Mt. 40,000 was used as an osmolyte. The
proteins were myoglobin (MY) from horse skeletal muscle and hemoglobin(HM) form bovine blood. The proteins were dissolved in 10 mM HEPES buffers
at pH 7.4 with different PVP concentration (0 % to 25 % (w/v)). WAXS andSAXS measurements were performed both at SPring-8 and PF. The temperatureof the samples were controlled from 25°C to 85°C.
[Results]
The radius of gyration of the protein at 25 °C showed a minimum at a low PVP− S126 −concentration (~15 % for MY; 5 % for HM), which is similar to that oflysozyme [1], where the osmotic pressure induces change of hydration shell.
Both proteins show cross-beta transition by the elevation of temperature. The
presence of PVP descends the transition temperature of the whole structure,suggesting the destabilization of the native structures. The cross-beta transition
was also influenced by PVP addition. The detailed results and discussion will begiven.
[Reference]
1. M. Hirai, K. Takeuchi, et al., Thermochimica Acta 532 (2012) 15.
2PT130

Motion Tree 法で解析された SERCA の大規模な構造変化

Large-scale conformational changes of SERCA analyzed by
Motion Tree method
Chigusa Kobayashi1, Ryotaro Koike2, Motonori Ota2, Yuji Sugita1,3,4(1RIKEN, ASI, Theoretical molecular science, 2Graduation school for
information science, Nagoya University, 3RIKEN, AICS, 4RIKEN, QBiC)
Sarcoplasmic reticulum Ca2+-ATPase (SERCA) is structurally and functionallythe best studied member of P-type ATPases. To uptake Ca2+ from the cytoplasm
to sarcoplasmic reticulum lumen against a large concentration gradient, SERCA
undertakes rearrangements of the transmembrane helices coupled with largescale cytoplasmic domain movements. Although atomic structures of SERCA in
different physiological states have been determined by X-ray crystallography,
the differences between two crystal structures are significantly large, leaving themolecular mechanisms underlying the structural transitions elusive. In thisstudy, we develop an approach to analyze such large-scale conformationalchanges of SERCA or other proteins, based on the motion tree algorithm
originally developed by Koike et al. (manuscript in preparation). Using themethod, we characterize structural transition pathways computed by Climber, amorphing software, and 100ns molecular dynamics (MD) trajectories startingfrom E2 and E2.Pi states. We conclude that the method is useful to observe
intermediate structures for describing large-scale conformational transitions inmore detail.
2PT131

H-ras GTP 複合体と H-ras GDP 複合体の分子動力学シミュレー
ションにおける水分子の解析

Analysis of water molecules in molecular dynamics simulationsof H-ras GTP and GDP complexes
Takeshi Miyakawa1, Ryota Morikawa1, Masako Takasu1, Kimikazu
Sugimori2, Kazutomo Kawaguchi3, Hiroaki Saito3, Hidemi Nagao3 (1Sch. Life
Sci., Tokyo Univ. Pharm. and Life Sci., 2Dept. Phys. Therapy, Faculty HealthSci., Kinjo Univ., 3Inst. Sci. and Eng., Kanazawa Univ.)
We study the structures of H-ras GTP complex and H-ras GDP complex insolution by molecular dynamics (MD) simulations in order to understand thehydrolysis of the GTP to GDP in the H-ras complex, which play a key role inovercoming human cancer. We have derived the potentials and the atomic
charges around Mg2+ in H-ras GTP complex and H-ras GDP complex byquantum chemical calculations using hybrid B3LYP functional with 631G** basis set. We performed MD simulations using potential parameters of
AMBER03 and our parameters around Mg2+. And we analyzed the properties ofwater molecules around GTP and GDP in each complex. Our analysis showsthat the diffusion of water molecules around GTP and GDP is nearly equal to the
diffusion of ordinary water molecules. The distance of the closest watermolecule from one of P or O atoms in GTP or GDP depends on the species ofthis atom in GTP or GDP. The probabilities that the water closest to one of P or
O atoms in guanine nucleotide is the same water closest to another P or O atom
in guanine nucleotide have large value for bonded P-O pairs. We also found thatthe probabilities in GTP are higher than the probabilities in GDP. This implies
that these high probabilities in GTP make the water molecule come to theappropriate position to activate GTP hydrolysis in H-ras-GTP complex.
2PT132

T4 ゲノムパッケージングモーターの分子ダイナミクス

Molecular Dynamics of T4 Genome Packaing Motor
Kazuhiro Takemura1, Akio Kitao1,2 (1IMCB, Univ. of Tokyo, 2JST,CREST)
Almost all bacteriophages have a process of packaging genome into vacant
procapsids. The packaging of DNA is powered by ATP hydrolysis and the
packaging motor. T4 genome packaging motor consists of gp20, gp17, andgp16. The cryo-EM reconstruction study reported that gp17 forms pentamer andthat the X-ray crystal structure of monomeric gp17 could not be fitted into cryoEM image which is expected to be related to its function. Molecular dynamics

simulations were performed to model the atomic structure of pentameric gp17
and to investigate the mechanism of the DNA packaging. Gp17 is composed of
N-terminal (1-360) and C-terminal (361-610) domains. A domain motion was
detected as the first mode of the principal component analysis for the gp17
monomer in solution which contributes 50 percent of its molecular fluctuation.
The observed domain motion might be related to its function and the structuraldifference between the cryo-EM image and the X-ray crystal structure. To
model the gp17 structure close to the EM image from the X-ray crystal
structure, we used the recently developed sampling method, PArallel Cascade
Sampling MD (PACS-MD, R. Harada and A. Kitao in preparation) in which
the snapshots close to the target structure are selected and velocities are
redistributed. Using PACS-MD, we successfully obtained the structure ofmonomeric gp17 which is close to the structure of EM image. Starting from theobtained structure of monomeric gp17, we are currently modeling the atomic
structure of pentameric gp17 using PACS MD.
2PT133タンパク質へのリガンド結合過程の粗視化シミュレーション：タン
パク質の立体構造変化の影響の解析

Coarse-grained MD simulations of ligand binding to proteins:
Effect of the conformational changes of the proteins
Tatsuki Negami1, Tohru Terada2, Kentaro Shimizu1,2 (1Department of
Biotechnology, Graduate School of Agricultural and Life Sciences, The
University of Tokyo, 2Agricultural Bioinformatics Research Unit, Graduate
School of Agricultutal and Life Sciences, The University of Tokyo)
Clarifying the mechanism of protein-ligand interactions is one of the mostimportant research subjects in the field of biophysics. However, most of theresearch efforts have been devoted to predicting the docked structure. The
process of ligand binding remains to be clarified. We have demonstrated thatcoarse grained simulations with the MARTINI force field can reproduce theprocesses of ligand binding to proteins. However, this method is not directly
applicable to proteins that change their conformations upon binding to ligands
because elastic network is used to conserve protein structures in the simulation
with the MARTINI force field. Since conformational changes of proteins often
occur upon ligand binding, it is necessary to extend the method to enable theconformational change of a protein in the coarse grained simulation. Here, we
combined a double-well network model with the MARTINI force field for thispurpose. First, we constructed Elnedyn elastic network potentials for the ligandfree and ligand-bound structures, and then connected them smoothly to make adouble-well potential. We observed frequent conformational transitions
between the two states during coarse grained simulations with the double-wellpotential for several proteins. Furthermore, we performed a few-microsecond
simulation randomly arranging ligand molecules around these proteins. We
found that the ligands correctly bound to the ligand-binding sites. We will
discuss the coupling between the conformational change and the ligand binding
affinity.
2PT134

分子動力学シミュレーションによるリジン 48 結合型ジユビキチンのコンパクト構造の解析

Molecular dynamics simulation study of Lys48-linked
diubiquitin in compact conformation
Sotaro Fuchigami1, Mitsunori Ikeguchi1, Akinori Kidera1,2 (1Grad. Sch. of
Nanobioscience, Yokohama City Univ., 2CSRP, RIKEN)
Protein ubiquitination is a post-translational modification involved in regulationof diverse cellular processes, including protein degradation, cell signaling, DNAdamage response and transport processes. Ubiquitin can be conjugated not only
to a target protein but also to another ubiquitin via one of its seven lysine
residues, resulting in formation of polyubiquitin chains with diverse types oflinkages. The most examined is Lys48-linked polyubiquitin chain, which acts asa tag for protein degradation by 26S proteasome. Several studies have shownthat Lys48-linked diubiquitin is in the compact conformation, in which thehydrophobic patches of both ubiquitins interact with each other. It is consideredthat the Lys48-linked diubiquitin changes its conformation to interact with its
receptors, whereas its mechanism is not well understood. In the present study,
we performed molecular dynamics simulation of the Lys48-linked diubiquitin in
the compact conformation and investigated its conformation stability. More than
one microsecond simulations revealed that the compact conformation was fairly
stable even though interaction between the hydrophobic patches was partiallydisrupted by local conformational changes of ubiquitins. We attributed the highstability of the diubiquitin to main-chain hydrogen bonds between ubiquitinsand confirmed this by mutant simulations.

− S127 −2PT135

粗視化モデルによる PPAR γの基質依存的な活性変化の考察

Coarse-grained molecular dynamics study of liganddependent reaction activity of PPARγ
Akinori Awazu (Dept. of Mathematical and Life Sciences, Hiroshima
University)
Peroxisome proliferator-activated receptors  (PPAR) is one of the nuclear
receptors of transcription factors controlling genes implicated in energy,
carbohydrate, and lipid metabolism. Recent experimental studies reported that
PPAR can bind with several ligands and show its activity. These ligands areclassified by Full agonist and Partial agonist depend on the activity of thetranscriptional response, where the binding with Full agonists induces muchhigher molecular activity than that with Partial agonists. On the other hand, therecent X-ray crystal structure analysis implies the structure of PPAR is
obtained almost the same independent of the types of agonists. This fact seems
the difference of transcriptional activity is originated by the difference ofmolecular motion and the motion inducing effective molecular structure, which
are encoded in the mechanical network consists of amino acids and the binding
ligand.
In the present study, we perform the Brownian dynamics simulation of thecoarse-grained model of PPAR to unveil the biding ligand shape dependencyof the molecular dynamical properties. Through the statistical analysis of theresults of GO-like model simulation, we extract the ligand-dependent effective
interaction network and compare the contribution of these networks to themolecular function.
2PT136

多剤排出トランスポーター AcrB の全原子分子動力学シミュレー
ションによる解析

All-atom molecular dynamics simulation of multidrug transporter AcrB
Tsutomu Yamane, Mitsunori Ikeguchi (Grad. Sch. Bio-nano sci. Yokohama
City Univ.)
In E. coli, the multi drag transporter AcrB resides in the inner membrane region.
Recently, x-ray structures provided that AcrB forms trimeric protein where each
protomer is different conformation, “binding”, “extrusion” and “access” state,
which are different from the shape of drug binding region (porter domain). In
addition, extrusion state protomer has the unique side chain conformations ofresidues which are essential for the proton transfer, “proton translation site”(Asp407, Asp408 and Lys940). These results suggest that AcrB exported drugs
outside of the cell by a three-step structural change involved in the proton
motive force which related with the conformational change in protonation sites.
In the present study, we performed all-atom molecular dynamics (MD)simulations of AcrB-membrane-water system. Our simulations with thedifferent protonation states of the titratable residues in the proton translation site
revealed the correspondence between the specific protonation states and theside-chain configurations observed in the crystal structure. Changing theprotonation state of one aspartate induced a spontaneous structural transition,suggesting that the stoichiometry of proton translocation might be one proton
per step of the functional rotation. Furthermore, our simulations demonstratethat alternating the protonation states in the transmembrane domain induces
functional rotation in the porter domain that is primarily responsible for drugtransport.
2PT137

キサンチン酸化還元酵素とリガンド複合体に関する分子動力学計算と結合自由エネルギー解析

Molecular dynamics simulations and binding free energy
analysis of xanthine oxidoreductase-ligand complexes
Hiroto Kikuchi1, Hiroshi Fujisaki1,3, Tadaomi Furuta2, Ken Okamoto4,
Takeshi Nishino5 (1Dept. of Phys., Nippon Med. Sch., 2Grad. Sch. of Biosci. &Biotech., Tokyo Inst. of Tech., 3Wako, Riken, 4Dept. of Biochem., Nippon Med.
Sch., 5Grad. Sch. of Agri. & Life Sci., Univ. Tokyo)
Xanthine oxidoreductase (XOR) is an enzyme which is found in a wide range oforganisms from bacteria to man and plays an important role in the catabolism of
purine substrates. In mammals and birds, XOR physiologically catalyzes thehydroxylation of hypoxanthine to xanthine, followed by the catalysis of thehydroxylation of xanthine to uric acid. As the excessive production of uric acidleads to gout and hyperuricemia, the inhibitors for XOR can be effective as thedrugs for these diseases. Febuxostat, which fills up most of the cavity of

mammalian XOR without being covalently bound to it, has been developed as a
non-purine selective inhibitor of XOR, showing a more potent and longerlasting urate-lowering effect than allopurinol which is another XOR inhibitor,
widely prescribed as a treatment of gout and hyperuricemia for more than 40
years. Clinical efficacy and tolerance to febuxostat have been confirmed, andthe drug is available as Adenuric (EU), Uloric (US), or Feburic (Japan). In ourprevious work [H. Kikuchi et al., Sci. Rep. 2, 331 (2012)], we studied thebinding interaction between febuxostat and two (mammalian and bacterial)
XORs, and clarified the role of the molecular details of the drug-enzyme
interaction using both molecular dynamics simulations and enzymologicalexperiments. In the present work, we further investigate the binding mechanism
using free energy calculations, employing different types of drugs and mutated
XORs.
2PT138Features of Amino Acids Changes Determining the Glycan
Specificities of Influenza Hemagglutinins: Interaction Energy
ProfilesKatsumi Omagari (Nagoya City University)
The interaction between hemagglutinin (HA) and glycan receptors exposed on
the host cell is a kernel in the study of host adaptation of influenza A viruses.
Receptor binding, the initial event in virus infections, is major determinant ofvirus transmissibility. HA's of avian viruses prefer sialic acid in 2,3-linkage togalactose, the avian receptor, while those of human viruses prefer sialic acid in
2,6-linkage, the human receptor. A conversion to human receptor specificity is
believed to be one of the changes required for the introduction of new HAsubtypes to the human population, which can lead to pandemics. Extensive
crystal structural studies have revealed that the human or avian receptors adopt avery similar conformation in both human and avian HA-receptor complexes.
Those structures revealed no preferential binding of avian receptor over that ofhuman receptor. In this poster, to understand the detailed interaction profile ofthe HA-receptor complex, I will describe why/how HA's recognize two type ofreceptor through those HA's bind receptors in similar fashion, using molecular
dynamics and MM-GBSA/PBSA methods. The examined systems comprise
explicitly solvated form of three H1 human virus HA's, avian and swine H1 HA
in complex with avian and human receptors, respectively. Effects ofsubstitutions at key positions on the HA and their possible contribution to
specificities were investigated too. Those results suggested the differences ofinteraction energy profiles between HA's in spite of similar binding
conformation.
2PT139筋小胞体カルシウムポンプの ATP 結合状態の分子動力学計算

Molecular dynamics simulations of SR Ca2+-pump in the ATPbound forms
Yasuaki Komuro1,2, Chigusa Kobayashi2, Eiro Muneyuki1, Yuji Sugita2,3,4(1Grad. Dept. Phys., Chuo Univ., 2RIKEN ASI, 3RIKEN QBiC, 4RIKEN AICS)
Sarcoplasmic reticulum (SR) Ca2+-pump is a P-type ATPase that transports
two Ca2+ ion across the SR membrane against a large concentration gradient,utilizing chemical energy released in ATP hydrolysis. This protein consists often transmembrane helices as well as three large cytoplasmic domains: A(actuator), N (nucleotide-binding), and P (phosphorylation) domains. In an
enzymatic reaction cycle, SR Ca2+-pump rearranges six out of tentransmembrane helices and thereby changes the affinity for Ca2+ at the bindingsites. In classical E1/E2 theory, the E1 state is defined as the high affinity state
for Ca2+, whereas the affinity is low in the E2 state. In this study, we focus on
the ATP or ADP bound states (E1ATP and E1PADP) for the SR Ca2+-pumpand perform atomistic molecular dynamics (MD) simulations with explicit
solvent and DOPC lipid bilayer, starting the X-ray crystal structures. Although
the X-ray structure for the ATP bound form is almost the same as that for the
ADP bound form, the differences of physiological conditions between these twostates have been pointed out. By performing the MD simulations, we investigate
the global conformational flexibilities of the pump as well as the Ca2+-binding
affinities at the transmembrane binding sites.
2PT140Computational design of small peptide inhibotrs of proteinprotein interactions in intracellular signaling

Junya Yamagishi1,2, Noriaki Okimoto2, Takuma Kasai3, Mariko Okada3, Akira
Imamoto4, Makoto Taiji1,2 (1Grad. Sch. of Fron. Sci., Univ. of Tokyo, 2QBiC,
RIKEN, 3Yokohama Inst., RIKEN, 4Ben May Dept., Chicago Univ.)
A CRK SH2 domain preferentially binds to peptides having the pYXXP motif− S128 −(where pY is a phosphotyrosine and X is any amino acids). CRK have been
considered to be indispensable for malignant features of human cancers, so that
it is important to develop novel inhibitors of CRK mediated protein-protein
interactions for new cancer therapies. In this work, we performed dockingbased screening of small peptides against the CRK SH2.
Conventional docking programs have been developed to design small moleculesand cannot be applied for peptide design because of their following
characteristics. (1) Peptides have many rotatable bonds, which makes difficult
to predict binding mode in a peptide-protein complex. (2) Peptides also havemore polar functional moieties than small molecules and no proper scoring
functions are available for peptide docking. Rescoring of docking poses using
MM (molecular mechanics) is a useful approach to estimate binding affinities of
peptides to their target protein. But the problem of inaccuracy of docking poses
still remains.
In this work, we developed our docking program using MM and the generalizedborn (GB) solvent. For large computational requirements of MM and GB, we
accelerated our program using GPU. After docking, a MM-PBSA rescoring was
conducted to estimate binding affinities. Furthermore, we performed additional
conformational search of peptides in unbound states to take account of ligand's
reorganization effects. Our methods showed high performances indiscrimination of known binding sequences.
2PT141タンパク質（DHFR）における局所変異が引き起こす運動性の変化に関する理論的考察

Theoretical study of change of motility by local mutation in
DHFRTomo Matsubara, Masashi Fujii, Hiraku Nishimori, Akinori Awazu (Dept. ofmath and Life Sci, Hiroshima Univ)
The dihydrofolate reductase (DHFR) is composed of some secondary structuresand fluctuating loops called Met20 loop, CD loop, FG loop, GH loop. The
recent experimental study indicates that the dynamical properties encoded bysuch molecular structure provide the dominant contribution to the function of
DHFR, where a small mutation of CD loop which does not give structual
variation of the molecule and far from the reaction site (around Met20 loop)
gives a drastic change in intra-molecular global motion and its reaction activity.
In the present study, we perform the normal mode analysis of an all-atom modelof DHFR and discuss the general features of the relations among the local andglobal motions of such molecules.
We employ the elastic network model as all-atom model of protein dynamics.In
this model, each atom is described as beads and each pair of beads connected byvirtual bonds with the natural length that equals to the native distance between
the corresponding atoms. We obtained the atomic coordinates from Protein Data
Bank (PDB code:1rx2, 1rx6) to construct the structure of DHFR. By the normal
mode analysis of this model, we investigate the correlation of motility among
characteristic local structures and discuss the mechanism of the suppression of
the reaction activity by the minor local mutation observed in the experiment.
2PT142

Theoretical evaluation of structural stability of the active siteof T1 lipase: cation-π vs. water-π interactions

Atsushi Nakamura, Jiyoung Kang, Masaru Tateno (Grad. Sch. Sci., Univ.
Hyogo)
Crystallographic data obtained by Matsumura, H., et al. (Osaka Univ.)suggested that the sphere electron density that was found in the active site of T1
lipase is corresponding to the interaction of Na+ and the aromatic ring of a Phe
residue (i.e., cation-π interaction). However, it was also augured that a watermolecule can interact with π electrons (i. e., water-π interaction). Thus,
experimental approaches could not dismiss the possible presence of waterinstead of Na+ in the active site of T1 lipase. We theoretically evaluated these
two types of interactions, employing molecular dynamics (MD) simulations.
The current force fields cannot estimate the cation-π interaction correctly,
requiring ab initio quantum mechanical calculations. So, we developed a novel
efficient scheme to calculate the potential energy of cation-π interaction, withthe high accuracy at the CCSD(T) level and the low calculation cost comparable
to the standard force field. Our MD calculations showed that a large enthalpy
gain of the Na + -π interaction can preserve the catalytic core structure. In
contrast, water-π interaction seriously induced the structural instability. Thus,
the possible presence of water may be excluded [1-2]. Our novel scheme iscurrently the only way to perform long-time MD simulations involving cation-πinteraction, with reasonable calculation cost. In the session, the functional rolesof cation-π interactions in T1 lipase will be discussed.
[1] JCTC 7 (2011) 2593
[2] JACS 132 (2010) 2751

2PT143

分子動力学シミュレーションによる緑色蛍光タンパク質の切断位置と蛍光回復の研究

Research of Split Point and Fluorescent Recovery of Green
Fluorescent Protein by Molecular Dynamics Simulation
Mashiho Ito1,2, Shoji Takada2, Takeaki Ozawa1 (1Dep. Chem., Sch. Sci., Univ.
Tokyo, 2Dep. Bio., Grad. Sch. Sci., Kyoto Univ.)
Split Green Fluorescent Protein (Split GFP) is a useful tool for investigating
protein-protein interaction in vivo. Each fragment of Split GFP are reconstructchromophore by binding and recover fluorescence. However, less is known
about the mechanism of folding and reconstructing of Split GFP. In particular,
the reason why fluorescence depends on the position of split point. To reveal
this, we performed molecular dynamics simulation of Split GFP, Coarse-Grain
simulation and All-Atom simulation. From Coarse-Grain simulation of Split
GFP, here we report three points about Split GFP Folding. First, N-terminal
fragment in Split GFP is folding earlier than C-terminal at all events. Second,
the folding of C-terminal fragment and whole GFP are at the same time. Finally,
the stability of N-terminal fragment is related to the speed of folding of whole
Split GFP. From these data we devised a model of Folding of Split GFP that firstN-terminal fragment becomes core structure of GFP, and then it will covered by
C-terminal fragment. From All-Atom simulation, we evaluate the stability of
Split GFP of each split point and show the difference of split point in loop
regions or -strand regions. These results provide a new perspective on split
protein folding.
2PT144SlyD 融合による人工三本鎖ベータへリックスの可溶化の試み

Solubilization approaches of artificial three-stranded betahelix by fusing SlyD protein
Kaname Nishijo1, Shuji Kanamaru2, Fumio Arisaka1 (1Dept of LifeScience,
Grad Sch of Biosci&Bioeng, Tokyo Institute of Technology, 2Dept of
Bioengineering, Grad Sch of Biosci&Bioeng, Tokyo Institute of Technology)
The C-terminal domain of gp5 of bacteriophage T4 has a unique three-stranded
parallel beta-helix and forms a triangular prism. The amino acid sequence of thisdomain has the characteristic 12 repeats of 8 amino acids VXGXXXXX. We
aim to create artificial triple beta-helix proteins whose length is controlled bytandemly connecting the building block of 6 repeats of VXGXXXXX((VXG)6).
In our previous studies of tandemly connected (VXG)6 by genetic engineering,
we were able to create and purify the artificial triple beta-helix which was
expected to consist of one or two building blocks. However, large amounts oflarger oligomers as well as the dimer of beta-helix were formed. Those larger
oligomers are formed by association the artificial three-stranded beta-helix.
In order to avoid the dimer formation of the beta-helix, we fused HisSlyD at the
N-terminus of the three-stranded beta-helix. SlyD is known to have the ability tosolubilize fused proteins. Sedimentation velocity analysis of the purified
proteins indicated that the main product was still the dimer of the beta-helix. In
addition, size exclusion chromatography of the products indicated the presenceof partially unfolded beta-helices. Thus we concluded that the dimerization of
the beta-helix spontaneously occurred at the N-terminus of the beta-helices
which is partially unfolded.
2PT145

Fluorescence Titration for finding the binding sites of Peptide
Aptamers on Calmodulin
Yasodha Manandhar, Wei Wang, Takanori Uzawa, Yoshihiro Ito, Toshiro
Aigaki (RIKEN, Advanced Science Institute)
Aptamers are in vitro selected nucleic acids or peptides which specifically bindto various target molecules. Although aptamers modified with a fluorescentmolecule are often used for diagnoses to signal out its target binding, suchmodifications sometimes interfere the specific binding. To avoid this wepreviously selected signaling peptide aptamers from a library of random
sequenced peptides including a non-natural fluorogenic amino acid, 7-Nitro-2,1,
3-benzoxadiazole modified aminophenylalanine (NBD-aa). We choose amultifunctional intermediate messenger protein, calmodulin (CaM), as a target.
CaM transduces calcium signals by binding calcium ions on its two
approximately symmetrical globular C and N domains. We found that one of theselected peptides, C5 (YWDKIKD*IGG; *NBD-aa) has a strong binding
affinity (Kd= 2) with the C domain of CaM. However the binding sites of theother selected peptides have not been clarified yet. Thus motivated here we
investigated the binding sites of those peptides from the titrations with the CaM-

− S129 −C5 complex.
The titration of B4’ with CaM-C5 exhibited signal increase in a similar way as
the titration with CaM, implying that the primary binding site of this peptide is
N domain of CaM. In contrast the titration of other peptides with CaM-C5 did
not exhibited significant signal change, implying that the primary binding site ofthose peptides are C domain of CaM where C5 occupies already. We further
examine the B4’ binding site by employing NMR.
2PT146

Characterization of norovirus RNA replicase in vitro for theautonomous protein evolution using in vitro virus

Hidenao Arai1, Manish Biyani2, Yuzuru Husimi3 (1Grad. Sch. Sci. Eng.,
Saitama Univ., 2Inst. of Engineering Innovation, Grad. Sch. Eng., Univ. ofTokyo, 3Innovation Research Organization, Saitama Univ.)
We have been trying to construct an autonomous protein evolution system,
combining the in vitro virus (IVV) and the one-pot isothermal RNA replication
reaction. In our system, the norovirus RNA replicase (3Dpol) is planned to be
displayed on the IVV.
In this work, we evaluated the activity of 3Dpol, which was synthesized in cellfree protein synthesis system. As indicated in the previous report, when a ss
(single-stranded) RNA was incubated with 3Dpol, it was replicated to a ds
(double-stranded) RNA in a primer-independent manner. And the isothermal
amplification of the dsRNA by eight-fold was realized. In the primerindependent replication reaction, the presence of C-stretch at the 3’-terminus
increased the replication efficiency by six-fold. Contrary to the previous report,
3Dpol also replicated RNA in a primer-dependent manner and a self-primed(small stem-loop) manner. Moreover, the initiation of replication was severely
dependent on the 3’-terminal structure of RNA, and especially, 3Dpol preferred
the very small self-primed structure than the single- or double-stranded
structure. And the initiation form may be dependent on the divalent ions (Mg2+or Mn2+). From these results, we were able to draw a scheme of the in vitro
primer-independent RNA replication by 3Dpol.
Using the IVV displaying 3Dpol, we designed the one-pot isothermal replication
reaction system, in which the IVV can amplify itself, and evolve autonomously.
This reaction system will evolve not only the RNA replicase but also theamplification algorithm itself.
2PT147In vitro selection of a protease using cDNA display
Shingo Ueno1,3, Yuka Mashio2,3, Takanori Ichiki1,3, Naoto Nemoto2,3 (1Grad.
Sch. Eng., Univ. Tokyo, 2Grad. Sch. Sci. and Eng., Saitama Univ., 3CREST, JST)The artificial evolution of enzymes is one of the ultimate goals of evolutionary
molecular engineering. In vitro display technologies, such as phage display,ribosome display, mRNA display and cDNA display have been widely used foraffinity selection against various targets in the last two decades1. Conversely,
although some groups have been able to evolve enzymes using phage display2,
there are only a few examples describing mRNA display3 and ribosome
display4.
Recently, we have started the challenge to evolve enzymes in vitro using cDNA
display, which is more stable and easy-to-use than mRNA display5. We
designed a selection system with a new puromycin linker of cDNA display forin vitro selection of a protease. The linker was designed to release the cDNA
coding the gene of a displayed protease, from a solid-phase support when apeptide substrate was cleaved by the displayed protease.
In this study, we demonstrated a model selection of subtilisin, one of wellidentified protease. We will discuss the model screening experiments andpresent some results.
References
1. Ullman CG, Frigotto L, Cooley RN (2011) Brief Funct Genomics 10: 125134.
2. Fernandez-Gacio A, Uguen M, Fastrez J (2003) Trends Biotechnol 21: 408414.
3. Seelig B, Szostak JW (2007) Nature 448: 828-831.
4. Yanagida H, Matsuura T, Kazuta Y, Yomo T (2011) Chembiochem 12: 962969.
5. Yamaguchi J, et al. (2009) Nucl Acids Res 37: e108.

Hinako Nakada, Nobuya Itoh, Yoshihide Makino (Grad. Sch. Eng., Toyama
Pref. Univ.)
Proteins have ability to bind other molecule specifically. The creation of
artificial specificity of proteins has been studied. The proteins developed bythese studies are expected to be applied to pharmaceuticals or drug carriers. Our
objective is to create the library mutant human retinoic acid binding protein 2(HCRABP2), and to screen novel binding ability to vascular endothelial growth
factor (VEGF). Sixteen amino acid mutations were introduced into HCRABP2
at the side surface of beta-barrel. Phage display library of the mutants wereconstructed. Display phage clones were selected through three rounds of affinity
panning. The affinity of concentrated display phages was analyzed; and thepositive signals were obtained by ELISA analysis. This result suggests existenceof clones which might bind to VEGF. Detailed analysis of VEGF-binding
affinity and sequence of mutant clones is in progress.
2PT149Microwave has shown its various features in various chemical reactions, thetime shortening, the reduction of reaction temperature, and so on. The
microwave effect can be also applied to biocatalysts and enzyme reactions. We
have studied to take advantage of the microwave heating to the field of chemical
biology and biotechnology. Among them, the polymerase chain reaction (PCR)
for gene amplification under microwave irradiation was obtained as a uniqueresult. In PCR method, the three processes are repeated, (1) the thermal
denaturation to the single-stranded DNA from the double strand DNA, (2) the
hybridization by annealing between the primer and the single-strand DNA, (3)the enzymatic DNA elongation from the primer by DNA polymerase. So that,
we demonstrated the microwave heating for the processes of the thermal
denaturation and the enzymatic elongation reaction. The Vent DNA polymerase
and ExTaq DNA polymerase were used in this study. The ExTaq polymerase
was decreased the fluorescence intensity by the microwave irradiation. On theother hand, the Vent polymerase was shown same intensity of fluorescence
between the microwave irradiation and the conventional condition. The ExTaq
polymerase showed the different phenomena between the microwave irradiationand the conventional heating in spite of same temperature. It is seems that theprotein structure is changed under the microwave irradiation. This fact mightmean a new state of protein denaturation, what we would call “Microwave
Denaturation”.
2PT150モルテングロビュール状態の蛋白質を利用した新規抗腫瘍複合体の作製

Development of novel anti-tumor complexes made from aprotein in the molten globule state
Takashi Nakamura1,2, Ryusho Kariya3, Seiji Okada3, Kunihiro Kuwajima1,2,4(1Okazaki Inst. Integr. Biosci., 2Inst. Mol. Sci., 3Center for AIDS Reseach,
Kumamoto Univ., 4Dept. of Funct. Mol. Sci., Grad. Univ. Adv. Studies)
Human -lactalbumin (-LA)-oleic acid complex, named HAMLET (humanalpha-lactalbumin made lethal to tumor cells), induces apoptosis-like death oftumor cells, and -LA in the HAMLET is in a molten globule (MG) -like
conformation. Our previous studies have suggested that -LA in the anti-tumor
complex may act as a carrier and that oleic acid in the complex may act as an
apoptosis inducer in tumor cells. We have thus asuumed that -LA in the MG
state can be used as a delivery carrier of anti-cancer drugs to tumor cells. To
investigate this possibility, first, we attempted to make complexes between LA and various anti-cancer drugs. As a results, certain anti-cancer drugs form acomplex with -LA in the MG state at pH 2.0, and these complexes were stable
under physiological conditions (pH 7.4). Next, we tested cell toxicity of thesecomplexes against various cells. Interestingly, these -LA-anti-cancer drug
complexes exhibited stronger toxicity against tumor cells, but weaker toxicity
against normal fibroblast cells than the free drugs. This finding demonstrates
that -LA in the MG state may be a potential drug carrier for cancer therapy.
2PT151

2PT148マイクロ波照射下での PCR 反応の DNA ポリメラーゼの特異性

The DNA Polymerase Specificity on Microwave Assisted PCR
Hiroya Osoegawa1, Seiji Higa1, Takaya Takei1, Kaori Tanaka1, Takeo
Yoshimura2, Shokichi Ohuchi1 (1Dept. Biosci. Bioinform., Kyushu Inst.
Technol., 2Dept. Appl. Bio. Sci., Tokyo Univ. Sci.)

計算化学と実験から考察した，黄色ブドウ球菌のヘム鉄結合・輸送機

ヒトレチノイン酸結合蛋白質をスカッフォールドとする蛋白質のス

構解析

クリーニングComputational and experimental studies on heme-binding andheme-transport mechanisms of Isd proteins of Staphylococcus
aureusScreening of mutant proteins scaffolding human cellularretinoic acid binding protein 2− S130 −Yoshitaka Moriwaki1, Tohru Terada1, Jose M. M. Caaveiro2, Yousuke
Takaoka3, Itaru Hamachi3, Kouhei Tsumoto2, Kentaro Shimizu1 (1Dept. of
Biotech., Grad Sch. of Agri. Life Sci., Univ. of Tokyo, 2Ins. of Med. Sci., Univ.
of Tokyo, 3Dept. Syn., Chem. & Biol. Chem., Kyoto Univ.)
Iron is an absolute requirement for nearly all organisms. Bacterial pathogens
have evolved the iron acquisition systems because iron is not readily available inhuman body. Staphylococcous aureus employs the iron-regulated surface
determinant (Isd) system as its primary heme-iron uptake pathway. In otherwords, this system is a promising target to battle S.aureus. Heme molecule isfirst extracted from humans’ hemoglobin and captured by IsdH or IsdB. IsdA
and IsdC relays heme to IsdE on the plasma membrane. Although the crystal
structures of Isd proteins have been solved with and without heme, themechanism of the one-way transport is still unclear because of the close
similarity of these structures. We examined the heme-binding and hemetransport mechanisms of Isd proteins not only in vitro, but also using in silico
methodologies. Isothermal titration calorimetry (ITC) experiments showed thatthe downstream Isd proteins have higher affinities against heme than the
upstream proteins. Qualitatively consistent results were obtained from the freeenergy calculations with the MM-PBSA method, which revealed enthalpic
contributions of the residues to the affinity to heme for each Isd protein. Basedon these results, we designed and synthesized a heme derivative. We will
examine its growth inhibition activity in vivo. In addition, the QM/MM(ONIOM) calculation is being performed for a model of a heme-transportcomplex composed of two Isd proteins and one heme. We will discuss thetransport mechanism between the Isd proteins.
2PT152

ヒトヘモグロビンの酸素親和性の制御における本質：四次構造、三次構造変化に共通して起こる Fe-His 結合の変化

Essence in oxygen affinity regulation of human adult
hemoglobin: Change of Fe-His bond length by quaternary or
tertiary structure change
Shigenori Nagatomo, Yasuhisa Yamamura, Kazuya Saito (Dept. Chem., Univ.
Tsukuba)
Human adult hemoglobin (HbA) possessing 2 2 tetramer structure exhibits
cooperativity in oxygen binding. X-ray crystallographic studies have demonstrated
the presence of two distinct quaternary structures, called T (tense) and R (relaxed)states, which correspond to the low- and high- O2 affinity forms, respectively. Boththe quaternary structures of T and R exhibit different Fe-His bond lengths: they areshorter in R state than T state in both the  and  subunits.
Extensive examinations of HbA in the presence of the allosteric effector
bezafibrate (BZF) showed a reduction of the oxygen affinity of oxyHbA in the R
structure and the importance of tertiary structure change for oxygen affinity isstressed. Recently, one of authors reported that the decrease in oxygen affinityof oxyHbA upon BZF addition accompanies the elongation of Fe-His bond in 
subunit [1]. Our finding is consistent with the molecular dynamics calculation of
Yonetani and Laberge [2], in which the main chain in E and F helices of HbA
fluctuates more in the presence than in the absence of BZF. This enhanced
fluctuation in the E and F helices results in weakening of the Fe-His bond.
Thus, the longation of Fe-His bond length is caused by not only quaternary
structure but also tertiary structure change. This suggests that the elongation of
Fe-His bond is essential in oxygen affinity regulation of human adult
hemoglobin. [1] S. Nagatomo, H. Hamada and H. Yoshikawa, J. Phys. Chem. B
115, 12971 (2011). [2] T. Yonetani and M. Laberge, Biochim. Biophys. Acta
1784, 1146 (2008).
2PT153Structural fluctuation and change around heme pockets ofliganded Hb induced by L35 binding

Kenji Kanaori1, Yusuke Tajiri1, Antonio Tsuneshige2, Takashi Yonetani3(1Grad. Sch., Kyoto Inst. of Tech., 2Hosei Univ., 3Univ. Pennsylvania)
Hemoglobin (Hb) takes two structures, depending on oxygen binding; Tquaternary structure with low oxygen-affinity and R-quaternary structure withhigh oxygen-affinity. Heterotropic effectors decrease the oxygen-affinity and its
cooperativity. In the presence of two potent effectors, L35 and inositol
hexaphosphate (IHP) at pH 6.8, 1H-NMR spectra of human adult Hb showed
oxy-T-quaternary structure. The NMR signals around heme pockets of the oxyT structure were rather broad, indicating that some structural fluctuations occur
around the heme, and the fluctuations may be related with the reduction of theoxygen-affinity. In order to clarify the structural fluctuations in the hemepocket induced by L35 and IHP, NMR spectra of liganded met-Hbs were
measured in the presence of the effectors. It has been known that the N3-metHbshows some IHP-induced hyper fine shifted (hfs) signals besides the heme
methyl signals around 15-35 ppm. The addition of L35 to the N3-metHb with
IHP showed that the heme signals were shifted and broadened, but the IHPinduced hfs signals were increased. The pH dependence of the hfs signals was
also examined and the pH increase from 6 to 8 showed that the IHP-induced
signals disappeared, suggesting that the spectral change should be related to thechange in the oxygen affinity. The quaternary structural change of metHb will
be discussed based on hydrogen bond signals around 12-13 ppm.
2PT154

ヒトヘモグロビンのβ 145Tyr の Thr または Leu への置換が機能と構造に与える影響

Effects of the substitutions of Thr or Leu for β145 Tyr on the
structure and oxygen binding properties of human hemoglobin
Momoko Ichii2, Yukifumi Nagai1, Kiyohiro Imai1,2, Masako Nagai1 (1Res.
Center for Micro-Nano Tech.Hosei Univ., 2Frontier Biosci. and Applied Chem.
Hosei Univ.)
In all the vertebrate hemoglobins and myoglobins, the penultimate position(HC2) is invariably occupied by tyrosine (Tyr). The natural and artificial
mutants of 145Tyr studied so far, show the extremely high oxygen affinity andlow cooperativity. To examine the role of 145Tyr on the structure and oxygenbinding properties, we expressed two new artificial mutant Hbs in E. coli,rHb(Y145L) and rHb(Y145T) and purified them as described previously [1].
These mutant Hbs exhibited a markedly high oxygen affinity and diminishedcooperativity (P50=0.6-0.9 mmHg, Hill's n=1.3-1.4). In the presence of inisitol
hexaphosphate (IHP), however, oxygen affinity of these mutant Hbs decreased
and exhibited significant cooperativity; P50 = 1. 7 mmHg, n = 1. 9 for Hb(Y145T); P50=2.4 mmHg, n=2.2 for Hb(Y145L). From the oxygen binding
properties of these mutant Hbs, the replacement of 145Tyr by Leu seems tostabilize the T-conformation more than that of Thr. DeoxyHb (Y145L)
exhibited a smaller negative CD band at 286 nm (a T-state marker band) thanthat of deoxyHbA, but exhibited an increase in intensity of the negative CDband upon the addition of IHP. These results indicate that the residue at HC2 of subunits contributes to stabilization of the T-conformation throughhydrophobic interactions with the surroundings in addition to hydrogen bonding
to FG5Val corbonyl group. [1]Nagai,M., Nagai,Y., Aki,Y., Imai,K., Wada,
Y., Nagatomo,S., and Yamamoto,Y. Biochemistry, 47, 517-525 (2008).
2PT155cbb3型チトクロム酸化酵素の酸素消費活性

Oxygen consumption activity of cbb3-type cytochrome coxidaseYui Iwamoto1, Yuriko Ando1, Kazumasa Muramoto1, Robert Gennis2, Yoshitsugu
Shiro1,3 (1Grad. Sch. Sci., Univ. Hyogo, 2Univ. Illinois, 3Harima Inst., Riken)
Terminal oxidases of the aerobic respiratory chain in inner mitochondrial
membrane and bacterial cell membrane catalyze oxygen reduction to water
coupling to electron and proton transfer. Terminal oxidases containing heme
and copper atom at catalytic sites are called as heme-copper oxidases, which areclassified into A-, B-, C- and several types of subfamilies. Although structuresof the oxygen reduction sites are similar between subfamilies, structures of the
proton pathways and energy transduction efficiency are different. To understandenergy transduction mechanism, in this study, we compared oxygen
consumption activity of cytochrome aa3 (A-type) and cbb3 (C-type) oxidases
purified from bovine heart mitochondria and Vibrio cholera cells, respectively.
The measurement was performed by using ascorbate as electron donor and
TMPD as mediator. In the presence of aa3 or cbb3, oxygen concentration wasalmost linearly decreased. When the enzyme concentration was changed, therate varied as aa3 up to 5uM and cbb3 up to 0.2uM. The rate increased withincreasing concentration TMPD, indicating that electron transfer from TMPD tothe enzyme is rate limiting step. In ascorbate/TMPD system, the specific activityof cbb3 was estimated to be 50 to 60 times higher than that of aa3. Inhibitory
effect by nitric monoxide was measured. Addition of nitrite decreased oxygen
consumption rate of both aa3 and cbb3, but in the case of cbb3 the rate wasdependent on the oxygen concentration suggesting change of rate limiting step.
2PT156cbb3型チトクロム酸化酵素の精製

Purification of cbb3-type cytochrome c oxidase
Yuriko Ando1, Yui Iwamoto1, Kazumasa Muramoto1, Hideo Shimada1,2,
Robert Gennis3, Yoshitsugu Shiro1,4 (1Graduate school of Life Science,
University of Hyogo, 2Picobiology Institute, University of Hyogo, 3University
of Illinois, 4RIKEN, SPring-8 Center)− S131 −The terminal oxidases of the aerobic respiratory chains in mitochondria andbacteria catalyze the oxygen reduction coupled to proton pumping, which
generates the proton-motive force. The terminal oxidases containing heme andcopper at the catalytic site form the heme-Cu oxidase (HCO) super family.
Although conformations of the catalytic sites are similar in HCOs, the protontransfer pathways are not conserved and the energy transducing efficiencies aredifferent. To identify structurally important elements and clarify common
mechanism on the energy transduction, we aim to investigate cbb3 -type
cytochrome c oxidase from Vibrio cholera by X-ray crystallographic analysis.
In previous study, we have examined culture condition for V. cholera cells
carrying His-tagged cbb3 oxidase expression plasmid and then performed
purification by metal affinity chromatography. We have obtained cbb3 oxidase
showing enough oxygen consumption activity, but SDS-PAGE showed the
preparation still contained some impurity. In this study, to improve purity andyield, we examined the conditions of metal ion resin and solution compositionon the chromatography. By using Ni ion resin, 5mg of cbb3 oxidase wasobtained from 30g cells. Purity was significantly improved by increasing salt
concentration in charge and wash solution on the chromatography. Currently,
we collect many preparations by this method for crystallization.
2PT157

水素原子レベルでの酸化還元に伴う構造変化同定を目的としたウシ
心筋チトクロム酸化酵素の X 線結晶構造解析

X-ray structural analysis of bovine heart cytochrome c oxidase
to clarify redox coupled structural changes at H atom level
Naomine Yano1, Kazumasa Muramoto1, Kyoko Shinzawa-Itoh1, Tomoko
Maeda1, Eiki Yamashita2, Tomitake Tsukihara2, Shinya Yoshikawa3,
Yoshitugu Shiro1,4 (1Dept. of Life Sci., Univ. of Hyogo, 2Inst. for protein Res.,
Osaka Univ, 3Picobio. Inst., Univ. of Hyogo, 4Harima Inst., Riken)
Cytochrome c oxidase (CcO) catalyzes the molecular oxygen reduction
coupling to the proton transfer across the inner membrane. To understand protontransfer mechanism in detail, it is necessary to determine orientations andprotonation states of amino acid residues (imidazole and carboxyl group etc.)
and waters in the proton pathways in both oxidized and reduced states. For thispurpose, we are currently carrying out high resolution X-ray structural analysisof bovine CcO.
To reduce X-ray damage and increase data accuracy, many data fromisomorphous crystals were collected. Low quality images whose Rmerge ormosaicity were larger than 0.3 were removed from data set. At present, data of
the oxidized state showed I/σ(I) of 1.6 at 1.42 to 1.40 Å resolution range. As aresult of structural analysis, many multi-conformations (Met residue etc.) were
found. Electron density map of phospholipids, waters and peroxide at the O2binding site were improved. To determine H atom positions, residual density of
Fo-Fc map must be reduced. 1) about 2800 waters and 36 hydrocarbons wereadded, 2) 88 multi-conformations were introduced to amino acids, 3) individual
anisotropic B-factor were introduced 50% amino acid residues. As a result,residual density of hydrophilic region was removed. R and Rfree values were also
reduced from 0. 199 to 0. 154 and 0. 213 to 0. 177, respectively. Structuralrefinement of reduced state carries out as well as oxidized state.
2PT158有機薄膜電極上での P450 反応の電気化学制御と応用

Electrochemically-driven cytorhcome P450 reactions at organic films and its bioprocess application
Yasuhiro Mie, Emi Tateyama, Yasuo Komatsu (AIST)
Cytochrome P450 enzymes catalyze a vast array of oxidative biotransformations
that are potentially useful for pharmaceutical synthesis. Generally, P450s
require reducing power in the form of expensive cofactors such as NADPH andone or two additional redox proteins to transfer electrons to the P450 active site
to limit their technological applications. Replacing the conventional electron
supply system with an electrode is a promising approach to develop inexpensive
P450-driven systems. Recently, we found that nanostructured gold electrode isuseful for the purpose. In the present study, we prepared several organic films
on nanostructured electrodes to investigate the effect of surface features ofelectrodes on the electrochemical P450 reactions. Nanostructured surfaces ofgold electrodes were modified with some thiolates by dip treatment.
Immobilizations of P450s on the thiolate films were examined. CYP modified
electrodes were investigated with voltammetry of a normal three-electrodeconfiguration. CYPs on hydrophobic electrode surfaces coated with aromatic
thiolates showed clear non-turnover signal, indicating rapid direct electrontransfer between P450 heme and electrode and gave the largest catalytic current.
On the other hand, electrode surfaces with coating materials containinghydrophilic functional groups were found to be mostly not suitable for the rapid
electron transfer, and poorer P450-catalytic currents were obtained, indicating
the importance of electrode surface nature for electrochemically-driven P450
reaction.
2PT159ヒトヘモグロビンα鎖の近位ヒスチジンのグリシンへの置換が構造と酸素結合機能に与える影響

Substitution effects of Gly for the proximal His of α subunits on
the structure and oxygen binding function of human hemoglobin
Yayoi Aki1, Yukifumi Nagai2, Kiyohiro Imai2, Shigenori Nagatomo3, Takashi
Ogura4, Teizo Kitagawa4, Masako Nagai2 (1Med., Kanazawa Univ., 2Res.
Center for Micro-Nano Tec., 3Chem., Univ. Tsukuba, 4Life Sci., Univ. Hyogo)
It has been proposed that the proximal histidine (His) in human adult
hemoglobin (HbA) plays a pivotal role in the transmission of oxygenationinduced conformational changes in one subunit to another and in the resultant
triggering of cooperativity [1]. We synthesized a mutant Hb in which theproximal His of  subunits was replaced by glycine (G), and examined its
quaternary structure change upon ligand binding by means of near-UV circulardichroism (CD) and UV resonance Raman (UVRR) spectroscopy as well as its
oxygen binding function. Mutant Hb(87His->Gly) (H87G) was produced in
E. coli, and purified as previously described [2]. Oxygen equilibrium curve(OEC) was measured by an automatic OEC recording apparatus or by atonometer method. The OEC of Hb(H87G) showed an unusual shape having
two components with a very high oxygen affinity and a distinctly low oxygen
affinity without cooperativity. In near-UV region, a small positive CD band wasobserved for oxyHbA while a distinct negative CD for deoxyHbA. The negative
CD of deoxyHbA at 287 nm, called “a T-state marker band”, was observed in
oxyHb(H87G), indicating that the substitution of Gly for the proximal His
failed to undergo the T-to-R quaternary structure transition. Changes of UVRR
spectrum upon ligand binding of Hb(H87G) also suggested lack of quaternary
structure transition.
[1] Perutz, M. F., Nature, 228, 725-734 (1970).
[2] Nagai, M., Nagai, Y., Aki, Y., Imai, K., Wada, Y., Nagatomo, S., andYamamoto, Y. Biochemistry, 47, 517-525 (2008).
2PT160

細菌 Vibrio alginolyticus 由来呼吸鎖末端酸化酵素の遺伝子ク
ローニングと同定

Gene cloning and identification of terminal oxidase of
respiratory chain in Vibrio alginolyticus
Naomine Yano1, Kazumasa Muramoto1, Seiji Kojima2, Michio Homma2,
Yoshitsugu Shiro1,3 (1Dept. of Life Sci., Univ. of Hyogo, 2Dept. of Biol. Sci.,
Nagoya Univ., 3RIKEN, Harima)
The terminal oxidase of the aerobic respiratory chain catalyzes reduction ofmolecular oxygen to water molecule. The terminal oxidases containing heme
and copper at the catalytic site are classified into several types which form theheme-Cu oxidase superfamily. Although conformations of the catalytic sites are
similar between different types, the proton transfer pathways are not conserved
and the energy transducing efficiencies are different. In many bacteria, the cellis known to expresses multiple terminal oxidases in different types dependingon growth environment. Genome sequence of Vibrio alginolyticus indicates that
there are several gene homologs of terminal oxidases, one of which is aa3-type
homolog. Primary sequence of core subunit I shows about 47% homology tobovine mitochondrial aa3-type and 46-47% homology to Paracoccus
denitrificans or Rhodobacter sphaeroides aa3-type, these are well studied ones.
Structural modeling suggests that hemes a and a3 can be built into the subunit I.
To identify the aa3-type terminal oxidase in V. alginolyticus, we cloned possible
aa3-type genes and constructed His-tagged expression system in V.
alginolyticus cell. Absorption spectrum of cultured cell suspension showedabsorption peak at cytochrome a region, although its height was much lower
than those of cytochromes b and c regions. Partially purified sample showedabsorption peak at cytochrome a region in the reduced minus oxidized
difference spectrum. These results indicate that the aa3-type terminal oxidase isin V. alginolyticus cell.
2PT161− S132 −硬骨魚類の祖先型ヘモグロビン遺伝子の設計・合成および大腸菌内
発現

Design and construction of ancestral Osteichthyes hemoglobin
genes and its expression in Escherichia coli

Sho Sugiyama1, Takatoshi Matsuo2, Kazuha Seki3, Taro Nakagawa4, Kiyohiro
Imai1,2,3 (1Department of Frontier Bioscience, Graduate School ofEngineering, Hosei University, 2Research Center for Micro-Nano Technology,
Hosei University, 3Department of Frontier Bioscience, Faculty of Bioscienceand Applied Chemistry, Hosei University, 4Department of Bioscience,
Nagahama Institute of Bio-Science and Technology)
The hemoglobin (Hb) of vertebrates including Osteichthyes is tetramericcomposed of two  chains and two  chains. Hbs from some Osteichthyes
species show the Root effect in which the oxygen capacity of Hb is halved upon
lowering of blood pH to an acidic range. We intended to learn by a reverse
phylogenetic analysis how those species acquired the Root effect andcooperativity in oxygen binding through molecular evolution of Hb. In thispresent study, we designed and constructed Node296 and Node203 genes
which are the globin genes of a common ancestor of Osteichthyes, and cloned
them into pET-22b(+). The resultant pET- Node296 and pET- Node203 wereexpressed in E.coli., yielding a definite quantity of the ancestral Hbs.
2PT162

Exclusion of the active site waters by substrate in cytochrome
P450cam
Ayaka Kishimoto1, Keisuke Sakurai2, Kenji Takagi1, Tsunehiro Mizushima1,
Takashi Hayashi3, Hideo Shimada1 (1Grad. Sch. Sci., Univ. Hyogo, 2Inst. Sci.
Ind. Res., Osaka Univ., 3Grad. Sch. Eng., Osaka Univ.)
Waters occupying the substrate-binding site of enzymes are expelled bysubstrate. Mechanistic study on the water exclusion is limited by difficulty inmonitoring the waters. Cytochrome P450cam, a heme b containing
monooxygenase of D-camphor, is a good model for such study because thewater exclusion accompanied by spin change of heme iron could be monitored
by UV/Vis spectroscopy. We have proposed a water-expelling pathway withgate formed by Asp297, the heme-7-propionate, Glu322, and Arg299. Here, wereport binding reactions of Asp297Leu-mutant enzyme with D-camphor and the
X-ray structures. Leu297 mutation dramatically retarded camphor binding; at
25 °C, bound form was ca 20% of the enzyme at 24 hrs after exposure to 1mM
camphor (Kd value of wild type is ca 1M). Binding still proceeded at a month
later (Under the same conditions wild type enzyme showed denaturation).
Binding, however, was greatly facilitated by glycerol, PEG4000, and sucroseand by temperature over 5 and 45 °C; At 65% (V/V) glycerol ca 75 % boundform was rapidly formed, while in wild type, glycerol lowered the affinity. In
wild type, temperature does not change Kd for camphor between 10 and 30 °C(temperature higher than 30 ° C denatured the protein). X-ray structure ofcamphor-free Leu297-mutant showed opened substrate channel that closed bycamphor binding, suggesting normal substrate channel. The present results
strongly suggest retardation of camphor binding by the mutation is caused byslow exclusion of the waters, supporting the water-expelling pathway.
2PT163

ヘモグロビンの自動酸化速度に対する糖類の効果

The effect of saccharide on autoxidation rate of hemoglobin
Wataru Uehara, Kiyohiro Imai (Department of Frontier Bioscience, Graduate
School of Engineering, Hosei University)
Hemoglobin (Hb) is a tetrameric heme protein composed of two  and two 
subunits. The ferrous heme iron of each subunit is capable of reversible oxygenbinding which becomes irreversible when the heme iron is oxidized to the ferricstate (met form). OxyHb is apt to autoxidize gradually to metHb. Recently it
was reported that aquometHb with trehalose and glucose added underwent heatinduced reduction at 70-80 ℃. In the present study we measured autoxidation
rate for horse oxyHb at 50 ℃ in the presence and absence of glucose, trehalose,sucrose and glucose-trehalose mixture. It was suggested that saccharide iseffective in preventing autoxidation of Hb.
2PT164

Norcamphor binding to cytochrome P450cam with a mutation
to block pathway for the active site waters excluded bysubstrate

Natsumi Kitamura1, Aya Amano1, Ayaka Kishimoto1, Keisuke Sakurai2, Kenji
Takagi1, Tsunehiro Mizushima1, Takashi Hayashi3, Hideo Shimada1 (1Grad. Sch.
Sci., Univ. Hyogo, 2Inst. Sci. Ind. Res., Osaka Univ., 3Grad. Sch. Eng., Osaka Univ.)
Active site waters of cytochrome P450cam, a heme b containing
monooxygenase of D-camphor, are excluded by camphor. We have proposedthat the waters are expelled to bulk waters through a pathway with gate formedby Asp297, heme-7-propionate, Glu322, and Arg299. Mutation of Asp297 to
Leu seems to block the water exodus, thereby interfering substrate bindings.

Here, we studied binding reaction between the Leu297-mutant and norcamphor,which lacks two methyl groups at C8 and C9 positions of camphor. Norcamphor
binding was dramatically retarded. However, high concentration of polyol suchas glycerol, PEG4000, and sucrose greatly facilitated binding, while in the wild
type protein polyol lowered the affinity for norcamphor. X-ray structures of
norcamphor bound wild type and Leu297 mutant proteins were determined at
1.6 and 1.8 Å resolution, respectively. Norcamphor at the active site of the bothproteins exhibited multiple conformations where the Tyr96-bound conformation
was not evident, contradicting to the X-ray structure by Poulos et al. where only
norcamphor H-bonded to Tyr96 was reported. When we compare norcamphor
bound mutant-structure with the previously determined camphor-free one,
norcamphor provides no repulsive effect on Leu297. However, Leu297
sidechain exhibited the same conformation with that of previously determined
camphor-bound form. Thus, sidechain conformation of Leu297 would not be
resulted from repulsive effects from C8 methyl group of camphor. The present
results support the proposed water-expelling pathway.
2PT165

ヒトヘモグロビンの複数酸素分子侵入経路の計算的解析

Computational analysis of multiple oxygen migration pathways in human hemoglobin
Masayoshi Takayanagi1,2, Ikuo Kurisaki1,2, Masataka Nagaoka1,2 (1Graduate
School of Information Science, Nagoya University, 2CREST, Japan Scienceand Technology Agency, Japan)
Hemoglobin expresses the function upon its binding to the ligand molecules at
the heme site. The ligand binding site of hemoglobin is buried in the interior of
the protein, indicating that the ligand must migrate inside of hemoglobin.
However, it is still under debate to clarify the ligand migration pathway. We
examined multiple ligand migration pathways in human hemoglobin andidentified the dominant ones. We executed 128 independent molecular
dynamics (MD) simulations of tetramer human hemoglobin with the time lengthof 8 ns under oxygen-rich condition and obtained the migration pathways inside
hemoglobin subunits. With the help of the clustering analysis, we visualized themultiple ligand migration pathways. The obtained rate of ligand entry into thebinding site is consistent with those observed by CO geminate recombination
experiments.
2PT166

Theoretical analysis of fully-hydrated structures of humanadult hemoglobin exploiting molecular dynamics simulations
Tetsuhiko Itagaki, Jiyoung Kang, Masaru Tateno (Grad. Sch. Sci., Univ.
Hyogo)
Molecular dynamics (MD) simulations of human adult hemoglobin (HbA) were
performed employing the highest resolution crystal structures (1.25 Å) of theR(oxy)- and T(deoxy)-forms, to compare the resultant fully-hydrated structures(1000-ns MD calculations) with the spectroscopic data obtained using theprevious NMR and infrared analyses. Previously, Perutz proposed the stereo
chemical mechanism of the O2 affinity regulation by comparing the crystal
structures in both forms of HbA. This is widely accepted. However, recent
experiments have shown that several heterotoropic effectors (e. g. L35) can
modulate the O2 affinity without significant structural changes of HbA.
Yonetani, et al. postulated that entropic/dynamical properties contribute to thismodulation. We found that the hydrated structures in both forms obtained using
the MD simulations are consistent with the spectroscopic data, some of which
are inconsistent with the crystallographic data. As a result of the analysis, thesolution structure of the T-form may be different from the crystal structure,
while the R-form is conserved in the solution and crystal structures. The
hydration/entropic properties could be closely relevant to this difference
between the solution and crystal structures of the T-form. Thus, our MD
simulations can be a solid basis to analyze the roles of the heterotoropic
effectors.
2PT167

Theoretical analysis of the electronic structure of CuB site ofbovine cytochrome c oxidase
Toru Matsuoka, Jiyoung Kang, Masaru Tateno (Grad. Sch. Sci., Univ. Hyogo)
Cytochrome c oxidase (CcO), which is the terminal enzyme of the electrontransfer system, reduces an oxygen molecule to two water molecules (4H+ + O2--> 2H2O). The trigger of this reaction is the binding of an oxygen molecule tothe bi-nuclear center (BNC), which is composed of CuBsite and heme a3 site).
Experimentally, to obtain the geometric structure of the complex with an
oxygen molecule is currently difficult; recently, the crystal structures of several− S133 −complexes with CO, NO, and CN ligands were revealed by Muramoto et al. In
this study, employing these crystal structures, we performed hybrid ab initio
quantum mechanics/molecular mechanics (QM/MM) simulations, and analyzed
the geometric and electronic structures of the BNC.
As a result of the analysis, we found that the configuration of CO ligand in the
crystal structure is different from that of the theoretically-optimized structure.
We investigated this discrepancy by mapping the potential energy values ofvarious positions of CO ligand in the BNC. The obtained potential field revealed
the major two types of configurations, for which the energy values are close toeach other, although the corresponding electron densities obtained by the
crystallographic analysis cannot be separated into the two CO configurations.
So, we calculated the IR spectrums, and found that they are consistent with theexperimental data. In the session, the roles of the CuB site in the reaction will be
discussed.
2PT168In-situ 光照射固体 NMR を用いた D96N-bR の M 中間体で生じる構造変化の解析

Conformational change in M-intermediate of D96N-bR as
studied by in-situ photo-irradiated solid-state NMR
Ryota Miyasa1, Izuru Kawamura1, Satoru Tuzi2, Akira Naito1 (1Graduate
School of Engineering, Yokohama National University, 2Department of Life
Science, University of Hyogo)
Bacteriorhodopsin (bR) is the membrane protein of H. salinarum. When bR isphoto irradiated, retinal in bR is photoisomerized and bR transfer proton fromcytoplasmic side to extracellular side during photo cycle starting from all-transretinal via K, L, M, N and O-intermediates. When M-intermediate generate,
Schiff base is deprotonated and occurred large dynamic conformationalchanges. Since these conformational changes play a key role for proton pumpmechanism, we aim to detect the conformational changes occurred in Mintermediate by applying in-situ photo-irradiated solid state NMR (P-SS-NMR)
to D96N-bR that has long life time of M-intermediate.
We measured 15N P-SS-NMR spectrum of [ε -15N] Lys-D96N-bR at thetemperature range from 0℃ to -60℃. As a result, we assigned new peaks
appeared with photo-irradiation at 300 ppm and, 201 and 399 ppm (firstspinning side bands) to deprotonated Schiff base of M-intermediate in D96NbR. In contrast, we assigned the peaks decreased significantly with photoirradiation at 154 and 147 ppm to the protonated Schiff bases of 13-cis, 15-synand all-trans retinals in D96N-bR, respectively.
Additionally, we measured 13C P-SS-NMR spectrum of [3-13C]Ala, [1-13C]Val
doubly labeled D96N-bR. We observed the changes of 13C NMR signal
intensities from dark adapted state to photo activated state. Consequently, we
detected local conformational changes in the protein side at M-intermediate of
D96N-bR.
2PT169LOV2 ドメインの中間体揺らぎの時間分解検出

Transient fluctuation of phototropin LOV2 domain
Kunisato Kuroi1, Francielle Sato2, Yusuke Nakasone1, Kazunori Jikihara3, Satoru
Tokutomi3, Masahide Terazima1 (1Univ. of Kyoto, 2Univ. of Maringa, 3Osaka
Prefecture University)
For biological function of proteins, their structural fluctuations could play animportant role. Recently we succeeded in detecting such transient volume
fluctuations by using TG and TrL techniques for a blue light sensor protein,
PixD. Here, we studied the fluctuation of reaction intermediates of phototropoin
LOV2 domain, which is one of the most versatile blue-light sensor domain.
Although the reaction have been investigated extensively so far, it remains still
unknown how LOV2 domain transmits its light signal toward the effector
domain linked with usually the C-terminal of LOV2 domain, because only aminor structural change around its chromophore is observed upon the blue-light
excitation. For example, it has been reported that the LOV2 domain except thechromophore region does not undergo a remarkable structural change upon thelight excitation, but it causes the unfolding of the C-terminal linker-helix
domain. We consider that the intermediate structural fluctuation of LOV domainshould be involved in this step. Therefore in the present study, we detected andcompared the intermediate fluctuation of the LOV2-linker and LOV2 domains
by extending the TG and TrL measurements. For the LOV2-linker domain, we
succeeded in observing the enhanced fluctuation for the unfolding event of thelinker-helix. By comparison of the fluctuation between the LOV2 domain and
LOV2-linker, we discuss the functional role of the fluctuation of the LOV2
domain in the signal transmission step.

2PT170

Photoactive Yellow Protein 中に存在する低障壁水素結合近傍のアルギニン 52 のプロトン化状態

Protonation State of Arginine 52 near the Low Barrier
Hydrogen Bond in Photoactive Yellow Protein
Kento Yonezawa1, Hironari Kamikubo1, Keito Yoshida1, Shigeo Yamaguchi1,
Tarou Tamada2, Kazuo Kurihara2, Yoichi Yamazaki1, Mariko Yamaguchi1,
Mikio Kataoka1 (1Grad. Sch. Mat. Sci., NAIST, 2Japan Atomic Energy Agency)
Low Barrier Hydrogen Bond (LBHB) is a special type of hydrogen bonds(HBs), in which a proton locates near the center between donor and acceptor
atoms. Although the bond energy of LBHB (ΔHLBHB) is generally higher thanthat of a normal HB, ΔHLBHB in a protein has not been evaluated. To estimate
ΔHLBHB, we carried out pH titration experiments at various temperatures upon
Photoactive Yellow Protein. We clarified that the HB between the chromophore(pCA) and E46 is LBHB. The LBHB is converted to an ordinary HB (NH-O) in
E46Q. Neither the LBHB nor the HB can be formed in E46A. Therefore,
Δ pKa (WT-E46A) and Δ pKa (E46Q-E46A) is assumed to reflect bond freeenergies of LBHB and HB, respectively. From the temperature dependence ofΔ pKa values, Δ HLBHB and Δ HHB were estimated to be 8 kcal/mol and 7
kcal/mol, respectively. ΔHLBHB is rather smaller than the expected value. We
found the substantial difference in Hill's coefficients. The Hill's constant for WT
is about 2, while those of E46Q, and E46A are close to 1, suggesting that there
exist another protonation site in WT than pCA. To identify the presumed
protonation site, we performed the neutron crystallographic analysis on E46Q,
revealing that R52 in E46Q is partly protonated. Together with the fact that R52
is deprotonated in WT, it can be proposed R52 was cooperatively protonatedupon the protonation of the pCA in WT. The apparent ΔHLBHB reflects both ofthe energy gain of LBHB and the energy loss due to deprotonation of R52 at thephysiological pH, resulting in the relatively small value.
2PT171

PYP Phytochrome Protein Related (Ppr) に存在する PYP
ドメインによるフィトクロムドメインの光反応構造変化の制御

Photoreaction of PYP domain regulates the structural change
during the photoreaction of phytochrome domain of Ppr
Keito Yoshida, Hironori Kamikubo, Kento Yonezawa, Yoichi Yamazaki,
Mariko Yamaguchi, Mikio Kataoka (Graduate school of Materials Science,
Nara Institute of Science Technology)
PYP-Phytochrome Related protein (Ppr) is a multi-domain protein comprised ofa PYP domain, a bacteriophytochrome domain (Bph), and a histidine kinasedomain. We previously reported that the pohoto-reactions of PYP and Bph areclosely coupled, where the intermediate state of PYP influences the photoreaction rates of Bph. To investigate the molecular mechanism of theinterdependency, we prepared holo-holo-Ppr and apo-holo-Ppr, and performed
solution x-ray scattering experiments. The scattering profiles of the holo-holo
and apo-holo Pprs were nearly identical, indicating that the overall molecularshapes of holo-holo and apo-holo Pprs are indistinguishable. Red lightabsorption brings that holo-holo-Ppr changes in the scattering profile, holoholo-Ppr does not. It can be suggested that the structural change is suppressedby the holo-PYP domain despite that holo-PYP does not absorb red light. Bluelight irradiation to holo-holo-Ppr causes the formation of PYPM. The
photointermediate of Bph was slowly accumulated by the following red lightirradiation. The scattering profile of the final product is largely different fromthe original state, the change of which is similar to that of apo-holo-Ppr by red
light absorption. From these results, we propose that the photoreaction of PYP
domain regulates the domain rearrangement during the Bph photoreaction. The
unphotolyzed PYP domain suppresses the structural change, but the PYPM state
enables Ppr to undergo the large structural change.
2PT172

PYP の構造形成に対する N 末端領域の効果
Effect of N-terminal region to the structure formation of PYP

Mitsuhiro Sakonji, Yoichi Yamazaki, Hironari Kamikubo,
Yamaguchi, Mikio Kataoka (Grad. Sch. Mat. Sci., NAIST)

MarikoStructure element is defined as an uninterruptable region on the amino acidsequence which is required for the structure formation of a protein [1].
Photoactive yellow protein (PYP) is a water soluble light absorbing protein.
PYP can be separated into the N-terminal region and the chromophore binding
region. Upon absorption light, large disorder is brought to the N-terminal
region. The deletion of the N-terminal region, however, does not affect the− S134 −structure of the chromophore binding region. The N-terminal lacking PYP isalso photoactive. These facts suggest that the N-terminal region of PYP does notcontain structure element. In order to analyze the effect of N-terminal region to
structure formation of PYP as well as the effect of the chromophore binding, weapplied systematic alanine insertion to the N-terminal region of PYP.
Wild type apo-PYP takes a unique tertiary structure, although its CD spectrum
is slightly different from that of holo-PYP, indicating that the further structureformation by chromophore binding. In apo-form, CD intensities of some alanine
insertion mutants decreased from that of wild type. The CD increase uponchromophore binding is commonly observed, even though the CD spectra ofholo-form are slightly different from that of wild type. These results indicatethat the N-terminal region does not contain structure element, although theeffect of chromophore binding to the structure formation is affected byinsertion.
[1] R. Shiba, et.al. Biophysics 7, 1-10 (2011)
2PT173

Rc-PYP の相互作用における発色団周辺水素結合の寄与

The role of hydrogen bonding network around the chromophore for the interaction of Rc-PYP
Yoichi Yamazaki, Mayu Shimada, Hironari Kamikubo, Mikio Kataoka (Grad.
Sch. Mat. Sci., NAIST)
Photoactive Yellow Protein (PYP) is a blue light receptor firstly isolated frompurple photosynthetic bacterium, Halorhodospira halophila (Hh-PYP), which
has a p-coumaric acid as a chromophore. The PYP photoreaction and structural
information has been analyzed in detail, but there has been little progress in theanalysis of signal-transduction mechanism, since the target protein of Hh-PYP
has not been identified. PYP from Rhodobacter capsulatus (Rc-PYP) is the only
protein that the interaction protein is identified so far. Therefore, Rc-PYP is
more suitable for the research of PYP signal-transductions. Rc-PYP shows two
absorption maxima at around 370 and 440 nm, and shows reactivity to UV-A
light. Rc-PYP’s reaction with its interaction protein is light dependent. UV-A
illuminated Rc-PYP binds its interaction protein. At the formation of the RcPYP complex the absorption maximum of cis-chromophore shows red-shift, thisis indicating chromophore environment changes in the complex state.
To verify relationship between the chromophore environment and complex
formation, we examined spectral difference and complex formation about
several amino-acid residues mutants around the chromophore which constructhydrogen bond network and Rc-PYP re-constituted by chromophore analog
which lacks chromophore hydrogen bond. All the mutants kept their interaction
ability, even though spectral features were different from that of wild-type. But
the Rc-PYP in which chromophore substituted with cinnamic-acid did not showcomplex formation.
2PT174

桂皮酸導入 Photoactive Yellow Protein の発色団由来水素結合の欠損効果

Roles of hydrogen bonds around chromophore in Photoactive
Yellow Protein studied by OH-defficient cinnamic acid
Masatoshi Narumi, Yoichi Yamazaki, Mariko Yamaguchi, Hironari
Kamikubo, Mikio Kataoka (Grad. Sch. Mat. Sci., NAIST)
Photoactive Yellow Protein (PYP) is a water-soluble photosensor protein
isolated from Halorhodospira halophila. It has a covalently attachedchromophore, p-coumaric acid (pCA). Oxygen atom of pCA phenol groupforms hydrogen bonds with the E46 and Y42. These two hydrogen bonds play
crucial roles in the structure formation and photoreaction of PYP. In order toclarify the roles of these hydrogen bonds, we have attempted to remove thesehydrogen bonds by using a cinnamic acid as a chromophore(CA-PYP). CAPYP was successfully reconstituted. Although free cinnamic acid showed its
absorption maximum at 265nm, it shifted to 316nm in CA-PYP, suggesting thatthe importance of hydrogen bonds for visible light absorption. Absorption
intensity of CA-PYP decreased by UV illumination, but intensity neverrecovered to its original state by shutting of the light. CA-PYP is light reactive
but not cyclic. FT-IR difference spectrum showed chromophore vibationalmode changes of trans-cis photoisomerization by UV-light illumination in CAPYP. In addition, back bone changes and carboxyl group changes were also
observed in FT-IR difference spectrum. CA-PYP did not show radius of
gyration change and secondary structural change. These results suggest thathydrogen bonds constructed from phenolic oxygen of pCA, Y42 and E46 arerequired for the proper photoreaction of PYP accompanying with the substantial
structural change, although the hydrogen bonds are not essential for thechromophore attachment.

2PT175青色光センサータンパク質 YtvA の LOV と LOV-linker の光化学反応の比較

Comparison of the photochemical reaction between the LOV
domain and the LOV-linker domain of a blue light sensor
protein YtvA
Seokwoo Choi1, Yusuke Nakasone1, Klaas Hellingwerf2, Masahide Terazima1(1Department of Chemistry, Graduate school of Science Kyoto University, 2Universityof Amsterdam, Swammerdam Inst Life Sci)
YtvA is a blue light sensor protein which consists of the N-terminal LOV(Light-Oxygen-Voltage) domain and the C-terminal STAS (Sulphate
Transporter and Anti Sigma factor antagonists) domain, linked by the linker
helix. Physiological studies have reported that YtvA is involved in the initiationof environmental stress response (light, salt, heat etc.) by regulating an activityof transcriptional factor σB. However, the molecular mechanism of its signaltransduction process has not been revealed yet. For understanding this signaltransduction mechanism, the photochemical reaction dynamics has to beelucidated. In this study, we investigated the photochemical reaction dynamics
of the LOV and LOV-linker domains of YtvA using the time-resolved transient
grating (TG) method. In particular, by comparing the photochemical reactions
dynamics of the LOV and LOV-linker domain, we try to find the importance of
linker domain in the signal transduction. From the concentration dependence,
we found that the LOV domain in the ground state exists in the equilibrium
between the dimer and the tetramer, whereas the equilibrium is between the
dimer and the higher oligomeric form for the LOV-linker domain. For bothcases, it was shown that the photoreaction of dimer seemed to be important; thatis, the dimer of the LOV domain associates into the tetramer, whereas the dimerof the LOV-linker domain occurs structural change without oligomerization.
We will discuss the reaction in more detail in the poster presentation.
2PT176Natronomonas pharaonis 由来のセンサリーロドプシン II の光誘起プロトン移動における塩化物イオンの役割

The role of chloride in the photo-induced proton transfer insensory rhodopsin II from Natronomonas pharaonis
Jun Tamogami1, Katsunori Iwano2, Atsushi Matsuyama1, Takashi Kikukawa2,
Makoto Demura2, Kazumi Shimono1, Toshifumi Nara1, Naoki Kamo2(1College of Pharmaceutical Sciences, Matsuyama University, 2Faculty of
Advanced Life Science, Hokkaido University)
Natronomonas pharaonis has a sensory rhodopsin II (NpSRII, or calledpharaonis phoborhodopsin) which functions as a negative phototaxis receptor.
This protein has a retinal as a chromophore and illumination induces thephotochemical reaction (photocycle) accompanied by the photo-induced proton
uptake and release. Previously, it was reported that the early proton release
originated from Asp193 in phospholipid-reconstituted NpSRII occurs in thepresence of chloride [1, 2]. In this study, the effect of Cl- on this proton releasefrom Asp193 was investigated by detail experiments with the indium-tin oxide(ITO) electrode. With increasing of Cl- concentration in the medium, the pKa'sof Asp193 in both of the dark and excited state increased. From these Cl- dependence of the pKa's in Asp193, Kd values in both of the photolyzed andunphotolyzed state were estimated. In the unphotolyzed state, two Kd’s were
estimated. One was estimated to ~24 mM, while the other was estimated to > ~1
M which indicates very weak Cl- binding affinity. On the other hand, Kd wasestimated to ~244 mM in the photolyzed state. This implies that the Cl- binding
affinity becomes about 10-fold weaker by the conformational change in the
excited state. In addition to these estimations, we verified the position of
putative Cl- binding sites. Finally, the role of Cl- in the photo-induced protontransfer was discussed.
[1] Iwamoto et al. (2004) Biochemistry 43, 3195-3203. [2] Kitade et al. (2009)
Biochemistry 48, 1595-1603.
2PT177

アナべナセンサリーロドプシンの光誘起プロトン移動反応の解析

Photo-induced proton transfer of Anabaena sensory rhodopsin
Takatoshi Hasemi1, Takashi Kikukawa1, Masakatsu Kamiya1, Tomoyasu
Aizawa1, Keiichi Kawano1, Kwang-Hwan Jung2, Naoki Kamo1,3, Makoto
Demura1 (1Grad. Sch. Life Sci.,Hokkaido Univ., 2Dept. Life Sci. & Inst. Biol.
Interfaces, Sogang Univ., 3College Pharm. Sci., Matsuyama Univ.)Anabaena sensory rhodopsin (ASR), found from freshwater cyanobacterium, isa novel photosensing rhodopsin, which activates the soluble transducer for the− S135 −chromatic adaptation. Unlike the most microbial rhodopsins, the cytoplasmic(CP) half of ASR is unusually hydrophilic due to many polar residues and watermolecules. Basically, ASR lacks electrogenic proton transport. However,unusual proton transfer was observed: the Schiff base proton is transferred toAsp217 in the CP domain upon the formation of M photo-intermediate (Shi etal., J. Mol. Biol. 2006). Thus, ASR has a potential to move proton in the
direction opposite to that of proton-pumping rhodopsins.
In this study, we examined the proton transfer reactions of ASR by using
indium-tin oxide electrode, which enables the time-dependent measurements oflocal pH changes by flash-activated ASR. At the neutral pH, ASR induces thetransient alkalization of the local medium, indicating that photolyzed ASR
accepts proton from the medium and then releases it at the same side. On theother hand, above pH 8, the sequence of the proton movement is reversed:
proton release occurs first, followed by uptake. This suggests that a certain
residue acts as proton releasing residue in the alkaline condition. These protontransfers were greatly modified by the mutations of Asp217 and Glu36 locating
at the CP domain. Thus, the proton transfers mainly occur through thishydrophilic channel. Based on this analysis, we will discuss the proton transfer
reactions of ASR.
2PT178ウシロドプシンの構造に関する理論研究

A theoretical study on the structure of bovine rhodopsins
Motoshi Kamiya, Shigehiko Hayashi (Grad. Sch. Sci., Kyoto Univ.)
Rhodopsin is a photoreceptor in the retina. It consists of the protein moiety
opsin and an all-trans-retinal which is derived from Vitamin A. The
photoisomerzation of the retinal induces the structural change of the protein andeventually activates the associated G-protein, transducin. The purpose of thisstudy is to elucidate the structures of early intermediates during thephotoreactions such as lumirhodopsin. Computationally, the structure around
Schiff base of the retinal is difficult to be described with the classical forcefield,
although accurate description of the structure around the retinal is criticallyimportant for the calculations of vibrational frequencies and the excitationenergy. Therefore, QM/MM calculations must be involved to obtain reasonablestructure. On the other hand, structural rearrangements/relaxations at finite
temperature (not only fluctuations) of the system, which are usually not
involved in QM/MM methods, shall not be ignored for the structural relaxation
which takes more than nanoseconds. In order to fulfill these two requisites, inthis study, we employed QM/MM-RWFE-SCF method [Kosugi and Hayashi,
JCTC, 2011]. The structures and vibrational frequencies of the Schiff base inrhodopsin, bathorhodopsin, and lumirhodopsin will be discussed.
2PT179

グロエオバクターロドプシンの三量体形成に関する研究

Homo-trimeric assembly of a cyanobacterial ion-pump
Gloeobacter rhodopsin
Takashi Tsukamoto1, Takashi Kikukawa1,2, Masakatsu Kamiya1,2, Tomoyasu
Aizawa1,2, Keiichi Kawano1,2, Kwang-Hwan Jung3, Naoki Kamo2,4, Makoto
Demura1,2 (1Grad. Sch. Life Sci., Hokkaido Univ., 2Fac. Adv. Life Sci., Hokkaido
Univ., 3Dept. Life Sci. & Inst. Biol. Interfaces, Sogang Univ., 4College Pharm.
Sci., Matsuyama Univ.)
Rhodopsin-like molecules in microorganisms (microbial rhodopsins) play roles
as an ion-pump or a photosensor. The four microbial rhodopsins from halophilicarchaea have been investigated well so far; bacteriorhodopsin (BR),halorhodopsin (HR), sensory rhodopsin I (SRI) and sensory rhodopsin II (SRII,
also called phoborhodopsin). These proteins form intrinsic oligomers dependingon their function. The phototaxis sensors SRI and SRII form a tetramer which iscomposed of two units of receptor-transducer hetero-dimer. On the other hand,
the ion-pumps BR and HR form a homo-trimer. This is a unique structuralproperty of them and recent studies have revealed several factors for the trimer
formation and stabilization (Tsukamoto et al., Biophys. J. 2012). Other than BR
and HR, however, little is known about the trimer formation of the ion-pumping
rhodopsins.
We have started to investigate the oligomeric structure of Gloeobacterrhodopsin (GR), which is an ion-pumping protein discovered from freshwater
cyanobacterium, by means of size-exclusion chromatography (SEC) andcircular dichroism (CD) spectroscopy. The analysis of SEC gave us aconclusion that GR formed a trimer as well as BR and HR. However, the CD
spectrum of GR ((+) 500 nm/(-) 567 nm, crossover point; 540 nm) indicated thatthe trimeric state of GR was different from that of BR and HR. Interestingly, thetrimeric state of GR altered in a pH-dependent manner. Based on these analyses,
we will discuss the homo-trimeric structure of GR.

2PT180

パルミトイル修飾とロドプシンのラフト親和性の関係

Relationship between palmitoylations and raftophilicity ofrhodopsin
Keiji Seno1, Fumio Hayashi2 (1Hamamatsu Univ. Sch. Med., 2Grad. Sch. Sci.,
Kobe Univ.)
As we reported in the last meeting of BPSJ, we have found that rhodopsin forms
dimer in the complex of retinal G protein transducin (Gt). The rhodopsin dimer
favors l o lipids and distributes in the detergent-resistant membrane (DRM)
prepared from vertebrate (bovine and frog) rod photoreceptor outer segment
membranes. Our single molecule observation of rhodopsin and transducin
revealed that the increased affinity to raft of rhodopsin upon dimerization
hinders the diffusion of rhodopsin. Here, we studied the contribution of tandem
palmitoyls at the C terminal end of helix-8 of rhodopsin to the raftophilicity ofrhodopsin dimer. We examined the effect of depalmitoylation of rhodopsin by
reducing agent (20 mM dithiothreitol) on the distribution of rhodopsintransducin complex or IgG-crosslinked (artificially dimerized) rhodopsin to the
DRM of frog rod photoreceptor outer segments. Depalmitoylation was
confirmed by MALDI-TOF MS. Rhodopsin-transducin complex in DRM
fraction was reduced to almost half by depalmitoylation. When we artificiallydimerized rhodopsin by anti-rhodopsin monoclonal antibody, significant portionof anti-rhodopsin IgG, which means rhodopsin dimer, was detected in DRM
fractions, however, when we used depalmitoylated rhodopsin, much less IgG
was detected in DRM fractions. These data support our idea that the palmitoyl
modifications on rhodopsin are indispensable for raftophilicity of rhodopsin
dimer.
2PT181

海洋性細菌におけるプロテオロドプシンの生理的役割および分子特性

Physiological role and molecular properties of proteorhodopsin from marine bacterium
Rei Abe-Yoshizumi1, Keiichi Inoue1, Susumu Yoshizawa2, Kazuhiro Kogure2,
Hideki Kandori1 (1Dept. of Frontier Materials, Nagoya Inst. Tech., 2AORI,
Univ. Tokyo)
Nature created two kinds of light-driven ion pumps, bacteriorhodopsin (BR) and
halorhodopsin (HR), functioning as outward proton and inward chloride pumps,
respectively [1]. Then, thousands of proteorhodopsin (PR) have been discovered
and are widely distributed among marine prokaryotes [2]. But its physiologicalrole was unclear, because very few strains carrying the PR gene have been
cultured. Recently, we demonstrated light-driven proton pump activity of PR in
native Flavobacteria cells [3].
Here we studied the molecular mechanism and the physiological role of PR from
Krokinobacter sp. in Flavobacterium that codes two microbial rhodopsin genes intheir genome. We first examined their pump activity in native cells. It was foundthat PR is expressed in stationary phase, suggesting that PR-mediatedelectrochemical proton gradient is use to ATP synthesis when nutrition isdepleted. We then studied the spectral and photochemical properties ofrecombinant PR from Krokinobacter (KroPR). KroPR shows λmax at 525 nm(pH 7.0), being similar to GPR. On the other hand, pKa of the counterion (4.3) islower than that of GPR (6.9), although the amino acid identity between KroPR
and GPR is 41 % and His75 (GPR) is conserved. Flash photolysis measurement of
KroPR is in progress and the molecular mechanism of KroPR will be discussed.
[1] Kandori, Mol. Sci. 5, A0043 (2011).
[2] Beja et al. Science 289, 1902 (2000).
[3] Yoshizawa et al. Environ. Microbiol. 14, 1240 (2012).
2PT182

時間分解赤外分光法によるファラオニスハロロドプシンの機能発現過程での水の水素結合ダイナミクス

Water hydrogen-bonding dynamics in the function of pharaonis halorhodopsin studied by time-resolved FTIR spectroscopy
Kuniyo Fujiwara1,2, Tetsunari Kimura1,2,3, Takashi Kikukawa4, Makoto Demura4,
Hideki Kandori5, Yuji Furutani1,2,6 (1SOKENDAI, 2IMS, 3JST CREST, 4Grad. Sch.
Life Sci., Hokkaido Univ., 5Grad. Sch. Eng. , NITech, 6JST PRESTO)
pharaonis Halorhodopsin (pHR) is an inward chloride ion pump protein. pHR
has a retinal as the chromophore, which is bound through the protonated Schiffbase and all-trans to 13-cis photoisomerization triggers conformational changesof the protein moiety for pumping a chloride ion. The ion transportation is
performed through the sequential formation of some intermediates which are
named K, L1, L2, N and O. The structural detail of each intermediate state is still− S136 −unclear, and especially, it is open question how water molecules involve in thetranslocation of a chlorine ion inside protein during the photocyclic reaction.
To analyze the hydrogen-bonding dynamics of water and protein, we performed
the time-resolved Fourier transform infrared (TR-FTIR) spectroscopy in thewhole mid-infrared region. The measurement was performed under thecondition of hydration with H2O or H218O, which allow us to assign the water
O-H stretching bands. As a result, we observed a water band at 3626 cm-1 which
exhibited down shift to 3605cm-1 upon the formation of the N and O states. We
also analyzed the dependency on the chloride concentration to discriminate
whether the water change occurs in the N or O intermediates, which are related
to release and uptake of chloride ion. Anion dependency (Br-, NO3- and SO42-)
was investigated as well. By comparing these experimental data, the relationshipbetween the water hydrogen-bonding dynamics and the conformational changesof protein will be discussed.
2PT183

全反射赤外分光法によるキメラチャネルロドプシンの構造変化解析

Structural changes in the chimeric Channelrhodopsins revealed by ATR-FTIR spectroscopy
Asumi Inaguma1,2, Hisao Tsukamoto1, Tetsunari Kimura1,3, Toru Ishizuka3,4,
Hiromu Yawo3,4, Yuji Furutani1,2 (1Inst. Mol. Sci., 2JST PREST, 3JST
CREST, 4Grad. Sch. Life Sci., Tohoku Univ.)
Channelrhodopsin 1 and 2 (ChR1 and ChR2) can function as light-gated cation
channels which were discovered from Chlamydomonas reinhardtii. Recently,
ChR2 and its derivatives have been utilized as a useful tool to control the neuralcircuit by light. ChR1/2 chimera such as ChR-wide receiver (ChR-WR) and
ChR-fast receiver (ChR-FR) have improved characters as theelectrophysiological tool. ChR-WR is sensitive to the light in the wider range of
wavelength compare to the wild type. ChR-FR exhibits faster ON/OFF kinetics
in the photocurrent. However, the molecular mechanisms behind these usefulproperties have not been understood well. Nowadays, the crystal structure of
ChR has been reported, but the structural changes connecting the closed andopen conformation have not been answered well. In this research, we applied
attenuated total reflection Fourier transform infrared (ATR-FTIR) spectroscopy
to reveal the structural changes of the chimeric ChRs during the formation of the
open/photostationary state and return to the closed state. The light-induced
difference FTIR spectra of ChR-WR and ChR-FR were very similar to eachother, suggesting that the structural changes are mostly same. Interestingly, therecovery from the photostationary state of ChR-FR was faster than ChR-WR.
We will discuss the relationship between the structural changes and thephotocurrent properties of these chimera ChRs.
2PT184

Crystal Structure of deltarhodospin from Haloterrigena
Thermotolerans
Jin Zhang, Katsuhide Mizuno, Mizuno Kouyama (Nagoya University)
Deltarhodopsin, a new member of the microbial rhodopsin family, functions as alight-driven proton pump. In this study, we investigated the crystal structure ofdeltarhodopsin (dR3) from Haloterrigena thermotolerans, a thermo-tolerantarchaeon isolated from a solar saltern in Puerto Rico. For preparation of a large
quantity of the target protein, the gene encoding deltaopsin-3 was transformed
into a bR-deficient host strain of Halobactrium salinarum. When the membranefusion method was applied, the purified sample of dR3 was crystallized into
trigonal crystals belonging to space group R32. In this crystal form, dR3 forms atrimeric assembly as observed for bR and other archaeal rhodopsins. It isdemonstrated that the inner part of the protein (the retinal binding pocket and theproton release and uptake pathways) is similar to that of bR and other microbial
rhodopsins. One of major differences is observed in the protein-protein contact
regions. Interestingly, the carotenoid bacterioruberin binds to the crevice
between the AB helices of one subunit and the DE helices of a neighboring
subunit. Another peculiar property of dR3 is a highly crowded distribution of
positively charged residues on the cytoplasmic surface. Structural comparisonof dR3 with other archaeal rhodopsins allows us to recognize the structural
motif that is relevant to the proton pumping activity, while also offering insight
into the formation mechanism of the trimeric structure and the recognition
mechanism of specific lipid components.

2PT185The chroride ion uptake mechanism in pharaonis halorhodopisin as studied by the energy representation method
Hiroyuki Tamura1, Shuntaro Chiba1, Shun Sakuraba2, Nobuyuki
Matsubayasi2, Minoru Sakurai1 (1Center for Biol. Res. & Inform, Tokyo
Tech, 2ICR, Kyoto Univ)

Halorhodopsin (hR) is a light-driven chloride ion pump exist in halophilicarchaea. hR uses the light energy to move chloride ions from extracellular side
to cytoplasmic side. The process is thought to be divided into the two steps: (i) achloride ion passively enters the binding site near retinal Schiff base linkage inthe protein from the extracellular medium with a high NaCl concentration(~4M), and (ii) the chloride ion is transported into the cytoplasmic side after thephotoisomerization of retinal. The purpose of this work is to clarify themechanism of the first step by calculating the free energy of a chloride ion in thefollowing two cases: it is located in the extracellular side (bulk) and at thebinding site. The free energies were calculated using the method of energy
representation combined with molecular dynamics simulations. First, we
calculated the free energy of a chloride ion in the aqueous solution of NaCl. As aresult, the free energy decreased from -88.6 to -122.0 kcal/mol as the NaCl
concentration was increased from 0M to 4M. Next, we calculated the freeenergy of a chloride ion at the binding site: the result was -120.3 kcal/mol,which is equal to that in the aqueous solution of 3.4 M NaCl. These results
indicate that a chloride ion in bulk passively enters the binding site if the NaCl
concentration in bulk is lower than 3. 4 M. The largest contributor to thestabilization of a chloride ion at the binding site is the chromophore (-49.3
kcal/mol) and the next is the nearby water molecule (-20.5 kcal/mol).
2PT186ATP ショットと caged-ATP の光分解により開始されるホタル生
物発光の時間発展及びその pH 依存性
Time Dependent Characteristics of Firefly Bioluminescence
Initiated by Two Methods with Usual ATP Injection andPhotolysis of Caged-ATP

Yuki Yanagisawa1, Takeshi Kageyama1, Masatoshi Tanaka1, Shin-ya Ohno1, Naohisa
Wada2 (1Faculty of Engineering, Yokohama National University, 2Department of Life
Sciences, Toyo University)
The time-resolved spectrum and the rise time course of firefly bioluminescence
initiated by ATP injection into the HEPES buffer containing luciferin,
luciferase, and Mg2+ were first measured with time resolution of 10 ms, andcompared with those of the bioluminescence triggered by photolysis of cagedATP. The time course is significantly dependent on pH; both time constants ofrise and decay increase with decreasing pH from 7.8 to 6.8. The observed timecourse is constituted by two components; the diffusion-controlled reaction
follows the homogenization of reactants. The usual ATP injection may prevent
ATP4- from falling into the catalytic center of Luc. Contrarily, in the case of thebioluminescence triggered by photolysis of caged-ATP, the intrinsic reaction
dominates the time course not correlative with pH [1]. The time-resolved
spectra can be decomposed into two Gaussians peaked at 2.0 eV and 2.2 eV.
The intensity of 2. 2 eV component decreases with decreasing pH, being
consistent with previous report [2]. Spectral shapes do not change from theinitiation of the enzymatic reaction up to 200 s for both methods of ATP
addition. After 200 s, the 2.0 eV component decays slower than the 2.2 eVcomponent in the case of the photolysis of caged-ATP, which may be caused by
the product of photolysis. Interestingly, above results are not influenced
regardless of the concentration ratio [Ln]/[Luc].
[1] T. Kageyama, et al., Photochem. Photobiol. 87 (2011) 653-658.
[2] Y. Ando, et al., Nature Photonics 2 (2008) 44-47.
2PT187水溶液中のホタルルシフェリン蛍光スペクトルの理論的研究

Theoretical Analysis of Fluorescence Spectra of Firefly
Luciferin in Aqueous Solutions
Miyabi Hiyama1, Hidefumi Akiyama2, Kenta Yamada3, Nobuaki Koga1(1Grad. Sch. Info. Nagoya Univ., 2ISSP Univ. Tokyo, 3Grad. Sch. Nanobio.
Yokohama City Univ.)
To understand the mechanism of firefly bioluminescence, researchers haveinvestigated the spectroscopic properties of firefly luciferin as an analog of
oxyluciferin [1-6]. Despite the previous experimental and theoretical studies,some peaks in the fluorescence spectra of firefly luciferin still remain
unassigned. To elucidate the origin of fluorescence spectra of firefly luciferin,
the excited states of firefly luciferin and its conjugate acids and bases were
investigated by performing the density functional theory calculations (DFT).
Time-dependent DFT calculations were performed for the excited states of thesemolecules. The solvent effect in aqueous solution was taken into account by thepolarizable continuum model. We obtained the energies and oscillator strengthsof excitation of firefly luciferin and its conjugate acids and bases [7]. Although
the wavelengths of energies in theoretical fluorescence spectra areapproximately 40 nm lower than those in the experimental data, the relative− S137 −theoretical energies are in good agreement with the experimental ones.
[1] Seliger et al., Proc. Natl. Acad. Sci. U.S.A. 47, 1129 (1961) [2] Morton et al.,Biochem. 8, 1598 (1969)[3] White et al., Bioorg. Chem. 1, 92 (1971) [4] Jung etal., J. Am. Chem. Soc. 98, 3949 (1976) [5] Gandelman et al., J. Photochem.
Photobio. B: Biol. 19, 187 (1993) [6] Ando et al., Jpn. J. Appl. Phys. 49, 117002(2010) [7] Hiyama et al., Photochem. Photobio. (2012) In press
2PT188

シアノバクテリア由来のフィトクロム様光受容体「PixJ1」における短波長シフトした光反応の分子基盤

Molecular Basis for the Blue-shifted Photoconversion of PixJ1,
Phytochrome-like Photoreceptor in Cyanobacterium, Studied
by FTIR
Kazunori Zikihara1,2, Shizue Yoshihara1, Takahiro Kitano1, Hitomi Katsura1,
Takayuki Kohchi3, Satoru Tokutomi1 (1Grad. Sch. Sci., Osaka Pref.
Univ., 2Grad. Sch. Med., Osaka Univ., 3Grad. Sch. Bio., Kyoto Univ.)
Plant phytochrome binds a linear tetrapyrrole, phytochromobilin (PΦB), as achromophore covalently to a conserved Cys residue (1st Cys) in the GAF
domain forming a thioether bond and has two absorption forms, named red
light-absorbing (Pr) and far-red light-absorbing (Pfr) forms, between which they
are photoconvertible by red and far-red light, respectively. Recently, aphytochrome-like chromoprotein, PixJ1, was found in cyanobacterium
Synechocystis sp. PCC 6803 that binds a tetrapyrrol chromophore to the 1st Cys
in the GAF2 domain and shows a photoconversion between blue lightabsorbing (Pb) and green light-absorbing (Pg) forms instead between Pr and Pfr.
To uncover the molecular basis for the blue-shifted photoconversion, Fouriertransform infrared (FTIR) spectra of the PixJ1-GAF2 domain were measured
and analyzed in reference to that of the photosensory domain of Arabidopsis
phytochromeB (PhyB-N651).
Both the PixJ1-GAF2 and the PhyB-N651 showed light-induced changes in thefingerprint region of the FTIR difference spectra that are reproducible uponsuccessive photoconversions. Notably, Pg minus Pb and Pg minus Pb difference
spectra of PixJ1 showed positive and negative peaks, respectively, in the S-H
stretching region indicating an association and a dissociation of H in a S-H group,
possibly of the 2nd Cys in PixJ1-GAF2. On the other hand, neither Pfr minus Pr nor
Pfr minus Pr difference spectra of the PhyB-N651 showed them. The details of themolecular basis for the blue shift in PixJ1-GAF2 will be discussed.
2PT189

視物質の熱雑音発生メカニズム

Mechanism of thermal activation of visual pigments in the dark
Daiki Kawata1, Kota Katayama1, Hiroo Imai2, Hideki Kandori1 (1Department
of Frontier Materials, Nagoya Institute of Technology, 2Primate ResearchInstitute, Kyoto University)
Visual pigments possess an 11-cis retinal as the chromophore, and its
photoisomerization to the all-trans form initiates our vision. Highly sensitive
vision originates from efficient photoisomerization and little thermal
isomerization. In fact, the cis-trans isomerization coordinate suggests highthermal activation barrier (> 180 kJ/mol), which is supported by the tightly
packed chromophore in the crystal structure of rhodopsin. However, thereported activation enthalpy of the dark noise is 80-110 kJ/mol, and mechanismof such low thermal activation has been a paradox in vision. We recently
challenged this paradox using FTIR spectroscopy and H/D exchange. Thr118 is
located in contact with the chromophore and isolated from hydrogen-bonded
network connecting to the outside of rhodopsin. We reported that the O-H groupis not deuterated in D2O at ambient temperature [1]. Nevertheless, we found that
H/D exchange occurs at 50-70 ℃, suggesting that protein fluctuation drives theretinal binding pocket transiently open. We proposed a quantitative two-stepmodel that can explain the observed low thermal activation in rhodopsin [2]. In
this study, we extend this analysis to other visual pigments. Temperaturedependent H/D exchange measurements of the O-H group are in progress for 9cis rhodopsin and monkey green-sensitive pigment, and molecular origin ofdark noise in vision will be discussed.
[1] Nagata et al. J. Phys. Chem. A 106, 1969-1975 (2002).
[2] Lorentz-Fonfria et al. J. Am. Chem. Soc 132, 5693-5703 (2010).
2PT190クモにおけるピンぼけ像を用いた新規距離知覚メカニズム

1YS1015

A novel depth perception mechanism based on image defocus
in a spider

Takashi Nagata1, Mitsumasa Koyanagi2, Hisao Tsukamoto2, Shinjiro Saeki3,
Kunio Isono3, Yoshinori Shichida4, Fumio Tokunaga5, Michiyo Kinoshita1,
Kentaro Arikawa1, Akihisa Terakita2 (1Dept Evol Stud Biol Sys, SokendaiHayama, 2Grad. Sch. Sci., Osaka City Univ., 3Grad. Sch. Information Sci.,
Tohoku Univ., 4Grad. Sch. Sci., Kyoto Univ., 5Grad. Sch. Sci., Osaka Univ.)
A pair of eyes of jumping spiders has a unique retina with four-tiered
photoreceptor layers, on each of which light of different wavelength is focused
due to chromatic aberration of the lens. Because the absorption spectrum ofvisual pigments is in general adaptively tuned to maximize visual information,
the visual pigment in each layer was thought to be sensitive to the wavelength ofthe light focused on the layer. However, interestingly, we previously found thatall photoreceptors in both the deepest and second-deepest layers contain agreen-sensitive pigment, although green light is only focused on the deepest
layer, indicating that the second-deepest layer always receives defocused
images. Given that defocused images contain distance information of the scene
in optical theory, we hypothesized that jumping spiders perceive distance fromthe amount of defocus in the second-deepest layer. In the present study, to test
the hypothesis, we conducted detailed behavioral analysis of distance perceptionof the spiders under monochromatic illumination by measuring distances ofjumps toward preys. Under red illumination, which generates a larger amount of
defocus in the second-deepest layer than green light, jumping spiders exhibitedshorter jumps than under green illumination. This result was in good agreementwith theoretical predictions of our model based on experimentally determined
optical parameters of the lens, strongly supporting our hypothesis that jumping
spiders perceive distance based on how much the retinal image is defocused.
2PT191酵母におけるニワトリクリプトクロム 4 の大量発現と特異的抗体を用いた精製

Overexpression of chicken cryptochrome4 in a budding yeast
and its purification using a specific monoclonal antibody
Hiromasa Mitsui, Toshinori Maeda, Chiaki Yamaguchi, Yusuke Tsuji, Yoko
Kubo, Keiko Okano, Toshiyuki Okano (Dept. Eng. and Biosci., Grad. Sch.
Adv. Sci. and Eng., Waseda Univ.)
A wide variety of living organisms on the earth have developed several
photoreceptive proteins for photosynthesis, visual perception, circadianphotoentrainment, seasonal reproduction, and so on. Cryptochromes (CRYs),
which are flavoproteins and are likely function as blue-light photoreceptor, havebeen found in bacteria, plants, and animals. Although CRYs are known to work
as central components in the circadian oscillator in mammals, many CRYs in all
kingdoms of life are considered to work as photosensors. The photochemistry
and photocycle of CRYs have been studied in plants and flies, but little is known
about the photoreception mechanism of vertebrate CRYs partly due to the lackof the expression and purification system of vertebrate CRY. We have tried toexpress vertebrate (chicken or zebrafish) CRYs in various host cells such as
Escherichia coli, insect cell line, mammalian cell line, and yeast, and here we
report a novel expression system for chicken CRY4 (cCRY4) using a budding
yeast, Saccharomyces cerevisiae. In this system, cCRY4 was constitutively
expressed and the expression level was approximately 500 g per liter, which iscomparable to a conventional system using E. coli. We also established apurification system of cCRY4 without any tag by immunoaffinity
chromatography using a monoclonal antibody to C-terminal region of cCRY4.
The purified cCRY4 was subjected to UV-visible absorption spectroscopy toanalyze its photocycle.
2PT192

青色光により誘起される転写因子 AUREO1 の構造変化

Blue-light induced conformational change of a transcription
factor, AUREO1
Osamu Hisatomi1, Ken Takeuchi1, Youichi Nakatani1, Fumio Takahashi2,
Hironao Kataoka3 (1Graduate School of Science, Osaka University, 2PRESTO,
JST, 3Botanical Gardens, Tohoku University)
Aureochrome 1 (AUREO1) found in Vaucheria has one basic region/leucine
zipper (bZIP) domain and one light-oxygen-voltage sensing (LOV) domain, andis believed to play a role as a blue light (BL)-regulated transcription factor1. To
clarify the molecular mechanisms of AUREO1, we prepared recombinant
AUREO1 proteins, containing full coding sequence (FL), bZIP and LOV
domains (ZL), and LOV domain. Recombinant AUREO1s showed absorption
and emission spectra typical of flavoproteins, and selectively bound to the target
sequence (TGACGT). The absorption around 450 nm decreased immediately
after the BL illumination, and was recovered to its original level with a half-lifeof about 6 min at pH 7 in the dark. Dynamic light scattering (DLS)− S138 −measurements demonstrated that the BL-induced change of the hydrodynamic
radius (RH) of LOV domain was slight. However, the RH of FL and ZL in thelight-adapted states were about 5% larger than those in the dark-adapted states.
The RH values were reverted to the dark-adapted level when illuminated FL andZL were kept in the dark. Our results suggested that FL and ZL underwent areversible conformational change upon illumination. Furthermore, the nonreducing SDS-PAGE assay indicated that ZL existed as dimers throughintermolecular disulfide bonds in bZIP domains. The conformational change
and dimer formation may play a crucial role for AUREO1 function.
1. Takahashi, F., et al. Proc. Natl. Acid. Sci. USA 104, 19625-19630 (2007).
2PT201

インシリコハイブリダイゼーション：新しい原核生物種の分類手法

In silico hybridization: A new tool for the classification ofprokaryotic cells
Kunio Ihara1, Tomomi Kitajima-Ihara1, Masahiro Kamekura2, Akinobu
Echigo3 (1Nagoya Univ., 2Halophiles Lab., 3Toyo Univ.)
What does species mean in prokaryotic cell? We, now, know even a single
mutation might cause a drastic phenotypic or biochemical changes. Therefore, it
is very difficult to delineate the species by comparing two prokaryotic cells onlyin phenotypic or biochemical point of view. At present, further including of
DNA-DNA hybridization (DDH) studies should be essential for establish thespecies concept. However, DDH studies are laborious and their reproducibility
among different laboratory would be marginal. Recently, it comes to easy to makecomplete genome sequencing by using next generation sequencer. We have tried
to make a quick and simple method for classification of prokaryotes using SOLiD(Sequencing by Oligonucleotide Ligation and Detection) sequencing system. For
the first trial, we have investigated the genus Haloarcula, because of ourinteresting of rhodopsin patchy distribution. We have sequenced 7 Haloarcula
strains including the type strain Haloarcula marismortui. Several millions of
50bp short reads produced by the SOLiD sequencer were de novo assembled
using the software velvet to produce thousands of contigs. Total short reads fromone strain were mapped against assembled contigs. Mapping process is to search
for the closely related region from the total genome, what we call, in silico
hybridization (ISH). The mapping values obtained in ISH were compared withthe experimental DDH values and their correlation constant was 0.97. We will
present in detail in the meeting.
2PT202

Homologous proteins with different fold: how were they raised ?
Shintaro Minami1, Kengo Sawada1, George Chikenji2 (1Grad. Sch. Eng.,
Nagoya Univ., 2Dept. Eng., Nagoya Univ.)
Currently some examples of protein pairs with evidence for evolutionary
relationships that fold into topologically different but share the same secondary
structure packing arrangement have been known. One of the most interesting
examples of such protein pairs is that of KH domains of hnRNP K andribosomal protein S3 [1]. They show statistically significant sequence similarity(38% identity) implying homology. However, their structures are quite different
in topology while converging to the same architecture.
Such examples suggest that proteins have changed their folds by segment
shuffling or rearrangment events while conserving the same core packing
arrangements. Further investigation of such protein pairs would give us a hint as
to how proteins can change their fold in the course of evolution.
In this work, we performed comprehensive database search for all possible pairs
which was classified into same superfamily in SCOP to encompass suchinteresting pairs of homologous protein. After the comprehensive search, we
found a number of homologous protein pairs which show non-topologicalstructural similarity. These pairs were investigated at a level of structure, aminoacids, and DNA, and we found some clues as to how these proteins can arise in
the course of evolution.
[1]N. V. Grishin, J. Str. Biol. (2001) 134:167-85
2PT203

全生物共通祖先以前のタンパク質のアミノ酸組成に実験的に迫る

Experimental approach to the amino acid usage of pre-lastuniversal ancestor
Masami Shimada, Satoshi Akanuma, Kozue Shinozaki, Yoshiki Nakajima,
Akihiko Yamagishi (Dept. of Mol. Biol., Tokyo Univ. of Pharm. Life Sci.)
Little is known about the nature of ancient proteins hosted by primitive
organisms that lived before the last universal common ancestor emerged.
However, it has been argued that primordial protein synthesis was much simpler

and involved less than 20 amino acids. Given simpler protein synthesis system,
the primitive proteins, which might have comprised a reduced set of aminoacids, must have had a sufficiently adequate structure for functional interactions
and catalysis. To address this issue experimentally, we used “protein
simplification engineering (1)” to examine whether a protein composed of lessthan 20 types of amino acids can retain its stable structure and biological
function. Using a thermally stable, ancestral-designed nucleoside diphosphatekinasase (Arc1) as the starting molecule, we constructed two reduced aminoacid set variants, Arc1-s1 and Arc1-s2, in which non-conserved M, Q, K, Y and
N and all of the five amino acid letters were replaced by other amino acids,
respectively. Because cysteine is absent from Arc1, Arc1-s2 consists of only 14
amino acid letters. Arc1-s1 and Arc1-s2 retain thermal stability similar to that of
Arc1. Arc1-s1 shows catalytic efficiency similar to that of Arc1; whereas, nodetectable level of catalytic activity was observed for Arc1-s2. Therefore, thefourteen amino acid types are sufficient to encode a thermally stable protein but
more amino acid types may be required for its function.
(1) Akanuma et al., PNAS 99, 13549 (2002)
2PT204

cDNA display 法による 4 種類のアミノ酸からなる機能ペプチドの創出

Generation of Functional Peptides Consisting of 4 Types ofAmino Acids by cDNA Display Method
Shigefumi Kumachi1, Miho Suzuki1, Koichi Nishigaki1, Yuzuru Husimi2,
Naoto Nemoto1 (1Grad. Sch. Sci. & ENG., Saitama Univ., 2Innovation
Research Organization, Saitama Univ.)
In general, proteins are composed of the 20 natural amino acids. However, howmany type of amino acids required for the creation of a functional protein
remains unresolved. Thus, we tried to design an experiment to examine thefunction of primitive proteins composed of a limited set of amino acids by invitro selection approach. Interestingly, it has been postulated by two different
approaches that the most primitive protein could consist of 4 types of aminoacids (i.e., glycine, alanine, aspartic acid and valine): 1) Eigen et al . (RNAworld hypothesis); and 2) Ikehara et al . (protein sequences analysis). So what
function would a primitive protein have in an early translation system? The firstprimitive protein would most likely be associated with the enhancement of
Ribozyme. Furthermore, in the early stages of protein synthesis, the genotypephenotype linking strategy, which is essential for molecular evolution, could
become a virus-type, because this strategy is the most efficient for protein
evolution of a single function. If this strategy is adopted, the interaction between
RNA and protein is indispensable. Thus, we investigated the possibility ofwhether peptides consisting of these 4 amino acids could interact with RNA by
cDNA display technology. Then, some peptides were selected to interact withtRNA with low affinity. This shows that a primitive protein consist of 4 types ofamino acids could evolve by the virus-type strategy in the early stage of
emerging translation system.
2PT205

Directed evolution of a self-encoding system using giant liposome
Takeshi Sunami1,2, Norikazu Ichihashi1,2, Takehiro Nishikawa1, Yasuaki
Kazuta1, Tomoaki Matsuura1,3, Hiroaki Suzuki1,2, Tetsuya Yomo1,2,4(1ERATO, JST, 2Grad. Sch. Info., Univ. Osaka, 3Grad. Sch. Eng., Univ.
Osaka, 4Grad. Sch. Biosci., Univ. Osaka)
Evolvability is a common property of living organisms, and gene replication
system is essential to allow the living system to evolve. We have constructed asimplified system, in which the genetic information is replicated by selfencoded replicase in liposomes. For the self-encoding system to evolve, thegenes must be compartmentalized and only a small number of them should bepresent in each compartment for genotype-phenotype linkage. The translated
replicase needs to amplify only the gene encoding itself. Here we demonstrate
the directed evolution of the self-encoding system by screening replicase
mutants. Cell-sized giant liposomes were used as a micro reactor, andfluorescence-activated cell sorter (FACS) was used to analyze and sort thereacted liposomes. The antisense of the beta-galactosidase gene was inserted
into the template DNA as a reporter gene. Beta-galactosidase was transcribedfrom the minus strand RNA replicated from plus strand RNA, and the green
fluorescence from the hydrolyzed products by beta-galactosidase was evaluatedas self-replication activity. Fluorescent liposomes with higher active replicase
gene were sorted by FACS. The DNA templates for next selection round were
amplified from the selected plus strand RNA in a test tube. After 69 rounds of
selection, the self-replication activity of the selected mutants was evaluated inliposomes, and the mutants were higher active than wild-type replicase. The
result indicates that the simplified gene replication system has evolvability as
well as extant cells.

− S139 −2PT206

枯葉に擬態した蛾・蝶の翅模様に実装された正確な擬態をもたらす
モジュールデザイン

Modular spatial stabilization of leafy moth/butterfly wing
pattrns for precise mimesis

2PT209

Takao K. Suzuki, Hideki Sezutsu (NIAS TG_SilkwormUnit)
In nature, where yuragi is pervasive, many features of animal’s structures are
assumed to be so perfectly suited to their function that they seem unlikely tohave arisen by chance. Among these features, mimetic patterns, resemblance tonatural objects such as leaves, seem to be a typical example implementing a
specific design to deal with yuragi. However, how the mimetic leafy patterns are
established to manage yuragi has been poorly understood. Here, we developed anovel method, morphometrical network analysis, to reveal how the leafy
moth/butterfly wing patterns are spatially coordinated. As model organisms, weused two leafy wing patterns: a moth, Oraesia excavata, and a butterfly,
Kallima inachus. Our quantitative analysis indicated that in both organisms theconstituent components of the leafy patterns are tightly coupled as modules,
each of which corresponds to each component of the leaf venation pattern (i.e.,the main, right-hand, and left-hand venation patterns). Interestingly,
phylogenetic analysis showed that the modules detected in two organisms
evolved independently, strongly suggesting that the modules of two leafy
patterns were needed for mimetic performance. The establishment of modules
allows within-module components to be put together, suggesting that the leafy
pattern is precisely stabilized by implementing the spatially-synchronizedarrangement of its subordinate leaf-venation components. Our results may
suggest that the modular design likely improve the visual trick of leafy mimesis
for predator avoidance.
2PT207

coefficient is usually underestimated. It was shown that many quasi-repetitive
sequences were locally found in proteins in addition to those found in theprevious work. These results would be a supporting data for our hypothesis on
the amino-acid sequences of the earliest proteins.

巨大化大腸菌(GP)からの大腸菌再生過程の観察

人工共生系の実験進化

Experimental evolution of a synthetic symbiosis
Kazufumi Hosoda1, Akihiro Asao2, Shingo Suzuki1, Tetsuya Yomo1,2,3 (1IST,Osaka-u, 2FBS, Osaka-u, 3ERATO, JST)
Most organisms belong to some mutually beneficial symbioses (mutualisms) innature. Despite their ubiquity, mutualisms are evolutionarily vulnerable,
because they are based on cooperative behaviors which do not necessarily
provide direct benefit to the own fitness. Roughly speaking, if organisms only
optimize their own fitness, mutualisms seem to collapse due to the emergence of“selfish” one that abandons the cooperative behavior. Various theories based on
the natural selection (ultimate factor) have been proposed to fill the gap between
the ubiquity and the vulnerability, but little is known about the insight into thephysical/structural property of organisms (proximate factor) that affects theevolution of mutualism. As it is very challenging to follow the evolution ofnatural mutualisms at the molecular level, studying experimental evolution of asynthetic symbiosis, which is composed of previously non-interacting different
populations, is effective to gain an understanding of the structural insight(Momeni et al. 2011). Here we show that a synthetic mutualism composed of
two genetically engineered Escherichia coli strains did not collapse but
developed in ~1,000 generation. The strains actually increased their own growthability in the evolution, but they did not lose their cooperative ability. With theresults of comprehensive analyses showing how their intracellular statechanged, we want to discuss why they developed, not being too selfish.
2PT210

束論を用いた種の遷移における頑健性と最適性の研究

Observation of reproducing process the E. coli bacterium froma giant protoplast
Kazuhito Tabata1, Takao Sogo1, Shoji Takeuchi2, Hiroyuki Noji1 (1App.
chem. Univ. Tokyo, 2IIS Univ. Tokyo)

The robust and the optimal adaptation in species-transition
network from the perspective of the lattice theory
Takayuki Niizato, Yukio Gunji (Grad. Sch. Sci., Univ. Kobe)

We report to reproduce into the original bacteria from a giant protoplast (GP),having a diameter in excess of 10 m and prepared from a single Escherichia
coli bacterium. Bacteria have long been known to be capable of gigantism.
When ampicillin is added after a lysozyme treatment in an isotonic solution for
culturing E. coli, cell wall synthesis is inhibited and division does not occur; thisresults in gigantism and the relatively easy formation of a GP with a diameter
larger than 10 m. A GP formed through such abnormal gigantism is believed tohave already lost the ability to divide and proliferate. However, when this GP
was confined in a microchamber and observed for more than 10h, we found thatthe spherical GP deformed and divided. This result may indicate that E. coli
maintains its life system and is in a functional state even after becoming a GP
and experiencing an extreme change in its intracellular environment. We
showed that the GP have an ability to divide and return to its original bacteriacell using the miriochamber. We investigated the extent to which the gene
expression system inside the GP was stable with respect to the duration of GP
transformation culturing and found that protein expression ceased
approximately 10 h after culturing. This finding is consistent with the fact that
E. coli also stopped increasing in size in approximately 10 h, suggesting that
10 h is the critical line for the E. coli life system.

We investigate the adaptive behavior of species to a given environment. We
here focus on two contradictory aspects of the adaptive process in evolution.
The one is the optimization implying that species increase their own fitness asmuch as possible in a given environment. The other one is robustness implyingthat species sustain the inherent diversity despite changing environments. We
address this issue to construct a model by using a lattice theory. A crucial pointin our model relies on the novel idea of "multiplicity of fitness" which can covermalfunctioned individuals. This idea is partly taken in nature of exaptation. The
multiplicity of fitness, in our model, would emerge as an overestimation of thefitness (each individual tends to overestimate own fitness compared with
underlying fitness). This overestimation is not random, but context-dependent,
represented by a lattice structure. We here show that species in our model
demonstrate the power law of the lifespan distribution and 1/f fluctuation for theadaptive process without any parameter tuning. We also discuss essential
difference between the behavior for adapting process and stochastic parametertuned process. Using a lattice theory enables us to construct a simple model ofthe adaptation equipped with the multiplicity of fitness, because we can addressa complex fitness landscape by an algebraic ordered set. Furthermore, we
provide new approach to the network theory by using lattice theory.

2PT208

天然のタンパク質のアミノ酸配列における隠れた局所的な反復性についての考察An investigation on local pseudo-periodicity in the amino-acidsequences of native proteins
Koji Inai, Masahito Oka (Osaka Prefecture University)
In the previous work, we tried to investigate the repetitive amino-acid sequencesin proteins by using currently available protein data base. A total of 8286132
proteins have the repetitive amino-acid sequences among whole 12619557
proteins, suggesting that repetitive amino-acid sequences are generally found in
the native proteins. Then, using autocorrelation function method, we also tried
to investigate the cryptic periodicity of amino-acid sequence, which cannot befound in the usual method aimed at the strict amino-acid sequence. It was shownthat many proteins have quasi-repetitive sequences by these analyses. In thiswork, it is further tried to investigate on local pseudo-periodicity which was
failed to notice in the full sequence analysis because its autocorrelation

2PT211

Study on competitive reaction between enzymes with different
diffusivity

Kenta Yashima1, Jun Nakabayashi2, Akira Sasaki3 (1Meiji Univ., 2Yokohama
City Univ., 3The Graduate University for Advanced Studies)
Diffusion process of molecules within heterogeneous medium shows a peculiar
behavior termed anomalous diffusion. Here the motions of molecules arehindered by the obstacles, which constitute the heterogeneity of theenvironment. Accordingly, compared to homogeneous medium, the area that amolecule moves for a given time is smaller for heterogeneous medium. Thisaffects the reaction process deeply (especially for diffusion limited reactions)and the classical formulation based on mass action law fails to describe theprocess and a formulation based on the Fractal reaction theory is necessary[Kopelman, Science 1988]. In this study we analyzed the competitive reaction
between enzymes with different sizes. We assume that for larger enzymes
obstruction due to molecular crowding makes the environment heterogeneous toit. On the other hand for smaller enzymes the environment is assume to be− S140 −homogeneous due to smaller obstruction effects from the molecular crowding.
Accordingly there is a difference in the diffusivity, one with normal diffusivity
and the other with anomalous diffusivity. We would like to discuss thephysiological implications of this effect using Lattice Monte Carlo simulationand theoretical analysis based on the Fractal reaction theory.
2PT212

Counting statistics for genetic switches
Jun Ohkubo (Grad. Sch. Informatics, Kyoto Univ.)
The number of switching between activate and inactivate states in a genetic
switch is investigated. Counting statistics is a scheme to study the number of
specific transitions in a stochastic system. While the counting statistics has beenstudied a lot for a stochastic system with a finite number of states, the genetic
switches are formulated as a system with an infinite number of states. Hence, itis needed to check the applicability of the counting statistics for the genetic
switches. Actually, it immediately becomes clear that a straightforward
application of the scheme derives intractable non-closed equations. We here use
an effective interaction approximation, and it is shown that the switchingproblem can be treated as a simple two-state model approximately, whichindicates that the switching obeys non-Poisson statistics.

2PT213

分化多能性を持つ細胞の分化初期段階における状態遷移

Transitions of the cell state in the early stage of differentiationfrom the pluripotent cell
Kou Makishi, Tomoki P. Terada, Masaki Sasai (Dept. of Computational
Science and Engineering, Nagoya Univ.)
Embryonic stem (ES) cells have the ability to differentiate into all three germlayers, mesoderm, endoderm and ectoderm. This capability of ES cells is called
pluripotency, and it has been shown that Sox2, Oct4, and Nanog (SON) are theimportant genes for maintaining pluripotency. We built a model of the networkof core genes including SON, and performed the stochastic simulation of theearly stage of differentiation starting from the pluripotent ES cells. We take into
account three types of processes to regulate the gene expression in the model;
binding/unbinding of transcription factors (TF), formation of the transcription
apparatus (TA), and the modification of epigenetic histone code. The simulated
population of pluripotent cells becomes phenotypically heterogeneous showing
the different patterns of gene activity in individual cells when the rate offorming TA is slower than the rate of change in the copy number of TF. We will
discuss the diversity of patterns of gene activity arising from the difference inthe time scale among the processes to regulate the gene expression.
2PT214

シロイヌナズナにおける植物ホルモンの制御ネットワーク解析

Regulation network analysis of plant hormones in Arabidopsis
thaliana
Mariko Miyamoto, Hiraku Nishimori, Akinori Awazu (Dept. of Mathematicaland Life Sciences, Hiroshima University)
The plant hormone is low-molecular-weight chemical that is produced in the plant
body and affects the plant activity in minute amounts. The plant produces several
kinds of plant hormones and hormone-like substances which play the roles of thesignaling molecules for gene expressions related to the growth, stress response,
etc... In general, each of these hormones and hormone-like substances does notwork independently but works cooperatively or competitively with each other,and the production of them are mutually regulated.
In this study, we extrapolate the regulation network among several plant
hormones and hormone-like substances by the statistical analysis of the databaseof gene expression levels of microarray. We analyze the data set released in theInternet produced by “At Gen Express” project through the statistical methods
such as Gaussian graphical modeling. Based on this analysis, we also construct
the dynamical system model of the plant hormone regulation network of
Arabidopsis thaliana to unveil physiological mechanism of the stress response
and adaptation against the environmental fluctuation.
2PT215

Toward simulation of epidermal growth factor (EGF) pathway
at the molecular resolution for K computer

Kazunari Iwamoto, Kazunari Kaizu, Koichi Takahashi (Laboratory for
Biochemical Simulation, QBiC, RIKEN)
Cellular environment consists of the localization of various proteins, clusteringof membrane molecules and cytoskeleton. Recently, the simulation at themolecular resolution indicated that characteristic of molecular network such asbistable and ultrasensitivity under such environment was different fromprediction by conventional non-space simulation, which was supported by invitro experiments. Therefore, the simulation at the molecular resolution is usefulto understand the characteristic of cellular signaling pathways. In this study, we
will implement the simulation at the molecular resolution of epidermal growth
factor (EGF) signaling pathway which regulated cell proliferation anddifferentiation, and elucidate the characteristic.
The simulation software at the molecular resolution, Spatiocyte, was developed
by Arjunan et al. (2010). However, it was difficult to execute the cellular-scale
simulation at the molecular resolution using Spatiocyte. Thus, we aredeveloping new software for supercomputer, especially K computer, based on
the algorithm of Spatiocyte. On the other hand, prototype model of EGF
pathway was constructed and preliminary simulation at one hundredth scale ofactual cell was implemented. The simulated results showed heterogeneity of
ERK activation, which qualitatively agreed with observed data. Further analysis
suggested that this heterogeneity resulted from the minority of specific species
in EGF pathway.
2PT216Emergence of a spatial structure with a minority molecule in a
catalytic reaction network
Atsushi Kamimura, Kunihiko Kaneko (Dept. of Basic Science, The Univ. ofTokyo)
It is crucial to explain the emergence of primitive cellular structure from a set ofchemical reactions to unveil the origin of life and to experimentally synthesize
protocells. Recently, we considered a hypercycle with two mutually-catalyzing
chemicals to demonstrate that the reproduction of a protocell with a growthdivision process occurs when the replication and degradation speeds of one
chemical are, respectively, slower than those of the other chemical, andmolecules are crowded as a result of replication. In this presentation, we discuss
the effects of the crowding molecule in details on the formation of primitive
structure and also show results when we consider more complex reaction
networks.
2PT217

Complex biological networks from the standpoint of thecategory theoretical duality
Taichi Haruna (Kobe University)
In general, there are two pictures on directed biological networks. The first
picture is structural one: a network is a collection of passages on whichsomething flows. In the second picture, networks are seen as more functional
entities: a network is a collection of local workings of the biological system
which the network represents glued together. In our previous work, we showedthat these two pictures can be described as a category theoretical duality and twopath notions corresponding to each picture emerge as a result of a category
theoretical universality. Conventional directed path and a path notion called
lateral path correspond to the first and second pictures, respectively. Here, toexamine how this duality is embedded in real directed biological networks, we
introduce lateral betweenness centrality and directed betweenness centrality ofarcs based on the corresponding path notions. Both centralities measureimportance of a given arc by counting the number of shortest paths in each pathnotion that pass through the arc. We found that there exists a significant tradeoff relationship between them in real biological networks. We show that thetrade-off relationship can emerge mainly from an optimization processmaximizing efficiency with respect to lateral path by numerical simulations. We
also found that the optimized networks have similar qualitative features withreal biological networks in the view of standard network analysis.
2PT218分子混み合いが小胞内反応系に与える影響の理論的考察

Theoretical study of influence of molecular crowding to
enzyme reaction system in vesicle
Masashi Fujii, Hiraku Nishimori, Akinori Awazu (Grad. Sch. Sci., Hiroshima
Univ.)
Several living systems contain a variety of macromolecules, and the large
number of such molecules is confined in small internal spaces of cells or
intracellular organs. Some recent studies pointed out that the reactions anddiffusions of these molecules are extremely restricted by their excluded volume.
In this study, we investigate the effects of the excluded volume of densely
confined molecules, commonly called `molecular crowding' effects, to thebiochemical reaction. In order to consider this issue, we propose a coarse-− S141 −grained model of the enzyme reaction in a small vesicle, where the reaction
process is inspired by the sugar-chain scission reaction of the lysozyme.
In this model, the enzyme/substrate is constructed by five/two large particles
constructing the chain-like molecular form with three small particles playing therole of binding sites to the substrate/enzyme. We assume when the each bindingsite of an enzyme binds to that of the substrate, the substrate becomes the
products.
By the Brownian dynamics simulation of the present model, the rate of the
product formation in the initial stages is obtained as a bell-shaped function of
the initial substrate density. We also observe that the reaction progresses
intermittently like “stick-slip motion” when the initial substrate density is high,while the reactions occur smoothly when the initial substrate density is low.
These results indicate that the molecular crowding provide the glassy state of thebiochemical reaction in vivo.
2PT219Temporal Decoding of MAP Kinase and CREB Phosphorylation by Selective Immediate Early Gene Expression
Takeshi Saito1, Shinsuke Uda1, Yu-ichi Ozaki1,2, Shinya Kuroda1(1Department of Biophysics and Biochemistry, Graduate School of Science,
University of Tokyo, 2Laboratory for Cell signaling Dynamics, Quantitative
Biology Center, RIKEN)
A wide range of growth factors encode information into specific temporal
patterns of MAP kinase (MAPK) and CREB phosphorylation, which are further
decoded by expression of immediate early gene products (IEGs) to exert
biological functions. However, the IEG decoding system remain unknown. We
built a data-driven model based on time courses of MAPK and CREB
phosphorylation and IEG expression in response to various growth factors to
identify systems. We found that IEG expression uses common decoding systems
regardless of growth factors and expression of each IEG differs in upstream
dependency, switch-like response, and linear temporal filters. Pulsatile ERK
phosphorylation was selectively decoded by expression of EGR1 rather than cFOS. Conjunctive NGF and PACAP stimulation was selectively decoded bysynergistic JUNB expression through switch-like response to c-FOS. Thus,
specific temporal patterns and combinations of MAPKs and CREB
phosphorylation can be decoded by selective IEG expression via distinct
temporal filters and switch-like responses.
2PT220

ショウジョウバエ翅原基における成長制御メカニズム

Growth control mechanism in the Drosophila wing discKen-ichi Hironaka1,2, Yoh Iwasa2, Yoshihiro Morishita1 (1CDB, RIKEN, 2Grad.
Sch. of Sys. Life Sci., Kyushu Univ.)
In the Drosophila wing disc, whereas BMP-homolog Dpp directs AnteroPosterior patterning, it also functions as a growth factor for the tissue. But
underlying mechanism of Dpp-dependent growth control is not considered asimple concentration-dependent manner, because tissue grows uniformly undergraded Dpp profile and grows nonuniformly under uniform Dpp profile.
Recently, Wartlick et al. (2011) proposed that cell division is triggered bytemporal change in Dpp concentration. We test the validity of this hypothesis bya constructive approach. In this presentation, we report that some combinationof network motifs can realize the hypothetical growth control mechanism of
Wartlick et al.(2011).
2PT221

ミドリゾウリムシの細胞内共生における最適行動

Optimal Behavior in Endosymbiosis in Green Paramecium
Sosuke Iwai (Faculty of Education, Hirosaki Univ.)
Paramecium bursaria, or green paramecium, is a unicellular ciliate that contains
several hundreds of chlorella-like green algae in its cytoplasm as endosymbionts.
It is generally believed that the symbiosis is mutualism, in which the hostparamecium provides the symbiont alga with nitrogen sources such as aminoacids, while the symbiont provides the host with photosynthetic products. To gain
insights into mutualistic endosymbiosis, here we have constructed a simple modelfor the endosymbiosis in Paramecium bursaria and determined optimal behavior
both for the host and for the symbiont. Assuming that the fitness of both of twoorganisms is linearly related to the amounts of carbon and nitrogen sources gainedby two organisms, we revealed the optimal behavior of both of two organisms andalso the conditions that allow the mutualistic endosymbiosis. The experimental
verification of the model will be discussed.

2PT222

2 次元セルオートマトンモデルによる細胞の自己形成及び自己複製のシミュレーション

Two-dimensional cellular automata simulation of the selfassembly and self-reproduction phenomenon of cellTakeshi Ishida (Nippon Institute of Technology)
Understanding the generalized mechanism of self-assembly and selfreproduction phenomenon of cell. is considered to be fundamental for
application in various fields such as mass-production of molecular machines ofnanotechnology and artificial synthetic of biology (synthetic biology). Thus wehave to elucidate not only the details of the real cellular reaction network but
also the necessary conditions for self-organized, self-replicating cells.
We developed a model for simulating cellular self-organization in twodimensional cellular automaton. We demonstrated that the following 3 functions
can be realized. (1) Emergence of cell (emergence of border (like cell
membrane) and information carrier (like DNA). (2) Self-replication is achieved
while maintaining a carrier containing information. (3) The division of the cell
membrane is achieved while maintaining the total structure.
The previous self-reproduction models were two types, first is Neumann modelthat was very complicated one, second is Langton model that was very simple
shape. Our study was able to show a self-reproduction phenomenon of the shapethat was nearer to a living cell for the first time. This is not study to clarify all thenecessary and sufficient conditions of the self-reproduction. It is thought that it
can simulate the self-replication phenomena in real dynamic, chemical reaction
environment applying the transition rules that became clear by this study.
2PT223神経細胞の過渡的ダイナミクスにより神経情報コーディングがどのように制限されるか

Restriction of the information-coding scheme in the nervous
system based on transient dynamics of neuronal activity
Takanobu Yamanobe1,2 (1Sch. Med., Hokkaido Univ., 2PRESTO, JST)In digital circuits, information is encoded on the basis of pulse patterns. To thisend, pulse generation is independent of previous device activity. The rise andfall times of a pulse corresponding to the duration of its transient regime should
be sufficiently short to achieve this independence. Otherwise, pulse generation
will be dependent on previous activity because the transient regime affects thegeneration of other pulses. If the nervous system uses spike patterns to transmit
information, as in the case of pulses in digital circuits, spike generation should
be independent of previous neuronal activity. Thus, understanding the transient
dynamics of a single neuron is crucial to elucidating the information carrier. In
this study, we analyze the transient dynamics of a stochastic neuron model.
First, we introduce a linear Markov operator that relates the density evolution
between two consecutive input impulses. We show that the products of theoperators (also linear Markov) reproduce the dynamics of the entire phase spaceof the neuron model in response to impulses. Next, we introduce a stochastic
rotation number of the neuron model, which makes it possible to relate thedensity evolution of the neuron model to the number of generated spikes.
Furthermore, the dynamics of the products of the operators is decomposed into
invariant and transient parts. Thus, we evaluate the amount of the transientcomponent, and we discuss the possible information carrier on the basis of its
properties.
2PT224

Spontaneous and Evoked Neural Activities Shaped through A
Sequential Learning Process

Tomoki Kurikawa, Kunihiko Kaneko (Univ. Tokyo, Grad. Sch. Arts. Scis.)
In neural science, neural response activities to external stimuli are generally
recorded in order to understand neural computation. In this scheme, spontaneous
activity in the absence of an input is disregarded as a back ground noise.
However, recent experimental studies have suggested that the spontaneous
activity plays a role in information processing and this role is not unclear. In
order to uncover the role, in our previous study, we proposed a novel memory
view and introduced a neural network model in which network structure isgiven. We found that when a neural network responds to a memorized input
significantly, the spontaneous activity wanders among the memorized patterns,
which is similar to the behavior typically observed in the neural system. In thisstudy, we introduce a model with a simple learning rule. By this rule, we cananalyze the spontaneous and evoked (memorized) activities for larger class of
network structures than the given structure in the previous model. As results, wefound that two types of behaviors are shaped: One behavior shows a stationary− S142 −activity both in the absence and presence of the learned input and does not
respond to the input. The other behavior shows chaotic behavior wandering
among the memorized patterns in the absence of the input and responds
significantly to the input. Remarkably, the network structure for the latter issimilar to that in the previous given model. It suggests that wandering
spontaneous behavior is helpful to respond to external familiar stimuli.
2PT225大腸菌の複数の鞭毛モーターの回転方向の協調的な切り換え現象の理論

A theory of the coordinated switching in the rotational
direction of multiple flagellar motors in a single bacterium
Toshinori Namba1,2, Tatsuo Shibata2 (1Department of Mathematical and Life
Sciences, Hiroshima University, 2RIKEN CDB)
Many living organisms have systems to respond to the change in external
environments, such as signal transduction systems. Such systems responsible formany important processes consist of complex interaction of molecularcomponents. In these systems, molecular signals need to propagate in thecytoplasm. Terasawa et al. have recently reported a coordinated switching in therotational direction of the multiple flagellar motors in an E.coli [1], suggestingthat signaling molecules could propagate in cytoplasm to coordinately regulate
the motors. The bacteria regulate the swimming behavior by controlling therotational direction of flagellar motors to respond to extracellular stimuli. Thus,
this coordination in the motor rotation may be important for the efficientregulation of the swimming behavior. This coordinated motor behavior can be
explained by a spatial propagation of response regulator molecule. In thispresentation, we propose a mathematical model of spatial regulation of response
regulator molecules. We show possible behaviors of the signal molecules in thecytoplasm, such as wavelike propagation. Based on such a theoretical results,
we will discuss the condition of the coordinated motor behaviors.
[1] S. Terasawa et al. (2011) Biophys. J. 100, 2193-2200.
2PT226

走化性真核細胞の変形と勾配認識

Gradient sensing of deformed eukaryotic chemotactic cells
Akinori Baba, Tetsuya Hiraiwa, Tatsuo Shibata (CDB, Riken)
Eukaryotic chemotactic cells sense the gradient of ligand molecules from thenoisy input of the ligand binding/unbinding of the receptors distributed around thesurface. For example, Dictyostelium Discoideum cells are possible to sense ashallow (about 1-2% of) gradient. To understand the eukaryotic chemotaxis, atheoretical model has been studied in terms of the maximum likelihood estimationof the chemical gradient from the noisy input. If the gradient is sufficiently
shallow and/or the ligand concentration is sufficiently dilute, it is hard for a cell tofind the correct direction from the ligand binding/unbinding of the receptors. In
this case, the estimated direction of the gradient is strongly affected by the input
noise. We consider the cell deformation to an elliptical shape in this model. For
the shallow limit of the gradient, the estimated direction for an elliptical cell isanisotropic due to the anisotropic distribution of the receptors. We will discuss thecomparison between the two scenarios of the gradient estimation: thesimultaneous estimation of the steepness and the direction, and the estimation of
only the direction with knowing the correct steepness.
2PT227

アメーバ運動による 3 次元経路探索の理論的考察

dimensional jungle gym that imitates complex structure within organs. This result
implies that the strategy of amoeboid migration is versatile for any environment.
2PT228

Daisuke Takagi, Kazumi Hakamada, Jun Miyake (Osaka university)
Genotype does not always agree with the phenotype, though cells can maintaintheir function. Such inconsistency leads us the idea that the system is maderobust. In recent study, many reports try to elucidate their robustness and stableof the system using single cell analysis. Taniguchi et al. reported the distributionof gene expression follow gamma distribution, and Ozbudak et al. reported themulti-stability of bacterial system. These suggest it is difficult to elucidate thesystems without understanding their distribution. In this study, four deletionmutants of Escherichia coli (Δ flhC, Δ yhjH, Δ yfiN, and Δ ycgR) and theirparental strain BW2513 were selected and distributions of motility of single
cells were observed. The peaks of their logarithmic distributions were observed
3. 7 (BW25113), 3. 2 (Δ flhC), 3. 5 (Δ yhjH), 4. 3 (Δ yfiN), and 5. 5 (Δ ycgR),
respectively and they had statistical significance (p <0.001). The peaks of ordercorresponds to the function of genes. In the case of using ΔycgR, the bimodal
distribution of was given and it means this system has multi-stability. On thecontrary, unimodal distribution was given in the case of using BW25113. This
result suggests ycgR plays important role of giving stability of the system offlagella. It is necessary to construct mathematical model to elucidate the system.
2PT229Shin I. Nishimura (Hiroshima University)
Amoeboid cells such as lymphocytes migrate within collagen web matrix andpass through narrow channels between cells. It is known that T lymphocytes
inhabiting lymph nodes migrate with searching antigen presenting cells (APC)that present acceptable antigens. Neutrophils appearing from vessels should findand catch external microbes within the body. Amoeboid cells in our body should
migrate within complex structure. It is not well known how those cells arrive atappropriate positions through complex maze-like substrata. Even if externalsignal guides them, amoebae sometimes turn their direction to avoid obstacles.
How they keep persistent migration even in such obstacle-rich space? To answer
those questions, I construct a model for a three-dimensional amoeba andobstacles. The model cell has membrane, actin and control factor (CF) thatprevent actin polymerization. CF is assumed to accumulate by retraction but
become diluted by protrusion. This mechanism amplifies fluctuation of CF
distribution and leads to directional migration of the cell. The simulated cellshows amoeboid-like migration not only on plane surface but also in a three-

時計タンパク質 KaiB-KaiC 相互作用間の ATP による制御

The regulation of cyanobacterial circadian clock proteins
KaiB-KaiC interaction by ATP
Risa Mutoh1, Atsuhito Nishimura2, So Yasui2, Kiyoshi Onai2, Masahiro
Ishiura2 (1Institute for Protein Research, 2Center for Gene Research)
The cyanobacterial circadian clock oscillator is composed of three clock
proteins called KaiA, KaiB and KaiC. The oscillator generates circadianoscillations in the phosphorylation level, ATPase activity of KaiC and complex
formation among the three Kai proteins in the presence of ATP. KaiC is ahomohexameric ATP-binding protein with autokinase activity and very weaktemperature-independent ATPase activity. The KaiC subunit has a duplicatedstructure, which is composed of N-terminal and C-terminal domains, and eachdomain has a series of ATPase motifs (a Walker's motif A, a Walker's motif B
and a catalytic glutamate (CatE)).
In the absence of ATP, KaiC was monomeric and formed a complex with KaiB.
In the presence of Mg2+ and ATP (Mg-ATP), KaiC became a homohexameric
ATPase with bound Mg-ATP and formed a complex with KaiB, whereas the
KaiCs hexamerized by unhydrolyzable substrates such as ATP and Mg-ATP
analogs did not. KaiC N-terminal domain protein formed a complex with KaiB,
whereas its C-terminal one did not, indicating that KaiC associates with KaiB
via its N-terminal domain. A hexameric KaiC mutant lacking N-terminal
ATPase activity did not form a complex with KaiB, whereas that lacking Cterminal ATPase activity formed the complex. Thus, the hydrolysis or release of
KaiC-bound ATP by N-terminal ATPase motifs is required for the complex
formation.
2PT230A theoretical study for three-dimensional path finding byamoeboid migration

一細胞分布解析を用いた大腸菌運動性の非遺伝的な影響評価

Single cell distribution analysis of motility reveals theheterogeneity of Escherichia coli

時計タンパク質 KaiC の ATP 放出

ATP release from cyanobacterial circadian clock protein KaiC
Keita Iwata1,2, Risa Mutoh3, Kiyoshi Onai1, Masahiro Ishiura1 (1Center for
Gene Research, Nagoya Univ., 2Sch. of Sci., Nagoya Univ., 3Institute forProtein Research)
Cyanobacteria are the simplest organisms that exhibit circadian rhythms. The
cyanobacterial circadian clock oscillator is composed of three clock proteins
called KaiA, KaiB, and KaiC. KaiC has autokinase and ATPase activities. KaiC
subunit has a duplicated structure, which is composed of N-terminal and Cterminal domains, and each domain has a series of ATPase motifs (a Walker's
motif A, a Walker's motif B, and a catalytic glutamate (CatE)). KaiC subunit has
two phosphorylation sites, Ser431 and Thr432, on its C-terminal domain. In thepresence of ATP, ATP binds to the ATPase motifs of KaiC and induces thehexamerization of KaiC.
Here, we investigated ATP release from KaiC by the real time monitoring of
bioluminescence using firefly luciferase. We measured the amount of ATP
released from KaiC, using wild-type KaiC and mutant KaiCs with a mutation in
the phosphorylation sites and ATPase motifs. We observed different ATP− S143 −releasing patterns between with and without KaiB, suggesting that KaiB may
enhance ATP release from KaiC.
2PT231概日周期の温度 / 栄養補償性に関する理論的研究

Theoretical study of temperature and nutrient compensationof circadian clock
Tetsuhiro Hatakeyama, Kunihiko Kaneko (Graduated school of Arts andScience, Univ. of Tokyo)
The circadian clock satisfies three criteria: persistence in constant conditions,
phase resetting by light/dark signals, and temperature/nutrient compensation of
the period, which is our concern here. In general, the period of chemical
oscillator is very much sensitive to kinetic rate constants, which increase
strikingly with temperature. Thus, the temperature compensation is an
uncommon property of biochemical oscillators. Here, we propose a novel
mechanism for this temperature compensation. Kondo and his colleagues
recently demonstrated that the period of circadian oscillation of Kai-proteinsindeed is temperature compensated. By simulating a model with phosphoration/dephosphoration cyclic reactions (extended from van Zon, et al., 2007), we first
demonstrated the temperature compensation of the period indeed works over abroad range of temperatures, without the need for tuning parameters in thereactions. We then unveiled the mechanism for it as " enzyme-limitedcompetition". We also showed that the nutrient compensation for ATP/ADP
ratio is achieved by same mechanism as temperature compensation.
2PT232

シアノバクテリアの概日時計は低温で Hopf 分岐を介して消失する

Cyanobacterial circadian clock is nullified by low temperature
through Hopf bifurcation
Yoriko Murayama1, Hiroshi Kori2,3, Takao Kondo4,5, Hideo Iwasaki1,3,
Hiroshi Ito2,6 (1Grad. Sch. Sci. Eng., Waseda Univ., 2Fac. Sci., Ochanomizu
Univ., 3PRESTO JST, 4Grad. Sch. Sci., Nagoya Univ., 5CREST JST, 6Fac.
Design, Kyushu Univ.)
Physiological rhythms with a period of approximately 24 hours are calledcircadian rhythms. One of the key characteristics of all circadian rhythms is thatthe free-running period remains stable under a relatively broad range of ambient
temperatures, referred to as “temperature compensation” of the period. Outsideof the range of temperature compensation, circadian clocks stop running and arearrested at a certain phase.
Dynamics of self-sustained oscillator such as circadian clocks has been studied
in the field of nonlinear physics. Qualitative changes in the dynamics asparameters of a system are varied are called “bifurcations”. Based on thebifurcation theory, Hopf bifurcation and saddle-node bifurcation are plausible
scenarios of circadian arrhythmia at low temperature.
The cyanobacterial circadian timing in Synechococcus elongatus PCC 7942
requires neither de novo transcription nor translation, and the posttranslational
oscillation can be reconstituted in a test tube using only three clock proteins,
KaiA, KaiB, and KaiC. The KaiC phosphorylation rhythm in vitro is best to
observe directly and precisely dynamics of circadian oscillator. We found thatthe phenomena of nullification of KaiC phosphorylation rhythm by low
temperature were explained by theory of Hopf bifurcation. We will reportinteresting phenomena predicted from the theory, and discuss fitness andphysiological implications of circadian clock under lower temperature
condition.
2PT233

チャコウラナメクジ嗅覚神経系に見られる時空間活動パターンとその非線形解析

Spatiotemporal Patterns of Neural Activities in the OlfactoryCenter of the Land Slug and the Nonlinear Analysis
Tomoya Shimokawa1, Yuuta Hamasaki1, Yoshimasa Komatsuzaki2, Minoru
Saito1 (1Graduate School of Integrated Basic Sciences, Nihon University, 2Collegeof Science and Technology, Nihon University)
Rhythmic patterns of electrical activity are a ubiquitous feature of nervous
systems. The modulation of oscillatory activity plays an essential role in theprocessing of sensory information. In the present study, we examined the odor
responses of the oscillatory activity in the olfactory center (procerebrum; PC) of
the land slug Limax valentianus by extracellular recording, and analyzed them bywavelet analysis. The local field potential (LFP) of the PC showed an oscillationof about 1 Hz, and the oscillatory activity was changed by various odor stimuli tothe tentacle. Prior to the odor stimuli, the wavelet energy was distributed intosome frequency ranges. After the aversive odor stimuli, the wavelet energy

concentrated mainly into two ranges and the wavelet entropy decreased. These
results suggest that the activities of neurons in the PC became more coherent inresponse to the aversive odor stimuli. To confirm it, we then examined thespatiotemporal patterns of neural activities in the PC by fluorescent voltage
imaging technique. The PC preparation was stained with a voltage-sensitive dye,
Di-4-ANEPPS. The stained preparation was illuminated by a LED (530 nm;
LEX2-G, Brain Vision), and the 705 nm fluorescent images were acquired
through a CCD camera (iXon X3 897, Andor). As a result, an oscillation offluorescent intensity was observed in the PC, and the oscillation had a phase delay
along the distal-proximal axis. After aversive odor stimuli, the phase delay
disappeared, which showed more coherent activities of neurons in the PC.
2PT234
Motion analysis of collective soldier crab swarms
Hisashi Murakami1, Yuta Nishiyama1, Takayuki Niizato1, Koichiro
Enomoto2, Masashi Toda2, Toru Moriyama3, Kojiro Iizuka3, Yukio Gunji1(1Kobe University, 2Hakodate Future University, 3shinshu University)

Previously, collective behavior just as bird flock, fish school and insect swarm is
explained by numerical model such as BOID (Bird-andrOID) and SPP (SelfPropelled Particle). These models can make collective motion by matchingvelocity of agent to neighbors in neighborhood, coupling well-turned noise
externally. Yet, there is a little field data that should be compared with themodels. We have observed the soldier crabs in Iriomote Island, Okinawa, Japan,
which live in tideland, making big swarm. By using video camera, we recorded
their swarms from above, and analyzed their collective motion by reconstructing
their positions on two-dimensional coordinates. The results of this analysis
show that the fluctuation of their motion positively contributes to maintenanceand/or formation of a swarm, standing in contrast to the fluctuation of classicalmodel, which is given as just random noise.
2PT235

Analysis of lateral connectivity in networks based on a latticetheoryTakenori Tomaru, Yukio Gunji (Kobe University)
In studying networks, there are many useful toolkits to analyze the properties.
However, most of ways of analysis focus on an amount and distribution ofvertices and/or edges to estimate structural properties of networks. We here
propose a novel method to analyze a network structure as a whole in a term ofalgebraic structure, lattice structure. A directed network is expressed as a latticeby using a formal context based on a relation between edges and vertices.
Through applying this method to some real networks, we show that a generated
lattice can reflect original structure well. In addition, we can extract a particular
structure called "overlapped lateral connection" of a network by the structure ofa lattice and/or the number of generated elements by completion. The
overlapped lateral connection can be related to robustness based on parallel
functionality. In biological networks, such as food webs, it is important that
networks evolve and maintain with diversity having overlapped target. This
notion was missed by previous analytical toolkits based on existence of
redundant pathways although it can play an essential role in a network evolving
with respect to quality.
2PT236

真性粘菌モデルを用いた感性の計算

Computation of emotional information by using numericalmodel of true slime moldIori Tani, Yukio-Pegio Gunji (Department of Earth and Planetary Sciences,
Graduate School of Science, Kobe University)
True slime mold Physarum polycephalum plasmodium is a unicellular cenocytic
amoebic organism which is used as model organism in the field of
biocomputation. Our numerical model of Physarum is able to construct various
morphological pattern of real plasmodium and we have tried to apply this modelto the emulation of visual perception. We already showed that the behaviors of
our model for two-valued ambiguous figures were similar to human’s ones infigure-ground sense (I. Tani, et al., 2011). We treat more simpler figure like
Kanizsa triangle and show our model can construct subjective contour of
Kanizsa triangle. In addition, we discuss relationship of emotional information
and biocomputation.
2PT237− S144 −Hill 式による非線形自励力学系の近似計算
Numerical investigation with Hill equation on approximatednonlinear autonomous dynamical systems

Eisuke Chikayama1,2 (1Niigata University of International and Information
Studies, 2RIKEN)

between the PRC property and the adaptive properties of cAMP oscillations.

We have shown that arbitrary nonlinear autonomous dynamical systems forchemical systems can be approximately expressed by using combinations of Hillequations or sigmoid functions. The numerical aspect of the theory, however,
have not been demonstrated enough. We thus report that the results of theinvestigation of numerical calculations between exact and approximatednonlinear autonomous dynamical systems. As preliminary results for dynamicalsystems in one variable, of which the right-hand sides are X, squared X, and sineX, we set the intervals, 1, 0.1, and 0.01 along each X axis for step functions.
They resulted in errors of -0.74, -0.0082, and -0.00010, respectively, for X; -5.9,-0.16, and -0.0017, respectively, for squared X; and 0.011, 0.015, and -0.00036,
respectively for sin X. We further investigated the interval 1 with sigmoid
functions and it resulted in errors of -0.70, -0.31, and 0.94, respectively, whichwere relatively good. We will offer further investigation including nonlinear
dynamical systems with two variables.

2PT240Feedback-enhanced active-passive microrheology in cells
Hiroshi Arimatsu, Marcel Bremerich, Daisuke MIzuno, Heev Ayade, Peijuan
Zhang (Univ.Kyushu)

2PT238非平衡人工細胞モデルのための油中水滴マイクロ流体システム

Droplet-based microfluidic system for nonequilibrium artificial cells
Masahiro Takinoue1,2, Haruka Sugiura1 (1Interdisciplinary Grad. Sch. Sci.
and Eng., Tokyo Tech., 2PRESTO, JST)
Understanding the essential mechanism of life systems is one of the mostimportant issues in biophysics. Although biological sciences and technologies
have clarified the molecular basis of life systems, the whole picture of life
systems as autonomous integrated molecular systems has not been revealed yet.
In recent years, biophysical technologies to construct artificial cells as
simplified models of natural living cells have been proposed, and they help us tocharacterize life systems as the autonomous integrated molecular systems.
However, most of them have limitations caused by the difficulty in transporting
exterior/interior biomolecules through the membrane interface of artificial cellvesicles. Therefore, it is difficult to achieve experiments of dynamic
nonequilibrium reaction systems in the artificial cell vesicles [1].
Here, we demonstrate a novel method for artificial cell study based on picolitersized water-in-oil (W/O) microdroplets (named "Cell-sized CSTR"). The cellsized CSTR can control influx and efflux of biomolecules into and out of theartificial cell vesicles by microfluidic and electric manipulations of molecularinterfaces of W/O microdroplets [2]. In this presentation, we show experimental
investigations of the cell-sized CSTR and discuss the possibilities of nonlinear
chemical reactions in the cell-sized CSTR. We believe that our method will
promote the construction of nonequilibrium artificial cells in future.
[1] Takinoue et al., Anal. Bioanal. Chem. (2011)[2] Takinoue et al., Small (2010)

The cytoskeleton is responsible for mechanics and various mechanical functionsof cells. Mechanics of intracellular environments are, however, still elusive
since living cells indicate a further complication due to the active force
generation mostly governed by molecular motors. Here we investigate thenon-equilibrium mechanical properties of cells using optical-trapping-based
active-passive microrheology. By combining "active" micromanipulation ofphagocytosed probe particles with an optical trap with high-resolution trackingof thermal(passive) motions of the very same particles by laser interferometry,
we can both measure the mechanical properties of and, at the same time, identify
nonequilibrium forces in biological materials as a violation of flluctuationdissipation theorem. In cells, however, stable microrheology experimetns arehampered due to the active athermal fluctuations which easily drives the probe
particles out of the laser foci. We therefore compensated the 3D active motion
by implementing a PID-controlled feedback to reposition the sample chamberwith a 3D-piezo stage. The piezo-control signal together with the optical trap
measurement recover the complete bead trajectory. This way we performed
active and passive microrheology in fibroblasts and analyzed the full
distribution of probe particle displacements, the lag-time dependent van Hove
correlation function, and discuss the results by comparing with those obtained
from active reconstituted cytoskeletons in vitro (actin/myosin gel).

2PT239Phase response of the collective cAMP oscillations in
Dictyostelium discoideum and its implication to the adaptive
properties
Daisuke Imoto1, Satoshi Sawai1,2,3 (1Graduate School of Arts and Sciences,
University of Tokyo, 2Research Center for Complex Systems Biology,
University of Tokyo, 3PRESTO, Japan Science and Technology Agency)
Oscillatory phenomena are widely found in biological systems such as heart
beating, flashing of fireflies, and development of Dictyostelium andMyxococcus. In most of these phenomena, it is important that the timing of
pulsing of each element is synchronized. Characterizing the responses of thesystems experimentally by the phase response curve (PRC) helps one identify
the underlying instability that gives rise to the periodic activity (T. Tateno et al.
(2007)). Earlier works on this aspect in cAMP oscillations in Dictyostelium
have been fragmentary. Gerisch predicted Type 0 PRC (discontinuous type) torelatively strong cAMP stimuli in his theoretical model (Gerisch et al. (1979)).
Experimentally, pH measurements on cell suspension also reported Type 0 PRC(Nanjundiah (1998)). However, PRC has not been obtained directly frommeasurements of cAMP. Moreover, there have been no work on measurementof Type 1 PRC (continuous type), thus it is still unclear how much stimuli areneeded for the onset of cAMP oscillations.
Here we report PRC obtained by direct measurements of intracellular cAMPusing a FRET sensor (Nikolaev et al. (2004)). By studying PRCs obtained atvarious concentrations of extracellular cAMP stimuli, we explored therelationship between the group-level PRCs and the single-cell input-output
relation (Gregor et al. (2010)). We also examined mutant cells harboring a nonphosphorylatable form of the cAMP receptor and discuss the relationship− S145 −3PS001

IC138 リン酸化によるクラミドモナスダイニン-f テールの形態変化
Configuration changes of Chlamydomonas dynein-f tailcoupled with IC138 phosphorylation

Hitoshi Sakakibara, Kazuhiro Oiwa, Hiroaki Kojima (KARC, Nat. Inst. Inf.
Com. Tech.)
Inner-arm dynein-f of Chlamydomonas is a hetero-dimeric dynein which has
two heavy chains (α and β), three intermediate chains and light chains on thetail. It has been shown that an axonemal casein kinase I phosphorylates the138K intermediate chain (IC138) and that this phosphorylation is involved inregulation of flagellar activity. To elucidate the regulation mechanisms ofdynein-f functions through the IC138 phosphorylation, we examined molecularconfiguration changes of dynein-f coupled with IC138 phosphorylation by
negative-staining EM and following single-particle analyses. For the analyses,
we picked up a dynein-f density map from the tomogram of axoneme, preparedprojections of the map from various angles, and used tail parts of the projections
as the templates for alignments. First, we found that the distribution of the twoheads' positions spread and distance between the α and β heads became closer
coupled with IC138 phosphorylation. Second, in ~10% classes, we found thatthe tail showed forked structure which consisted of thick stem and the thinbranch linking to the beta head. This thin branch became smearing in
phosphorylation conditions, suggesting that the branch positioning became
unstable. From these observations, we suppose that the IC138 phosphorylation
could first affect the thin branch positioning, then the β-head position should beshifted. In axonemes, position shift of the β head could affect the interactions ofdynein-f and adjacent microtubule to change their operation mode.
3PS002鞭毛・繊毛運動の研究に向けたヒト軸糸ダイニンの発現系の開発

Expression and purification of human axonemal dynein forstudying cilia/flagella beating
Akane Furuta, Ken'ya Furuta, Misako Amino, Hiroaki Kojima (Bio ICT lab,
NICT)
Motile cilia and flagella are crucial component for wide variety of cells.
Previous studies have shown that the mutation in axonemal dyneins often
impairs beating of cilia, leading to serious diseases known as primary ciliary
dyskinesia. Normal axonemal dyneins in the cilia and flagella generate force
that is highly regulated in time and space to generate regular waveform. Given
the timescale of the regulation which is much shorter than that of signaltransductions, it is natural to expect that individual dynein molecules act as amechanical sensor for external loads, forming feedback loop to generate theoscillations. However, how dynein responds to external loads is unclear. To
address this problem, it is critical to measure the force of both normal andmutated axonemal dyneins upon an applied external load. These assays require
specific probes and mutations at desired sites on the axonemal dynein. A
limiting issue in the study of axonemal dynein is that the recombinant proteins
are not available so far. Here we used a transient expression system in HEK293
cells to express human recombinant axonemal dyneins. We isolated full-lengthand truncated human inner arm dyneins (DNAH2 and DNAH7) in a soluble
form. Currently, we are working with functional analysis of these recombinant
axonemal dyneins.
3PS003Tubulin polyglutamylation regulates axonemal motility bymodulating the function of a specific inner-arm dynein species
Tomohiro Kubo1, Toshiki Yagi2, Ritsu Kamiya1 (1Department of Biological
Sciences, Graduate School of Science, University of Tokyo, 2Department of
Cell Biology and Anatomy, Graduate School of Medicine, University of Tokyo)
Tubulin polyglutamylation is a post-translational modification that affects
various microtubule-dependent cellular functions. The tpg1 mutant ofChlamydomonas lacks a homologue of mouse TTLL9 (a tubulin polyglutamylase) and has a reduced level of polyglutamylated alpha tubulin in theouter-doublet B-tubule. It displays lowered flagellar motility because of deficient interaction between inner-arm dynein and doublet microtubules (Kubo et
al. Curr. Biol. 20, 441-445, 2010). Chlamydomonas has seven major species of
inner-arm dyneins, designated dynein a~g. Dynein f (dynein I1) is a two-headed
dynein containing two heavy chains, while all others are one-headed. In thepresent study, we produced double mutants of tpg1 and various inner-armdeficient mutants, and found that the tpg1 mutation did not affect the lowered
motility of mutants lacking inner-arm dynein e, while it greatly reduced the
motility of mutants lacking dyneins other than dynein e. This observationsuggests that tubulin glutamylation most sensitively affects the function ofdynein e. The exceptionally basic microtubule binding site (the stalk tip) of
dynein e may account for its high sensitivity to the tpg1 mutation, which should
greatly change the microtubule surface charge.
3PS004

高速 AFM による細胞質ダイニンの機能動態の観察

High-Speed-AFM Observation of Processive Movement of
Cytoplasmic Dynein
Shuji Fujita1, Karunakaran Aathi2, Uchihashi Takayuki1,3, Vale Ronald D.2,
Toshio Ando1,3 (1College of Science and Engineering, Kanazawa
University, 2Department of Cellular & Molecular Pharmacology, University ofCalifornia, San Francisco, 3Bio-AFM Frontier Research Center, College of
Science and Engineering, Kanazawa University)
Cytoplasmic dynein is a microtubule-based motor protein which transports acargos toward microtubule minus end. So far, several studies based on structural
analysis and single molecule observations have been reported to reveal dynein’s
mechanims1-3). For examples, the structural analyses revealed the manners inwhich the mechanical elements interacts with the ATPase ring and a long-range
allosteric communication takes place between the primary ATPase site and themicrotubule-binding site. The single molecule analysis revealed the step-wise
motions during the processive movement and what the stepping behavior varied
as a function of interhead separation. However, there is no study by which direct
observation of the conformational state of the functional dynamic because of alack of technique. While we have recently succeeded in directly visualizing
hand-over-hand movement of myosin V.4) This observation provided
straightforward and detailed information on how myosin V moves along an
actin filament. Here, we applied to high-speed AFM to observation ofcytoplasmic dynein moving along microtubules. In this presentation, we will
discuss what information about the dynnein’s mechanisms can be gained withhigh-speed AFM .
1) A. Carter, C. Cho, L. Jin and R. D. Vale, Science 331, 1159-1165 (2011).
2) M. A. DeWitt., A. Y. Chang, P. A. Combs, A. Yildiz, Science 335, 221-225(2011).
3) T. Kon, T. Oyama et al., Nature 484, 345-350 (2012).
4) N. Kodera, D. Yamamoto et al., Nature 468, 72-76 (2010).
3PS005鞭毛軸糸ダイニンによって駆動される微小管配列の振動運動

Oscillation of a microtubule array driven by axonemal dynein
Susumu Aoyama, Yuichi Hiratsuka (Sch. Matl. Sci., JAIST)
Flagellar axoneme produces high speed oscillatory movements, which is essential
for many eukaryotes to survive. Details of the mechanism of flagellar beating is
not clearly elucidated, although some studies have shown that the minimal
elements of oscillation is motor protein dynein and microtubules in an axoneme.
To solve the mystery of flagellar movements, we tried to fabricate oscillatory
units by arranging microtubules and dynein molecules with some accessory
structures. In an axoneme, nine microtubules are arranged in the same polarity,and the minus ends of them are tightly bound with each other. We fabricated suchmicrotubule arrays by polymerizing tubulin from polarity-arranged microtubules
fixed on a glass surface. Polarity of microtubules was arranged by kinesin motility
guided by a micro-track. A typical microtubule array was composed of five tofifteen microtubules. Next, we made microtubules on which axonemal dynein
molecules were aligned, and incorporated them into microtubule arrays. In the
microtubule array, dynein-bound microtubules interacted with polarity-arranged
microtubules and displayed cyclical attachment and detachment. As a next step of
this study, we are now trying to produce bending oscillation in our experimental
system by loose cross-links between microtubules. We expect that the basis offlagellar beating can be produced in a simple microtubule-array system driven bydynein motors.
3PS006Structural and Biochemical Properties of the Outer-DyneinArm Docking Complex (ODA-DC) in Chlamydomonas flagella

Mikito Owa1, Takahiro Ide1, Ritsu Kamiya1,2, Kenichi Wakabayashi1 (1Dept.
Biological Sciences, Grad. school of Science, Univ. Tokyo, 2Dept. Life Science,
Faculty of Science, Gakushuin Univ.)
Outer dynein arms in cilia and flagella are bound to specific loci on outerdoublet microtubules with a regular spacing of 24 nm. Their binding to the
doublet microtubules is mediated by the outer dynein arm docking complex(ODA-DC) present at the base of the outer arm. In Chlamydomonas, the ODADC is composed of three subunits, DC1 (ODA3), DC2 (ODA1) and DC3− S146 −(ODA14). ODA-DC binds to specific loci on the outer-doublets of mutant
axonemes that lack outer arm dynein, suggesting that it is responsible for thebinding of outer arm dyneins to specific sites. To understand the basis for thiscrucial function of the ODA-DC, we examined the structural and biochemical
properties of ODA-DC using both recombinant and native proteins. Low-angle
rotary shadowing indicated that the recombinant ODA-DC has an oval shape,~25 nm in length. The recombinant ODA-DC binds to cytoplasmic
microtubules in an all-or-none manner, suggesting a cooperative binding event.
Chemical crosslinking of axonemes from a mutant lacking outer-arms revealed
that the native ODA-DCs associate with each other on the doublet microtubules.
These data suggest that the periodic and cooperative binding of the ODA-DC
molecules on the doublet microtubules is based on an end-to-end association of
the ODA-DCs. Furthermore, chemical crosslinking also revealed that the ODADC is associated with three uncharacterized novel proteins on the axoneme.
These proteins possibly specify the binding site of the ODA-DC on the outerdoublet microtubules.
3PS007

クラミドモナス軸糸微小管上の外腕ダイニンの組み立てに必要なタ
ンパク質間相互作用の解析

Analysis of protein-protein interactions required for theassembly of outer arm dyneins on the axonemal microtubules
in Chlamydomonas
Takahiro Ide1, Mikito Owa1, Kaoru Yoshida3, Manabu Yoshida2, Ritsu
Kamiya4, Ken-ichi Wakabayashi1 (1Dept. Biological Sciences, Grad. School of
Science, Univ. Tokyo, 2Misaki Marine Biological Station, Graduate School of
Science, University of Tokyo, 3Biomedical Engineering Center, Toin University
of Yokohama, 4Department of Life Science, Graduate School of Science,
Gakushuin University)
Outer arm dynein (OAD) in cilia and flagella are bound to specific loci on outerdoublet microtubules with a regular spacing of 24 nm. Previous studies showedthat OAD has two sites for binding to microtubules: intermediate chain 1 (IC1)and the outer dynein arm docking complex (ODA-DC) located at the base of
OAD. Studies using Chlamydomonas mutants have shown that OAD cannot
bind to the microtubule at all without the ODA-DC, and the ODA-DC can bindto MTs only in a reduced amount without OAD. These data suggest that the twosites have weak binding affinities to microtubules, and therefore they possibly
cooperate with each other to enable strong OAD attachment to microtubules. To
test this idea, we quantified binding affinities of IC1, IC2 (the other intermediatechain located at the base of OAD) and the ODA-DC using recombinant proteins,and also tested whether there is a direct interaction between ICs and the ODADC. The recombinant IC1 and IC2 were found to form heterodimers, and theIC1-IC2 heterodimer further formed a complex with ODA-DC in vitro, in which
IC2 was in contact with DC1 (an ODA-DC subunit). In addition, the binding
affinities of ICs and ODA-DC to cytoplasmic microtubules, measured with aquartz crystal microbalance (QCM), were similar to each other. From theseresults, we propose a new model of protein-protein interactions for the dockingof outer dynein arms to the axonemal microtubules.
3PS009In vitro analyses on two adjacent inner-arm dyneins,subspecies e and c
Youské Shimizu, Hiroaki Kojima, Kazuhiro Oiwa, Hitoshi Sakakibara(Advanced ICT Research Institute, NICT)

Subspecies of inner-arm dyneins are aligned periodically and specifically inflagella. Though each of dynein subspecies “e” and “c” is able to be purified asmonomer, these two dyneins are adjacent in flagella so that there may beinteraction and/or cooperation between them. To study the properties, dynein eand c were highly purified. ATPase activity of dynein e (kcat ~ 0.3) was low
compared with that of dynein c (kcat ~ 1.8). MT-binding affinity of nucleotidesensitive binding domain of dynein e was higher than that of dynein c. In vitro
motility assay indicated that dynein e was relatively slow (Vmax ~ 2 μm/s) anddynein c was relatively fast (Vmax ~ 17 μm/s) in MT-sliding. To obtain clues on
the mechanism of spontaneous work of two dyneins, we analyzed properties of
mixture. From the MT-sliding by the mixture of dyneins, it was suggested thatslow dynein e did not cause a drag force on fast MT-sliding by dynein c. On theother hand, direct interaction between these dyneins was not confirmed, so theinteraction was thought to be limited or weak. Dynein e and c may be working
in flagella in cooperation with each other.

3PS010

生細胞内における温度分布のイメージング

1YS0915

Imaging of temperature distribution in a living cell
Kohki Okabe1, Seiichi Uchiyama1, Noriko Inada2, Yoshie Harada3, Takashi
Funatsu1 (1Grad. Sch. Pharm. Sci., Univ. Tokyo, 2NAIST, 3iCeMs, Kyoto
Univ.)
Temperature is a fundamental physical quantity that governs every biologicalreaction within living cells, and temperature distribution reflects cellular
thermodynamics and function. In medical studies, the cellular pathogenesis of
diseases (e. g., cancer) is characterized by extraordinary heat production.
Therefore, intracellular temperature imaging of living cells should promotebetter understanding of cellular events and the establishment of novel diagnoses
and therapies. However, imaging of temperature distributions in living cells has
never been achieved. Here we demonstrate the first intracellular temperature
imaging based on a fluorescent polymeric thermometer and fluorescence
lifetime imaging microscopy (FLIM). The spatial and temperature resolutions ofour thermometry were at the diffraction limited level (200 nm) and 0.2 degree
Celsius, respectively. The intracellular temperature distribution we observed
indicated that the nucleus and centrosome of a COS7 cell both showed asignificantly higher temperature than the cytoplasm and that the temperature gap
between the nucleus and the cytoplasm differed depending on the cell cycle. The
heat production from mitochondria was also observed as a proximal local
temperature increase. These findings demonstrate an intrinsic connection
between temperature and organelle function. Thus, our intracellular temperature
imaging has a significant impact on the comprehension of cell function and will
provide insights into the regulatory mechanisms of intracellular signaling.
3PS011

走査電子顕微鏡を用いた非染色生物試料の高コントラスト・低ダメー
ジ観察方法

A high-contrast and low-damage observation method of theunstained biological samples by scanning-electron microscope
Toshihiko Ogura (AIST Biomedical Research Institute)
Scanning electron microscope (SEM) is a useful technique for the investigationof surface structure of biological samples. To allow observations under the highvacuum conditions of SEM, many preparations of biological samples have beendeveloped, e. g., glutaraldehyde fixation, negative staining, the Sputter-Cryotechnique, and coating with gold or osmium. These preparations have somepositive effects on the biological samples; for instance, they enhance contrast,
reduce damage, and are uncharged up by the electron beam. However, these
preparations require many steps and/or special equipments. Therefore, a moreconvenient preparation method for biological samples is needed. Here, weshow that the unstained biological samples under a thin film give a high contrast
detected by the secondary electron (SE) signal at a low accelerating voltage. The
unstained biological samples are attached to the lower surface of a 30-50 nm
thin film. The high-contrast images are created from the SE signal under the thinfilm. These are very low damage to the samples, and the images are similar to
the transmission image. Our novel method can be easily utilized to observevarious biological samples of bacteria, viruses and protein complexes.

3PS012低温電子顕微鏡による細菌べん毛キャップ連結部の 3 次元再構成

The structure of the flagellar cap-junction complex by electron
cryomicroscopy
Fumiaki Makino, Takayuki Kato, Tomoko Miyata, Keiichi Namba (Frontier
Bioscience)
The bacterial flagellum is a biological nanomachine for the locomotion ofbacteria. The cap at the growing end of the bacterial flagellum is essential for its
growth. Flagellar axial proteins travel through the central channel of theflagellum to the distal end where self-assembly occurs. After flagellar hook hasreached its mature length, the two junction proteins FlgK and FlgL assemble toform the hook-filament junction, and then the filament cap protein FliD forms a
cap structure at the distal end of the junction to form the cap-junction complex.
When FliC molecules are exported to the distal end, the FliD pentamer works as
the filament cap for filament growth. It has been revealed form the cap-filament
structure that five leg-like anchor domains of the pentameric cap flexibly adjust
their conformations to keep just one FliC binding site open at a time. This has
suggested a cap rotation mechanism to promote FliC self-assembly. However,the boundary between the cap and the filament end has remained unclear.
In this study, we report 3D structure of the cap-junction complex, in which we− S147 −can see how the leg-like anchor domains of FliD bind to FlgL in a similarmanner to the structure of the D0-D1 domain of FliC over the symmetry
mismatch as well as the depression and space inside the cap complex for
promoting assembly.
3PS013

バイメタルカンチレバーを用いた褐色脂肪細胞の発熱量の定量的解析

Quantitative analysis of heat production of brown adipocytes
using bimetal cantilever
Masaaki Sato1, Masaya Toda2, Naoki Inomata2, Yuichi Inoue1, Takahito Ono2,
Akihiko Ishijima1 (1IMRAM, Tohoku Univ., 2Grad. Sch. Eng., Tohoku Univ.)
Brown adipocyte is a cell specified for metabolic heat production. From acalorimetry using 108 numbers of the cells, the cell is thought to produce nWordered heat. However, the character of each cell cannot be investigated by thecalorimetry. To reveal the character, the measurement technique for heatproduction of a single cell is needed. So we developed a bimetal cantilever (750×40 μm), which is composed of gold (100 nm in thickness) and silicon nitride(200 nm in thickness). When the cantilever senses heat, it bends due to thedifference of the coefficient of thermal expansion of the metals. We tried tomeasure the heat produced by a small number of the cells using the cantilever.
The displacement of cantilever was measured under a conventional microscope
when the 5-6 cells reached about 2 μm away from the tip of cantilever. After the
addition of norepinephrine (final 1 μ M), the displacement of the cantileverincreased as time passed on. After 60 min from the addition, the displacement of
cantilever became maximum value (60 nm). If the heat which the cantileverdetected has escaped only at the end of the cantilever, the value corresponds tothat the temperature of the tip of the cantilever has increased 10 mK. After
further 90 min, the displacement was not observed. We would like to discuss theactivation and inactivation of heat production of the cell together with lipid
consumption of the cell and expression level of protein which is involved in heatproduction.
3PS014細胞シート 1 軸延伸測定法による時間依存力学測定

Time-dependetn Poisson's ratio and Power-law rheology of cellsheet in uniaxial stretching experiment
Masahiro Tsuchiya, Yusuke Mizutani, Takaharu Okajima (Graduate Schoolof Information Science and Technology, Hokkaido Univ.)
Mechanical properties of living cells are strongly related to their physiological
functions such as migration, division and restructuring of their intracellular
structures. Rheological properties of living cells have been extensively
investigated by various microbead techniques, atomic force microscopy andsingle cell stretching method. These studies showed the single cell exhibited a
power-law behavior in frequency domains. The Poisson’s ratio of living cells is
one of the important modulus and related with some time dependent modulus.
Moreover, studies regarding polymer materials revealed that the Poisson’s ratio
was a function of time. However, the time-dependent Poisson’s ratio of cells has
not been measured. In this study, we measured the Poisson’s ratio of cells as afunction of time during stress relaxation of cell sheet composed of NIH3T3 cellswith a size of ca.500 × 400 μm. We found that the Poisson’s ratio of cellincreased with time and became time constant. As the actin filaments were
disrupted, the time dependent Poisson’s ratio reduction is rapidly changed. The
results indicated that the time dependent Poisson’s ratio was strongly related tothe contraction of actin structures.
3PS015

原子間力顕微鏡を用いた単一細胞レオロジーの高速・精密測定法の開発

High-speed measurement of single cell rheology with an atomic
force microscope
Ryosuke Takahashi, Satoshi Ichikawa, Takaharu Okajima (Graduate Schoolof Information Science and Technology, Hokkaido University)
Single cell measurements of cell mechanics are crucial not only forunderstanding various cell functions such as migration and proliferation but also
for distinguishing different types of cells. It has been reported that the elastic
property of cancer cells is smaller than that of normal cells [1, 2]. We obviously
observed that cancer cells are softer and more fluid than normal cells and foundthe difference of the ensemble distribution of rheological properties between
normal and cancer cells such as human mammary epithelial cells (MCF-10A) as
normal cell and human mammary adenocarcinoma cells (MCF-7) as cancer cell[7]. To improve throughput of cell measurements, we developed an AFM
system for the measurement of rheological properties of a large number cells,

the shape of which was controlled with micropatterned substrates [3], in thefrequency domain [4, 5]. The observed AFM data could be analyzed using a
power-law structural damping model [6]. We will present the detailed AFM
system and discuss the ensemble distribution of power-law rheology parameters
between normal and cancer cells.
[1] J. Guck, et al., Biophysical Journal. 88, 3689 (2005)[2] S. E. Cross, et al., Nat. Nanotech. 2, 780 (2007)[3] C. Y. Park, et al., Am J Physiol Cell Physiol 298, 1245 (2010)[4] Y. Mizutani, et al., Jpn. J. Appl. Phys. 47, 6177 (2008)[5] S. Hiratsuka, et al., Ultramicroscopy 109, 937 (2009)[6] B. Fabry, et al., Phys. Rev.E, 68, 041914 (2003)[7] R. Takahashi, et al., in preparation
3PS016"ホッピングモード"高速原子間力顕微(HS-AFM)の開発

Development of “hopping-mode” high speed atomic force
microscopy (AFM)
Hiroki Watanabe1, Mikihiro Shibata2, Takayuki Uchihashi1,4, Ryohei
Yasuda3, Toshio Ando1,4 (1Department of Mathematics and Physics, Grad
School of Natural Science and Technology, Kanazawa University, 2Departmentof Neurobiology, Duke University Medical Center, 3Max Planck Florida
Institute, 4Bio-AFM Frontier Research Center, College of Science andEngineering, Kanazawa University)
High-speed AFM (HS-AFM) can directly visualize dynamic behaviors of
single protein molecules in action and has recently been proven to be powerfulin the elucidation of functional mechanisms of proteins1, 2). However, it is hardto observe large and soft biological samples such as live cells and organelles
using our tapping-mode HS-AFM because the AFM cantilever tip exerts
relatively large lateral forces to the sample during scanning and thus damages
soft samples.
To overcome this problem, we attempt to combine a new operation mode of AFM
called hopping mode3) with the HS-AFM. In hopping-mode AFM, we no longer
use continuous feedback operation. Instead, at each imaging point, the AFM tip
approaches the sample from a starting position that is above any of the surface
features. The cantilever is freely oscillating when the tip is well away from thesurface. The tip then approaches until the amplitude is reduced to a predefinedvalue. When the amplitude experiences this reduction, the position of the z-piezo
is recorded as the sample height at this imaging point. Owing to this operation
principle, the tip lateral forces applied to the sample can be significantly reduced
even for tall samples. In this presentation, we will show the capability of this new
AFM system by presenting captured images of HeLa cells.
1) M. shibata et al., Nature Nanotechnol. 5, 208-212 (2010)2) T. Ucihashi et al., Science 333, 755-758 (2011).
3) P. Noval, C. Li et al., Nature Method 6, 279-281 (2009).
3PS017

超音波高速 AFM の開発に向けた基礎研究

Pilot study for the development of high-speed ultrasonic AFM
Yasuto Nagashima1, Tomofumi Saito1, Noriyuki Kodera2, Toshio Ando1,2(1Sch. Math. & Phys., Inst. Sci. & Eng., Kanazawa Univ, 2Bio-AFM Frontier
Research Center, Inst. Sci. & Eng., Kanazawa Univ)
We have developed high-speed AFM (HS-AFM) that enables us to directly
visualize dynamic structural changes of protein molecules at highspatiotemporal resolution. So far, various biological processes performed byprotein molecules have been directly filmed as molecular movies, whichallowed us to gain insights into their functional mechanisms much morestraightforwardly than other techniques [T. Ando, Nanotechnol (2012)].
However, the observations by HS-AFM are generally limited only tophenomena occurring on relatively hard surfaces, meaning that we can observe
neither objects placed on a soft surface (e.g., protein molecules on mammaliancell surfaces) nor objects placed under a surface (e. g., organelles andcytoskeletons in the interior of cells).Ultrasonic techniques must be useful inbreaking this limitation because they have been widely used for noninvasiveness imaging of objects lying on/under a surface, whereas their spatial
resolution is limited by the wavelength of ultrasonic used. However, AFM
which is combined with ultrasonic techniques is recently claimed to be able toperform subsurface imaging of objects in the interior of cells at nanometer
resolution [GS. Shekhawat & VP. Dravid, Science (2005)]. Thus, we are
currently attempting to find a way to combine our HS-AFM with ultrasonictechniques. In the presentation, we will report our pilot study on this subject.
3PS018− S148 −原子間力顕微鏡を用いたタンパク質界面膜の研究

derived cardiomyocyte clusters by electrical stimulation with extracellularpotential measurement.

AFM studies on the spread films of protein
Taiji Furuno (Dept. Phys., Keio Univ. Sch. Med.)The spread films of protein at the air/water interface hold almost hundred yearsof research history. The surface properties of them have been mainly discussed
based on the pressure-area isotherms. However, the study lacks the direct
structural information on the state of the spread protein molecules.
The invention of atomic force microscope (AFM) has opened the avenue to study
single proteins or protein assemblies in water if they were deposited on smoothsolid surfaces. In the present study, a number of proteins have been directly
spread on the water surface using a 10uL microsyringe with its needle end
touching the water surface. The surface films were transferred onto hydrophobic
silicon surface and imaged by tapping mode AFM under water. They wereclassified into three groups. Catalase, carbonic anhydrase, ferritin and apoferritin,which denatured gradually after spreading, formed two-dimensional random
packing in the spread films. In the second group to which bovine serum albumin
and thermally treated carbonic anhydrase belonged, the unfolding was very quickat the air/water interface, and almost no granular particles corresponding to the
spread protein molecules were not found bound to or embedded in the film. The
third group consisted of lysozyme and streptavidin, which resisted to be spread at
the interface, and were mostly lost into the subphase. All these classifications
were possible by AFM imaging of the transferred films.
3PS019カーボンナノチューブ電極を用いた神経伝達物質の高感度記録法の開発

Development of Carbon Nanotube Electrode For HighSensitivity Recordings of Neurotransmitters
Mao Fukuda, Ikurou Suzuki, Masao Gotoh (Department of Bionics, Graduate
School of bionics, computer and media science Tokyo University ofTechnology)
We have developed a microelectrode array chip and paste electrode that canmeasure neurotransmitters. In this study, we focused on using carbon nanotubes
because they indicate high conductivity, and changeable in physical properties.
A 64 planner electrodes were fabricated by etching an indium-tin oxide (ITO)
layer on a glass slide, then covered with insulating film. The surface ofrecording terminals was formed by etching the insulating film to 50 μ m indiameter and was then coated with single-wall carbon nanotube (SWCNT) andmulti-wall carbon nanotube (MWCNT) of bamboo-type or HC-type byelectroplating. On the other hand, the paste electrode was fabricated by filling itwith mixed carbon nanotube dispersion. Using these carbon nanotube
microelectrode array chip and paste electrodes, we successfully detected their
response to (10nM) of dopamine, acetylcholine, and serotonin. These results
suggest that the microelectrode array chip have the potential for real-time
recordings of neurotransmitters.
3PS020心毒性検査での細胞外電位記録における心筋細胞拍動周期制御のた
めの電気刺激方法の最適化

Optimization of electrical stimulation protocol for cardiac beat
control with extracellular potential measurement in cardiotoxicity test
Tomoyuki Kaneko, Fumimasa Nomura, Tomoyo Hamada, Kenji Yasuda(Dept. Biomed. Inform., IBB, TMDU)The QT prolongation in electrocardiogram is recognized as a potential risk
associated with the use for cardiotoxicity testing. Recently, cardiomyocytes
derived from human embryonic stem cell (hESC) have been applied for in vitro
cardiotoxicity testing, in which QT prolongation risk is estimated by the
prolongation of drug-induced change of the field potential duration (FPD) in onchip extracellular potential measurement assay. As this in vitro assay usuallyuses unstable spontaneous beating cardiomyocytes, the precise beat-to-beatcorrection procedure of in vivo QTc measurement was not applied for thisunstable FPD data. To overcome this problem, we have examined the effective
electrical stimulation protocols for cardiomyocyte beating interval control withsmallest influence to continuous extracellular potential measurement. First, byapplying the simple bipolar stimulation with same amplitude of positive andnegative voltages, the artifacts in electrical stimulation were too large and longto record the extracellular field potentials of cardiomyocytes after eachstimulation. In contrast, the sequential short rectangular pulses with thebalanced positive and negative pulses caused to record the sharp negative
artifact only, which was little and short influence to measure the FPD of
cardiomyocytes. The results indicate that the balanced applied voltages andpulse numbers are desirable to minimize the artifacts and to respond the hESC-

3PS021

イオン移動ボルタンメトリーに基づく微量ペプチドの物質量決定

Determination of mole number for trace amount of a peptide
based on the ion transfer voltammetry
Yumi Yoshida, Yoshiro Morita, Shotaro Nakamura, Junya Uchida, Kohji
Maeda (Department of Chemistry and Materials Technology, Kyoto Institute ofTechnology)
Most of quantitative analytical methods require a calibration curve made of thereference material of analyte, of which mole number is usually determined byweight measurement. For trace amount of biological compound such as peptides
and proteins, its weight is too little to determine its mole number. Usually, thesecompounds are decomposed into short peptides, amino acids or N element, andthese decomposition products are determined using a calibration curve of low
molecular substances which is weighable. These procedures are complicate andshould be carried out with respect to each compound. In contrast, electrochemical method is the most powerful method to determine mole number
without weight measurement, in which mole number is estimated according toFaraday’s law. In the present work, we propose mole number determination
based on the ion transfer voltammetry, in which an ionic molecule transfer from
aqueous sample, W, to organic phase, Org, by applying interfacial potential at
the W|Org interface, and the ion transfer is detected as current. Total charge, Q,of current gives mole number, n, of the ionic molecule based on Faraday’s low,n = Q / zF. Here, z and F are valence of the ionic molecule and Faraday
constant. As an ionic molecule, we tried to determine mole number of someshort peptides such as Lys-Leu-Val-Phe-Phe.
3PS022

リング状微小電極上の心筋細胞の環状細胞ネットワークを用いた心毒性検査技術On-chip quasi-in vivo cardiac toxicity assay using ring-shaped
closed circuit microelectrode with lined-up cardiomyocyte
network
Fumimasa Nomura, Tomoyuki Kaneko, Tomoyo Hamada, Kenji Yasuda(Inst. of Biomaterials & Bioengineering, Tokyo Medical & Dental Univ.)
Re-entry of excitation in heart is one of the abnormal phenomena to cause thelethal arrhythmia and is thought to be induced by the looped structure of
excitation conduction pathway. For adaptable preclinical strategies to evaluate
global cardiac safety, an on-chip quasi-in vivo cardiac toxicity assay for lethal
arrhythmia measurement using ring-shaped closed circuit microelectrode chiphas been developed. The ventricular electrocardiogram (ECG) -like fieldpotential data, which includes both the repolarization and the conductanceabnormality, was acquired from the self-convolutied extracellular fieldpotentials (FPs) of a lined-up mouse embryonic cardiomyocyte network on acircle-shaped microelectrode in an agarose microchamber. When E4031 (hERG
blocker, positive control) and Astemizole (false-negative drug in vitro assay)
applied to the closed-loop cardiomyocyte network, self-convoluted FP profile ofnormal beating changed into an early afterdepolarization (EAD) like waveform,
and then showed ventricular tachyarrhythmias and ventricular fibrilations(VT/Vf). QT-prolongation-like self-convoluted FP duration prolongation and its
fluctuation increase ware also observed according to the increase of Astemizole
concentration. The results indicate that the convoluted FPs of the quasi-in vivo
cell network assay includes both of the repolarization data and the conductanceabnormality of cardiomyocyte networks has the strong potential to prediction
lethal arrhythmia.
3PS023

細胞外電位解析と光学イメージング解析を組み合わせた心機能計測
解析システムの開発

Development of Dual Recording Assay for Cardiac Function
Measurement using Electrical Field Potential and Optical
Image Analysis
Toru Shoji, Tomoyuki Kaneko, Fumimasa Nomura, Akihiro Hattori, Kenji
Yasuda (BMI, IBB, TMDU)
In drug assessment of cardiac safety, development of mechanophysiologicaleffects on cardiomyocytes caused by false positive/negative compounds has
becoming more important to acquire more precise prediction of cardiotoxicity.
For the evaluation of correlation between mechanophysiological effects andelectrophysiological effects against drugs, we have developed a dual

− S149 −measurement system that records the extracellular field potentials and thecontractile motions in lined-up cardiomyocyte network simultaneously.
Cardiomyocytes were isolated from embryonic mouse heart by collagenase
treatment and seeded in rectangular microchambers fabricated on the multielectrode array (MEA) chip for field potential recordings. To detect contractilityof cardiomyocytes, the cells were labeled with the fluorescent polystyrene beads
and the motions of beads were treated with image-based analysis. Hence,electrophysiological properties (interspike intervals and field potential
durations) and mechanophysiological properties (displacements and velocities
of beads) were obtained simultaneously. In the presence of verapamil, typical Ltype calcium channel blocker, beating rate decreased companying withshortening of field potential duration (FPD) and its fluctuation increase, andcontractility was weakened significantly with its fluctuation increase. The
results indicate that the fluctuation of force generation should also become abiomarker of cardiotoxicity risk more correctly in drug development, also might
be used for the clarification of mechanisms.
3PS024

白色 X 線源を用いた X 線 1 分子計測法の開発

Development of Diffracted X-ray Tracking measurement
system using various wide-band energy X-ray sources
Kouhei Ichiyanagi1,2, Hiroshi Sekiguichi2,3, Masato Hoshino3, Kentaro
Kajiwara3, Shunsuke Nozawa4, Tokushi Sato4, Shin-ichi Adachi4, Naoto Yagi3,
Yuji Sasaki1,2 (1Graduate School of Frontier Sciences, 2JST CREST Sasaki
team, 3Japan Synchrotron Radiation Research Institute, 4High energy
acceralation research organization)
Diffracted X-ray tracking (DXT) enable to measure milliradian resolved rotating
and tilting motion of single protein molecules using high-brilliant and wide-band
energy X-ray source. The tilting (θ) and rotating (χ) angle resolution and temporal
resolution in this measurement are 0.9 mrad, 6 mrad and from 5 μs/frame to 36
ms/frame corresponding to the photon flux. Tracking the diffraction spot fromgold nanocrystals attached functional group with protein was developed forrevealing the detailed fluctuation and functionalized motions in protein reaction
processes at the beamline BL28B2, and BL40XU at the SPring-8 and NW14A at
the PF-AR, KEK. (1) We installed the X-ray toroidal mirror at the beamline
BL28B2 because the wide-angle diffraction spot tracking of gold nanocrystal
markers in the measurement range θ= 7.4° can be observed in this beamline. (2)The undulator beamline BL40XU (the measurement range, θ= 4.2°) allows highspeed DXT in microsecond time region using the CMOS CCD camera. (3)NewDXT measurement system which was synchronized with pulse laser system (Nd:YAG laser, pulse-width 10 ns, 10 Hz, UV-Vis) has succeeded to observe theopen-close motion of membrane protein in reaction process at the beamline
NW14A (measurement range,  θ = 2. 1°). Thus, the biological science
applications of DXT can high profit from observation of the in-vivo
functionalized motions of single protein molecules. Here, we will present thedevelopment of DXT measurement system and the future plan.
3PS025

3PS026XFEL を用いた 3 次元分子イメージングのための雑音下における2 次元回折像の類似度自動同定

Automatic similarity identification of 2D diffraction patterns
with noisy background for 3D coherent x-ray diffractive
imaging
Atsushi Tokuhisa1, Yasumasa Joti2, Hidetoshi Kono3, Nobuhiro Go1(1Harima Inst., Riken, 2JASRI, 3KPSI,JAEA)
We proposed a method for classifying and assembling two-dimensional
diffraction patterns to reconstruct the three-dimensional diffraction intensity
functions (A. Tokuhisa, et. al., Acta Crystallographica, A68, 366 (2012) ).In theclassification process, similarity of each pair of patterns is judged based on thecorrelation of corresponding pixels along the concentric circles When two
diffraction patterns are similar to each other, a straight line with highcorrelations appears from the origin to a certain high scattering-angle region.
Theoretically, the integrated value of the correlation value along this line is
proportional to the extent of the similarity and can be used as threshold for theclassification. This correlation line disappears with increasing scattering-angle
due to the quantum noise and the structural irregularity. In our estimation,
diffraction images of 106orders will be required to construct the 3D structurewith 3 Å resolution for a molecule with a radius of 200 Å. In this case, thenecessary number of pattern comparisons is ~ 1010order. To achieve such a huge
number of comparisons, we have to develop an automatic system. In thismeeting, we will report how to deal with such a huge amount of data and ourcurrent status of the system.
3PS027植物細胞の飛行時間型二次イオン質量分析法と多変量スペクトル分離法による評価

Evaluation of plant cells using time-of-flight secondary ion
mass spectrometry and multivariate curve resolution
Satoka Aoyagi1, Noriko Kodani1, Katsushi Kuroda2, Kazuhiko Fukushima3,
Isao Kayano4, Seiichi Mochizuki4, Akira Yano1 (1Facul. Life & Environmental
Sci., Shimane Univ., 2Forestry & Forest Products Res. Institute, 3Dept.
Biosphere Resources Sci., Nagoya Univ., 4Dept. Medical Eng., Kawasaki Univ.
Medical Welfare)Time-of-flight secondary ion mass spectrometry (TOF-SIMS) is one of the mostsensitive surface analysis methods providing submicron-scale chemical
mapping. It is, however, often difficult to interpret TOF-SIMS spectra ofcomplex samples such as tissues and cells. In this study, multivariate curve
resolution (MCR) was employed in order to extract less complex TOF-SIMS
spectra from original TOF-SIMS data of plant tissue. As a result, components
related to intracellular tissues and cell wall tissues, respectively, were extracted
from both samples. By comparing the component of the main sample to that of
the reference sample, peaks specific to the main sample were indicated.

CT 撮影用投影型 X 線顕微鏡の開発
3PS028Development of Projection X-ray Microscope for MicroTomography

Shannon エントロピーの可視化による質量顕微鏡データの情報解析
Information analysis by visualizing Shannon entropy using
Imaging Mass Spectrometric data

Hideyuki Yoshimura (Meiji Univ.)
The projection X-ray microscope makes use of a very small X-ray light source
excited by the focused electron beam on a thin metal target. If an object isplaced just below the metal target, the expanding X-rays enlarge the shadow of
the object with infinite focal depth. We have developed the microscope bymodifying a scanning electron microscope (SEM) and achieved a resolution
about 0.1 &mum. It is advantageous to investigate an opaque sample with better
resolution than an optical microscope. And also has additional benefit toinvestigate three-dimensional structure of the thick specimen which can notstudied by an electron microscopy. Taking these advantages, we have applied
projection X-ray microscope for three-dimensional (3-D) structure analysis bymeans of cone-beam computed tomography (CT). To get higher resolution andbetter contrast, the choice of the metal target is important. The size of X-ray
source determines the resolution, thus the electron beam should be focused as
small as possible by condenser lens of SEM. And it should have a good balance
to get enough strength of X-ray. The electron beam irradiated at the metal target
is scattered in the metal and the scattered area is smaller in heavy metal. To getbetter contrast the wave length of X-ray should be longer utmost it penetrate thesample. In this case light metal target works better because of longer wavelengthof Kα characteristic X-rays. By accounting these conditions we have optimized
the metal target for biological specimens.

Noritaka Masaki, Mitsutoshi Setou (Hamamatsu University School of
Medicine, Department of Cell Biology and Anatomy)
Microscopic Imaging Mass Spectrometry (MIMS), which we have developed
cooperatively with Shimadzu Corporation and others, enables us to analyze
spatial distribution of enormous molecules in the resolution of single cell level.
MIMS has demonstrated its successful performance by revealing key molecules
and their distribution in various research fields, e. g., medical development,
agriculture, and material analysis. On the other hand, ageing and someinflammation are difficult to understand using a few key molecules. Principal
component analysis and machine learning are also inadequate to elucidate suchphenomena overlaying high-dimensional space. In this study, we try to figureout biological state in the framework of overall behavior of molecules, citing thefamous argument by Erwin Schrödinger who first utilized entropy for biology tounderstand living organisms as system that autonomously maintaining theirorder against disruption.
In our previous presentation in this biophysical society, we visualized Shannon
entropy in mouse brain to quantify in situ information held in molecularcomposition. Entropy image reveals characteristic distribution irrespective toanatomical representation. To confirm and discuss such spatial variation in
information entropy, we propose a new data processing that takes advantage of− S150 −spatial distribution by binning mass spectrum. This bundle of mass spectrum
corresponds to maximum likelihood estimation and entropy image calculated
from bundled spectrum shows more distinct pattern than normal entropy image.
3PS029シャペロニン GroEL フットボール型複合体を介したタンパク質
フォールディングの直接観察

Direct observation of protein folding mediated by the footballshaped GroEL-GroES complex
Yodai Takei, Ryo Iizuka, Taro Ueno, Takashi Funatsu (Grad. Sch. of Pharm.
Sci., The Univ. of Tokyo)
The chaperonin GroEL is an essential molecular chaperone that mediates
protein folding together with its cofactor GroES in Escherichia coli. It has beencommonly believed that GroES binds alternatively to each ring of GroEL,
which is composed of two identical rings stacked back to back, during thefunctional cycle. In other words, an asymmetric GroEL-GroES complex (thebullet-shaped complex) is formed throughout the cycle, whereas a symmetric
GroEL-(GroES)2 complex (the football-shaped complex) is not formed. We
have recently shown that the football-shaped complex coexists with the bulletshaped complex during the functional cycle. However, how protein folding
proceeds in the football-shaped complex remains poorly understood. Here, weused green fluorescent protein (GFP) as a substrate to visualize the folding in the
football-shaped complex by single-molecule fluorescence techniques. First, we
directly counted the number of refolded GFP molecules in the football-shaped
complex, and showed that GFP can fold in both rings of the complex. Next, we
performed the real-time imaging of GFP folding in the football-shaped complex.
Remarkably, the folding was a sequential two-step reaction, and the kinetics wasin excellent agreement with that in the bullet-shaped complex. These results
demonstrate that the same reactions take place in both cavities of the footballshaped complex in the same manner to facilitate protein folding. These findings
shed new light on the functional cycle including the football-shaped complex.
3PS030等方性と偏光変調性の 2 つの全反射型蛍光顕微鏡を組み合わせる

Combining of isotropic TIRFM and polarization-modulation
TIRFM
Shoko Fujimura1, Yuh Hasimoto1,2, Kengo Adachi1,3, Rinako Nakayama1,
Tomoko Masaike1, Takayuki Nishizaka1 (1Gakushuin Univ.,Dept.Physics, 2Present
Dept. Hamamatsu Photonics K.K., 3Present Dept. Waseda Univ.)
Two different types of total internal reflection fluorescence microscopy,
TIRFM, have been independently developed in our research group. The first one
is an isotropic TIRFM, iTIRFM (Adachi et al., Cell 2007), in which thehollow-cone light generated by a diffractive diffuser is parallelized and focusedto the back focal plane of the objective. Single flurorephores at the sample plane
are almost uniformly excited even when their orientation are randomly
distributed, because the incident light comes from all directions and so theresultant evanescent field contains all (X, Y and Z) polarization-components.
The second one is a polarization-modulation TIRFM, pmTIRFM (Nishizaka etal., Nat. Struct. Mol. Biol., 2004; Masaike et al., Nat. Struct. Mol. Biol., 2008),
which enables us to detect orientation of single flurorephores in XY-plane byanalyzing a phase of the oscillation of their intensity.
We here combined these two techniques on the same Nikon microscope
equipped with a commercial ‘perfect-focus system.’ Two different illuminations
are switchable in a second by translating two filter cubes, which are located at
the different points on the optical axis, with electric motors. This experimental
setup allows to characterize single fluorophores quantitatively in terms ofintensity, orientation and anisotropy, which uncover the change in local
environment of multiple catalytic sites in a single protein molecule, such as F1ATPase, by directly visualizing repetitive binding and hydrolysis of 2’-Cy3EDA-ATP.
3PS031

炎症時におけるヒト単球サイトカイン分泌のライブセルイメージング

Live-cell secretion imaging assay of the inflammatory cytokine
from human monocytes
Yoshitaka Shirasaki1,2, Nanako Shimura1,3, Nobutake Suzuki1, Kazushi
Izawa4, Asahi Nakahara1,2, Mai Yamagishi1, Yoshie Harada5, Shuichi Shouji2,
Ryuta Nishikomori4, Osamu Ohara1,6 (1RCAI, RIKEN, 2Grad. Sch. Adv. Sci.
Eng., Waseda Univ., 3Grad. Sch. Med. Pharm., Chiba Univ., 4Grad. Sch. Med.,
Kyoto Univ., 5iCeMS, Kyoto Univ., 6KAZUSA DNA Res. Inst.)
Inflammatory cytokines play a key role in the initiation of inflammation.
However, their secretion dynamics especially at the single cell resolution still

remain unclear. For example, IL-1β is a leaderless protein, lacking the signal
peptide that typically directs secreted proteins through the classical endoplasmic
reticulum-Golgi secretory pathway. Therefore the mechanisms of cellularrelease into the extracellular environment remain the subject of muchinvestigation. To overcome this, we developed live-cell secretion imaging assay
by total internal reflection fluorescence illumination with micro-fabricated wellarray chip. Here we tried to monitor the release dynamics of LPS activatedhuman monocyte before and after additional ATP stimulation. Forty wells wereobserved every 1 minute for 4 hours. While little signals of IL-1 β could
observed before ATP stimulation, the fraction of the IL-1 β secreting cells
largely increased after ATP stimulation. Moreover they showed strong IL-1βsignal and increased in their membrane permeability indicated with SYTOX
dead cell staining reagent. This change seems to represent a “Terminal release”,
which is usually associated with cell death called “pyroptosis”. This work thus
succeeded in imaging of IL-1β secretion from living cells at a high temporal
resolution, which will enable us to investigate acute-phase inflammatory
reactions of monocytes at the single-cell level for the first time.
3PS032

大腸菌異物排出タンパク質複合体コンポーネントの細胞内動態解析

Intracellular dynamics of the xenobiotic efflux proteins inEscherichia coli
Kentaro Yamamoto1, Takehiko Inaba2,4, Yoshiyuki Sowa2,3, Ikuro
Kawagishi1,2,3 (1Dept. Frontier Biosci., Grad. Sch. Eng., Hosei Univ., 2Res.
Cen. Micro-Nanotech., Hosei Univ., 3Dept. Frontier Biosci., Fac. Biosci. andAppl. Chem., Hosei Univ., 4Present: RIKEN Adv. Sci. Inst.)
The rising occurrence of multidrug-resistant bacteria is a serious worldwide
issue. One of the major causes of the multidrug resistance is the elevatedexpression of an RND-type xenobiotic efflux transporter complex, which
consists of three components, the inner membrane transporter, the membranefusion protein, and the outer membrane channel. Among the five inner
membrane transporters of Escherichia coli, only AcrB, which forms a complex
with the membrane fusion protein AcrA and the outer membrane channel TolC,
is constitutively expressed. Interestingly, the other inducible transporters arealso coupled to TolC. This raises an interesting question: does a newly
synthesized inner membrane transporter replace the pre-existing transporter incomplex with TolC or form a ternary complex de novo? In this study, we aim at
visualization of the construction processes of the efflux protein complex. First,
we constructed a strain expressing a chromosome-encoded fusion protein AcrBGFP. We observed with total internal reflection fluorescence microscopy(TIRFM), most fluorescent foci of AcrB-GFP did not show lateral movement,
whereas they were moving incessantly in the absence of TolC. Second, a residue
in the extramembrane loop of TolC was replaced by Cys, and the resulting
mutant was stained with the thiol-reactive fluorescent reagent to demonstrateco-localization of AcrB-GFP with labeled TolC. These results suggest that AcrBis fixed when it forms a complex with TolC, which penetrate through the
peptidoglycan layer.
3PS0331 分子蛍光イメージングを用いた転写活性化による Arp4 の動態変化の定量解析

Quantitative analysis of changes in molecular dynamics of
Arp4 upon transcriptional activation using single-molecule
fluorescence imaging
Katsuo Ichinomiya1, Masahiko Harata2, Kumiko Sakata-Sogawa1,3, Makio
Tokunaga1,3 (1Grad. Sch. Biosci. Biotech., Tokyo Inst. Tech., 2Grad. Sch. Agr.
Sci., Tohoku Univ., 3RCAI, RIKEN)
Actin-related protein 4 (Arp4) is a component of a chromatin-remodelingcomplex. Chromatin-remodeling is thought to occur with direct binding of Arp4
to histones, indicating that Arp4 is involved in transcription control.
Consequently, molecular dynamics of Arp4 is expected to change in correlation
with transcriptional activity. However, the details of relationship between
molecular dynamics of Arp4 and transcriptional activity remain unclear. In thisstudy, we performed quantification of molecular dynamics of Arp4 both before
and after stimulation with the phorbol ester PMA, as a transcriptional activator.
We carried out single-molecule fluorescence imaging of the nucleus of CHO
cells expressing TagRFP-T fusion proteins of Arp4 at a low level. The residence
time of individual Arp4 molecules, the time from appearance to disappearanceof the fluorescence spot, was measured in the images. We found that a residence
time after stimulation tends to become shorter than before. It means thattranscriptional activation causes dissociation of Arp4-containing chromatinremodeling complexes from the target sites. In yeast, the number of the− S151 −chromatin-remodeling complexes is estimated to be much less than that of thetarget sites. Upon transcriptional activation, Arp4 needs to cover the increased
target sites with the limited number of the molecules. Our result may reflect
this molecular mechanism. We will also discuss how Arp4 is involved intranscription with regard to RNA polymerase II.
3PS034

ATP 結合部位変異体 Arp4 βのイメージング解析

Imaging analysis of Arp4β mutants in ATP-binding site
Naomichi Inaba1,2, Yuma Ito1,2, Masahiko Harata3, Makio Tokunaga1,2, Kumiko
Sakata-Sogawa1,2 (1Grad. Sch. Biosci. Biotech., Tokyo Inst. Tech., 2RCAI,RIKEN, 3Grad. Sch. Agr. Sci., Tohoku Univ.)
Actin related proteins (Arps) are evolutionarily and structurally similar withactin, and they have ATP binding activity like actin. Actin and Arp4β are criticalcomponents of ATP-dependent chromatin remodeling complex, which regulates
the dynamic modification of chromatin architecture. Arp4β found to play a keyrole as a regulator of the activity. However, the detail of the regulatory
mechanism remains unclear.
To clarify whether ATP binding to Arp4β handles the regulation, we planned toanalyze dynamics of the Arp4β mutant which have the defect of ATP binding
activity. We constructed GFP fusion protein of Arp4β (GFP-Arp4β) and GFPArp4 β -G173D by single nucleotide substitution to deprive ATP binding
activity. We established CHO cell lines expressing GFP-Arp4β or GFP-Arp4βG173D at a low level.
When we observed the cells with fluorescence microscopy, wild type Arp4β
were localized mainly in the nucleus. In contrast, Arp4β-G173D was scarcely
localized in the nucleus, and most of them were localized in the cytoplasm. To
check the effect of nuclear export or actin polymerization to this cytoplasmic
localization of the mutant protein, we observed the mutant under nuclear export
inhibitor (LeptomycinB), or actin polymerization inhibitor (LatrunculinB)
existence. However, the localization of the mutant did not change. It issuggested that ATP binding activity may regulate its localization in the nucleus.
3PS035遺伝的にコードされたカルシウムインジケーターを用いた細胞内カ
ルシウム分布の動態解析

Dynamics of intracellular Ca2+ distribution measured bygenetically encoded Ca2+ indicators
Hirofumi Oyama1,2, Yuma Ito1,2, Satoshi Ikeda1,2, Kumiko SakataSogawa1,2, Makio Tokunaga1,2 (1Grad. Sch. Biosci. Biotech., Tokyo Inst.
Tech., 2RCAI, RIKEN)
Ca2+ mediates the signal transduction pathways in cells as a second messenger.
By stimulus, the distribution of intracellular Ca2+ changes dramatically. After
Ca2+ is released to the cytoplasm from the endoplasmic reticulum, calcium
influx occurs via calcium ion channel on the cell membrane to the cytoplasm.
Ca2+ is thought to diffuse into nucleus from the cytosol through nuclear pores.
Free calcium in the nucleus regulates important functions such as transcriptionof some genes. There could be some mechanism for precise regulation of Ca2+
concentration in the nucleus. Recently genetically encoded Ca2+ indicators havebeen developed. The fusion proteins of the indicator enable us to analyze thelocal Ca2+ concentration. To analyze the Ca2+ distribution after stimulation, weconstructed fusion proteins of membrane protein or nuclear protein to Ca2+
indicator GCaMP3 and established the HeLa cells expressing these Ca2+
indicators. Upon stimulation with Histamine, We observed the propagation of
Ca2+ over the cytoplasm and into the nucleus. We will discuss the difference ofCa2+ distribution pattern between in the cytoplasm and the nucleus.
3PS036

大腸菌二成分制御系センサーおよび応答調節因子の蛍光イメージング

Fluorescence imaging of histidine kinases and response
regulators of Escherichia coli
Daigo Nakamura1, Yuka Iritani2, Mitsuyasu Fukushima2, Akiko Yamakawa1,
Hiroyuki Sawaki1, Takehio Inaba3,5, Satomi Banno4, Ikuro Kawagishi1,2,3(1Dept. Frontier Biosci., Grad. Sch. Eng., Hosei Univ., 2Dept. Frontier Biosci.,Fac. Biosci. Appl. Chem., Hosei Univ., 3Res. Cen. Micro-Nano. Tech., Hosei
Univ., 4Natl. Inst. Infect. Dis., 5RIKEN Adv. Sci. Inst.)
Escherichia coli has many two-component regulatory systems (TCS) that
respond to specific environmental signals. Each TCS consists of a sensor
histidine kinase (HK) and a response regulator (RR), together forming a signaltransduction pathway via histidyl-aspartyl phospho-relay. We previously
constructed GFP fusions to each HK and classified their localization patterns
into six groups. In this study, to explore the physiological significance of

localization of HKs, we constructed a series of plasmids encoding mCherry
fusions to each RR. Unexpectedly, expression of mCherry fusions to some RRs
resulted in occurrence of elongated cells. This appears to be due tooverexpression because cell sizes were normal when the expression levels of therelevant RR-mCherry fusions were tightly controlled. Out of 24 RRs tested,mCherry fusions to AtoC that is involved in acetoacetate metabolism and TorR
that is involved in regulation of anaerobic respiration showed (polar and/or
lateral) dotted patterns of localization. These RR-mCherry fusions appeared toco-localize with the GFP fusions of the cognate HKs (AtoS and TorS,
respectively). We next examined the effect of the cognate HKs (AtoS and TorS)
on the localization of these RR-mCherry fusions. Localization of AtoCmCherry was lost when the atoS gene was deleted, whereas TorR-mCherry
retained its localization pattern in the torS deletion background. These results
suggest that AtoC (and possibly TorR also) localize to a pole or lateral
membrane regions by interacting with its cognate HK.
3PS037

ラマン分光顕微鏡を用いて細胞の分化状態を識別する

Raman microscopy distinguishes the status of differentiatingcell
Muneki Yoshida1, Taro Ichimura1,2, Chiu Liang-da3, Katsumasa Fujita3,
Tomonobu Watanabe1,2,4, Hideaki Fujita1,2 (1Grad. Sch. FBS, Unive.
Osaka, 2QBiC, Riken, 3Grad. Sch. Eng., Unive. Osaka, 4Precursory Research for
Embryonic Science and Technology)
We demonstrate that Raman microscopy distinguishes the status of celldifferentiation without any labeling. Raman scattering arises from molecularvibration of sample molecules. When a molecule is exposed to photons, a small
fraction of photons are scattered with a different frequency from that of incident
photons. The shift of the frequency is equal to the molecular vibration
frequency. Spectra of Raman scattering are considered as “molecularfingerprints”, because one can reveal molecular species, structures, andsurrounding environment by the shift. We expect that the Raman scattering
spectrum of a cell can be considered as “cellular fingerprint”, and that we candistinguish, in particular, the status of cell differentiation without any labeling.
In order to prove it, we observed the transition of the status of cells during
differentiation by Raman microscopy. We built a confocal slit-scanningmicroscope with excitation wavelength of 532 nm, and continuously obtained
Raman images of ES cells from the beginning of differentiation. In analyzing
the spectra of nuclei of the ES cells, we found a significant difference between
before and after differentiation. Furthermore, to analyze the spectra quantitatively, we employed principal component analysis. The plot of principal
component scores showed the translation of the scores in the principal
component hyperspace with progression of the differentiation. We believe thisresult verifies the applicability of Raman microscopy to monitoring of thetransition of cellular status.
3PS038

VSFG 検出赤外超解像顕微鏡を用いた毛髪サンプルの分子構造解析
Molecular structure analysis of the human hair samples byusing VSFG detected IR super-resolution microscope

Katsuya Kikuchi1, Takaki Sato1, Tomoki Tajima1, Masaaki Fujii1, Shinobu
Nagase2, Yuuji Hirano2, Takashi Itou2, Makoto Sakai1 (1Tokyo Tech, 2KaoCorp)
We have developed vibrational sum-frequency generation (VSFG) detected IRsuper-resolution microscope with a sub-micrometer spatial resolution until now,and have reported the applications to various biological samples such as onion
root cell, A549 cultured cell, bio-cellulose, etc. In this study, we applied an IRsuper-resolution microscope to human hairs. Human hair is a complex
biological material consisting of distinct morphological components and is wellknown to compose of the medulla, cortex, and cuticle regions from inside tooutside. At the cortex area occupying 90% of human hair, keratin protein, which
is primary component of human hair, forms a line in the shape of fiber along thelongitudinal direction of human hair, and controls physical properties such as
flexibility, strength, softness and curliness. To understand the relationshipbetween physical properties and the internal nanostructure, we attempted IR
imaging at IR super-resolution in the 6-9 μm mid-IR region.
As a result, we succeeded in obtaining VSFG image in the amide III band at
1250 cm-1 with sub-micrometer spatial resolution, and clearly observed strong
VSFG signals only from the cortex area. VSFG image seems to be contributed
to the IR mapping of the distribution of α-helix structure at the cortex. This is
supported by very weak emission from the cuticle and medulla, those do nothave an α -helix structure. In the presentation, the internal nanostructure ofhuman hairs will be discussed in detail.

− S152 −3PS039

希土類材料を用いた膜蛋白質の高感度イメージング

Highly sensitive imaging of cell membrane proteins by usinglanthanide materials
Tatsuya Nakamrua1, Shin Mizukami1,2, Kazuya Kikuchi1,2 (1Graduate
school of Engineering, Osaka University, 2Immunology Frontier ResearchCenter, Osaka University)
Since cell membrane proteins play important roles for membrane transport andsignaling, it is important to analyze functions of cell membrane proteins. So far,fluorescent proteins have been utilized to study the protein behaviors. On theother hand, protein labeling technique with organic dyes and protein tags has
been focused because it yields temporal labeling and excellent fluorescent
properties. Our group developed protein labeling system based on a mutant βlactamase (BL-tag), which specifically forms covalent binding with β-lactam
compounds. We have developed the fluorogenic probes, which consist oforganic fluorescent dyes and β-lactam compound.
In this study, we further developed new probes that utilize luminescentlanthanide complex with silica nanoparticles to achieve higher measurement
sensitivity. Luminescent lanthanide complexes show long lifetime luminescencethat reaches millisecond order. Since fluorescence lifetime of organic dyes is of
the order of nanoseconds, luminescent lanthanide complexes enable timeresolved fluorescence measurement to eliminate short-lifetime fluorescencesuch as autofluorescence from biological samples. Silica nanoparticles arebiocompatible materials and can include a large number of dyes. By combining
lanthanide complex with silica nanoparticles, we developed highly luminescentlanthanide silica nanoparticles. Additionally, we attempted labeling of BL-tag
expressed on cell surface.
3PS040ストレス顆粒内 mRNA 微小構造体の超解像イメージング

Super-resolution imaging of mRNA nano-structures in stressgranuleKo Sugawara, Kohki Okabe, Akihiko Sakamoto, Takashi Funatsu (Grad. Sch.
Pha. Sci., Univ. Tokyo)
mRNAs play critical roles in gene expression with various regulations. Understress, cytoplasmic mRNAs assemble and form stress granules (SGs), where
they are remodeled for repression of translation. However, the mechanisms ofaction remain elusive. The aim of this study was to visualize the fine structure of
SGs and to investigate the detailed physiological role of them. Stochastic opticalreconstruction microscopy (STORM) provided us super-resolution images withspatial resolution of ~20 nm in the lateral direction and of ~60 nm in the axial
direction. Three-dimensional super-resolution imaging was performed using
cylindrical lens. To visualize endogenous cytoplasmic mRNAs, we havemicroinjected Cy5-labeled linear antisense 2'-O-methyl probes into thecytoplasm of COS7 cells. After the injection, cellular stress was induced byaddition of 0.5 mM arsenite in a culture medium. To examine the maturation ofSGs, STORM images were captured at various time-points during SG
formation. Three-dimensional super-resolution images showed that endogenous
mRNAs located in spherical compartments with a diameter of 200 nm, which
could not be observed in diffraction-limited imaging. We termed this structure“mini-granule”. With stress duration, mini-granules increased in number, while
they maintained the same size. The results demonstrated that the growing
process of SGs was resulted from the assembly of mini-granules. The result of
this study indicated that mini-granules were responsible for the physiological
functions of SGs.
3PS041

位相変調型微分干渉顕微鏡を用いたミトコンドリアのイメージング
解析

The imaging analysis of mitochondria with retardation
mediated differential interference microscope (RM-DIC)
Keisuke Haseda, Keita Kanematsu, Yoshihiro Ohta (Div. Biotechnology andLife sciences , Tokyo University of Agriculture and Technology)
Mitochondria produce ATP by utilizing the membrane potentials. In this study,
we measured the mitochondrial internal density with RM-DIC and examined theinfluence of the internal density on mitochondrial membrane potentials (MMP).
Both the internal densities and the MMPs are heterogeneous in a single cell.
Though it is considered that increase in the internal densities affects the MMPs
through the concentration of proton pumps, the correlation between themremains to be clarified. To examine the effects of the mitochondrial internal
densities on the MMPs, we have improved RM-DIC and examined eachmitochondrion in terms of the optical length and TMRE fluorescence asmeasures of the internal density and MMP, respectively. To eliminate the effectsof the mitochondrial size on the optical length and TMRE fluorescenceintensity, we corrected these values with the mitochondrial sizes obtained fromtransmittance images of individual mitochondria. When we measured isolated
mitochondria, the corrected optical length showed a close correlation with thecorrected TMRE fluorescence. These results suggest that the increase in internal
densities of mitochondria stimulates mitochondrial activity. Next we examined
mitochondria in neurites but we did not observe the correlation between theinternal density and the activity. These results indicate that the surrounding
environments of mitochondria are more important for the mitochondrial activity
than the internal density.
3PS04219

F MRI monitoring of Gene Expression in living cells

Hisashi Matsushita1, Shin Mizukami1,2, Yuki Mori2,3, Fuminori Sugihara1,2,
Masahiro Shirakawa4, Yoshichika Yoshioka2,3, Kazuya Kikuchi1,2 (1Grad. Sch.
Eng., Univ. Osaka, 2Immunology Frontier Research Center, Univ.
Osaka, 3Grad. Sch. Frontier Biosci., Univ. Osaka, 4Grad. Sch. Eng., Univ.
Kyoto)
It is highly significant to accurately determine spatiotemporal expression
patterns of each gene in vivo for the establishment of new medical treatments,
development of diagnostic methods, and discovery of pharmaceuticals. The
development of 19 F MRI probes as a powerful in vivo imaging tool formonitoring biological phenomena has attracted increasing attention, since 19 F
MRI has many advantages such as no detectable intrinsic MRI signals inbiological samples and the relatively high sensitivity, which corresponds to 83%
of 1H sensitivity.
Recently, we successfully developed off/on switching 19F MRI probes to detect
enzyme activities on the basis of the paramagnetic relaxation enhancement(PRE) effect. In the previous study, we detected β-galactosidase activity as areporter enzyme for gene expression in the cytosol using a specific 19 F MRIprobe. However, the cells required fixation to image intracellularly expressed βgal activity, because the probe molecules were not permeable through the intact
plasma membrane. For the in vivo applications, it is necessary to detect gene
expression in living cells. In this report, we describe a novel method formonitoring gene expression in living cells by measuring extracellularly
expressed enzyme activity by 19F MRI. We constructed a new imaging system
by exploiting β -lactamase displayed on the plasma membrane as a reporter
enzyme, a 19F MRI probe was rationally developed to monitor β-lac activity.
Development of nanocapsule probes for sensitive 19F MRI
Hiroaki Mukai1, Shin Mizukami1,2, Yosuke Nakanishi1, Fuminori Sugihara2,
Kazuya Kikuchi1,2 (1Grad. Sch. Engin., Osaka univUniv., 2iFReC)
3PS043

MRI is suitable for in vivo imaging because it can non-invasively visualize theliving body with high spatial and temporal resolution. Thus, recently manysmart 1 H MRI probes have been reported. However, such 1 H MRI signal
enhancement needs to be discriminated from the background 1H MRI signals
of water, fatty acids, and other biomolecules. To avoid the limitation, we havefocused on the use of 19F MRI. Since almost no intrinsic 19F MRI signals areobserved in animal bodies, a high signal-to-noise ratio measurement of
extrinsic 19 F-containing compounds could be achieved. Thus, we havedeveloped the 19F MRI probes to detect protease activity on the basis of PRE
effect. 1) However, further improvement of the sensitivity is still required forpractical usage. Thus, we developed nanoparticles (NPs) containing
perfluorocarbon as highly sensitive 19F MRI contrast agents.
When the nanoparticles were injected into live animals, the 19F NMR signals
were clearly detectable and accumulation was observed in the liver after 1 day.
Protein-tag ligands were modified on the surface of NPs, then specific cellularlabeling was achieved. In summary, we developed a highly sensitive 19F MRI
nanoparticle that can be visualized in vivo, and this novel material would lead tonovel molecular imaging method using 19F MRI.
1) J. Am. Chem. Soc., 2008, 130, 794-795
3PS044

hiPSCs 細胞培養のための polydimethylsiloxane への細胞外基質コート

ECM coating on polydimethylsiloxane for hiPSCs culture
Ryosuke Yoshimitsu1, Koji Hattori2, Shinji Sugiura2, Toshiyuki Kanamori2,
Kiyoshi Ohonuma1 (1Nagaoka University of Technology, 2National Institute ofAdvanced Industrial Science and Technology)− S153 −Human induced pluripotent stem cells (hiPSCs), which can be cultured inmonolayers under a feeder- and serum-free condition, provide a good model forstudying cell differentiation by imaging. However, cell differentiation on aconventional culture dish faces problems such as spatio-temporal unevenness of
medium components caused by autocrine and paracrine factors and byintermittent medium change. To address such problems, we have developed aperfusion culture microchamber array chip that enables the control of culture
conditions. Most previously reported microchips have been made ofpolydimethylsiloxane (PDMS). However, only limited cell types are cultivable
on PDMS without extracellular matrix (ECM) coating. Here, we show that ECM
coating (fibronectin, laminin, collagen, and gelatin) is determined by theadhesion, growth, and differentiation of hiPSCs on the PDMS surface under afeeder- and serum-free condition. The number of adhered cells and the growthrate of hiPSCs that were cultured on the PDMS surface coated with fibronectin
and laminin were larger than that on the surface coated with collagen andgelatin. Flow cytometry analysis revealed that undifferentiated marker positive
cells on the PDMS surface coated with fibronectin and laminin were larger thanon the one coated with collagen and gelatin. These results suggest that the
optimal ECM for culturing hiPSCs on a PDMS surface is fibronectin andlaminin. This study enables the establishment of differentiation process imaging
systems using hiPSCs on perfusion culture chips.
3PS045

Perfusion device to observe of single ES cells

Shougo Nakamura1, Atushi Maruyama4, Yuichi Wakamoto2, Shin-ichi
Sakai3, Bayar Hexig3, Toshihiro Akaike3, Kiyoshhi Ohnuma4 (1Nagaoka
university of Technology, 2Grad. Sch. Arts. & Sci., Univ. Tokyo, 3Grad. Sch.
Biosci. & Biotech., Tokyo Inst of Tech, 4TRI, Nagaoka Univ of Tech)
ES and iPS cells characterized by both pluripotency and self-renewal are apromising source of regenerative medicine, as well as a good model for studying
cell transition to multiple states. It is believed that cell population differentiation
is a result of individual cell differentiated sub-population. To determine which
hypothesis is more reasonable, we developed a single cell observation system,
which we reported at the previous meeting. The system consists of threeelements: GFP reporter mouse ES cells used to monitor the differentiation state,
serum- and feeder-free culture conditions, and an E-Cad-Fc coated plate thatenables us to dissociate cells into single cells. However, the number of dataacquired is very small, because cells at seeding were heterogeneous in theirstate, and many cells were lost during the medium change. In this study, wedeveloped a perfusion device for observing a single-cell reaction to medium
stimulation to improve data acquisition efficiency. This device has twofunctions: to change the culture medium automatically by using a perfusionculture system, and to seed the cells directly in the device using a cell sorter.
This device may enable us to elucidate mechanisms of cell transtion to multiple
states.
3PS046

アルギン酸シートを用いた初代培養細胞セルソーティング法の開発

A Non-destructive Culturing and Cell Sorting Method for
Cardiomyocytes and Neurons Using an Alginate Layer
Hideyuki Terazono1, Hyonchol Kim1, Akihiro Hattori2, Fumimasa Nomura2,
Tomoyuki Kaneko2, Kenji Yasuda1,2 (1Yasuda " On-chip cellomics" project,
KAST, 2Biomed. Info., Biosys., Biomat. Bioeng., Tokyo Medical and Dental
Univ.)
It is proposed that a non-destructive collecting method of cultured cells after
identifying their functional characteristics. Embryonic stem (ES) or induced
Pluripotent stem (iPS) cells are widely expected to be used in clinicaltherapeutics for transplantation or as drug screening tools. For each passaging
and collecting of cells, cultured cells are usually detached from the culture dishusing collagenase or trypsin, which degrades the extracellular matrix orproteins. These methods are harmful to cells, so that fragile cells cannot be
recultured. Alginate is a useful polymer for use as a culturing scaffold, because
it is non-perturbing to cells. In our method, cells are cultivated on a thin alginate
layer in a culture dish and released by spot application of a calcium chelatebuffer that locally melts the alginate layer and allows collection of cultured cells
at the single-cell level. Primary hippocampal neurons, beating humanembryonic stem (hES) cell-derived cardiomyocytes, and beating hES cellderived cardiomyocyte clusters cultivated on an alginate layer were successfully
released and collected with a micropipette. The collected cells were recultured
while maintaining their physiological function, including beating, and elongatedneurites. The results suggest that this method may allow to sort cells thatphysiological properties are aligned, and eventually facilitate transplantation of
matured ES- or iPS-derived cardiomyocytes and neurons differentiated inculture.

3PS047

様々な粒径の超常磁性ヤヌス微粒子の作製と非侵襲的細胞回収技術への応用

Fabrication of Superparamagnetic Janus Particles Having
Various Sizes and Its Application for Non-Destructive Cell
Sorting
Hyonchol Kim1, Hideyuki Terazono1, Hiroyuki Takei1,2, Kenji Yasuda1,3(1KAST, 2Facul. Life Sci., Toyo Univ., 3Inst. Biomat. Bioeng., Tokyo Med.
Dent. Univ.)
Janus particles, which are composed of two chemically distinct hemispheres, areused for sensors, new devices, or for a model to study self-assembly physics.
Fabrication of various Janus particles is therefore fundamental, and we propose
a method for fabrication of superparamagnetic Janus particles and its application
for target cell sorting without serious damages. Janus particles were fabricatedusing polystyrene spheres as casts. The sphere was placed on a substrate, andmagnetic materials were deposited on the sphere by thermal evaporation. In
addition, thin Au layer, typically 2 to 5 nm thick, was formed on the particle foreasy immobilization of biomolecules. Janus particles whose half was composedof deposited metal and the other was polystyrene were then fabricated. By using
this method, we fabricated superparamagnetic Janus particles with strict controlof magnetic material thickness. One advantage of our method is easy fabricationof particles having various sizes using different polystyrene casts, which
achieve particle size-dependent collection of target molecules. For a practicalapplication of fabricated magnetic particles, a DNA aptamer was immobilized
onto the particle to use it for specific cell sorting. The particle was reacted withcells and collected using magnet, then its target cell can be specificallycollected. Moreover, the magnetic probe can be easily detached from the cellwith nuclease treatment. These results indicate that fabricated magnetic particles
can be used for non-destructive cell sorting.
3PS048

一細胞時系列解析を用いた定量的プロモータ活性評価法の開発

Event driven time lapse image analysis reveal the promoter
activity
Kazumi Hakamada, Yusaku Somei, Jun Miyake (Osaka University)
Modeling and understanding cellular dynamics is the holy grail of biology.
Many researchers addressed to this target and developed a lot of methods. It is
still difficult to elucidate cellular dynamics quantitatively due to cellular
heterogeneity. Here, using time lapse images of both phase-contrast andfluorescent images of single cell image analysis, we investigate the promoter
activity. HeLa cells were transfected with AcGFP, which encodes AcGFP under
the control of promoter of CMV, EF1-α, and SV40 by NanoJuice, respectively.
After transfection of them, phase-contrast image and fluorescent images of cells
in each well were recorded at interval of ten min for 24h by using Programmable
Cellular image Tracer. By recording the timing of cell division by using phase
contrast image and that of onset of fluorescent by using fluorescent image, we
calculate the difference between cell division and onset of gene expression
about three promoters. The fluorescent intensity was observed in order of CMV(248. 5 min), EF1-α (461. 3 min), SV40 (339. 4 min), respectively and time
periods between cell division and onset of three promoters had statisticallysignificance (p <1.8 x 10-20, welch’s t-test). Because these three vectors havethe same genes (AcGFP) and they were expected to have the same translation
period, those differences suggested the differences of promoter activities. By
using single cell time lapse image analysis, we can quantitatively compare thepromoter activity.
3PS049画像認識型オンチップ・セルソーターによる形状認識に基づいた細胞分離法の定量的評価

Quantitative evaluation of cell separation method based onshape recognition using on-chip imaging cell sorter
Akihiro Hattori1,2, Tomoyuki Kaneko1, Fumimasa Nomura1, Kenji Yasuda1,2(1Dept. Biomedical Information, Div. Biosystems, Inst. Biomaterials andBioengineering, Tokyo Medical ana Dental Univ., 2Yasuda “On-chip
Cellomics“Project, Kanagawa Academy of Science and Technology)
We have evaluated the method for the label-free optical microscopic image-based
separation of cardiomyocytes from the mixture of collagenase-digested
embryonic mouse heart cells using the on-chip imaging cell sorter system, inwhich real-time high-speed camera-based phase-contrast cell image recognition
was performed. To separate the cardiomyocytes from other cells including− S154 −fibroblasts, we distinguished the roughness of the cell surface quantitatively as theindex of sorting. The selected cells with smooth surface (roughness index not
more than 1.1) for cardiomyocytes, and confirmed that the more than 80% of thecollected and recultivated cells were self-beating and the 99.2% of them wereidentified as cardiomyocytes by immunostaining. The results indicates thepotential of the label-free cell purification using imaging cell sorting system,
especially for cardiomyocytes by the index of their cell surface roughness, whichmight be applicable for purification of cariomyocytes for regenerative medicine.
3PS050マイクロ波照射下での微生物培養とプロテオーム解析

Microorganism cultivation under microwave irradiation andits proteomic analysis
Wataru Nagayoshi1, Rintaro Hoshino1, Yuki Kurita1, Arata Shiraishi1, Takeo
Yoshimura2, Toru Kodama3, Shokichi Ohuchi1 (1Dept. Biosci. Bioinform.,
Kyushu Inst. Technol., 2Dept. Appl. Bio. Sci., Tokyo Univ. Sci., 3Vecell Inc.)
We have studied to take advantage of the microwave heating to the field ofchemical biology and biotechnology. In this study, we applied the microwave
irradiation for microorganism cultivation. The bacteria such as Escherichia coli,
Flavobacteria, and Bacillus subtilis were cultivated on various temperatures andmicrowave irradiations. The microwave culture system was composed ofmultimode microwave apparatus with electrical transformer, thermocouple, andair pump. The microwave apparatus by modified domestic microwave ovencould be controlled the temperature by thermocouple at 2.45 GHz frequency.
The air bubble was flowed to the culture medium instead of shaking the tube.
Escherichia coli JM 109 was cultured at 37 degree C for 12 hours under
microwave irradiation in LB and M9 culture media. Conventional culture withbubbling was incubated by water bath at 37 degree C. The culture medium was
sampled to measure the optical density (OD660) at each time. A comparison of
optical density between conventional and microwave cultivation showed that
optical density remained unchanged. On the other hand, the expressed proteinfrom cells of E. coli JM 109 was estimated by two-dimensional electrophoresis
through the treatment for the proteomic analysis. As the results, several proteins
under microwave cultivation were very different from conventional incubation.
Their proteins from microwave culture was analyzed and identified by methodof peptide mass fingerprint (PMF).
3PS051PDMS 基盤表面上でのタンパク質混合物の吸着による hiPS 細胞のパターニング

Patterning for human iPS cells by adsorption mixture ofproteins on PDMS surface
Ryotaro Yamada1, Koji Hattori2, Shinji Sugiura2, Toshiyuki Kanamori2,
Kiyoshi Ohnuma1 (1Nagaoka University of Technology, 2Agency of Industrial
Science and Technology)
Human induced pluripotent stem (hiPS) cells derived from human somatic cells
have pluripotency, and they are therefore expected to be a suitable model forstudying early development. Cell differentiation is affected by cell-cellinteraction, which is difficult to control. A micro-patterning technique makes it
possible to create cell patterns in order to control cell-cell interaction. However,fabrication of these micro-patterns generally requires a lot of time and labor,
as well as expensive reagents and robotics. Here we report a simple
micropatterning technique to create patterns of hiPS cells on a polydimethylsiloxane (PDMS) surface under a serum- and feeder-free culture
condition. PDMS has translucency, gas permeability, low cytotoxicity, and can bemolded easily, so it is therefore typically used as a substrate material for
fabricating micro structures for cell cultures. We found that cell adhesion
patterning was achieved by coating a mixture of proteins, rather than a single
purified protein, on a PDMS patterned surface. This suggests the cell adhesion on
a PDMS surface is controlled by multiple proteins. We coated the PDMS surfaces
with a combination of purified proteins and succeeded in controlling hiPS celladhesion. We will apply this coating on a PDMS surface treated with patterned
plasma. This technique enables simple and low-cost patterning of hiPS cells.
3PT001

飢餓的ストレス下での 100S リボソーム形成による細菌の生存戦略

Survival strategy of bacteria under the starvation stress by
100S ribosome formation
Takayuki Kato1, Masami Ueta2, Tomoko Miyata1, Hideji Yoshida3, Akira
Wada3, Keiichi Namba1 (1Grad. Sch. of Frontier Biosci Osaka Univ, 2Yoshida
Biol. Lab., 3Dept. Phys., Osaka Med. Coll.)
In most bacteria, protein biosynthesis is suppressed by the formation of 100S

ribosome, a dimmer of 70S ribosome, under the starvation stress. In the case ofgram-negative bacteria, such as Escherichia coli (E. coli), the 100S ribosome is
promoted by ribosome modulation factor (RMF) and hibernation promoting
factor (HPF) binding to 70S ribosome. On the other hand, a protein named
SaHPF, which is a homolog of HPF, causes the formation of 100S ribosome ingram-positive bacteria, such as Staphylococcus aureus (S. aureus). To
understand the suppression mechanisms of protein biosynthesis, we tried toanalyze both structures by cryoEM image analysis. The numbers of particle
images used for the structural analyses of 100S ribosome from E. coli and S.
aureus were 11,889 and 15,885, respectively. We treated the images of two 70S
ribosome particles individually for 3D image reconstruction, and the structuresof 70S ribosome were solved at 18 Å and 15 Å, respectively. Then the atomicmodel of 70S ribosome was fitted into the 3D density map to build the models of100S ribosomes. In both cases, 100S ribosome is formed by the face-to-facecontacts between the 30S subunits of the two 70S ribosomes. Surprisingly,
however, the binding interfaces are completely different between E. coli and S.
aureus. We are now trying to determine the binding site and the structure of
SaHPF on ribosome. I will discuss different mechanisms of 100S ribosome
formation and the structural difference between RMF/HPF and SaHPF
responsible for the different mechanisms.
3PT002

ラミノパチー発症に関する A 型ラミンの構造学的研究

Electron microscopy of oligomerization function of nuclear
lamin A and the laminopathic mutants
Kazuhiro Mio1, Toshihiko Sugiki2, Muneyo Mio1, Chie Matsuda1, Yukiko
Hayashi3 (1AIST, 2Dep. Pharm, Musashino Univ., 3NCNP)
The nuclear lamins are type V intermediate filament proteins that constitutelamina at the inner nuclear membrane, critically important for the structuralproperties of the nucleus. Mutations in A-type lamins (LMNA) cause a variety
of diseases from muscular dystrophy and lipodystrophy to systemic diseases
such as premature aging syndromes. The molecular basis of these diseases isstill unknown. To understand the lamin-associated network at the inner nuclear
membrane and the mechanisms of laminopathies onset, we analyzed thestructures of lamin A ant its mutants using electron microscopy and otherstructure determining methods. We obtained wild type and mutant lamin A, that
include R453W (Emery-Dreifuss muscular dystrophy; EDMD), R527P(EDMD) and R527H (mandibuloacral dysplasia; MAD) from recombinantly
expressed E. Coli and analyzed their oligomerization function in terms of headto-tail filamentous assembly and the paracrystal formation. NMR andbiochemical analysis was performed for addressing the association modificationof lamin A with LEM family proteins (Lap2, EMD, Man1).
3PT003

NMR 法による APOBEC3C デアミネース活性に関する研究

A study on deaminase activity of APOBEC3C by NMR
Ryo Iwaoka1, Shingo Kitamura2,3, Keisuke Kanba1, Ayako Furukawa1,
Hideyasu Okamura1, Wataru Sugiura2,4, Takashi Nagata1, Yasumasa
Iwatani2,4, Masato Katahira1 (1Inst. Of Adv. Energy, Kyoto Univ., 2CRC,
NMC, 3Biotech., Grad. Sch. Of Eng., Nagoya Univ., 4Prog. In Integ. Mol.
Med., Grad. Sch. Of Med., Nagoya Univ.)
APOBEC3 (A3) proteins are responsible for the restriction of exogenous viruses
by its cytidine deaminase activity against minus strand DNA produced byreverse transcription of the virus genome. We have been focusing on A3G, apotent restrictor against HIV-1, and been analyzing its deaminase activity.
Recently, we started analyzing cytidine deamination by A3C, another antiviralprotein, which has different target with A3G. A3G prefers 5’-CCC-3’ context on
viral minus strand DNA, while A3C primarily recognizes 5’-TC-3’ context.
Detailed knowledge on the molecular mechanism of the deaminase activity ofA3C together with that of A3G would contribute to better understanding of how
A3 family proteins recognize and catalyze the different targets.
In this study, we have applied our established NMR method (EMBO J., 2009) tomonitor the time-course of cytidine deamination by A3C. The length of thesubstrate single-strand DNA (ssDNA) reportedly affects the affinity and activityof A3G. A3G shows similar affinity against 10-mer and longer ssDNAs,
however the affinity reduces against shorter ssDNAs. In addition, the activity of
A3G is highly influenced by the neighboring nucleotides of the 5’-CCC-3’.
Accordingly, we have carefully designed and synthesized two 11-mer ssDNAs,
TTTTTCTTTTT and AAAATCAAAAA. Using these substrates, we havechased, respectively, the decreasing and increasing signals of cytidine anduridine with time. The comparison of the deaminase activity between A3C andA3G will be discussed.

− S155 −3PT004

蛍光相互相関分光法を用いた転写因子-DNA 間相互作用の定量

Determination of dissociation constant between glucocorticoid
receptor and DNA using FCCS
Shintaro Mikuni1, Masataka Kinjo2 (1Research Center for Cooperative Projects,
Graduated School of Medicine, Hokkaido University, Japan, 2Laboratory of
Molecular Cell Dynamics, Faculty of Advanced Life Science, Hokkaido University,
Japan)
Glucocorticoid receptor (GR) is a transcriptional factor that controls broad rangeof physiological gene networks. By stimulation of ligand, not only GR activates
gene transcription by association to the specific genomic glucocorticoid
response elements (GRE) as a dimer, but also GR represses transcription by
association to negative GRE as a monomer in nucleus. Hence, it is important tounderstand quantitatively association of GR to GRE.
For measurement of affinity between DNA and DNA binding protein, such as atranscriptional factor, the electrophoresis mobility shift assay (EMSA) is wellused in vitro. However, in EMSA, their interaction is enhanced by the cage
effect of gel matrix. Therefore, it is difficult to discuss about the affinity
acquired from EMSA as in living cells, although EMSA is excellent as a methodfor detecting their interaction.
So, we established the determination of the affinity (dissociation constant, Kd)
between GR and DNA contained GRE sequence using fluorescence crosscorrelation spectroscopy (FCCS). For FCCS measurement, the full length
human GR was expressed in insect cells as EGFP fused protein (EGFP-GR) andpurified by using combination of His-tag and cation exchange column
chromatography. By FCCS measurement of purified EGFP-GR with Alexa647labeled GRE, the dissociation constant of GR-GRE was estimated as 2.71 μM.
Furthermore, we will discuss the differences of affinity of GR to several
different sequence of GRE.
3PT005

Quantitative study of Homo-Dimer Glucocorticoid Receptor
interaction by using Fluorescence Cross Correlation Spectroscopy in living cell
Manisha Tiwari1, Shintaro Mikuni2, Masataka Kinjo3 (1Graduate School ofLife Science, Hokkaido University, Japan, 2Research Center for Cooperative
Project, Graduate School of Medicine, Hokkaido University, Japan, 3Facultyof Advanced Life Science, Hokkaido University, Japan)
The Glucocorticoid Receptor (GR) is a member of a superfamily of ligand
inducible transcription factors that control variety of physiological andmetabolic processes. Upon ligand binding, GRs translocate from cytoplasm tonucleus, then associate with glucocorticoid response element (GRE) as a dimer.
This dimerization involves in regulation of variety of genes; however it is notclear that GR dimerization takes place in cytoplasm or nucleus in living cell. To
understand the dimerization mechanism of GR, Fluorescence Cross Correlation
Spectroscopy (FCCS) was performed in living U2OS cells transiently expressedmCherry tandem dimer and EGFP fused GR wild type. The positive crosscorrelation was obtained after addition of Dexamethasone (Dex) which is anagonist for GR. Then, the Kd value of dimerization of GR wild type wasestimated as 13.2 μM. Moreover, we measured A458T and C421G mutants
which were mutated to disable for dimerization and DNA binding, respectively,
for further understanding dimerization of GR. In nucleus after addition of Dex,
almost no cross correlation was observed in A458T mutant. On the other hand,
positive cross correlation was obtained in C421G mutant as wild type. Then, theKd value of dimerization of C421G was estimated as 12.3 μM. These results
suggest that DNA binding is unnecessary to dimerization for GR, and GR is
dimerized before association with GRE. Furthermore, we will discuss thedimerization mechanism in cytoplasm by FCCS measurement of nuclear
translocation deficiency mutant.
3PT006

Transcription factor p53 searching dynamics on nucleosomal
DNA: Coarse-grained molecular dynamics simulation study

Tsuyoshi Terakawa, Shoji Takada (Grad. Sch. Sci., Univ. Hyogo)
Transcription factor p53 has to search its specific binding site on DNA to
activate transcription of several proteins that are involved in tumor suppressing
process. Two of four major domains of p53 [Core domain (Core) and Cterminal domain (CTD)] are known to bind to DNA non-specifically. The roles
and interplay of these two DNA binding domains during the search have been
controversial. Furthermore, the searching mechanism on nucleosomal DNA in
which histone proteins bind to DNA has been largely unknown. In this work, we

performed coarse-grained molecular dynamics simulations of tetrameric fulllength p53 on duplex DNA with and without histone proteins to elucidate suchsearching mechanisms.
First, we conducted coarse-grained simulations of p53 on DNA without histone
proteins at various ion concentrations. The result shows that only CTDs keepbound to DNA and the Cores frequently hop on DNA during the search. This isin perfect agreement with a recent single molecule experiment. The result also
shows that the sliding via the CTD does not follow the helical pitch of DNA(rotation-uncoupled diffusion mechanism) at physiological ion concentration.
This finding indicates that p53 can bypass the obstacles on DNA such as histone
proteins.
Second, in order to get insight into how p53 bypasses the histone proteins, we
conducted coarse-grained simulations of p53 on DNA with histone proteins.
The result indicates the importance of rotation-uncoupled diffusion mechanism
to bypass the obstacles on DNA.
3PT007

粗視化分子シミュレーションを用いた T4 sliding clamp の DNA
上での動きの解析

Movement of T4 sliding clamp on DNA studied by coarsegrained molecular simulations
Itaru Narihara, Tsuyoshi Terakawa, Hiroo Kenzaki, Shoji Takada (Biophy,
Grad Sch Sci, Kyoto Univ)
DNA sliding clamp proteins increase enzymatic processivity of DNA polymerases
during DNA synthesis. DNA sliding clamp also play a key role in controlling
several reactions, such as DNA repair, translesion DNA synthesis, DNA
methylation and chromatin remodeling, through the coordination and organisationof different proteins. DNA sliding clamp forms a closed circle around double
stranded DNA and is able to move along DNA in a random walk, but the molecularnature of this diffusion process along DNA is poorly understood.
Then we studied movement of the DNA T4 sliding clamp (bacteriophage) on
double strand DNA at different ion strength by using a coarse-grained molecular
dynamics method of CafeMol. We used Clementi's Go model for proteins, dePablo model for DNA and included electrostatic and excluded volumeinteractions between DNA and protein. From a series of simulations, wemeasured the diffusion constant of the DNA T4 sliding clamp, its dependence
on the ion strength, coupling between the rotation around DNA and the
diffusion. We also analyze the tilt angle toward DNA.
3PT008分子シミュレーションと電子顕微鏡像を用いて解析された tRNA
転位の自由エネルギー地形

Free-energy landscape of tRNA translocation through ribosome
analysed using MD simulations and cryo-EM density maps
Hisashi Ishida, Atsushi Matsumoto (Quantum Beam Science Directorate,
Japan Atomic Energy Agency)
Ribosome is one of the supra-biomolecules used in the process of translating
genetic information for the synthesis of polypeptides. In the course of its
synthesis, two tRNA molecules move with mRNA through ribosome. This
process, called translocation of tRNAs, is catalyzed by the elongation factor G(EF-G). Recent results from cryo-electron microscopy (cryo-EM) suggest that
there are large-scale structural rearrangements of both ribosome and EF-G
occur. However, the dynamic mechanism of translocation is unclear at theatomic level. We used all-atom molecular dynamics (MD) simulations including
water molecules to direct 70S ribosome complexed with EF-G at the posttranslocational state towards the pre-translocational states by fitting 70S
ribosome into cryo-EM density maps. Additionally, the simulations wereassisted by umbrella sampling simulations, in which biased potentials were
imposed on the centers of masses of the protein molecules in the 70S ribosome,to relax the transitional conformations and to construct the free-energy
landscape of the translocation. Multistep structural changes, such as a ratchetlike motion between the small and large ribosomal subunits were observed
during the translocation. The free-energy landscape shows that there are semistable states between two stable states at the pre- and post-translational states. It
was shown that a loop of nucleic acids from the small ribosomal subunit, which
is located near the P- and E-sites, plays an important role in the translocation of
P-tRNA and E-tRNA.
3PT009− S156 −粗視化シミュレーションによる単 / 多ヌクレオソーム系の構造ダイ
ナミクス

Mono- and poly-nucleosome structural dynamics by coarse-grained simulations
Hiroo Kenzaki1, Shoji Takada1,2 (1Dept. of Biophysics, Graduate School of
Science, Kyoto Univ, 2CREST-JST)

tendency is observed in each of zinc fingers in TFIIIA, and thus this may be
applied to other proteins.

Nucleosome is a basic unit of chromatin structure in which DNA duplex wraps
about 1 and 3/4 times around histone octamer. Poly-nucleosome takes higher
order chromatin structure to store the very long genome into compact space. On
the other hand, nucleosome is needed to be unstructured dynamically for
function; recognized by transcription factor and transcription to mRNA.
Recently, many experiments have investigated mono-nucleosome dynamics.
Unwrapping of DNA duplex from histone octamer is observed by equilibrium
and force unwrapping experiments. We simulate these nucleosome dynamics byusing coarse-grained DNA-protein model, in which one amino acid isrepresented by one bead and one nucleotide is represented by three beads
corresponding to phosphate, sugar, and base. Simulation results reproduce
partial/full unwrapping of DNA. We also simulate model poly-nucleosome
systems to investigate higher-order structure of chromatin.

3PT101

3PT010Zero-dipole summation 法を用いた Est1-DNA 複合体の分子
動力学シミュレーション

Molecular Dynamics Simulations of Ets1-DNA complexes
using Zero-Dipole Summation Method
Takamasa Arakawa1,2, Masaaki Shiina3, Kazuhiro Ogata3, Narutoshi
Kamiya2, Ikuo Fukuda4, Haruki Nakamura2 (1Dept. of Biol. Sci., Grad. Sch. ofSci., Osaka Univ., 2IPR, Osaka Univ., 3Dept. of Biochem., Yokohama City
Univ. Sch. of Med., 4Riken)
For understanding the phenomenon of life, it is important to investigate
ingenious transcription mechanisms. Ets1 is a transcription factor, which has ahighly conserved DNA binding domain, the Ets domain, and functions invarious biological important procedures, such as the regulation of developmental processes in response to extracellular signals. The DNA binding
activity of Ets1 is in an autoinhibited state enhanced by phosphorylation. This
fact clearly suggests that phosphorylation of transcription factors plays a criticalrole linking signal transduction to transcriptional regulation. Here, we attempted
to unravel how phosphorylation affects protein's DNA-binding ability by
applying molecular dynamics (MD) simulations in explicit water moleculeswith small ions using zero-dipole summation method (Fukuda et al., 2011) toseveral transcription complexes having Ets1. Zero-dipole summation is a novelidea for calculations of long-ranged Coulombic interactions as a kind oftruncation methods, and it achieved good accuracy and high parallelization
efficiency in several biological systems even with highly charged DNA doublestrands. In addition, this method is formally free from the periodicity
assumption, and thus we can avoid the relevant artifacts. The effect ofphosphorylation for DNA-binding will be discussed based on the results of long
MD simulations.
3PT011分子シミュレーションによる TFIIIA ジンクフィンガーの DNA
上の拡散

Diffusion of TFIIIA zinc fingers along DNA studied bymolecular simulations
Yohei Miyoshi, Tsuyoshi Terakawa, Shoji Takada (Dept Biophysics, Div
Biology, Grad School Science, Kyoto University)
TFIIIA is a transcription factor which recognizes a site-specific sequence ofDNA. Generally, transcription factors search on DNA sequence by various
diffusion mechanisms. As one of search mechanisms, one-dimensional diffusion
along DNA, or sliding, is proposed. Sliding is that a transcription factor moves
along DNA sequence by keeping contact with DNA. It has been suggested that
one-dimensional diffusion coefficient is independent of ion concentration whena transcription factor slides on DNA. To investigate if this phenomenon is trueor not, we performed a series of coarse-grained (CG) simulations. The system ofsimulations includes one zinc finger motif of TFIIIA and 200-bp DNA. We used
Go model for proteins, de Pablo model for double-strand DNA, and electrostaticand excluded-volume interactions for protein-DNA interactions.
According to our simulation, the one-dimensional diffusion coefficientincreases when ion concentration increases from 1 mM to about 200 mM.
Moreover, the diffusion coefficient is constant when ion concentration is higher
than 200 mM. Hence, it is realized that electrostatic interaction between atranscription factor and DNA influences the diffusion coefficient when ion
concentration varies from diluted condition to physiological condition. Thisphenomenon suggests that experimental results often done at dilute solution are
not necessarily the same as the results of physiological solution. The same

溶液環境が決定付ける単一 DNA 分子の凝縮速度

How dose Environmental Solution Conditions Determine theCompaction Velocity of Single DNA Molecules?
Ken Hirano1, Ishido Tomomi1, Katsuhisa Nakamichi2, Naoki Ogawa2,
Masatoshi Ichikawa3, Kenichi Yoshikawa2 (1Health Res. Inst., AIST, 2Fac. Life
Med. Sci., Doshisha Univ., 3Dep. Phys., Kyoto Univ.)
Understanding the mechanisms of DNA compaction is becoming increasingly
important for gene therapy and nanotechnology. The kinetics of the compaction
velocity of single DNA molecules was studied using two non-protein
condensation systems, poly (ethylene glycol) (PEG) with Mg2+ as a representative of crowding effect and spermine as a polycationic condensation
agent. The compaction velocities of single tandem λ -DNA molecules were
measured at various PEG and spermine concentrations with video fluorescent
microscopy. Single DNA molecules were observed using a molecular stretching
technique under microfluidic flow. The results show that the compaction
velocity of a single DNA molecule was proportional to the PEG or spermine
concentration to the power of a half. Theoretical considerations indicate that thecompaction velocity is related to differences in the free energy of a single DNA
molecule between the random coil and compacted states. In the compaction
kinetics with PEG, acceleration of the compaction velocity occurred above theoverlap concentration while considerable deceleration occurred during thecoexistence state of the random coil and the compacted conformation. This
study demonstrates quantitative approach to determine the kinetics of DNA
compaction by use of environmental conditions as the control parameters.
Further study on such kinetics on the change of the higher-order structure in
genomic giant DNA molecules would exhibit of biological importance tounderstand the essential feature of living matter. [Ref] NAR,40(2012)284.
3PT102

新規なフォトクロミック ATP アナログによるキネシン / 微小管系の駆動と光可逆的な運動制御

Driving and Reversible Photo-control of Motility of aKinesin/Microtubule System by a Novel Photochromic ATP
Analogue
Takashi Kamei, Tuyoshi Fukaminato, Nobuyuki Tamaoki (RIES, Hokkaido
Univ.)
Adenosine triphosphate (ATP) is a biogenic molecule that functions as anenergy donor and interacts with various proteins, ATPases. Therefore an ATP
analogue having the potential to artificially control the functions of ATPases
would be a promising molecule to control various biochemical processes. In thisregard, a photo-controllable ATP analogue has the advantage of using an
external stimulus, light, with high spatiotemporal resolution.
We designed and synthesized novel nucleotide analogues, ATP-Azos, consist of
ATP and photochromic compounds, azobenzene derivatives. It is confirmedwith a spectrophotometer that ATP-Azos exhibit trans-to-cis and reverse cis-totrans isomerizations in the azobenzene moieties through alternating irradiation
with UV (λ = 365 nm) and visible (λ = 436 nm) lights, respectively.
Kinesin, an ATPase bio-nano-motor moving along a microtubule filament, isallowed to be driven by ATP-Azos without ATP molecules confirmed by an invitro motility assay. Furthermore, in the presence of the ATP-Azo possessing
tert-butyl group in the azobenzene moiety (ATP-tBuAzo), the gliding velocity(at ≤ 300 μ M ATP-tBuAzo condition) and binding manner (1 mM ATPtBuAzo) of the kinesin-microtubule system in the assay was reversibly photocontrolled by alternate irradiations with UV and visible lights.
We anticipate that ATP-Azos might find wide applicability in nanobiotechnology using bio-motors and other ATPases. Furthermore, replacing ATPwith other nucleotide might allow related systems to apply to othernucleotidases.
3PT103ナノメートルサイズの軸で巻くことにより DNA の曲げ弾性を測定

Bending stiffness of double-stranded DNA measured bywinding single-molecule on a nanometer-sized reel
Huijuan You, Ryota Iino, Rikiya Watanabe, Hiroyuki Noji (Department ofApplied Chemistry, School of Engineering, The University of Tokyo)
The bendability of double-stranded DNA (dsDNA) with curvature radius of 220 nm is important in many cellular process such as genomic DNA packaging,transcriptional regulation, and also important in fabrication of DNA− S157 −nanostructures. Previous measurements were based on the analysis of thermal
bending of dsDNA. However, sharp bending to curvature radius of 2-20 nm wasrarely occurred thermally. Therefore, methodology that controls the bending
curvature of dsDNA is required to explore the micromechanics of sharply bent
dsDNA. For mechanically bent dsDNA to nanometer curvatures, we developed
a molecular system of a nanometer-sized reel based on F1 -ATPase, a rotary
motor protein. By combination with magnetic tweezers and optical tweezers
system, single-molecule dsDNA was wound around the molecular reel. The
bending stiffness of dsDNA was estimated based on the winding tension (0.9-6.
0 pN) and the diameter of the wound loop (21.4-8.5 nm). Our results were ingood agreement with the conventional worm-like chain model and were used toestimate a persistence length of 54 ± 9nm. This molecular reel system offers a
new platform for single-molecule study of micromechanics of sharply bent
DNA molecules and is expected to be applicable to the elucidation of themolecular mechanism of DNA-associating proteins on sharply bent DNA
strands.
3PT1041 分子機械的アンジッピングが示すエントロピー駆動的 DNA 構造
安定性の GC と AT 塩基対における違い

GC and AT pairs show different entropic stabilization of DNA
structures found in single-molecule mechanical unzipping
Akihiro Fukagawa1, Michio Hiroshima2, Makio Tokunaga1,3 (1Grad. Sch.
Biosci. Biotech., Tokyo Inst. Tech., 2Adv. Sci. Inst. RIKEN, 3RCAI, RIKEN)
Unzipping of double-stranded DNA is involved in several biological processes.
We have performed a quasi-static mechanical unzipping of single-molecule
DNA using intermolecular force microscopy, modified atomic force microscopy
with high resolution. We obtained force-extension (FE) curves of two oligonucleotides with a stem-loop containing six tandem repeats of a 6-bp sequence.
To reduce noise, FE curves of the unzipping region were averaged using
correlation analysis. FE curves showed a dependence on the ruptures of singlehydrogen bonds.
To reveal the molecular mechanism of stabilization of DNA structures byhydrogen bonds, we have carried out the molecular dynamics (MD) simulationsof mechanical unzipping of DNA. Cross-correlation analysis of FE curves
showed the force-peaks of single hydrogen bonds more clearly than those by theunzipping experiments. Furthermore, we depicted the two-dimensional (2D)energy landscape, trajectory density map to visualize the behavior of DNA
unzipping from MD simulations. Trajectories tended to escape the energetic
mountain and took heterogeneous pathways on rugged energy landscape. We
calculated entropy from trajectory densities and total energy. The entropy of the
AT pair was negatively correlated with the number of hydrogen bonds. In
contrast, not the entropy but the total energy of GC pair was correlated with thenumber of hydrogen bonds. The result suggests that the formation of GC pair isdriven by mainly energy and that of AT pair is driven by not only energy but
also entropy.
3PT105Controllable adsorption-desorption of double-stranded DNA
onto single-walled carbon nanotube functionalized with
polyethylene glycol
Daisuke Nii, Takuya Hayashida, Kazuo Umemura (Grad. Shc. Sci. univ ofscience. Tokyo)
Since the discovery of the hybridization of DNA and the single-walled carbon
nanotube (SWNT), various researches have been conducted on the mechanismof the interaction between single-stranded DNA (ssDNA) and SWNTs during
the hybrid formation. However, the adsorption mechanism of double-stranded
DNA (dsDNA) onto SWNTs has not yet been studied as comprehensively.
Further, there are few reports on the detachment of DNA molecules from a
DNA-SWNT hybrid, although it may be important to understand the desorption
technique of ssDNA from the ssDNA-SWNT hybrids in order to employ thehybridization for medical applications such as biodevices or drug delivery. In
this study, we employed an SWNT functionalized with polyethylene glycol(PEG SWNT) for controlling ssDNA adsorption and desorption onto the
SWNT. Firstly, we achieved the hybridization of dsDNA and the PEG SWNT inaqueous solution for the first time. Secondly, we succeeded in the detachment of
dsDNA from the PEG SWNT by annealing at 95°C; the desorption was notobserved in the use of usual SWNT. Atomic force microscopy, electrophoresis,
and Raman spectroscopy indicated the occurrence of dsDNA adsorption ontoand desorption from the PEG SWNT. Our results demonstrated the potential ofcontrolling the interaction between ssDNA and PEG SWNT in aqueous
solutions.

3PT106

分子動力学シミュレーションによる dGMP、8-oxo-dGMP のコ
ンフォメーション比較：イオンの影響

Comparison of dGMP and 8-oxo-dGMP conformation bymolecular dynamics simulations: effect of ionsShin-ichi Fujiwara, Takashi Amisaki (Fac. Med., Tottori Univ.)
8-oxo-7, 8-dihydroguanine (8-oxo-G) is produced by the oxidation at C8
position of G and is the most abundant species among bases in DNA modified
by oxygen radicals. 8-oxo-G causes transversion as well as transition mutation.
Human MutT homologue-1 (hMTH1) hydrolyzes oxidized 8-oxo-dGTP to 8oxo-dGMP, thereby preventing the misincorporation of oxidized nucleotides
during replication. Although the “syn” conformation predominates in 8-oxodGTP, the crystal structure of hMTH1 indicates that the protein binds themononucleotide in the “anti” conformation with no direct interaction between
hMTH1 and the 8-oxo position of the oxidized base. It is unclear how theprotein structurally distinguishes 8-oxo-dGTP from dGTP. To elucidate thespecific interaction of 8-oxo-dGTP by hMTH1 on the basis of mononucleotide
conformation, molecular dynamics (MD) simulations were carried out for
deprotonated/protonated dGMP and 8-oxo-dGMP in explicit water. The effectof ions on the mononucleotide conformation was specifically examined, by
including Li+, Na+, Mg2+, or Ca2+ in the system. In a series of MD simulations,dGMP took both the stable “anti” and “syn” conformation. Although 8-oxodGMP took the stable “syn” conformation in most cases, 8-oxo-dGMP with
Mg2+ took the stable “anti” conformation as well as the stable “syn”
conformation. We will discuss the “anti” conformational stability of 8-oxodGMP with ion in detail.
3PT107

粗視化モデル分子動力学法を用いた RNA 立体構造予測

RNA 3D structure prediction by using coarse-grained
molecular dynamics simulation
Tomoshi Kameda (CBRC, AIST)
During the last two decades, significant developments have showed theimportance of RNA in biological functions. To perform their functions, many
RNA molecules adopt well defined tertiary structures. Hence, there is an interestin accurate ab initio prediction of three-dimensional (3D) structure of RNAs.
However, RNA 3D structure prediction is not studied well, compared to protein
three dimensional (3D) structure prediction. The one of reasons is that the size of
nucleic acid is larger than amino-acid, which needs quite a long time formolecular dynamics (MD) simulation even if we deal with small RNA molecules.
Hence, to predict RNA 3D prediction, we developed a simplified, coarsegrained RNA model for molecular dynamics simulation. In our model, one baseconsists of 3 beads (P; phosphate, S; sugar, B; base). When our model is applied
to small RNA (<60bases), it takes only ~5 minutes for the structure prediction
using single processor (1 core). The details will be shown in the presentation.
3PT108

分子動力学シミュレーションによる DNA 構造に対する溶媒条件の影響の研究

The influence of solvent condition on DNA structures studied
by molecular dynamics simulations
Satoshi Fujii1, Hidetoshi Kono2, Akinori Sarai1 (1Dept. Bioscience andBioinformatics, Kyushu Institute of Technology, 2Computational Biology,
JAEA)
The conformational properties of DNA play important roles in various
biological processes. For example, interactions of proteins with DNA sequences
are known to be affected by the conformational properties of DNA. It has also
been known that the transitions among various conformational states are
induced by the change in environments: Variations in ionic strength andidentity, sequence, water activity, and ligand/protein binding all can modulate
DNA structure. In this study, we analyzed how the sequence and theenvironments such as water activity influence the DNA conformations. We havecarried out a series of molecular dynamics (MD) simulations to explore theconformational preferences and dynamical behavior of DNA duplex. We
prepared the initial structure models with two different sequences in two kindsof conformations; d(CGCGTATACGCG)2 and d(CGCGTATACGCG)2 in Band A-forms. The MD simulations were carried out in four different
environments: in only water solvent and in 75, 85 and 95 % (v/v) ethanol / water
mixture solvent. We analyzed the differences of the DNA backbone
conformation, the base-pair step parameter and the localization of water− S158 −molecules in the groove on each structure. The variability of the conformation
was different depending on the solvation condition. The DNA conformation
seemed to be influenced by the localization of the water molecules in thegroove.

of T7-RNAP on the activity of T7-Tile.
3PT112

リポソームへの DNA リングの吸着

Adsorption of DNA ring onto Liposome
3PT109Model for self-assembly of flexible DNA motifs using stacking
interactions
Keitel Cervantes-Salguero, Shogo Hamada, Satoshi Murata (Tohoku
University)

Yumiko Kumagai, Shogo Hamada, Shin-ichiro Nomura, Satoshi Murata(Department of Bioengineering and Robotics, Graduate School of Engineering,Tohoku University, Japan)

Molecular Clusters (MCs) with variable number of constituents seem to bepossible if building units are properly designed. Recently, concentrationdependent self-assembly of MCs was demonstrated by utilizing both flexibilityof DNA motifs and inter-molecular binding by sticky-ends (He, Y. et al., Nature2008). Here we propose a cluster of DNA motifs by using DNA blunt-end
stacking and flexibility introduced into the motif. First, we study an abstract
model of flexible planar monomers (triangle with sticky sides A and B, andshrinkable side C. Angle between A and B is about 60 deg.) that move randomly
on a surface colliding against each other. Sides A and B of different monomers
can stack, thus forming a flexible dimer. Over time we expect to obtain clusters
from dimers up to hexamers. We simulated the dynamics of the cluster
formation based on chemical kinetics successfully applied to rigid monomers(Hosokawa, K. et al. Artificial life 1995). Our simulation shows that the amountof trimers overcomes the tetramers. However, contrary to the work of Hosokawa
et al., dimers and pentamers seem to be equally formed. Second, to realize theconcept, we have designed a DNA motif with blunt-end stacking interactions,which were proved to be a potential tool for self-assembling larger DNA
structures (Woo, S. et al., Nature Chemistry 2011).

The various molecular devices made of biomolecules such as nucleic acid,protein and lipid are reported. We are now able to design each devices withcertain complexity, although it is still difficult to combine multiple devices into
one functional system. Embedding various molecular devices within a givensize compartment is one of the potential solutions to solve this problem andenable us to achieve programmed behavior of the system. A key component torealize this concept is a channel device integrated into the compartment, whichallows materials pass through the compartment.
Here, we adopt a phospholipid as material for micron-sized compartment, andDNA nanostructure for the channel device embedded on the compartment
surface. In the design of the DNA channel, we have been inspired by themacrocyclic resorcin channel, which is an artificial mimic of a natural ion
channel. We have adopted the T-motif DNA ring that has a similar structure tothis. The surface of the DNA ring is hydrophilic, therefore, has affinity to thelipid membrane surface. In order to integrate the ring into the membrane,hydrophobic groups are attached to the DNA rings. We adopt Cholesteryl-TEG
as the hydrophobic groups and confirmed the formation of those DNA rings. We
also found that DNA structures tend to rest on the lipid membrane surface bygiving positive charge to the lipid. Currently, we are working on the
optimization of the lipid composition to give an appropriate charge condition.

3PT110

3PT113A Growth mechanism of DNA tile array
Yuki Matsubara, Nao Otani, Miho Tagawa, Akira Suyama (Univ Tokyo, DeptLife Sci)
We report a growing mechanism of DXAB (DNA double-crossover ABstaggered) tile array known to form planar structures and grow to micro-scale.
DXAB tile array is a kind of DNA tile arrays and can work as a scaffold tocontrol positions of nano-particles. Growth mechanisms of the array areimportant information to set its properties, such as size, accuracy of position
control, yield, or the required time of annealing. Here, we report that the growthof array is a co-operative phenomenon in narrow temperature range. Thistransition width is shorter than a prediction by model based on lengths of the
sticky-ends. This has been visualized by fluorescence optical microscope,
because, unlike the tiles’ assembly, growth of the tile arrays cannot be observedin melting curve. From this knowledge we show two applications, making big
isolated sheets and shortening the annealing time in protocol, as examples.
These experimental results demonstrate that such knowledge about growthmechanisms is useful to control properties of DNA nanostructures.

3PT111

DNA ナノ構造を用いた DNA-RNA ポリメラーゼ・ハイブリッド
ナノマシンの構築と機能評価

Construction and functional analysis of DNA origami base
DNA-RNAP hybrid nanomachine
Takeya Masubuchi1, Hisashi Tadakuma1, Masayuki Endo2, Hiroshi
Sugiyama2, Yoshie Harada2, Takuya Ueda1 (1Graduate School of Frontier
Science, The University of Tokyo, 2iCeMS, Kyoto University)
DNA nanotechnology has recently developed a new nanoscale device called as
DNA origami tile (DNA origami). DNA origami is a method for folding longsingle strand DNA into arbitrary two or three dimensional structures and can beused as a molecular platform which allows proteins and other molecules to placewith precisely controlled patterns at nanometer order. Here, we applied DNA
origami technology into transcription system. Transcription system is thought tocouple to other system in E. coli (e. g. translation and replication). And
reconstitution of such sequential reactions in vitro is important for science andengineering, for which the DNA origami technology may provide platform. As afirst step, we assembled T7 RNA polymerase (T7-RNAP) on DNA origami.
Rectangle DNA origami (90 x 60 nm) are prepared as reported (Rothemund
Nature 2006), and SNAP-tag protein fused T7-RNAP (T7-RNAP-SNAP) weresite-specifically anchored on DNA origami through its specific ligand(benzylguanine (BG)-ligand) included on the DNA origami. With the optimized
assembly condition, we succeeded in measuring the activity of T7 assembled
DNA origami (T7-Tile). We will check the effect of the number and the layout

脂質二重膜上での DNA ナノ構造の基板上成長

Substrate-Assisted Self-Assembly of DNA Nanostructure on
lipid bilayer
Shogo Kudo, Shogo Hamada, Shin-ichiro Nomura, Satoshi Murata(Department of Bioengineering and Robotics, Graduate School of Engineering,Tohoku University, Japan)
Substrate-Assisted Self-Assembly (SASA) by Hamada et al. enables us to makelarge 2-D DNA nanostructures that are difficult for conventional self-assemblyin free solution. So far, limited materials, e.g., cleaved mica, are known forsuitable substrate for the method. It is necessary to extend the method to utilize
various materials for the substrate. However, the process of SASA is very
complex and not yet thoroughly understood.
To model SASA, we divide it into adsorption and self-assembly processes. The
adsorption process is described by DLVO theory. Following Sushko, we cancalculate energy of DNA adsorption by DLVO. We also measured minimal
length of DNA, or equivalently energy per base pair, to be absorbed on mica
substrate. In order to extend SASA to other materials, measured absorptionenergy between DNA and mica surface roughly indicates the feasible condition
for SASA. Based on this consideration, we chose lipid bilayer (DOPG) for thesubstrate, because the calculated energy between 100 base pairs of DNA andthe bilayer is enough to absorb when magnesium ion concentration is 12.5 mM.
However, we haven’ t confirmed DNA adsorption under this condition.
Currently, we are working on experiments to search appropriate conditions for
SASA.
3PT114

中性膜に結合した LFampinB の膜結合構造と膜親和性の固体
NMR と QCM による解析

Structure and affinity of bovine lactferrampin bind to neutralmodel membrane as studied by by solid state NMR and QCM
Masayoshi Imachi1, Javkhlantugs Namsrai1, Atsushi Kira2, Izuru Kawamura1,
Akira Naito1 (1Graduate School of Engineering, Yokohama National
University, 2Reasearch and Development Division, ULVAC Inc.)
Recently antimicrobial peptide is focus attention on a potential alternative for
antibiotics. Analysis of membrane bound structure of peptide provides
important information on efficiency and side effect of medicine.
Bovine lactoferrampin(LFampinB) is an antimicrobial peptide found in the N1domain of bovine lactoferrin (268-284) and consists of 17 amino acid residues.
Bovine lactoferrin (bLF) is an iron binding glyco protein with innate immunity
factor in mammalian secretory fluid for example, tear saliva and milk.
In this study, the structure of LFampinB bound to the neutral membrane wasdetermined using chemical shift oscillation analysis by observing the chemical− S159 −shift anisotropy of the carbonyl carbon of Leu4, Ala7, Gly12 amino acidresidues. These results indicate that LFampinB forms α-helix in the N-terminal
region inserting into the membrane and the α-helical axis rotates rapidly about
the bilayer normal with the tilt angle of 12 degree to the axis
The association constant (Ka) of LFampinB with the neutral lipid wasdetermined which was smaller than that with the acidic membrane. The
difference of the Ka of LFampinB with neutral and acidic membrane explain thereason why the α-helical structure of LFampinB in neutral membrane is more
distorted than that in acidic membrane. This also explain that LFampinB may
selectively interact with the acidic bacterial membrane and shows the hemolytic
activity to the neutral membrane.
3PT115

大腸菌異物排出タンパク AcrB のペリスタポンプ機構の解明

Structures of the multidrug exporter AcrB reveal a proximal
multisite drug-binding pocket
Keisuke Sakurai1, Ryosuke Nakashima1, Seiji Yamasaki1,2, Kunihiko
Nishino1, Akihito Yamaguchi1,2 (1Institute of Scientific and Industrial
Research, Osaka University, 2Graduate school of pharmaceutical sciences,
Osaka University)
AcrB and its homologues are the principal multidrug transporters in Gramnegative bacteria and are important in antibiotic drug tolerance. AcrB is a
homotrimer that acts as a tripartite complex with the outer membrane channel
TolC and the membrane fusion protein AcrA. Minocycline and doxorubicin
have been shown to bind to the phenylalanine cluster region of the binding
monomer. Here we report the crystal structures of AcrB bound to the highmolecular-mass drugs rifampicin and erythromycin. These drugs bind to theaccess monomer, and the binding sites are located in the proximal multisite
binding pocket, which is separated from the phenylalanine cluster region (distal
pocket) by the Phe-617 loop. Our structures indicate that there are two discrete
multisite binding pockets along the intramolecular channel. High-molecularmass drugs first bind to the proximal pocket in the access state and are thenforced into the distal pocket in the binding state by a peristaltic mechanism
involving subdomain movements that include a shift of the Phe-617 loop. By
contrast, low-molecular-mass drugs, such as minocycline and doxorubicin,
travel through the proximal pocket without specific binding and immediatelybind to the distal pocket. The presence of two discrete, High-volume multisite
binding pockets contributes to the remarkably broad substrate recognition of
AcrB.
3PT116

エントロピー力による円筒状容器からの大球の放出

Entropic release of a big sphere from a cylindrical vessel
Hirokazu Mishima1, Hiraku Oshima1, Satoshi Yasuda1, Ken-ichi Amano2,
Masahiro Kinoshita1 (1Inst. of Adv. Energy, Kyoto Univ., 2Dept. of Chem.,
Kobe Univ.)
Insertion of a large solute into a vessel comprising biopolymers followed byrelease of the same solute from it is a fundamental function in biologicalsystems. A typical example of such insertion/release processes is found in TolC.
TolC is a cylindrical vessel possessing an entrance at one end and an exit at theother end for the solute. In earlier work [1], we showed that the solute-vessel
potential of mean force (PMF) formed by the solvent plays imperative roles in
the processes. The PMF can be decomposed into its entropic and energetic
components denoted by ΦS and ΦE, respectively. ΦS is insensitive to the soluteand vessel properties (i.e., solvophobic or solvophilic) whereas ΦE is largely
dependent on them. A significant feature of TolC is that a variety of solutes areinserted into and released from the vessel, which is known as “multidrug
efflux”. This feature can be exhibited when ΦS with the insensitivity mentioned
above is dominant. However, our earlier work [1] suggested that ΦS acts only
for the insertion. Here we explore the possibility of releasing the solute by ΦS.
We calculate the PMF between a big sphere and a cylindrical vessel with open
ends immersed in small spheres using the three-dimensional integral equation
theory [1] combined with rigid-body models. It is shown that the solute inserted
through the entrance can entropically be released by a continuous variation of
the vessel geometry to squeeze out the solute from the exit.
[1] K. Amano, H. Oshima, and M. Kinoshita, J. Chem. Phys. 135, 185101(2011).
3PT117

2 者タンパク質間を認識するライト抗体を利用したバンド 3-グリコフォリン A 複合体の精製
Use of a dual specificity Wright antibody for the purification of

structurally unstable band 3-glycophorin A complex
Yohei Ikeda1, Hinako Hatae2, Hiroyuki Kuma2, Naotaka Hamasaki2, Teruhisa
Hirai1 (1Harima Inst., RIKEN, 2Fac. Pharm. Sci., Nagasaki International
Univ.)
Band 3 (also known as anion exchanger 1 or SLC4A1) and glycophorin A(GPA) are the most abundant membrane proteins in human erythrocyte and bothpresent at ~1×106 copies per cell. Biochemical and genetic analyses suggested
a close relationship between band 3 and GPA during and after biosynthesis(Auffray et al., Blood 97 pp.2872-2878, 2001, Telen and Chasis, Blood 96 pp.
842-848, 1990). Bruce et al. reported that band 3 and GPA were associatedthough an interaction residues Glu658 of band 3 and Arg61 of GPA (Bruce etal., Blood 85 pp.541-547, 1995). However, it was difficult to maintain theirstructure as a complex during purification, because of loose binding. In thisstudy, we purified band 3 and GPA (B3-GPA) complex with Wrightmonoclonal antibody (or Fab fragment), which recognized Wright blood groupantigens. We also characterized the biochemical properties of the complex.
After we confirmed the binding of Wright antibody Fab fragment against theerythrocyte membrane, we solubilized that erythrocyte membrane with C12E8
directly, then C12E8-solubilized membrane were loaded on a size-exclusion
column. Elution peak of B3-GPA-Fab complex appeared at a position predicted
for a dimeric form together with a small uncharacterized shoulder. Two
dimensional polyacrylamide gel electrophoresis (blue native/SDS-PAGE)
analysis showed that purified complex contains three subunits. We will discuss
the polypeptide composition and stoichiometry of the complex.
3PT118カルシウム活性化クロライドチャネルの in vitro でのイオン透過活性測定系の構築

Functional in vitro reconstitution of Calcium-activated
Chloride channel, TMEM16A/anoctamin1
Hiroyuki Terashima1,2, Alessandra Picollo1, Alessio Accardi1 (1Departmentof Anesthesiology, Weill Cornell Medical College, 2Department ofMacromolecular Science, Graduate School of Science, Osaka University)
Calcium-activated chloride channel is widely expressed and involved in various
physiological functions, for example, electrolyte secretion in epithelia andgrands, contraction of cardiac muscle, olfactory signal transduction andmaintain/amplification of membrane potential in nervous system. At 2008, threeof independence groups demonstrated TMEM16A/anoctamin1 is a componentof calcium-activated chloride channel. TMEM16A protein is thought to haveeight transmembrane segments and form at least dimer in vivo from results ofchemical cross-linking and native PAGE. Although TMEM16A is activatedintracellular calcium, there is no well-known calcium-binding motif (e.g., EF
hand) in TMEM16 family protein. Recently, mutation of two other glutamate
residues dramatically decreased calcium-sensitivity, implying that two
glutamate residues are involved in calcium-binding or activation. We expressed
the protein in insect cell and solubilized it by digitonin. The reconstituted
liposomes showed chloride-conducting activity. The purified TMEM16A
protein was activated at sub-micromolar of calcium concentration and inhibitedby well-known chloride channel blocker. Chloride flux activity was changed
depending on applied voltage, suggesting that it has voltage-dependency. Fromthese results, we could construct in vitro flux assay system for TMEM16A.
3PT119

COS7 細胞と酵母細胞を用いた筋型 CPT1 の発現系の比較

Comparison of two expression systems using COS7 cells andyeast cells for expression of heart/muscle-type CPT1
Takuya Hada1,2, Yumiko Kato1,2, Eriko Obana1, Naoshi Yamazaki2, Takenori
Yamamoto1, Yasuo Shinohara1,2 (1Inst Genome Res, Univ Tokushima, 2Fac
Pharm Sci, Univ Tokushima)
Carnitine palmitoyltransferase 1 (CPT1), catalyzing the transfer of the acylgroup from acyl-CoA to carnitine to form acylcarnitine, is located at the outermitochondrial membrane. Because it is easily inactivated by solubilization,
expression systems using living cells are essential for its functional
characterization. COS7 cells or yeast cells are often utilized for this purpose;
however, the advantages/disadvantages of the use of these cells or the question
as to how the CPT1 enzyme expressed by these cells differs are still uncertain.
In this study, we characterized the heart/muscle-type isozyme of rat CPT1
(CPT1b) expressed by these two cellular expression systems. The mitochondrial
fraction prepared from yeast cells expressing CPT1b showed 25% higher CPT1
activity than that obtained from COS7 cells. However, the expression level of
CPT1b in the former was 3.8 times lower than that in the latter; and thus, under
the present experimental conditions, the specific activity of CPT1b expressed in− S160 −yeast cells was estimated to be approximately five times higher than that
expressed in COS7 cells. Possible reasons for this difference are discussed.
3PT120紫膜表面の隆起構造およびバクテリオロドプシン分子構造に与える
紫膜中の古細菌型脂質除去の影響

Effects of archaeal lipids removal in purple membrane uponbump structures of membrane surface and bacteriorhodopsin
conformations
Kosuke Yamada, Yasunori Yokoyama, Shigeki Mitaku (Department ofApplied Physics, Graduate School of Engineering, Nagoya University)
Purple membrane (PM) is a membrane patch found in cytoplasmic membrane of
halophilic archaebacteria. Since PM consists of a two-dimensional crystal of
membrane protein bacteriorhodopsin (bR) with some archaeal lipids, it ispresumed that surface of PM is uniform planner. However, atomic force
microscopy (AFM) observations for PM have revealed a lot of bump structuresin the surface, showing that PM surface is not a uniform. The molecular packingof bR and the lipids, including the bump structure formation, should bedetermined by physicochemical interaction between bR molecules as well as bR
and lipids. Our studies against the bump structures of PM have showed a large
contribution of electrostatic interaction because bump curvatures were affectedby pH and ionic concentrations of medium. In this work, we focus on the lipids
in PM because of its highly negatively charged headgroups, and investigate
removal effects of the lipids upon bump structures of membrane surface by
AFM observations. AFM topography of delipidated PM demonstrated bump
structures had larger curvature than those in native PM. A mean curvature waslargest at 25 % delipidated PM samples, and decreased with increasing
delipidation level. AFM analysis in the presence of KCl with various
concentrations showed KCl concentration dependence of the bump curvature ofdelipidated PM was different from that of native PM. The removal effect of thelipids upon bump structures will be discussed together with theses results aswell as delipidation effects on bR conformations.
3PT121

EGF 受容体の活性化機構：EGFR はリガンド結合前に 2 量体として存在する

Molecular mechanism underlying activation of the EGF
receptor: preformed, inactive dimer and positive cooperativity
Hiraku Miyagi, Ichiro Maruyama (OIST)
Epidermal growth factor receptor (EGFR) is an integral membrane protein thathas an extracellular ligand binding domain, a single-path transmembrane
domain, and an intracellular kinase domain. The ‘dimerization model’ haswidely been believed as a molecular mechanism underlying the activation of
EGFR, in which ligand binding induces EGFR dimers from monomers.
However, we have recently shown that EGFR and its family proteins exist as apreformed, yet inactive, dimers prior to ligand binding, and have proposed a“rotation/twist model”, in which ligand binding induces rotation/twist of thereceptor’ s transmembrane domains. The rotation/twist breaks the inactive
dimeric structure, and rearranges the domains to take active forms. To
distinguish the two models, we examined the dimeric structure based on
FLIM/FRET before ligand binding, and found that EGFR has a preformed
dimeric structure. We also constructed a series of EGFR mutants in which asmall point mutation or large deletion was introduced into the receptor
molecule, and found that EGFR mutants with the deletion at its C-terminal
region were spontaneously activated. In contrast to the wild-type EGFR withbiphasic affinity for EGF, further, the mutant showed a single affinity for theligand. These results are consistent with the ‘rotation/twist’ model for themechanism of the EGFR activation. In the meeting, we shall report the results
described above, and shall discuss about the positive cooperativity between theligand-binding sites required for the activation of EGFR.
3PT122

表層ストレス応答における、PDZ ドメインによる RseP プロテ
アーゼ機能制御機構の解析

Analysis of a regulatory mechanism of the proteolytic function of
RseP by the PDZ domains in extracytoplasmic stress response
Yohei Hizukuri, Yoshinori Akiyama (Institute for Virus Research, Kyoto Univ.)
The Escherichia coli σE extracytoplasmic stress response monitors and responds
to folding stress in the cell envelope. A protease cascade directed at RseA, amembrane-spanning anti-σ that inhibits σE activity, controls this critical signaltransduction system. Stress cues activate the DegS proteases to cleave RseA; asecond cleavage by the RIP protease releases RseA from the membrane,
enabling its rapid degradation. Stress control of proteolysis requires that RseP
cleavage is dependent on DegS cleavage. In vitro and structural studies foundthat RseP cleavage requires binding of RseP PDZ-C to the newly exposed
carboxyl terminal residue (Val148) of RseA, generated by DegS-cleavage,
explaining dependency (Li et al., 2009, Proc. Natl. Acad. Sci. U. S. A.). We
tested this mechanism in vivo. Neither the C-terminal residue of DegSprocessed RseA nor mutations in the putative PDZ ligand-binding regions oreven deletion of entire RseP PDZ domains had significant effects on RseA
cleavage in vivo. Indeed, strains with a chromosomal rseP gene deleted for
either PDZ domain grew normally at all temperatures and exhibited almost
normal σE-activation in response to stress signals. We conclude that recognitionof the cleaved amino acid by the PDZ domain of RseP is unable to explain
sequential cleavage in vivo. To explore the possibility that the PDZ domains are
involved in regulation of the RseP functions through ligand-binding we aretrying to identify a physiological ligand of RseP PDZ by using site-directed invivo photo-cross-linking experiment.
3PT123部分フッ素化ホスファチジルコリン小胞に再構成されたバクテリオ
ロドプシンの液晶相における分子集合体

Molecular assembly of bacteriorhodopsin reconstituted into
partially fluorinated phosphatidylcholine liposome in liquid
crystalline phase
Masaru Yoshino1, Kenji Kanayama1, Takashi Kikukawa2, Toshiyuki Takagi3,
Hiroshi Takahashi1, Toshihiko Baba3, Toshiyuki Kanamori3, Masashi
Sonoyama1 (1Grad. Sch. Eng., Univ. Gunma, 2Grad. Sch. Sci., Univ.
Hokkaido, 3R.C. Stem Cell Eng., AIST)
Fluorine-containing lipids are expected to be very useful in the research areaof membrane proteins. Our recent studies on physical properties of anovel partially fluorinate phosphocholine, 1, 2-di (11, 11, 11, 12, 12, 13, 13, 14,
14-nonafluorotetradecanoyl) -sn-glycero-3-phosphocholine (diF4H10-PC), an
analog of a common phosphatidylcholine DMPC, demonstrated that diF4H10PC membrane shows the gel-to-liquid crystalline phase transition at ~5 oC andthe fluidity in the interface region is higher than that of DMPC. We succeeded inreconstituting a photoreceptor membrane protein, bacteriorhodopsin (bR), into
diF4H10-PC liposome. The reconstituted bR retained the trimeric structure evenafter the chain melting phase transition of lipid bilayers at least up 40 oC andshowed a photocycle similar to native purple membrane, which is in a striking
contrast with the previous studies on bR reconstituted in DMPC*. In order toaddress what is responsible for retaining the trimeric structure even in the fluid
phase of diF4H10-PC, bR reconstituted in a binary immiscible membrane of
diF4H10-PC and DMPC was investigated by some spectroscopic methods. It
has been shown that most of bR molecules are selectively reconstituted into
DMPC-rich domain, suggesting bR is mostly immiscible to diF4H10-PC. It is
plausible that the immiscibility between bR and diF4H10-PC is essential for theformation of bR trimers.
*Sonoyama et al., (2009), Yokoyama et al., (2010)
3PT124

Elucidation of the membrane microdomain assembly
mechanism via reconstituted artificial lipid membranes using
high performance proteomics
Lumi Negishi1, Satoshi Hosoya1, Osamu Toda1, Tsuyoshi Oosawa1, Ken
Hirosaki1, Thanai Paxton2, Nobuhiro Hayashi1 (1Grad. Sch. of Life Sci. andBiotech., Tokyo Inst. Tech., 2Solution Center, Nihon Waters K.K.)
Plasma membrane microdomains are associated with a variety of biomoleculesand considered to play an important role in effective response to external stimuli.
The physicochemical basis controlling the association remains incompletely
understood. In this work, we investigate the role of intermolecular interactions in
the assembly of proteins and lipids into the microdomain using reconstituted
membranes of varying composition and physiochemical properties. We carriedout high performance proteomics of the membrane microdomain andidentification of the proteins to investigate component proteins in the
microdomain. Using a standard procedure, the membrane microdomain was
extracted from Jurkat cells (Human T-cell lymphoblast cell line) because
membrane microdomains play an important role in the recognition of antigenpresenting-cells in immune system. The results of the 2D-gel electrophoresis
showed about 150 spots and of these approximately 60 proteins have so far been
identified using in-gel tryptic digestion and LC-MS/MS measurements. Some
identified proteins such as actin and V-type ATPase have already been reported asmembrane microdomain components. However, original proteins such as
interferon-induced GTP-binding protein, tumor necrosis factor and sphingolipid− S161 −desaturase have not yet been reported as microdomain components and they are
possibly involved with the immune system. Finally, important factors in theantigen recognition system and the construction of the in vitro system using
artificial membranes will also be discussed.
3PT125

無細胞膜タンパク質合成におけるリポソームのシャペロン機能

Liposomes as chaperone in cell-free membrane protein
synthesis
Minato Akiyama1, Jun-ichi Yasuoka2,3, Shin-ichi Sawada1,3, Kazunari
Akiyoshi1,3 (1Grad. Sch. Eng., Kyoto Univ., 2Inst. Biomater. Bioeng., Tokyo
Med. Dent. Univ., 3JST-ERATO)
Membrane proteins play significant roles in organism, such as signaltransduction and cellular communication. However, studies of membraneproteins are not enough established because most of them are water-insoluble
and likely to aggregate in water. Recently, we reported direct reconstitution of
membrane proteins into liposomes using cell-free protein synthesis system. In
this study, we focused on a membrane protein, connexin-43 (Cx43), which form
hexameric hemichannel named connexon. Two connexons on neighboring cells
join and form gap junction that mediates intercellular communication. We
report the synthesis of Cx43 using cell-free protein synthesis and chaperonelike function of liposome.
Cx43 was successfully synthesized with the reconstituted cell-free protein
synthesis system (PURE system), as shown by western blotting analysis.
Synthesized Cx43, however, was observed to completely aggregate in theabsence of liposomes. In the presence of liposomes, a certain amount ofsynthesized Cx43 was detected in the soluble fractions containing liposomes.
Aggregated Cx43 was removed by sucrose density gradient centrifugation. The
supernatant was collected as Cx43-reconstituted liposome fraction. The
chaperone-like function of liposomes prevents the aggregation of the waterinsoluble membrane protein in solution and aids the oligomerization of Cx43 in
the membrane. Hemichanel formation and the effect of concentration, size andlipid composition of liposome as a chaperone for the membrane protein
synthesis were investigated.
3PT126

可溶化系における膜タンパク質ハロロドプシンとバクテリオルベリンの複合体形成

Formation of a complex between membrane protein
halorhodopsin and carotenoid of bacterioruberin in thesolubilized system
Takanori Sasaki, Nur Wahida Abdul Razak, Noritaka Kato, Yuri Mukai (Sch.
Sci. and Tech., Univ. Meiji)
Halorhodopsin is a retinal protein with a seven-transmembrane helix and acts as
an inward light-driven Cl- pump. In this study, structural state of the solubilized
halorhodopsin (NpHR) from the biomembrane of mutant strain KM-1 of N.
pharaonis in non-ionic detergent was investigated. A gel filtration chromatography monitored absorbances at 280, 504 nm corresponding to the protein
and a lipid soluble pigment of bacterioruberin (BR) respectively has clearly
detected an oligomer formation of the NpHRs and a complex formation between
the NpHR and BR in the solubilized system. From a BR titration experiment for
the NpHR, a molar ratio of NpHR:BR in the solubilized complex was estimated to
be close to 1: 1. Further SDS-PAGE analysis of the solubilized NpHR crosslinked by glutaraldehyde has revealed that the NpHR forms homotrimer indetergent system. Although this trimeric structure was stable in the presence ofNaCl, it was dissociated to the monomer by the heat treatment at 45 °C in thedesalted condition. Interestingly, the trimer dissociation on the NpHR was
accompanied by the complete dissociation of the BR molecule from the protein,indicated that the cavity formed by the NpHR protomers in the trimericconformation is important for tight binding of the BR. The trimer dissociation
ratio of the NpHR-BR complexes (about 25%) after heat treatment for 3h at 45 °C
was much lower than that of the NpHRs not bound the BR (about 65%),
suggested the physiological role of the BR to stabilize the trimeric structure.
3PT127Structure, orientation and interactions of bovine lactoferrampin in membrane bilayers
Namsrai Javkhlantugs, Kazuyoshi Ueda, Akira Naito (Department ofAdvanced Materials Chemistry, Graduate School of Engineering, Yokohama
National University)acid residues. It is important to determine the orientation and the interactions of
LFampinB with lipid molecules in acidic dimyristoylphosphatidylglycerol(DMPG) and zwitterionic dimyristoylphosphatidylcholine (DMPC) membranes
to reveal the antibacterial mechanism. LFampinB formed alpha-helix at Nterminus region with the tilted angle of 42 degrees in DMPG and of 38 degrees
in DMPC to the bilayer normal after the simulation. We analyzed the interactionenergy between hydrophilic side chains of peptide and lipid head groups ofmembrane bilayers. It is found that the interaction energy between peptide andDMPG is higher than that of DMPC membrane. Structure of LFampinB in
mimetic bacterial membrane was investigated by experimentally using solidstate NMR method. It showed that the LFampinB specifically interacts withacidic phospholipids and disrupts the bacterial membrane. Solid-state NMR
clarified that peptide keeps helical structure and its helix were tilted to themembrane normal which is in good agreement with both methods.
3PT128Laser flash photolysis study on the decay kinetics of the M
photointermediate of bacteriorhodopsin in diheptanoylphosphocholine micelles
Masashi Sonoyama1, Yumiko Kuwabara1, Takashi Kikukawa2 (1Grad. Sch.
Eng., Gunma Univ., 2Grad. Sch. Life Sci., Hokkaido Univ.)
Diheptanoylphosphocholine (diC7-PC), a short chain phospholipid that has thesame basic chemical structure as native long chain phospholipid inbiomembrane, is a promising detergent available for solubilization of membraneproteins. We have recently reported that diC7-PC is actually a very powerfuldetergent for solubilization of a membrane protein bacteriorhodopsin (bR), andthe solubilized bR was much more stable than that with other conventional
detergents, especially in the functional state under illumination with visible
light*. Interestingly, laser flash photolysis of the solubilized bR at neutral pH
demonstrated that the decay curve of the M intermediate can be well expressedwith two kinetically different components. In this study, further laser flash
photolysis measurements were performed for bR in diC7-PC micelles in thewide pH range, in order to address the origin of the two kinetically different M
species. The obtained experimental results that the ratio of the two components
is highly dependent on pH of the surrounding media indicates that pKa of Asp
96 in the dark is perturbed by solubilization with diC7-PC and is estimated as~8.5. Further works on the activation volume for the decay of the two M species
are under progress by analyzing the hydrostatic pressure dependence of the
decay at neutral pH. These results will be also discussed.
*M. Sonoyama, M. Fukumoto and Y. Kuwabara, Chem. Lett. 39, 876-877(2010).
3PT129Characterization by mutagenesis analysis of putative protontransfer pathway, D-pathway of bovine heart cytochrome coxidaseRyohta Aminaka1, Mai Itoh1, Kunitoshi Shimokata2, Yukie Katayama3,
Tomitake Tsukihara1, Shinya Yoshikawa1, Hideo Shimada1 (1Grad. Sch. Life
Sci., Univ. Hyogo, 2WORLD INTEC CO., LTD., 3Grad. Sch. Agric. Life Sci.,
Univ. Tokyo)
X-ray structures of the bovine heart and bacterial cytochrome c oxidase suggest
three proton transfer pathways (D-, K-, and H-pathways), each homologous
between the two. Glu242Gln and Asp91Asn mutations of the bacterial Dpathway (bovine numbering) abolish the O2 reduction activity, while Asn98Asp
and Asn163Asp abolish proton-pumping activity with keeping high O2
reduction activity. These results support for a proposal that D-pathway transfer
both water-forming and pumping protons. Conservation of amino acid residues
essential to D-pathway functions suggests bovine D-pathway proton pumping,
contradicting to our H-pathway proton-pumping proposal. Here, we analyzed
bovine D-pathway by mutagenesis employing HeLa cell bovine/human hybrid
enzyme expression system developed by us. The Asn98Asp and Asn163Asp
mutations did not change the enzymatic activity. All subunit genes of the hybrid
enzymes including isoform genes were found not to have any other mutations,eliminating possible back mutation to restore the enzymatic activity. Immunoblot analysis demonstrated that Glu242Gln and Asp91Asn mutant did not
dominate over the endogenous human enzyme in contrast to expression of thewild type and other mutant hybrid enzymes. However, proton pumping andcytochrome c oxidation activities of the mitochondrial samples relative to thoseof the samples expressing only the human enzyme indicated the mutations
abolished the enzymatic activity. The present results did not support D-pathway
proton pumping but support the water forming proton transfer.

One of the antibacterial peptide is bovine lactoferrampin (LFampinB) whichfound in the sequence of bovine lactoferrin (268-284), and consists of 17 amino− S162 −3PT130

陰溶媒膜モデルとレプリカ交換シミュレーションによるα-ヘリカル型膜タンパク質の構造予測

Prediction of α-helical membrane protein structures by
replica-exchange simulations with implicit membrane model
Ryo Urano1, Yuko Okamoto1,2,3 (1Dept. of Phys. Grad. Sch. of Sci. Nagoya
Univ., 2Bio. Research Center Grad. Sch. of Sci. Nagoya Univ., 3Center for
Comput. Sci. Grad. Sch. of Eng. Nagoya Univ.)
Membrane proteins are essential for life. Their structures are related to theirfunctions. Their 3-dimensional structures are typically in α-helical structures.
Although the determination of the structures is usually done by x-ray
diffraction, solid-state NMR, or electron microscope experiments, it takes a longtime to prepare the samples. The purpose of the present work is to predict the 3dimensional structure of α-helical membrane proteins by computer simulation.
The procedure is as follows. At first, we extract trans-membrane amino acidresidues from membrane proteins by prediction tools based on bioinformatics.
Next, the ideal helix structure is formed for the trans-membrane helix regions.
During simulations, the replica-exchange method and a simple implicit membrane model to represent restrictions of membrane proteins from membrane
environment are applied. The Monte Carlo move sets include backbone torsion
angles for the deformation of helix structures from ideal helix formation. Thisimplicit membrane model represents the restriction of trans-membrane helices
within the membrane regions. A principal component analysis is used to
examine the simulation trajectory and divide structures into local- minimum freeenergy states. We applied our method to the prediction of the structures of small
membrane proteins including bacteriorhodopsin. In this poster presentation, weshow the results of these structure predictions.
3PT131

Homology modeling and molecular dynamics study of proton
permeation pathway in a channel protein
Susumu Chiba1, Kota Kasahara1, Matsuyuki Shirota1,2, Kengo Kinoshita1,2,3(1Grad. Sch. of Information Sci., Tohoku Univ., 2Tohoku Medical Megabank
Organization, Tohoku Univ., 3IDAC, Tohoku Univ.)
Hv1, voltage sensitive proton channel is known as voltage sensor only protein(VSOP). In contrast to other voltage sensitive ion channels, VSOP doesn’t havea pore domain but has only a voltage sensor domain (VSD), through which
proton permeate. Recent simulation studies based on homology models of
VSOP showed the possibility that proton permeation is caused by proton“hopping” (Grotthuss mechanism), from water to water in the crevice oftransmembrane region, but details of permeation mechanism is still unclear. To
analyze proton pathway, molecular dynamics (MD) simulation is powerful
method. Since the proton pathway is formed by the interaction between waterand the charged residues in transmembrane region, precise modeling of
membrane region is important to simulate and estimate characteristic of proton
permeation. VSD of voltage-gated potassium channel (Kv channel) was used fortemplate of homology modeling in the preceding studies. On the other hand,
voltage-gated sodium channel (Nav channel) NavAb, which was recently
entered in PDB database, share higher sequence identity with VSOP than Kv
channel. Therefore we made VSOP-NavAb alignment considering sequence oftransmembrane regions, and subsequently we modeled structure based on thealignment. We performed the MD simulation of the predicted model embedded
in POPE membrane for 200 ns. It was observed that a continuous pathway ofwater molecules exist in the crevice of the channel. Finally we discuss the
behavior of waters and charged residues, and proton permeation mechanism.
3PT132

大腸菌機械受容チャネル MscL の開口過程におけるタンパク質と水との協調過程の役割の分子動力学的解析

Molecular Dynamics Analysis of the Role of Protein-Water
Interplay in the Opening Process of E-coli Mechanosensitive
Channel MscL
Yasuyuki Sawada1, Masahiro Sokabe1,2 (1Dept. Physiol. Nagoya Univ. Grad.
Sch. Med., 2FIRST Res. Ctr. for Innovative Nanobiodevice, Nagoya Univ.)
The bacterial mechanosensitive channel MscL is constituted of homopentamerof a subunit with two transmembrane inner (TM1) and outer (TM2) α-helices.
Neighboring TM1s cross and interact with each other near the cytoplasmic sidethrough hydrophobic interaction, forming the most constricted hydrophobic partof the pore. The crossings stabilize the closed state of MscL with hydrophobic
interactions, thus called gate. The major issue of MscL is to understand thegating (opening) mechanism driven by tension in the membrane. We performed

molecular dynamics (MD) simulations of tension-driven opening of MscL,
particularly focusing on the conformational change of MscL associated withwater. Upon membrane stretch, Phe78 in TM2 helix, which is suggested to be amajor tension sensor in our previous work, was dragged by lipids and TM1swere gradually tilted accompanied by outward sliding of the crossings between
TM1 helices. While TM1s were tilted, a breakage of the helical structure aroundthe gate was shown and an exposure of oxygen atoms of the backbone to theinner surface of the gate occurred, followed by formation of hydrogen bonds
between water and the exposed oxygen. This allowed further access of water tothe gate and weakened the hydrophobic interaction at the crossings, leading to afurther opening of the gate. It is suggested that the combination ofconformational changes of MscL by membrane stretch and following hydrogenbond formation forms a positive feedback cycle, which seems to be critical toinduce rapid channel opening.
3PT133

ATP-Binding induced conformational change in CFTR as
studied by MD simulation
Tomoka Hagiya1, Tadaomi Furuta1, Shuntaro Chiba1, Yoshiro Sohma2,
Minoru Sakurai1 (1Grad. Sch. of Biosci. & Biotech., Tokyo Tech., 2Dept ofPharmacol., Sch. Med., Keio Univ.)
The Cystic fibrosis transmembrane conductance regulator (CFTR, ABCC7) is achloride channel and belongs to the ATP-binding cassette (ABC) protein
superfamily that binds and hydrolyzes ATP to drive substrate transport acrossmembranes. For the gating mechanism, the following hypothesis has been
proposed: after phosphorylation of the regulatory domain, ATP molecules bindto the two nucleotide-binding domains (NBDs), which in turn induces thedimerization of the NBDs and the concomitant opening of the channel gate. To
clarify its mechanism, we first built a homology model of CFTR (with aninward-facing conformation) by using several X-ray crystal structures of ABC
transporters as templates. Subsequently we explored its stable conformations
and dynamics using molecular dynamics (MD) simulations, which were
performed for Mg and ATP-bound (MgATP) state and nucleotide-free (Free)
state. Interestingly, in the MgATP state, NBD1 and NBD2 were dimerized sothat the ATP molecules were sandwiched between Walker A and the signature
motif. Additionally we found the closing of the TMDs internal cavity in theintracellular loop regions. On the other hand, the NBDs remained monomers in
the Free state. This study suggests that ATP-binding not only causes the NBD
dimerization, but also exerts a significant influence on the TMD structure.
3PT134

リポソームと機能性ペプチドを用いた酵素活性検出法の開発

Development of enzyme activity detection system using
liposome and functional antimicrobial peptide
Masayoshi Kashibe1, Shin Mizukami1,2, Kazuya Kikuchi1,2 (1Graduate
School of Engineering, Osaka University, 2Immunology Frontier ResearchCenter, Osaka Univ.)
The development of enzyme activity detection system has attracted wide
attention in the field of biology, pharmacology, and medicine. We developed a
new system for enzyme activity assay by using anionic liposome and thederivatives of an antimicrobial peptide, temporin L (TL). It was reported that
TL has membrane-damaging activity against anionic membranes of microorganisms. Previously, we reported that modification of the Lys amino group of
TL by a photosensitive protective group suppressed its membrane-damaging
activity. Thus, we hypothesized the membrane-damaging activity of TL
derivatives can be modulated also by modification of other amino acid residues.
We designed several TL derivatives modified with a phosphate group at thevarious amino acid residues. The membrane-damaging activities in some of thederivatives were suppressed and liposome disruption does not occur. When the
peptides were treated with a phosphatase, these activities were recovered. This
conversion induced the inclusion release from liposome, in which fluorophores
were quenched, and the inclusion release induced the fluorescenceenhancement. This result indicates that this system can be used for the detectionof phosphatase activity.
Since various modifications of peptides for many types of enzyme assay ispossible, it could lead to detection system for various enzyme activities.
Moreover, since liposomes can include various compounds such as proteins, anddrugs, this system would be applied not only to diagnosis but also totherapeutics.

− S163 −3PT135

モデル細胞間に形成された脂質 2 分子膜の吸着力測定

Adhesive force of a lipid bilayer between contacted cell-sized
dropletsTaka-aki Yoshida1, Miyuki Furuta2, Yui Matuda2, Satoshi Nakata2, Masayuki
Tokita1, Miho Yanagisawa1 (1Grad. Sch. Sci., Kyushu Univ., 2Grad. Sch. Sci.,
Hiroshima Univ.)
Cell-sized water/oil droplet coated by a lipid monolayer has been shown to be auseful system for examining the interaction between lipid membrane andencapsulated proteins [1-2]. The droplets can easily connect with each other andform a continuous structure like a cell tissue where lipid bilayers bound them. In
the case of adhesive two droplets, the adhesive force is calculated from F = 2γ(1-cosθ), where θ and γ are contact angle and surface tension at the oil/waterinterface [3]. We have evaluated the lipid dependence on θ and γ independently
by using three different types of lipids, unsaturated lipids: DOPE
(dioleoylphosphatidylethanolamine), DOPC (dioleoylphosphatidylcholine), anda saturated DMPC (dimyristoylphosphatidylcholine). Then, the contact angle
term 2(1-cosθ) differs among lipid species and fails in the order: DMPC <DOPC ~ DOPE, which might reflect the molecular structure of hydrophobic
tails. On the other hand, γ follows the order: DOPC <DMPC <DOPE, whichis consistent with non-lamellar PE unlike PC. Finally, we obtained thelipid dependence on F: DMPC <DOPC <DOPE. Based on these values, we
elucidated the effects of membrane or cytoplasmic structures on F by theaddition of polyethylene glycol (PEG) conjugated DOPE or encapsulation ofgelatin gel. We believe that this system is useful to clarify the effects of proteinson adhesive lipid mono-layers.
[1] A. Kato, et al., 2012 Sci. Rep., 2:283.
[2] M. Yanagisawa, et al., 2011 J. Am. Chem. Soc., 133:11774.
[3] P. Poulin et al., 1997 Phys. Rev. Lett., 79:3290.
3PT136

ジェット流による細胞サイズリポソームの作製

Preparation of cell-sized liposomes by pulsed jet flow
Koki Kamiya1, Ryuji Kawano1, Toshihisa Osaki1, Syoji Takeuchi1,2(1Kanagawa Academy of Science and Technology, 2Institute of Industrial
Science, The University of Tokyo)
Giant liposomes are composed of phospholipid membranes similar to thosecommonly found in living cells. A giant vesicle is typically ~10 μm in diameter,
which is sufficient observation by optical microscopy. Therefore, such artificial
liposomal systems have been used in biochemical and biophysical studies suchas the microencapsulation of gene expression systems. To effectively form
uniformly-sized giant vesicles and encapsulate biological molecules into giant
vesicles, in our previous study, we created giant vesicles by the microfluidicjetting-induced deformation of a planar lipid bilayer including an organic
solvent. However, this method has limitations; for instance, the giant vesicle is a
W/O/W emulsion having a thin shell of an organic solvent. Further, it is unstableand very large (over 300 μm in diameter). In the present study, we prepared cellsized lipid vesicles using an improved double-well device and a pulsed jet
flow. When the jet was applied for 4.2 ms at 250 kPa, vesicle formation could
be observed using a high-speed camera. Vesicles of two different sizes
were formed by the jet flow. The vesicles with a diameter of over 100 μm
immediately collapsed in the glucose solution. However, the vesicles with thediameter of approximately 5 μm sank in the glucose solution and their shape
was maintained. The average diameter of the vesicles formed immediately andafter 24 h was 4.4 μm.
3PT137タンパク質内包リポソームの熱安定性に関する小角・広角 X 線散乱研究

SWAXS study of thermal structural stability of liposomes
entrapping proteins
Ryota Kimura, Mitsuhiro Hirai (Grad. Sch. Eng., Gunma Univ.)[Introduction]
By using synchrotron radiation small-angle and wide-angle X-ray scattering(SR-SWAXS), we studied the structural characteristics of liposomes entrapping
proteins. The aim of this study is to clarify the thermal stability of liposomes
entrapping proteins that mimic an intracellular environment.
[Experimental]
Liposomes measured were large uni-lamellar vesicles (LUVs) of lipid-mixtures
composed of glycosphingolipid, cholesterol and phospholipid lipids (egg-PC or
DPPC). The protein occluded in liposmes was myoglobin. LUVs were prepared

as follows. The lipid mixtures dissolved in protein solutions in 10 mM HEPES
buffer at pH 7. 5 were subjected to natural swelling, ultra-sonic dispersion,
freeze-throw, extrusion using a extruder system, and spin-filtration,
successively. SR-SWAXS experiments were done SPring-8 and at PF The
temperature range of the samples was 25 - 85 °C.
[Results]
In the case of the empty DPPC-LUV, the bilayer structure changed evidently inthe range from 40 ° C to 45 ° C due to the gel-to-liquid crystal transition,
whereas, both the diameter and the radius of gyration were mostly unchanged.
In the case of the filled DPPC-LUV, the transition temperature became higher inaround 5 degrees, suggesting that the thermal stability of liposome was
improved by the protein occlusion. Both the diameter and the radius of gyration
increased once, and suddenly decreased. Above the transition temperature, both
values were mostly constant. Further experimental results and analyses will beshown.
3PT138

溶質含有脂質フィルムから調製した巨大リポソームのサイズ、形態と膜多重度

Size, morphology and lamellarity of giant liposomes generated
from solute-containing lipid films
Kanta Tsumoto, Shota Kimura, Masahiro Tomita (Grad. Sch. Eng., Mie
Univ.)
Giant liposomes are adopted for studies on cell membrane andmicrocompartment models, and a lot of methods have been developed to
improve various properties of them, including formation efficiency, size
distribution, lamellarity, etc. Of course, all protocols are useful; however, thehydration methods, such as the gentle hydration (natural swelling) which isbased on the Bangham method, and the electroformation, are often used for
preparation of giant liposomes in a buffer saline. Especially, the natural swellingis still popular probably because it is so simple and reasonable. Previously, we
and collaborators reported improvement of this method, in which hydration andswelling processes in liposome formation may be enhanced through promoted
osmosis due to water- and methanol-soluble solutes such as NaI andmonosaccharide sandwiched between lipid lamellae of films [1,2]. In the present
work, we further studies detailed properties of such giant liposomes with respect
to changes in size, morphology and lamellarity. Here, DOPC films with fructose
in various molar ratios were hydrated, resulting in transition of liposome sizes
from micron to submicron at the higher ratio of Fru/DOPC (60:1 and more). An
NBD quenching assay showed the transition from multi- to unilamellar
liposomes. We also tried to investigate if the liposome morphology would be
dependent on the kind of solute, using polymer as a model.
[1] Yamada, N.L., et al. EPL, 80, 48002 (2007). [2] Tsumoto, K., et al. Colloids
Surf. B, 68, 98-105 (2009)
3PT139

表面修飾ナノ粒子の巨大リポソームへのエンドサイトーシス様取込み

Endocytosis-like Uptake of Surface-Modified Nanoparticles
into a Giant Liposome
Kohei Tahara1,2,3, Satoshi Tadokoro1, Yoshiaki Kawashima2, Naohide
Hirashima1 (1Grad. Sch. Pharmaceut. Sci., Nagoya City Univ., 2Sch. Pharm.,
Aichi Gakuin Univ., 3Gifu Pharmaceut. Univ.)
Cell-sized giant unilamellar vesicles (GUVs) have been used as artificial cellsystems that mimic various cellular events. We previously reported an artificial
exocytosis model by encapsulating small unilamellar vesicles in a GUV.
In the present study, we observed the uptake of surface-modified nanoparticles(NPs). We prepared a cell-sized GUV that mimics a cell membrane toinvestigate the interaction between cell membranes and NPs. As NPs, we used
poly (D,L-lactideco-glycolide) (PLGA) NPs modified with nonionic surfactant
P80 or cationic polymer chitosan (CS) on their surface. To observe interaction
between GUV and NPs with a fluorescence microscope, GUV was preparedwith rhodamine-conjugated phosphatidylethanolamine, and NPs were labeledwith 6-coumarin.
Average particle diameters of unmodified NPs, CS-modified NPs and P80modified NPs were 221 ± 1. 4 nm, 350 ± 17. 1 nm, and 210 ± 5. 1 nm,
respectively. Zeta-potentials of unmodified NPs, CS-modified NPs and P80modified NPs were -27.4 ± 0.6 mV, +27.5 ± 0.6 mV, and -28.3 ± 0.7 mV,
respectively.
Endocytosis-like uptake of NPs into a GUV was observed when the NPs weremodified with P80. In contrast, unmodified NPs and those modified with CS
were not internalized into a GUV, although large number of NPs with CS
adhered to the surface of GUV due to their positive charge. These results− S164 −suggest that surface properties of PLGA-NPs are important for endocytosis-likeuptake into a GUV.
3PT140トランスポータン 10 が DOPC 膜の巨大リポソームの膜透過性と構造に与える効果

Effects of Transportan-10 on Membrane Permeability and
Structure of Single Giant Unilamellar Vesicles of DOPC
membranes
Hirotaka Ariyama, Masahito Yamazaki (Integrated Bioscience Section,
Graduate School of Science and Technology, Shizuoka Univ.)
Transportan 10 (TP 10) is one of the cell penetrating peptides. So far thepermeation of TP 10 into cells and the interaction of TP 10 with lipid
membranes using LUV suspension have been investigated. However, the detailcharacteristics and mechanism of the interaction and its permeation throughmembranes remain unclear. In this report, we have investigated the effect of TP
10 on membrane permeability of lipid membranes using the single giant
unilamellar vesicle (GUV)method.
First, we investigated the interaction of TP 10 with single GUVs composed ofdioleoylphosphatidylcholine (DOPC) and PEG2K-DOPE (molar ratio, 98: 2)
containing the fluorescent dye, calcein, in a physiological buffer using phase
contrast, fluorescence microscopy. The interaction of 0.8 μM TP 10 with single
DOPC-GUVsinduced the rapid monotonous leakage of calcein from the insideof the most GUVs, but in some GUVs step-wise leakages were observed.
During the calcein leakage, the size of most GUVs decreased a little (less than 5%), but in some GUVs their radius decreased greatly and some small highcontrast particles appeared on the GUV membranes. The fraction of leaked
GUVincreased with time, and also with an increase in TP 10 concentration.
Second, the effect of cholesterol on the TP 10-induced leakage of calcein wasinvestigated. 0.2 μM TP 10 did not induce calcein leakage from DOPC/chol(6/4) -GUVs. On the basis of these data, we discuss the mechanism of theinteraction of TP 10 with DOPC-GUVs and DOPC/chol-GUVs.
3PT141

F-BAR によるリポソームのチューブレーションのリアルタイム観察

Real-time observation of liposome tubulation by F-BAR
Yohko Takiguchi1, Toshiki Itoh2, Kingo Takiguchi1 (1Grad. Sch. Sci., Univ.
Nagoya, 2Grad. Sch. Med., Univ. Kobe)
The Fer-CIP4 homology-BAR (F-BAR) domain has been identified as a
biological membrane-deforming module. The F-BAR domain has been reported
to transform lipid bilayer membranes into tubules. However, the process oftubulation still remains unknown. Here we monitored the entire tubulation
process induced by the F-BAR domains or by the full lengths of four different FBAR domain proteins, PSTPIP1, FBP17, CIP4 and Pacsin2, using direct realtime imaging, and show that each F-BAR domain or protein induces tubules
through a unique kinetics from model membranes.
FBP17 and CIP4 develop many projections simultaneously throughout thesurface of individual liposomes, whereas PSTPIP1 and Pacsin2 develop only afew projections from a narrow restricted part of the surface of individualliposomes. The results provide striking evidence that a nucleation process isinvolved in the F-BAR-induced tubulation, and individual F-BAR domains havea unique nucleation rate and/or property even though essentially they have thesame crescent-shaped structure. The differences in process of tubulationinduced with those F-BAR domain proteins may reflect their unique
physiological roles, and function favorably to build networks of various androbust membrane trafficking processes observed in cells.
3PT142

PEG 担持ジャイアントベシクルにおける膜組成と分裂様式の関係
Correlation between Membrane Composition and Mode of
Division of PEG-grafted Giant Vesicles

Yumi Kan1, Kensuke Kurihara2, Taro Toyota2,3, Masayuki Imai4, Tadashi
Sugawara3,5 (1Ochanomizu University, 2The University of Tokyo, 3ResearchCenter for Complex Systems Biology, 4Tohoku University, 5Kanagawa
University)
Recently we realized a model protocell using a giant vesicle (GV) as acompartment, comprised of phospholipids, cationic synthesized membrane
molecule (V) and an amphiphilic catalyst (C). Encapsulated DNA in GV wasamplified by polymerase chain reaction, and then the GV grew and divided
when the incorporated membrane-precursor was converted to V by theassistance of C. We also revealed that the attractive interaction between
amplified DNA and the inner surface of the GV membrane accelerated the GV

division. Here we prepared a new model protocell including 5 mol% ofpolyethylene glycol (PEG5000) -grafted phospholipid: a PEG-chain of thephospholipid not only increased the tolerance of GV to high ionic strength andhigh temperature but also affected the interaction between DNA and the GV
membrane containing the cationic membrane molecule V. In order to elucidate
the morphological change of the GV membrane throughout the selfreproduction process, we stained the membrane by Texas Red-tagged
phospholipid and observed the dynamics in terms of a confocal laser scanning
fluorescence microscope. Whereas the division mode of non PEG-grafted GV
was a budding-type exclusively, the birthing-type deformation occurred mainlyin the case of PEG-grafted GV. We speculated that the interaction between theamplified DNA and the PEG-grafted inner surface of the membrane influences
the division mode of GV self-reproduction. In this symposium, we will discuss
the effect of the length and the content of PEG-grafted phospholipid on themode of GV division.
3PT143

静置水和法による均一径リポソームアレイ

Size-controlled Giant Liposome Array System with Gentle
Hydration
Toshihisa Osaki1, Koki Kamiya1, Kaori Kuribayashi2, Ryuji Kawano1, Shoji
Takeuchi2 (1Kanagawa Academy of Science and Technology, 2Institute ofIndustrial Science, The University of Tokyo)
This work presents a methodology that realizes size-controlled, solvent-free giant
liposome arrays with simple gentle hydration technique. Gentle hydration andelectroformation have been the most commonly used methods to prepare giant
liposomes, yet they have difficulty controlling the size distribution of the formed
liposomes as well as their shape and lamellarity. We therefore set the goal ofdeveloping an alternative method that allows the formation of uniform-size giant
liposomes with high reproducibility. Our system consists of a dried lipid pattern
on a substrate that is a micropatterned polymer thin film on an ITO glass slide. An
electrospray deposition (ESD) method was applied to obtain the selective lipiddeposition at the polymer pattern where the ITO surface was exposed. With asimple hydration process of the dried lipid, we succeeded in formation of giant
liposomes on top of the pattern. The diameter of the liposomes was similar to thepatterned size, achieving a narrow range of size distribution. We also report theapplicability of the system for various biological assays such as object
encapsulation into the liposomes, buffer exchange during the assays, andreconstitution of membrane proteins into the liposome membranes.
3PT144REMD シミュレーションによる粗視化された脂質膜の相転移の研究

Study on phase transition of coarse-grained lipid bilayer by
REMD simulations
Tetsuro Nagai, Yuko Okamoto (Grad. Sch. Sci., Nagoya Univ.)
A replica-exchange molecular dynamics simulation of a lipid bilayer system
was performed in order to study sol-gel phase transitions. The replica-exchange
molecular dynamics method enables one to enhance conformational sampling
efficiency and to study the system at a wide temperature range at once. We
employed a coarse-grained model MARTINI. The results show abrupt changes
in internal energy, bilayer thickness, and area of bilayer around 296 K. A peakin specific head capacity was also observed around this temperature. Thesesuggested that the sol-gel phase transitions. The bilayer has two states in the gelstate. One is a tilted gel state and the other is an un-tilted gel state. A previous
work with MARTINI force filed reported only the un-titled gel state. This
indicates that conformational sampling efficiency is not trivial even with coarsegrained model, which has smoother energy landscapes and reach longer timescales than atomistic model. The un-tilted gel phase is a little thicker than thetilted gel phase, while the area of lipid bilayer is quite similar. We will show
more detail results in the presentation.
3PT145長鎖リン脂質 DMPC と短鎖リン脂質 DHPC で構成される複雑な相挙動のダイナミクス

Dynamics of complicated phase behavior on the mixturesconsisting of long-chain phospholipids DMPC and short-chain
phospholipids DHPC
Ryota Kobayashi, Tetsuhiko Ohba (Department of Physics, Tohoku
University.)
The binary phospholipid mixtures of long-chain DMPC and short-chain DHPC
show a variety of complicated phase behavior, depending on the mixing ratio of
two lipids, the total lipid concentration in water, and temperature. At low− S165 −temperature, the mixture is transparent and has a low viscosity, and its structureis thought to be disk-like micelles, which is often called "bicelles". At hightemperature, the mixture is turbid and also has a low viscosity, and its structureis thought to be multi lamellar vesicles (MLV). In intermediate temperature
range, the mixture has high viscosity. This phase is called nematic phasebecause the polarized optical microscopy observation exhibit optical anisotropy.
The structure of nematic phase is not yet clear although some models, such as
worm-like or disk-like aggregates, had been proposed.
We measured membrane fluidity and fluorescence anisotropy of DMPC/DHPC
mixtures, using fluorescence GP imaging with Laurdan or fluorescence
spectroscopy with DPH. The results support stability of the structure of bicelles
at low temperature and mechanism of generating MLVs at high temperature.
However, in intermediate temperature range, we obtained conflicting results
between microscopic and macroscopic behavior.
Now, we are observing this system using Muller Matrix microscopy which wasdeveloped in our laboratory. Muller Matrix microscopy could detect, inprinciple, any change of polarization states induced by the sample and therefore
it is expected to elucidate structural dynamics around the nematic phase.
3PT146

細胞毒性を有する酸化コレステロールのホスファチジルコリン膜分子充填への影響

Effect of cytotoxic oxysterols on the molecular packing ofphosphatidylcholine membranes
Tatsuya Hoshino1, Takaaki Hikima2, Masaki Takata2, Toshihide Kobayashi3,
Hiroshi Takahashi1 (1Grad. Sch. Eng., Gunma Univ., 2Harima Inst., Riken, 3Wakou
Inst., Riken)
Oxidative stress induces the conversion from cholesterol (CH) to various
oxysterols in biomembranes. Some oxysterols are cytotoxic. For example, 7 βhydroxycholesterol (7 β-OH) and 25-hydroxycholesterol (25-OH) have beenreported to be associated with neurodegenerative diseases and arteriosclerosis,
respectively.
The oxysterol-related diseases presumably result from the difference between
CH and oxysterols on ability to modulate the biophysical properties of themembrane bilayer. In this work, to gain fundamental insight into the effects ofoxysterols on biophysical properties of biomembranes, we investigated themolecular packing of binary mixtures of dipalmitoylphosphatidylcholine(DPPC)/CH, DPPC/7 β-OH, and DPPC/25-OH.
The apparent specific volumes estimated by neutral flotation method were samefor all three mixtures, at least, from 0 to 40 mol% sterol concentrations.
Meanwhile, X-ray diffraction analysis of the mixtures containing 30 mol%sterols showed that the order of the bilayer thickness was DPPC/CH <DPPC/25-OH <DPPC/7 β-OH.
These results indicate that the addition of OH group by oxidation decreases CH’
s capacity of condensing the bilayer in the lateral direction and the OH group on
the middle position more strongly perturbs the molecular packing of bilayers
than that on the edge position.
We are also currently studying palmitoyloleoylphosphatidylcholine/oxysterol
mixtures.
3PT147ジヘキサデシルホスファチジルコリンの指組ゲル相二層膜間の氷点下温度における水和斥力Hydration repulsive force of the interdigitated phase ofdihexadecylphosphatidylcholine at subzero temperatures
Hiroshi Takahashi (Grad. Sch. Eng., Gunma Univ.)
The clarification on the interactions of lipid bilayers is one of the most important
issue of membrane biophysics, however, many aspects of these interactions still
remain mysterious. One of such examples is “hydration repulsive forces” andseveral different explanations on its origin have been proposed. Here we
investigated the hydration repulsive force between the interdigitated (LβI) phasebilayers of dihexadecylphosphatidylcholine (DHPC) at subzero temperatures.
The number of headgoups per an unit area at the bilayer surface of theinterdigitated phase is about twice as compared with normal gel (Lβ' ) phase.
The measurement of the force was carried out by using our previously proposed
method [1] based on the combination of ice formation free energy values andstructural information obtained by X-ray diffraction. The estimated hydration
repulsive force curve of DHPC in the Lβ I phase was different from that ofdipalmitoylphosphatidylcholine (DPPC) in the Lβ' phase. This suggests thatheadgroup (dipole moment) density affects the hydration repulsive force
profiles. In addition, we are planning to present the result of
dipalmitoylphosphatidylethanolamine (DPPE) and will also discuss the effectsof the difference of headgroup on the hydration repulsive forces.
[1] H. Takahashi & P.J. Quinn, Mol.Cryst. Liql.Cryst. 367 (2001) pp.427-434
3PT148架橋脂質二分子膜における秩序液体相／液晶相の分離

Localization of Liquid-Ordered/Liquid-Crystalline Phase
Separation at a Lipid Bilayer Suspended over Microwells
Koji Sumitomo1, Yukihiro Tamba2, Aya Tanaka1, Touichiro Goto1, Keiichi
Torimitsu1 (1NTT Basic Research Labs., 2Suzuka Nat. Coll. of Tech.)
The localization of liquid-ordered (Lo) and liquid-crystalline (L α) phasedomains is investigated on a silicon substrate with a microwell array. The
localization of Lo and Lα phase separation as a model of a lipid raft plays animportant role regarding the arrangement and function of membrane proteins.
When we fabricate a nanobiodevice that works with membrane proteins,
controlling the function of proteins is a key technique. A microwell array with asealing lipid bilayer, whose phase separation can be controlled, will be a
promising platform for nanobiodevices.
Suspended lipid bilayers were formed by rupturing giant unilamellar vesicles(GUVs) on the substrate. The GUVs were prepared from ternary mixtures of
DOPC, DPPC, and cholesterol. We found that the Lα phase is preferable at thesuspended membrane over microwells because of its curvature. On the other
hand, the Lo phase domain disappeared from the suspended membrane. Phase
separation and localization are governed by the stiffness and line tension of themembrane. Lateral diffusion at the molecular level occurs quickly, althoughdomain movement is restrained at the supported membrane by the interaction
with the substrate. Unsaturated lipid is condensed at the suspended membrane,
and saturated lipid and cholesterol are excluded. Additional phase transition anddomain rearrangement occurs. By modification of surface roughness, charge,
and/or chemical state, the localization of phase separation on the suspended
lipid bilayer will be controlled.
3PT149ラウリル硫酸ナトリウム(SLS)溶液塗布によるヒト皮膚角層の構造
変化

Effect of sodium lauryl sulfate on the human stratum corneum
structures
Hironori Yoshida1, Satoe Azechi2, Hiromitsu Nakazawa1, Yasutami Shigeta2,
Satoru Kato1 (1Sch. Sci. Tech., Kwansei Gakuin Univ., 2SUNSTAR INC.)
The stratum corneum (SC), a thin envelope surrounding our body, is known tobe responsible for the skin barrier function. It consists of corneocyte cells filledwith keratin filaments and intercellular lipid layers. In this study we examinedthe effect of a substance inducing rough skin on the SC structures by electron
diffraction measurements to clarify its permeation path in the human SC. Layersof corneocyte cells were stripped off from the surfaces of human forearms offive subjects with glue-coated grids for electron microscopy after administrationof a 1% sodium lauryl sulfate (SLS) aqueous solution on the skin surface.
Control specimens were collected from the opposite forearms without the SLS
treatment. Acquisition of electron diffraction patterns was carried out undervery low electron dose conditions (~3 e/nm2) with an electron microscope(JEM1400) equipped with a high-sensitivity CCD camera. One-dimensional
diffraction profiles were calculated for analysis of the structural change inducedby the SLS permeation. Our results suggested that the lipid packing structures
remain intact during the SLS permeation though the transepidermal water loss
increases significantly. Furthermore, the SLS treatment seemed to slightly
reduce the diffraction intensity around 2.0 nm-1, which comes from the structureother than the well-established orthorhombic or hexagonal domains in theintercellular lipid layers. We speculate that the applied SLS molecules
penetrated into the corneocyte cell body to affect the keratin filament structure.
3PT150

高感度 DSC によって求めたジトリデカノイルホスファチジルコリ
ン-コレステロール系の詳細な相図

Detailed Phase Diagram of DitridecanoylphosphatidylcholineCholesterol System Determined by High-Sensitivity DSC
Yasushi Kamitani, Fumihiro Okazaki, Yasuo Saruyama, Haruhiko Yao (Gra.
Sch. Sci. Tech., Kyoto Inst. of Tech.)
It was recently found that the Lx phase exists in ditridecanoylphosphatidylcholine (DC13PC)[1]. Besides, the Lx phase is the liquid-orderedphase reported in phosphatidylcholine-cholesterol system, because there is nophase line between the Lx phase of pure DC13PC and the liquid-ordered− S166 −phase of DC13PC-cholesterol mixtures in the binary phase diagram. However,
our previous phase diagram was determined every 5 mol% cholesterol
concentration. Thus, there is some risk that we might miss the phase line if it isnearly normal to the concentration axis. Therefore, we have performed heat
capacity measurements on DC13PC-cholesterol binary mixtures to make thephase diagram more detailed using high-sensitivity DSC developed in ourlaboratory. As a result, it is confirmed that phase transition was not observed
between the Lx and liquid-ordered phases, and that the Lx phase is the liquidordered phase. Moreover, it is found that the temperature range of the liquidordered phase drastically increases at cholesterol concentrations higher than 23
mol%. The detailed phase diagram will be presented.
[1] F. Okazaki, Y. Saruyama, H. Yao: Abstracts of 46th Annual Meeting of theJapan Society of Calorimetry and Thermal Analysis, (2010)2B1420.
3PT151

高感度 DSC によるヒト皮膚角層の熱容量測定

Heat Capacity Measurement of Human Stratum Corneum
using High-Sensitivity DSC
Keisuke Oki1, Ichiro Hatta2, Yasuo Saruyama1, Haruhiko Yao1 (1Gra. Sch.
Sci. & Tech., Kyoto Inst. of Tech., 2Nagoya Ind. Sci. Res. Inst.)
The outermost layer of skin called stratum corneum (SC) is composed ofcorneocytes and intercellular lipids. Intercellular lipids have the long and shortlamellar structures (LSs). It has been proposed that the short LS plays animportant role in control of water content in skin [1]. Therefore, we have
performed DSC measurements of human SC at various water contents between
0-40wt% using high-sensitivity DSC developed in our laboratory. Human SC
was dried under vacuum overnight. The water content of the dried SC was
defined as 0 wt%. Disk-shaped pieces (3 mm diameter) were punched out fromthe dried SC and hydrated in a container where relative humidity was controlledwith glycerol aqueous solution until they had reached a desired water content.
Then, they were sealed into a gold pan. DSC measurements were performed in
the temperature range of -20-130 °C. For human SC with a water content of 20
wt%, thermal anomalies were observed at 9, 20-60, 75 and 89 °C on 1st heating.
The phase transition at 9 ° C, that had not been reported previously, wasobserved at all water contents between 0-40 wt%. On 1st and successive
cooling, thermal anomalies were observed at -2, 12, 32, and 70 °C. On 2nd andsuccessive heating thermal anomalies were observed at 5, 15, 33, and 71 °C. At
higher water content, it is found that the anomaly at 70-71 °C splits into two
anomalies. To identify the phase transitions, we have performed x-ray
diffraction measurements and the results will be presented.
[1] I. Hatta et al., Biochim. Biophys. Acta 1758 (2006) 1830.
3PT152モデル細胞内における相分離とミクロゲルのパターン形成

Gel pattern formation in model cells coupled with phaseseparation
Shinpei Nigorikawa, Masayuki Tokita, Miho Yanagisawa (Grad. Sch. Sci.,
Kyushu Univ.)
It is known that huge amount and great variety of macromolecules areencapsulated in cells, and they maintain their appropriate spatial distribution
inside of the cellular membranes. We used cell-sized water/oil droplet coated bya lipid layer as a cell model, and studied phase separation of binary polymer
solution: gelatin and polyethylene glycol (PEG) aqueous system. This systemshows phase separation and gelation of gelatin with decreasing temperature,
however, the mechanism to bring the phase separation has been unclear. Using
fluorescence-labeled gelatin, we observed the pattern formation of gelatin-richdomains upon phase separation by a fluorescence microscope. At first, the timedevelopment of domain size was analyzed in PC (phosphatidylcholine) droplets,and found that the growth rate was decreased with an increase in droplet size.
Therefore, at a given time, the number of domains in small droplets <30μm islarger than that in large droplets > 100μm. This acceleration of domain growthin smaller droplets may be originated from the depletion effect between rigid
gelatin and soft PEG molecules under micro-scale confinements. On the other
hand, in the case of PE (phosphatidylethanolamine) droplets, a thin layer ofgelatin gel was observed at the interface. It is illustrated that the negatively
charged PE membrane has an electrostatic attractive force with electrolyte
gelatin gel. Furthermore, shape deformation of droplets coupled with the gelpatterns was observed, and was discussed from the Young-Laplace equation.
3PT153

脂質膜へテロ界面はナノ物質をサイズ依存的に識別する

1YS1045

Size-dependent selective localization of nano-particles onheterogeneous membrane surfaces

Tsutomu Hamada, Masamune Morita, Makiyo Miyakawa, Masahiro Takagi(Japan Adv. Inst. of Sci. and Tech.)
It is of biological importance that we understand the biophysical mechanismsthat govern the interaction between nanoparticles (NPs) and heterogeneous
cellular surfaces because of the possible cytotoxicity of engineered
nanomaterials. In this study, we investigated the lateral localization of NPs
within a biomimetic heterogeneous membrane interface using cell-sized twophase liposomes. We found that lateral heterogeneity in the membrane mediates
the partitioning of NPs in a size-dependent manner: small particles with adiameter of <200 nm were localized in an ordered phase, while large particles
preferred a fluidic disordered phase. This partitioning behavior was verified bytemperature-controlled membrane miscibility transition and laser-trapping of
associated particles. In terms of the membrane elastic energy, we present aphysical model that explains this localization preference of NPs. The calculated
threshold diameter of particles that separates the particle-partitioning phase was
260 nm, which is in close agreement with our observation (200 nm). Thesefindings may lead to a better understanding of the basic mechanisms that
underlie the association of nanomaterials within a cell surface.
3PT154

UV irradiation driven deformation of photoresponsive molecule doped membrane
Kazunari Yoshida, Yasuhiro Fujii, Izumi Nishio (Sci. and Eng., Aoyama
Gakuin Univ.)
The phospholipid bilayer has been widely studied as a model system of
biological membrane since its structure is the main component of biomembrane.
In particular, the deformation of the vesicles has been intensively investigated inrelation to the exocytosis and endocytosis.
In the previous study, we doped the photoresponsive molecule pseudogem-Bis(diphenylimidazole) [2. 2] paracyclophane (abbreviated as “pseudogem-BisDPI[2.2]PC” hereafter) to DOPC multilamellar vesicles, and performed UV lightirradiation experiments. The pseudogem-BisDPI [2. 2] PC has one [2. 2]paracyclophane that bridges two diphenylimidazoles and one C-N bond thatconnects two imidazole rings. The C-N bond is easily cleaved by UV
irradiation. It was found that the shape of the doped membrane began to change
after about 10 seconds of UV irradiation, and its surface area per volume
increased with time. Shape of the vesicle was changed into rippling form. We
assumed that increasing of surface area per volume of the vesicle was caused bypseudogem-BisDPI[2.2]PC that excluded lipids of around the molecule with UV
exposure.
In this study, we improved the experimental condition of forming vesicles sothat the unilamellar structure became dominant because we can analyze
unilamellar spherical vesicles easier than multilamellar vesicles with irregularform. In this presentation, we discuss the membrane deformation of unilamellar
vesicles.
3PT155

フリップフロップのダイナミクスの直接的観察

Direct observations of flip-flops in membranes
Takuma Akimoto1, Noriyoshi Arai2, Eiji Yamamoto1, Kenji Yasuoka1, Masato
Yasui3 (1Department of Mechanical Engineering, Keio university, 2Department of
Mechanical Engineering and Intelligent Systems, University of ElectroCommunications, 3Department of Pharmacology, Keio university)
How do lipid molecules in membranes perform a flip-flop? Flip-flop of a lipid
molecule plays a crucial role in membranes. However, little is known regarding
the way of flip-flop by experiments as well as molecular dynamics simulations.
Here, we observe single molecules performing filip-flops in membranes using
DPD simulations. There are three different ways of flip-flop, which can beclearly characterised by paths in a free energy surface. Furthermore, we find thatinterevent times of flip-flop are distributed according to the exponential
distribution with cutoff. The rate of flip-flop depends on temperature.
3PT156

Low-frequency dynamics of phospholipid bilayers studied byterahertz time-domain spectroscopy
Tomoyo Andachi1, Naoki Yamamoto2, Atsuo Tamura1, Keisuke Tominaga1,2(1Grad. Sch. Sci., Univ. Kobe, 2Molecular Photoscience Reserch Center, Univ.
Kobe)
Phospholipid is a main component of cell, which forms bilayers by selfassembling. Lipid bilayers, which are usually hydrated, continuously fluctuate
due to thermal activation in the terahertz (THz; 1 THz ~ 33 cm-1) frequency
region. These fluctuations originate from delocalized vibrational motions of− S167 −lipid molecules and/or hydrogen-bond making and breaking dynamics withwater molecules. Therefore, terahertz time-domain spectroscopy (THz-TDS) isa powerful tool to investigate lipid molecule dynamics. In this study, we focus
on dependence of the low-frequency dynamics of lipid bilayers on hydration,
temperature, and their structure by using THz-TDS. As a sample, we selected 1,
2-Dimyristoyl-sn-glycero-3-phosphoglycerol (DMPG), which is known to form
several different structures like crystalline or gel phase states. We measured thetemperature dependence of the low-frequency spectra of the DMPG under
different hydration degrees and structures. The absorption coefficients of eachsample were obtained in the frequency region from 10 cm-1 to 50 cm-1. We
discuss about the dynamics of DMPG from the changes of the absorption
coefficients.
3PT157

表面張力レプリカ交換法による脂質二重膜の構造サンプリング

Surface tension replica exchange molecular dynamics method
for an efficient sampling of lipid bilayer structures
Takaharu Mori1, Yuji Sugita1,2,3 (1RIKEN Quantitative Biology Center, 2RIKEN
Advanced Institute for Computational Science, 3RIKEN Advanced Science
Institute)
Generalized ensemble algorithm is one of the efficient sampling methods forbiomolecular systems. Recently, various replica exchange molecular dynamics
methods (REMD) in different parameter spaces have been developed. However,simulations for membranes or protein-membrane systems are still difficult withgeneralized ensemble algorithms, because the membrane can easily collapse insuch enhanced sampling methods like temperature REMD. To overcome thisproblem, we propose a new replica exchange molecular dynamics method,where the surface tension of the system in an NPγT ensemble is exchanged (γREMD). Exchanging the surface tension can moderately induce the deformationof lipid bilayers. We introduced γ REMD in our laboratory-developed MD
program (GENESIS), and it was applied to a fully hydrated 1,2-di-palmitoylphosphatidylcholine (DPPC) lipid bilayers. We found that the area per lipid andmembrane thickness can change significantly in the γ REMD simulation,compared to the single NPγT simulation. We believe that our method is usefulfor investigating structural properties of lipid bilayers, conformational changesof membrane proteins in membranes, and protein-lipid interactions.
3PT158

膜厚変化とゲル、アミノ酸、水素化アモルファスシリコンを用いたイオン伝導整流素子の特性

Film thickness and property of ion conductive rectification
element using amino acids, gel and hydrogenated amorphoussiliconTakahiko Sano1, Hideaki Sugawara1, Takaaki Ichikawa1, Yuuki Hiramitsu1,
Hiroshi Masumoto2, Takashi Goto3, Yutaka Tsujiuchi1 (1Department ofMaterial Science and Engineering, Akita University, 2Center forInterdisciplinary Research, Tohoku University, 3Institute for Materials
Research, Tohoku University)
This report mainly focuses an electro-potential-current property correlated withfilm thickness on ion conductive rectification element using amino acids, geland hydrogenated amorphous silicon. In general, ionic conduction, in solution
as well as in intermediate states between a liquid and a solid, such as gels, is aphenomenon key to inter-conversion between light energy, chemical energy,
and electric energy. As such, this phenomenon is essential for devices that store,
generate, and convert electric energy, including batteries, photovoltaics,
capacitors, and actuators. This report secondly focuses on ionic conduction in
nontoxic materials to discuss the creation of elements coated with multi-layer
functional thin-films using a combination of different technologies, such as therectification property of ionic conduction, photo-induction control, and the useof wavelength conversion materials, and the potential application of thesematerials to biological tissue. Ion conductive behaviors of neutral, acidic, orbasic amino acid dispersed in agarose gel are characterized. We report an
attempt of measuring a photo-controlled effect of a-Si:H film on ion conducting
behavior of bio-molecules in detail, comparing several thickness of a-Si:H film.
3PT159BLM を介したイオン性蛍光プローブの膜透過性―電流と蛍光に基づいた動的解析―

Permeability of an ionic fluorescent probe through a BLM dynamic analysis based on an electric current and afluorescent change-

Yasuhiro Naka, Yumi Yoshida, Megumi Shimazaki, Kazunari Sakai, Kohji
Maeda (Department of Chemistry and Materials Technology, Kyoto Institute ofTechnology)
Transport of an ionic species through a bilayer lipid membrane, BLM, isinfluenced not only by its hydrophobicity but also by a coexisted counter ion,and the transport mechanism has been considered to be complicated. Recently,
new opinion for the BLM transport of an ion has been reported by Shirai et al.
[1, 2]. In the opinion, a BLM was recognized as like a thin liquid membranewith two aqueous/membrane interfaces. The transferring ion distributes to a
BLM with coexisted counter ion and transports across the BLM keeping
electroneutrality in the BLM. Electrochemical results were given as
experimental evidences for the transport mechanism, but not enough forsupporting the cotransport mechanism which is not detected by electrochemicalmeasurement. In the present work, an ionic fluorescent probe was employed as
the transferring ion through a BLM. Transport of the probe was detected not
only as the ion transfer current but also as fluorescent change near to the BLM
induced by the ion transport. Comparing the transport current with thefluorescent change, whether the transferring species is ionic or neutral was
estimated. According to the observation, the mechanism of ion transport througha BLM was discussed. [1] O. Shirai, S. Kihara, Y. Yoshida, M. Matsui: J.
Electroanal. Chem., 389, 61-70 (1995) [2] O. Shirai, Y. Yoshida, M. Matsui, K.
Maeda, S. Kihara: Bull. Chem. Soc. Jpn., 69, 3151-3162 (1996)
3PT160ABC ト ラ ン ス ポ ー タ ー の ヌ ク レ オ チ ド 結 合 ド メ イ ン に 対 す る
ATP 結合自由エネルギー計算

Calculation of the binding free energy of ATP to the nucleotide
binding domain of an ABC transporter
Yusuke Kaneta, Shuntaro Chiba, Tadaomi Furuta, Minoru Sakurai (Center for
Biol. Res. & Inform., Tokyo Tech)
ABC (ATP-binding Cassette) transporters exist in almost all kinds of living
things and use ATP as an energy source to transport a wide range of substrates
across the membrane. ABC transporters have the common 3D structural
architecture: the whole molecule is divided into two domains, that is,Transmembrane Domain (TMD) and Nucleotide-Binding Domain (NBD). NBD
has two highly conserved ATP binding sites (Walker A&B and LAGGQ). It has
been proposed that binding of ATPs to these sites induces the dimerization of
two NBDs, eventually leading to a large structural change from the inward- to
outward-facing conformation of TMD.
Our study is to elucidate this structural change mechanism from energetic
aspects. In this study, as a first step, we evaluated the free energy gain obtained
from binding of the ATP to NBD. We examined this for NBDs in the maltose
transporter (MalFGK2) from Escherichia coli. For this purpose, molecular
dynamics (MD) simulations were performed starting from the crystal structureof the NBD dimer and subsequently the above free energy was calculated
according to the MM/PBSA method.
We first evaluated the binding energy of one ATP molecule to an NBD
monomer. The resultant values were -37.6 and 4.1 kcal/mol when ATP binds tothe Walker A&B motif or the LSGGQ one, respectively. Next, the NBD
dimerization energy was evaluated to be -5.91 kcal/mol. We are now calculating
the binding free energies of ADP and inorganic phosphate (Pi) to an NBD toelucidate the role of ATP hydrolysis in the substrate transportation process.
3PT161

パターン化モデル生体膜へのロドプシンの再構成

Reconstitution of rhodopsin into micropatterned model
biological membrane
Keisuke Okada1, Kenichi Morigaki1,2, Fumio Hayashi3 (1Grad. Sch. Agr., Univ.
Kobe, 2Res. Cent. Env. Genomi., Univ. Kobe, 3Grad. Sch. Sci., Univ. Kobe)
Rhodopsin (Rh) is a photoreceptor that forms a complex with transducin (Gt)upon activation by light. It has been reported that the affinity of Rh*-Gt complex
to lipid rafts (raftophilicity) was higher than that of Rh (Hayashi et al. ARVO
meeting (2011)). We are developing a methodology to evaluate theraftophilicity quantitatively with a supported phospholipid bilayer (SPB).
Micropatterned SPBs composed of photo-polymerized lipid bilayer and fluid
lipid bilayer. By changing the compositions of polymeric and fluid bilayers,liquid ordered (lo) domains and liquid disordered (ld) domains in the fluid
bilayer were separated into a pre-determined pattern. To measure thelocalization of Rh and Rh*-Gt in lo regions, we incorporated Rh into SPB by
rapid dilution of Rh/ octylglucoside micelles. We confirmed single Rh
molecules in SPB by TIRF-microscopy. Furthermore, we observed binding offluorescently labeled α-subunit of Gt (Gαt) upon activation of Rh and changes in− S168 −the mobility of Rh*-Gt upon subsequent addition of GTP. Hence, we concluded
that Rh and Gt in SPB were active. At the same time, we also observed some
immobile Rh molecules in SPB, which could interfere with the analysis of
raftophilicity. We discuss on the reconstitution strategies of Rh to establish themeans to measure the raftophilicity in micropatterned lo domains.
3PT162カリウムチャネル KcsA の開閉によるリン脂質の flip-flop 誘起能の評価

Induction of phospholipid flip-flop by KcsA channel gating
Hiroyuki Nakao1, Masaki Wakabayashi1, Yasushi Ishihama1, Minoru Nakano2(1Grad. Sch. Pharm. Sci., Kyoto. Univ, 2Grad. Sch. Med. Pharm. Sci., Fac.
Pharm. Sci., Univ. Toyama)
Regulation of phospholipid flip-flop is important for homeostasis of biologicalmembranes. Flip-flop of phospholipids in the endoplasmic reticulum (ER) isvery fast, compared to that in other membranes. However the flippases thatgovern the fast flip-flop in the ER have not been identified, and the mechanism
by which the flip-flop is induced is still unknown. It has been reported that
KcsA induces flip-flop of phospholipids. KcsA is a pH-dependent potassiumchannel, activated by acidic pH. Conformational change of transmembraneregion during channel gating was recently observed at a single channel level.
We therefore hypothesized that the fluctuation of transmembrane region
influences flip-flop of phospholipids.
Dithionite reduction assay was used to measure flip of C6 -NBDphosphatidylcholine (C6 -NBD-PC) in proteoliposomes, composed of PC andKcsA. KcsA was found to induce flip of phospholipids at acidic pH, while lessaffected at neutral pH. To confirm whether the fluctuation of transmembraneregion of KcsA induce flip, we measured flip of C6-NBD-PC at acidic pH in thepresence of tetrabutylammonium, which locks KcsA in the open state. The
results showed that KcsA mediates flip of phospholipids even in the openlocked state. These results suggest that KcsA enhances flip of phospholipids notby the fluctuation of transmembrane region, but by the open structure or thechange of charged state of amino acids in transmembrane region.
3PT163人工細胞膜アレイを用いた全自動イオンチャネル計測システム

Automated Drug Screening System for Ion Channel Proteinsusing Artificial Cell Membranes
Ryuji Kawano1, Yotaro Tsuji1,4, Koki Kamiya1, Toshihisa Osaki1, Minako
Hirano3,5, Toru Ide3,5, Norihisa Miki1,4, Shoji Takeuchi1,2 (1Kanagawa Academyof Science and Technology (KAST), 2IIS, Univ. of Tokyo, 3Riken, 4Keio
Univ., 5The Graduate School for the Creation of New Photonics Industries)
This paper describes an automated drug screening system for Ca2+-activated K+ channel (BK channel). BK channels are pharmacological targets for thetreatment of stroke. We have been developing multiple lipid bilayer membranes(BLMs) system for reconstituting the membrane proteins. In this study, we aresucceeded in simultaneous reconstitution of BK channels in the BLMs arrayusing an injection robot, and the channel inhibition behavior was examinedusing K+ channel blocker. Our system can be applied to a high-throughput
screening (HTS) system for drug discovery. Membrane proteins account for
approximately 25% of the polypeptides encoded within the human genome. Of
these, ion channels are an important class of proteins that mediate ion flow
across cellular membranes and which, among other things, are responsible forthe excitability of neurons. In the drug discovery, the high-throughput
screenings of the ion channels are highly desirable. We have been developing
the lipid bilayer membrane array for the HTS of the ion channels using MEMS
technology. BK channels are essential for the regulation of several key
physiological processes including smooth muscle tone and neuronal excitability.
We have therefore attempted to reconstitute BK channel within the artificial
lipid bilayers. In this study, BK channels were automatically reconstituted in the
BLMs array device by using an injection robot, and also the channel activity
was examined using its inhibitor.
3PT164

KcsA カリウムチャネル活性に影響を及ぼすリン脂質作用部位の同定

Identification of the interaction site with the effective
phospholipids for the KcsA potassium channel activity
Masayuki Iwamoto, Shigetoshi Oiki (Dept. Mol. Physiol. Biophys., Univ.
Fukui Facult. Med. Sci.)
Membrane lipids are known to affect the function and activity of ion channels.
Specific lipid molecules are indispensable for some ion channels to become
active. How the specific lipids regulate the ion channel activity is essential tounderstand the structure-function relationship of ion channels in the membrane
environment. For the KcsA potassium channel, anionic phospholipids such as
phosphatidylglycerol (PG) have been thought to play an essential role in its
activity as the KcsA channel exhibits quite low open probability in the absenceof these lipids. Our previous study revealed by means of single-channel current
recordings in the asymmetric lipid bilayer that the KcsA channel requires PG
molecules in the inner leaflet to exhibit high open probability. In this study wetried to identify the interaction site with PG within the KcsA molecule forfurther understanding of lipid-mediated regulation of the KcsA channel activity.
Potential positive charges such as Arg, Lys and His were expected to be
candidates for the interaction site with PG. We found that mutation of several
sites out of 24 potential positive charges in the monomeric KcsA molecule weresensitive to the PG effect on the KcsA channel activity. Mechanism underlie
PG-mediated regulation of the KcsA channel opening will be discussed.
3PT165

Kv1.2 チャネルでの選択的イオン透過

Selective Ion Permeation though the Kv1.2 Channel
Takashi Sumikama1, Shinji Saito2, Shigetoshi Oiki1 (1University of
Fukui, 2Institute for Molecular Science)
Ion selectivity is a crucial function for ion channels. So far, it has been
considered the K+ channels discriminate for K+ over Na+ more than a thousandfold due to the destabilized binding of Na+ at the selectivity filter. However,
several recent researches showed that Na+ can enter into the selectivity filter
and permeate: the x-ray crystallographic structure of the K+ channel with Na+
was resolved, where Na+ ions form six-coordination structure in the selectivity
filter. Its structure was then supported by theoretical calculations. The electrophysiological measurement also suggested that a certain amount of Na+ ionscan permeate through the K+ channel by punch-through mechanism. Thus, there
still exists controversial arguments concerning the ion selectivity. Here weobserved the efflux of ion in the mixed ion solution, 200 mM KCl and 200 mM
NaCl, through the Kv1.2 channel by the molecular dynamics simulation. It isfound that K+ ions permeate at rates at about five times higher than Na+ ions,which is approximately the same ratio the punch-through mechanism reported.
We also found that once ions, regardless of ion species, enter into the central
cavity, they cannot return to the cytoplasmic side solution. This means theselectivity is caused by the free energy barrier at the entrance of the channel.
Therefore, the selectivity is a part of ion permeation issues rather than thestability of ions in the channel; kinetics, instead of thermodynamics, determines
the ion selectivity.
3PT166

Controlling an ion channel’s voltage sensing domain without
voltage
Morten Bertz, Kazuhiko Kinosita (Waseda University, Dpt. of Physics)
Voltage gating - the opening and closing of ion channels on response to changes
in membrane potential - is fundamental to signal transduction in livingorganisms. Voltage gating is achieved by four voltage sensing domains (VSDs)
that surround the ion-conducting pore of the channel protein. In these VSDs,
conserved positively charged residues located in the S4 transmembrane helix
move according to the transmembrane electrical field, and the resulting
conformational change is transmitted to the channel pore. Up to date, however,direct structural information on the deactivated, closed state of a channel has
remained elusive. Consequently, the extent and direction of the VSD movement
remain controversial, with both small translations and large-scale movements
reported in the literature depending on the technique used in the investigation.
Here, we attempt the manipulation of voltage sensor conformation using
engineered probes as a substitute for voltage to shed light on the transitions
involved in voltage gating.
3PT167

ガラス針による人工平面膜へのイオンチャネルの再構成

Reconstitution of ion channel into lipid bilayer using glass
needleDaichi Okuno1, Minako Hirano2, Yukiko Onishi1, Toshio Yanagida1, Toru
Ide2 (1Quantitative Biology Center, Riken, 2The Graduate School for theCreation of New Photonics Industries)
Ion channels are membrane proteins that regulate cell functions by controlling
ion flow through cell membrane. To elucidate the molecule mechanism of theregulation for the ion flow, it is necessary to investigate the relationship between
the conformation and function and interaction with some factors. To aim thistask, ion channel is kept suppressing lateral diffusion in the membrane for− S169 −detecting conformational change, ligand desorption and current at the same timeand it is preferable to fix ion channel to solid support in the lipid bilayer. We
have previously reported that the reconstitution of ion channel fixed onto thesolid support such as agarose bead and the tip of AFM cantilever into lipid
bilayer. In this study, we conducted reconstitution of ion channel into lipid
bilayer using glass needle as a solid support. The tip of glass needle (<100 nm)
was modified with Ni-NTA and ion channel (KcsA) was fixed on it throughinteraction between His-tag and Ni-NTA. It was also available for reconstitutionof ion channel into lipid bilayer as well as AFM cantilever. There is possibility
that simultaneous optical and electrical recording system for single ion channel
may be built without complicated optics when using optical fiber instead ofglass needle.
3PT168

生物物理学的解析を目指した、哺乳培養細胞を用いた哺乳類カリウ
ムイオンチャネルの大量発現の試み

Trials of large amount expression of mammalian potassium ion
channels using cultured mammalian cells for biophysicalanalysesHisao Tsukaoto1, Koichi Nakajo2, Yoshihiro Kubo2, Yuji Furutani1 (1Institutefor Molecular Science, 2National Institute for Physiological Sciences)
Ion channels are very important membrane-spanning proteins, which regulate
permeability of ions between outside and inside of living cells. Thus, it isimportant to understand how ion channels function using various biophysicaltechniques. For example, infra-red (IR) spectroscopy on ion channels is
expected to reveal how the channels discriminate different ions and undergo
conformational changes in the channel functions. In fact, IR spectroscopy isapplicable to analyses of a bacterial potassium channel KcsA, and the ionprotein interactions at the selectivity filter (1). It is natural to suppose that you
could do similar studies on other ion channels, in particular mammalian ones.
However, there is a problem on studies of mammalian ion channels. IR
spectroscopy requires only several μg of purified proteins for the measurement,
but in general, even such amount of mammalian ion channels cannot be
expressed in E. coli. In this study, we tried to prepare sufficient amount of
mammalian potassium channels using mammalian cultured cells. We made aseries of constructs for expression of GFP-tagged ion channels. Based on
profiles of fluorescence-detected size-exclusion chromatography (FSEC), we
screened various potassium channels derived from human and mouse, andselected channels that can be highly expressed in the cultured cells. Currently,
we are trying to purify large amount of the screened channel proteins.
(1) Furutani et al., submitted., please see his presentation in this meeting.
3PT169テトラエーテル型人工リン脂質とアポリポタンパク質からの膜ディ
スク形成

Membrane disc formation from tetraether-type artificial
phospholipids and apolipoproteins
Teruhiko Baba1, Toshiyuki Takagi1, Toshiyuki Kanamori1, Daisuke Handa2,
Tatsuya Oka3, Hiroyuki Saito2,3 (1Res. Center Stem Cell Eng., AIST, 2Fac.
Pharm. Sci., Univ. Tokushima, 3HBS, Univ. Tokushima Grad. Sch.)
Plasma apolipoprotein A-I (apoA-I) molecules form a membrane disc in which astraight-chained phosphatidylcholine (PC) bilayer is accommodated. In order toapply this unique structure to membrane-protein reconstitution matrices, we
tried to prepare membrane discs by employing a tetraether-type artificial PC(PTEPC), which forms stable monolayer membranes, and obtained the optimal
molar ratio of lipid to apoA-I yielding relatively homogeneous membrane discs.
The size distribution of membrane discs was examined by GPC and TEM
techniques, and the average diameter of PTEPC membrane discs was 10-12 nm.
The helices content of apoA-I in PTEPC membrane discs was comparable tothose in bilayer-forming PC (e.g., egg yolk PC and diphytanoyl-PC) membranediscs. By evaluating membrane disc stability from the viewpoint of guanidineinduced denaturation of apoA-I, PTEPC was found to form more stable
membrane discs than straight-chained egg yolk PC. We will also discuss theusefulness of PTEPC membrane discs for membrane-protein reconstitution
matrices.
3PT170

AFM Probing Opioid Signalosome on Neuroblastoma

Lara Villaruz1, Junhua Li1, Catherine Tardin2, Daisuke Mizuno1 (1Kyushu
Universiy, 2IPBS/CNRS)
Opioid receptors responsible for sensing pain signals are the major target of pain
treatment. The opioid and other proteins confined in domains could play the roleof signalosome that permits the encounter of various proteins involved in thefast response of receptor signaling to extracellular activation. Thus it is likelythat the receptor function would coincide with dynamic physical properties ofthe signalosome. Geared towards this motivation, we aim to detect and quantify
the interaction of the AFM probe modified with antiT7 and the T7-tagged
receptor on SHSY5Y neuroblastoma. Forces between the tip and receptor weremonitored by indenting and retracting the tip at different points. Molecular
recognitions were evident from the attractive signals of a unique shape during
the retract phase which increase from stretching the linker after binding until thebond breaks. In a single event, several bonds may be formed so that theunbinding force is a multiple of the elementary bond strength. To obtain a good
statistical value in estimating the fundamental force, the force volume mode wasused to conveniently record successive force curves in one file. The data
analyzed with fuzzy logic for the event detection showed that single unbinding
force is 123 pN. Since this is in the range consistent with those reported inliterature, this indicates the potential of the sensor for localizing receptor sites infuture studies on the effect of agonist stimulation on the receptor distribution
and its association to changes in biophysical properties.
3PT171Analysis of protein translocation in a reconstituted Dictyostelium cell membrane on a solid substrate

Kei Takahashi1, Nao Shimada1, Taro Toyota1,2, Satoshi Sawai1,2,3 (1Grad. Sch.
Arts Sci.,Univ. Tokyo, 2Res. Center as Complex Sys. Bio., Univ. Tokyo, 3PRESTO,
Japan Science and Technology Agency)
Rigorous analysis of spatio-temporal dynamics of cell motion, membrane
deformation and their underlying biochemical reactions remain to be a challenge
due to their complexity. From yeast to human, localized signaling at the plasma
membrane such as activation of cdc42, rac and phosphatidylinositols signaling
breaks the intracellular symmetry and plays a crucial role in generating
polarized membrane extension and retraction. In an attempt to clarify andevaluate some of the critical dynamics involved in these processes, wedeveloped a two-dimensional reconstituted membrane on a glass-substrate using
lipid extracted from Dictyostelium discoideum. We have tested whether thesolid-supported membrane in a microchamber containing extracted cytosol
provides an appropriate microenvironment for localized signaling by observing
translocation of PH-domain protein and PTEN which were tagged with RFP and
GFP respectively. Signal intensities were analyzed by a confocal laser scanning
fluorescence microscopy. We found that both the PH-domain protein and PTEN
attached to the membrane within 20 seconds after application of the cytosol
containing these proteins. Localization of these proteins appeared as transient
reciprocal patches of micrometer order indicating that there was heterogeneityof phosphatidylinositols in our reconstituted membrane. We will discuss
implications of our present observations to the in vivo dynamics of cell signaling
during cell movement and chemotaxis of Dictyostelium.
3PT172

ラフト親和性 GPI アンカー型分子プリオンタンパク質の神経細胞膜でのダイナミクス

Dynamics of normal prion protein, a raft-associated GPIanchored molecule, in the live neuronal plasma membrane
Yuri L. Nemoto1, Chieko Nakada1, Hiroko Hijikata1, Takahiro K. Fujiwara1,
Rinshi S. Kasai1, Yoshiro Ishikawa1, Akihiro C. E. Shibata1, Ankita Chadda1,
Roger J. Morris2, Akihiro Kusumi1 (1Institute for integrated Cell-Material
Sciences (WPI-iCeMS), Institute for Frontier Medical Sciences, Kyoto
University, 2Wolfson Centre for Age Related Disease, King's College London)
The prion protein (PrP) is a glycosylphosphatidylinositol (GPI) -anchoredprotein located in the outer leaflet of the plasma membrane (PM) of neuronalcells in the brain. Misfolded forms of PrP cause prion diseases, such as
Creutzfeldt-Jakob disease, by inducing abnormal folding of other normal PrP
molecules and the subsequent clustering of the misfolded PrP molecules, which
creates brain damage. The behaviors of these normal PrP in the PM and theirfunctions remain unknown. Since GPI-anchored proteins are generally believed
to be raft-associated, one of the general hypotheses for the propagation of
misfolding involves concentration of both misfolded and normal PrP in raftdomains, which enhances their close and prolonged interactions, somehow
generating more misfolded molecules. Here, we investigated PrP dynamics inthe neuronal PM. Using single fluorescent-molecule imaging, we found that: (1)the diffusion coefficient of PrP (0.22 square microns/s) was 2.4-times smaller
than that of a control GPI-anchored protein Thy1 (0.53 square microns/s) in 1week-old rat neurons, (2) this difference is unchanged in older neurons (2week-old) or in CHO-K1 cells, and (3) immobilized fractions of these molecules
represent 29% and 19% for PrP and Thy1, respectively. In addition, live PALM

− S170 −observation revealed that some areas in the PM excluded PrP or Thy1 in theneuronal PM. These results suggest that there are two different-types of raftdomains, dynamically concentrating PrP or Thy1 in the PM of non-stimulated
cells.
3PT173

マガイニン 2 が誘起するポア形成の初期過程

The Initial Stage of Magainin 2-Induced Pore Formation in
Lipid Membranes
Victor Levadny1,2, Tomoki Takahashi3, Jahangir Md. Alam1, Masahito
Yamazaki1,3 (1Int. Biosci., Grad. Sch. Sci. Tech., Shizuoka Univ., 2Rus. Acad.
Sci., 3Dept. Phys., Fac. Sci., Shizuoka Univ)
Antimicrobial peptide magainin 2 (M2) forms pores in lipid membranes to
induce leakage of internal contents of cells, which is a main cause of its
bactericidal activity. However, the mechanism of its pore formation remains
unclear. In this report, to reveal the mechanism of the initial stage of the M2induced pore formation in lipid membranes, first we examined the area changeof lipid membranes during the interaction of GUVs of dioleoylphosphatidylglycerol (DOPG) and dioleoylphosphatidylcholine (DOPC)
mixture with M2 solution using the micropipet aspiration method. The areas of
40%DOPG/60%DOPG- and 30%DOPG/70%DOPC-GUVs increased with anincrease in M2 conc. in buffer. After the conversion from the M2 conc. in buffer
to the M2 surface conc. in GUVs, we found that the fractional changes of thearea of these GUVs were almost proportional to the M2 surface conc. Next wedeveloped a simple theoretical model that describes the variations in the freeenergy of the system upon the binding of M2 into the outer monolayer of GUVs.
Assuming that M2 molecules don’t transfer into the inner monolayer of GUVs
before the pore formation, the inner monolayer is stretched to produce tension,
resulting in production of a local rarefaction in the lateral density of the innermonolayer, i.e., half-pores. When a half-pore attains a critical size, it transforms
into a transmembrane pore. The lifetimes of intact GUVs were determined using
the mean first passage time concept, which agreed with experimental data [1].
[1] J. Phys. Chem. B. 113, 4846, 2009
3PT174

外力が誘起する脂質膜の張力によるポア形成の速度定数

Rate constants of constant tension-induced pore formation insingle GUVs
Taka-aki Tsuboi1, Victor Levadny2,3, Masahito Yamazaki1,3 (1Dept. Phys. Fac.
Sci. Shizuoka Univ., 2Rus. Acad. Sci., 3Grad. Sch. Sci. Tec. Shizuoka Univ.)
When external forces are applied to cells or liposomes, tension is induced inbiomembranes or lipid membranes. It is considered that above a critical value ofthe intensity of tension a pore formation occurs to cause rupture of cells or
liposomes. On the other hand, antimicrobial peptide, magainin 2 (M2), induces
pore formation in single giant unilamellar vesicles (GUVs) at and above a
critical surface concentration of M2 [1]. To compare the tension-induced pore
formation with the M2-induced pore formation, we investigated the constanttension-induced pore formation in GUVs of dioleoylphosphatidylglycerol(DOPG) and dioleoylphosphatidylcholine (DOPC) mixture using the micropipet
aspiration method.
First we applied aspiration pressure on a 40%DOPG/60%DOPC-GUV using a
micropipet to produce a constant tension of 5.5 mN/m and kept this state for along time. Suddenly a pore was formed to cause rupture of the GUV. When we
made same experiments using many single GUVs, we found that the pore
formation occurred stochastically. The time course of the fraction of intact GUV
without rupture, Pintact(t), was well fitted by a single exponential decay function.
By this fitting, we obtained the rate constant of the pore formation, kp, which
was 6.3×10-3 s-1. We made the same kinds of experiments under different
constant tensions, and found that kp increased with tension. We analyzed theseresults theoretically, and compared them with the M2-induced pore formation[1,2].
[1]J. Phys. Chem. B, 113, 4846, 2009, [2]ibid, 114, 12018, 2010
3PT175

ミトコンドリアの密集が ATP 合成に及ぼす影響

Effects of the crowding of mitochondria on their ATP
production
Daiki Yoshimatsu, Yoshihiro Ohta (Dept. of Biotech. and Lifesci.,Tokyo Univ.
of Agric. and Tech.)
Mitochondria are organelles which produce ATP through the transport of H+
across the membrane. They are considered to locate at the regions where ATP isrequired. Intracellular distribution of mitochondria is non-uniform. Since thecrowding of mitochondria will lead to the local increase in ATP concentrationand the lack of substrates around mitochondria, the mitochondrial crowding
might be unfavorable for ATP production. In the present study, to investigate
the effects of mitochondrial crowding on their ATP production, we adsorbed
isolated mitochondria on cover slips in different densities and measured ATP
production and membrane potentials of mitochondria. Membrane potential ofeach mitochondrion was measured with TMRE fluorescence. To investigate
ATP production rate of single mitochondria, ATP content in the supernatant andthe number of mitochondria on a cover slip were measured. Interestingly, when
we adsorbed mitochondria in the higher density, each mitochondrion produced
ATP at a higher rate. Oligomycin, an inhibitor of Fo F1 -ATPase, negated theeffects of mitochondrial crowding on their ATP production. In addition,
mitochondrial crowding induced the polarization of mitochondria. When
mitochondria were suspended in the buffer, we did not observe the dependenceof ATP production on the mitochondrial concentration. The mechanism bywhich mitochondrial crowding stimulates their ATP production on cover slips
will be discussed.
3PT176

Promotion of DNA delivery into nucleus dramaticallyenhances the transfection efficiency mediated by biosurfactant-containing liposomes
Yoshikazu Inoh1, Tadahide Furuno1, Naohide Hirashima2, Dai Kitamoto3,
Mamoru Nakanishi1 (1Sch. Pharm., Aichi Gakuin Univ., 2Grad. Sch. Pharm.
Sci., Nagoya City Univ., 3AIST)
Gene transfection using cationic liposomes is a promising approach for the safe
gene therapy. However, transfection efficiency by cationic liposomes has notbeen equal to those of by viral vectors yet. For successful gene transfection,
there are several barriers to be overcome, such as process that the foreign gene
passes through the plasma membrane, escapes from lysosomal degradation, andtranslocates into the nucleus. We previously showed that the biosurfactant
mannosylerythritol lipid-A (MEL-A) -containing cationic liposomes coulddeliver foreign gene into the target cells through the membrane fusion between
liposomes and plasma membrane. Using MEL-A-containing cationic liposomes,
the foreign gene was rapidly and efficiently delivered into the nucleus because it
was released into the cytosol directly and could escape from lysosomal
degradation. Here, we investigated the effect of pre-condensation of plasmid
DNA by a cationic polymer protamine on gene transfection. The precondensation of plasmid DNA significantly increased the transfection efficiency
by cationic liposomes. The promotion was more effective in cationic liposomeswith MEL-A than without MEL-A because MEL-A could accelerate greatly thedelivery of pre-condensed plasmid DNA into the nucleus. Our findings indicatethat combination of MEL-A-containing cationic liposomes and pre-condensed
DNA might be a useful system for gene delivery to non-dividing cells and forclinical application.
3PT177マスト細胞の開口放出様の膜融合におけるカルシウムの役割

Effects of Ca2+ on liposomal membrane fusion that mimics
mast cell exocytosis
Satoshi Tadokoro, Yumiko Nagai, Hiroki Sakiyama, Naohide Hirashima(Graduate School of Pharmaceutical Sciences, Nagoya City University)
Mast cells involved in allergic reactions. Antigen stimulation causes elevationof the intracellular Ca(2+) concentration, which triggers the exocytotic releaseof inflammatory mediators. Recent researches have revealed that SNARE(soluble N-ethylmaleimide-sensitive factor attachment protein receptor)
proteins such as syntaxin-3, -4, SNAP-23, and VAMP-8 are involved in mast
cells exocytosis. Although mast cells exocytosis is Ca(2+) dependent, the roleof Ca(2+) is not clear. Synaptotagmins are candidates of Ca(2+) sensor for
exocytosis. Since synaptotagmin 2 is the most abundant isoform ofsynaptotagmins expressed in mast cells, in this study, we focused on the role ofCa (2 +) in mast cell exocytosis by a liposome-based fusion assay. SNARE
proteins (SNAP-23, syntaxin-3, VAMP-8) and synaptotagmin 2 were expressedin E. coli and purified as GST-tagged or His-tagged fusion proteins. These
SNARE proteins were incorporated into liposomes by a detergent dialysis
method. Membrane fusion between liposomes was monitored by fluorescenceresonance energy transfer between fluorescent-labeled phospholipids. Ca2+ did
not show any significant effects on SNARE mediated membrane fusion by
itself, however, in the presence of synaptotagmin 2, Ca2+ induced membranefusion. This action of synaptotagmin 2 required phosphatidylserine as amembrane component. These results suggest that Ca2 + regulates SNARE
dependent membrane fusion in mast cells, and that this regulation is dependenton synaptotagmin 2 and phosphatidylserine.

− S171 −3PT178

脂質二重膜上における Def6 PH ドメインの高次構造の解析

Conformational properties of the Def6 PH domain located at
the lipid bilayer surface
Naomi Tokuda1, Michikazu Tanio2, Katsuyuki Nishimura2, Toshiyuki Kohno3,
Daisuke Yokogawa4, Takahisa Ikegami5, Satoru Tuzi1 (1Grad. Sch. Life Sci.,
Univ. Hyogo, 2Inst. Mol. Sci., 3Kitasato Univ., Sch. Med., 4Grad. Sch. Sci.,
Nagoya Univ., 5Inst. Prot. Res., Osaka Univ.)
Conformations of the pleckstrin homology (PH) domain of SWAP-70-like
adapter of T-cells (SLAT, also known as Def6) in solution and at the membranesurface were investigated by using circular dichroism (CD), fluorescence and
NMR spectroscopy. Previously, we reported that the switch-associated protein(SWAP) -70 PH domain undergoes conformational alteration including the αhelix to random coil transition at the lipid bilayer surface. The random coiltransition of the C-terminal α -helix was expected to allow water-exposed
conformation of nuclear localization signal (NLS) sequence located in the Cterminal α -helix, and consequently regulates intracellular relocalization of
SWAP-70. The Def6 PH domain shares high sequence homology with the
SWAP-70 PH domain, but lacks NLS in the C-terminal α-helix. The CD and
NMR spectra show that the β-sandwich core of the Def6 PH domain undergoes
similar conformational change to that of the SWAP-70 PH domain at the lipid
bilayer surface. On the contrary, the random coil transition of the C-terminal αhelix was not observed for the Def6 PH domain. The difference in theconformational properties of the C-terminal α-helices of the Def6 and SWAP70 PH domain would related to the absence and presence of the functional NLS
in the C-terminal α-helix.
3PT179ErbB シグナル伝達過程において Shc と Grb2 は個別に反応する

Independent response of Shc and Grb2 in the ErbB signaling
revealed by FCS/FCCS and single molecule analysis
Chang-Gi Pack, Yuko Saeki, Michio Hiroshima, Mariko Okada, Yasushi Sako
(RIKEN, ASI)
The signaling pathway of ErbB receptors including ErbB1 - 4 is involved indiverse physiological processes such as cell proliferation, differentiation,
apoptosis, and carcinogenesis. Systems-level analyses have been recently
carried out for this pathway, combining conventional biochemical methods
in vitro with computational simulation. However, information about
spatiotemporal dynamics and reaction-kinetics of related proteins are largely
lacking. It will be worthwhile to directly quantify the changes of localization,diffusion, and interaction of related proteins during the process in living cells.
We have established a live cell FCS/FCCS and single molecule (SM) analysis of
the proteins in the ErbB networks. Firstly, we focused on the behavior of
diffusions of Shc52 and Grb2 and direct interaction between them. Complex
formation between them is known to be necessary for activating Ras in the EGF(ErbB1 ligand) and HRG (ErbB3, 4 ligand) signaling process. Our analysis
using live cells indicates that diffusoinal responses of the two cofactors to ligand
stimulations were very different. Moreover, direct interaction between them was
hardly found in living cells, even though interactions with ErbBs were detected
for the factors. These results suggest that spatiotemporal responses of Shc52 andGrb2 with ErbB receptors are independently regulated in living cells, evendetails of the independent response have not been described until now. Our
study will pave the way toward a new insight for the true role of Shc52 in the
ErbB signaling.
3PT180

クロストークを利用したべん毛回転制御系の構築

Control of bacterial flagellar rotation via crosstalk from a noncognate histidine kinase to the response regulator CheY
Tohru Umemura1, Chiho Hara2, Yoshiyuki Sowa1,3, Ikuro Kawagishi1,2,3(1Res. Cen. Micro-Nano Tech., Hosei Univ, 2Dept. Frontier Biosci., Fac. Eng.,
Hosei Univ, 3Dept. Frontier Biosci., Fac. Biosci. Appl. Chem., Hosei Univ)
Escherichia coli has various two-component regulatory systems (TCS),
typically consisting of a histidine kinase (HK) and a response regulator (RR) for
responses to environmental changes. For instance, the chemotaxis of E. coli
employs, as a central processing unit, a TCS consisting of CheA (HK) and CheY
(RR). The activity of CheA is regulated but the four chemoreceptors in thecytoplasmic membrane in response to environmental stimuli. CheA
autophosphorylates at its histidine residue and then donates its phosphoryl group
to an aspartate residue of CheY. Binding of phospho-CheY to the flagellar
motor induces its clockwise (CW) rotation. An in vitro study showed that 22 outof 30 HKs of E. coli transfer their phosphoryl groups to non-cognate RRs(Yamamoto et al., 2005). In this study, we examined in vivo whether the activityof CheY can be regulated by non-cognate HK, BaeS. We first established a
CheY expression system under the control of the arabinose-controllable
promoter. In a strain (HCB437), in which all the chemotaxis genes are deleted,the more CheY is expressed, the more population of cells tumbled. We then coexpressed BaeS with CheY in the same strain. The tumbling frequency of cells
co-expressing BaeS and CheY was significantly higher than that of cells
expressing only CheY. Furthermore, the addition of indole enhanced tumbling
frequency of cells co-expressing BaeS and CheY, but not those expressing
CheY only. Thus, crosstalk between BaeS and CheY appears to occur in vivo,
providing a system to regulate flagellar rotation.
3PT181

T 細胞活性化におけるカルシウムシグナリングと細胞内構造変化の時空間定量解析

Spatial-temporal dynamics of calcium signaling and organelles
in T cell activation
Masahiro Shimozawa1,2, Yuma Ito1,2, Makio Tokunaga1,2, Kumiko SakataSogawa1,2 (1Grad. Sch. Biosci. Biotech., Tokyo Inst. Tech, 2RCAI, RIKEN)
When cells are subjected to the specific signals, Ca2+ is released from theendoplasmic reticulum (ER) resulting store-operated calcium entry (SOCE) via
calcium-release activated calcium (CRAC) channels in the plasma membrane.
These channels are opened by STIM1, ER-resident calcium sensor, after
depletion of intracellular Ca2+ stores. In T cell, the duration of this cytoplasmic
Ca2+ increases controls the efficiency and specificity of the transcription of
immune-related genes. STIM1 and Orai1, an essential pore subunit of the
CRAC channel, accumulate in the area of contact between T cell and antigen
presenting cells. Recently mitochondria are reported to translocate to the contact
area and allow sustained Ca2+ entry. As STIM1 resides in ER membrane, theaccumulation can cause the translocation of ER. Actually, we reported last yearthat ER redistributed in T cell activation. However, the relationship between
Ca2+ release from ER, SOCE, and local cytosolic Ca2+ elevations in T cell
activation is not known in detail. Here aiming to analyze the relationshipbetween ER redistribution and local Ca2+ elevations, we visualized CRAC
channels or ER redistribution using GFP fusion proteins. In addition, we
visualized local Ca2+ entry via CRAC channels or Ca2+ dynamics in ER using
genetically encoded calcium indicator. We will discuss spatial regulation of
Ca2+ signaling by structural changes in T cell.
3PT182低分子量 G タンパク質 Ras のフォトクロミック分子 PAM を用いた光制御

Photo-regulation of small G protein Ras using photochromic
molecule
Seigo Iwata1, Shinsaku Maruta2 (1Division of Bioinformatics, Graduate schoolof Engineering, Soka University, 2Department of Bioinformatics, Faculty ofEngineering, Soka University)
Ras is one of small G-proteins known as a molecular switch. Ras performs
intracellular signal transduction. Interestingly, the core nucleotide-binding motif
is considerably conserved in Ras and the ATP driven motor proteins: myosin
and kinesin. Therefore, it is believed that these bio-molecular machines sharecommon molecular mechanism utilizing nucleotide hydrolysis cycle.
Previously, we have incorporated photochromic molecules, 4-phenylazophenyl
maleimide (PAM), into the functional site of kinesin as a photo-switching
device and succeeded to regulate kinesin ATPase activities reversibly upon VIS
and UV light irradiation.
In this study, we performed basic study to control the Ras function reversibly by
light using photochromic molecules. First, to observe the nucleotide exchange
reaction of Ras GTPase, we synthesized a new fluorescent GTP analogue, NBDGTP that changes its fluorescent intensity along the formation of Ras-GTP, RasGDP-Pi and Ras-GDP states. Experimental data suggested that NBD-GTP bindto the nucleotide-free Ras with burst kinetics and subsequently the Ras-GTP
transit to Ras-GDP-Pi state slowly. Second, to photo-regulate the GTPase
activity of Ras, we designed the Ras mutants, which have an additional reactive
cysteine residue at the functional region. In the preliminary experiment, theGTPase activity of Ras mutants modified with PAM was reversibly alternatedupon ultra-violet and visible light irradiation.
3PT183
Gradient sensing limit in Dictyostelium discoiduem cell
Masaki Watabe, Kazunari Kaizu, Koichi Takahashi (RIKEN)− S172 −Dictyostelium discoideum cell can sense local gradient in cAMP concentration
across the cell body, and move with directional preference toward or away fromthe source of cAMP in a wide concentration range from 10 pM to 10 uM. In
shallow gradient less than 10^-3 nM/um, the cells showed no directional
response and exhibit a constant basal motility. In the steeper gradient, the cells
can spatially integrate information about 1-2% increase in cAMP concentrationon the cell surface, but their directional response is largely fluctuated in therange of 60 to 180 degrees. Here we simulate single particle-based cellular
dynamic model and quantitatively analyze the simulated data to determine
threshold response of sensing the gradient signals at an upper-stream part of thechemotactic signaling pathway.
3PT1843-クロロ安息香酸による 3-クロロ安息香酸分解細菌 Burkholderia
sp. NK8 外膜ポリン遺伝子の転写促進
Enhanced transcription of a porin gene in a 3-chlorobenzoate
degrading bacterium, Burkholderia sp. NK8, with 3-chlorobenzoate

Kimiko Yamamoto1,2, Ikuro Kawagishi2, Takeshi Fujii1 (1Natl. Inst. AgroEnviron. Sci., 2Dept. Frontier Biosci., Grad. Sci. Eng., Hosei Univ.)
Chlorobenzoates are the major intermediate products of aerobic catabolism ofpolychlorinated biphenyls (PCBs). In most PCB-degrading microorganisms,
however, chlorobenzoates are not further metabolized. Therefore, chlorobenzoate degradation is important for the complete degradation of PCBs.
Burkholderia sp. NK8 can utilize 3-chlorobenzoate (3CB) as a sole source ofcarbon and energy. The (chloro)benzoate dioxygenase of strain NK8 converts
3CB to 3- and 4-chlorocatechols, and then the resulting chlorocatechols may be
decomposed into β -ketoadipate by the products of the chlorocatechol
degradation genes tfdT-CDEF on the catabolic megaplasmid (pNK8) of strain
NK8. We demonstrated by capillary assay that cells of strain NK8 show
chemotaxis toward 3CB and the degradation compounds. We also found that thechemotaxis toward β-ketoadipate was lost in the absence of pNK8, and wasrecovered by the introduction of a plasmid carrying a porin gene, located next
to the tfdT-CDEF gene cluster of pNK8. By contrast, 3CB-taxis was
not complemented by the porin gene, named ompNK8. RT-PCR analyses
demonstrated that the transcription of the ompNK8 gene was enhanced whencells were grown in the presence of 3CB or β -ketoadipate. These findings
indicate that the outer membrane permeability and chemotaxis of strain NK8
may be regulated in response to the presence of 3CB or β-ketoadipate.
3PT185

ペリプラズム領域を欠失した走化性受容体 Tar の温度受容能

Thermosensing abilities of the mutant aspartate chemoreceptor Tar lacking the periplasmic domain
So-ichiro Nishiyama1,2, Masaaki Jinguji1, Ikuro Kawagishi1,2 (1Dept. Frontier
Biosci., Hosei Univ., 2Res. Cent. Micro-nano Tech., Hosei Univ.)
The aspartate chemoreceptor Tar of Escherichia coli can mediate
thermoresponses: attractant and repellent responses to increases and decreases
in temperature. When methylated upon adaptation to a chemoattractant, it
mediates thermoresponses with opposite polarity: repellent and attractant
responses to increases and decreases in temperature. Tar is an integral
membrane protein with two transmembrane (TM) helices as well as theperiplasmic sensing and the cytoplasmic signaling/adaptation domains. Our
previous studies suggest that the second TM region and the adaptation domain
are involved in temperature sensing, regulation of signal output or both.
However, whether there is a specific region of the chemoreceptor forthermosensing, is still unclear. Here we examined thermosensing ability of amutant version of Tar [ (44-183), named Taro], which lacks the entire
periplasmic domain [Pham & Parkinson (2011) J. Bacteirol. 193: 6597-6604].
Taro did not mediate any chemotactic or thermotactic response. However, Taroderivatives (S272L, T264I), when expressed in an otherwise receptor-less strain,
mediated repellent responses to glycerol, suggesting that they retained sufficient
activities to regulate the receptor-coupled histidine kinase CheA. Taro -S272L
mediated thermoresponses with opposite polarity. Taro-S272L showed elevatedmethylation without any stimuli, which can account for the inverted
thermoresponse. These results suggest that the periplasmic domain of thechemoreceptor is not essential for its function as a thermosensor.
3PT186

大腸菌アスパラギン酸走性受容体 Tar による忌避物質 Ni2+感知機
構の解析

Exploring the mechanism underlying sensing of the repellent
Ni2+ by the aspartate chemoreceptor Tar of Escherichia coli

Takaya Inui1, Hirotaka Tajima2, Yosiyuki Sowa3,4, Ikuro Kawagishi1,3,4(1Dept. Frontier Biosci., Grad. Sch. Eng., Hosei Univ., 2Dept. Micro-Nano
Systems Eng., Grad. Sch. Eng. Nagoya Univ., 3Dept. Frontier Biosci., Fac.
Biosci. Appl. Chem., Hosei Univ., 4Res. Cen. Micro-Nano Tech., Hosei Univ.)
In the chemotaxis of Escherichia coli, four methyl-accepting chemotaxis
proteins (Tsr, Tar, Trg and Tap) mediate attractant responses to some aminoacids, sugars and other organic compounds, and repellent responses to somemetal ions and organic compounds. In contrast to attractant sensing, repellent
sensing by the chemoreceptors is poorly understood in terms of ligand
recognition and structural changes triggered by ligand binding. In this study, we
focused on the ability of the aspartate chemoreceptor Tar to sense Ni2+ as a repellent. Taking advantage of the fact that the serine chemoreceptor Tsr, thoughclosely related to Tar, does not mediate a response to Ni2+, we tried to determine
the residues responsible for Ni2+ sensing by examining a series of chimeras
between Tar and Tsr. Responses of otherwise receptor-less cells expressing eachchimera to Ni2+ and amino acids, and methylation patterns of each chimera in
the presence or absence of Ni2+ and amino acids indicate that the region 157256 of Tar is critical for Ni2+ sensing.Iso-thermal calorimetry measurements
demonstrated direct binding of Ni2+ to the periplasmic domain of Tar. However,
we also found that a mutant version of Tar [(44-183), named Tar°], whichlacks the entire periplasmic domain [Pham & Parkinson (2011) J. Bacteirol.
193: 6597-6604] can mediate a response to Ni2+ and becomes less methylated in
the presence of Ni2+. To understand these apparently contradictory data, further
analyses are in progress.
3PT187走化性受容体 Tcp によるクエン酸と金属-クエン酸複合体の識別機
構の解析

Characterization of the bacterial chemoreceptor Tcp that
discriminates citrate and the citrate-metal ion complex as
distinct attractants
Tetsuya Shiroi1, Ikuro Kawagishi1,2,3, Hirotaka Tajima4 (1Dept of Frontier
Biosci., Grad. Sch. Eng., Hosei Univ., 2Res. Cen. Micro-Nano. Tech., Hosei
Univ., 3Dept of Frontier Biosci., Fac. Biosci. Appl. Chem., Hosei Univ., 4Dept.
Micro-Nano Systems Eng., Grad. Sch. Eng. Nagoya Univ.)
The chemoreceptor Tcp of Salmonella enterica mediates chemotactic responses
to citrate as well as a metal ion-citrate complex by recognizing them as different
attractants. Mutant Tcp receptors with substitutions for residue Asn67 fell intoeither of the four possible phenotypes in terms of their abilities to mediate
responses to citrate and metal-citrate (referred to as Cit and Mec, respectively):(i) Cit+ Mec+; (ii) Cit+ Mec−; (iii) Cit− Mec+; (iv) Cit− Mec−. This lead to a
proposal of two models for the mechanism of discrimination; (a) citrate binding
to one of the two binding pockets would alter the shape of the other binding
pocket, the induced conformation of which accommodates metal-citrate but notcitrate alone; (b) a metal ion binds to citrate that is already bound to one of thetwo binding sites to modify the conformation of the bound citrate, which would
then trigger a conformational change of the ligand-binding domain to generate
an attractant signal. In this study, we aimed at testing these models by
examining binding of citrate and metal ions to Tcp. Direct binding of citrate toTcp was suggested previously by our chemical modification assay. Here
wecarried out isothermal calorimetry measurements of a periplasmic fragmentof wild-type Tcp or its derivative with a cysteine residue at the dimer interface.
Neither fragment bound citreate or Mg2+. However, membrane vesicles of anotherwise receptor-less strain of Escherichia coli expressing full length Tcp
appeared to bind citrate. Further analyses are in progress.
3PT188

コレラ菌新規アミノ酸走性トランスデューサーの同定と機能解析

Identification and characterization of novel Vibrio cholerae
transducers for amino acid chemotaxis
Tetsuya Kawaguchi1, Kimiko Yamamoto1,2, So-ichiro Nishiyama3,4, Ikuro
Kawagishi1,3,4 (1Dept. Frontier Biosci., Grad. Sci. Eng., Hosei Univ., 2Natl.
Inst. Agro-Environ. Sci., 3Dept. Frontier Biosci., Fac. Biosci. Appl. Chem.,
Hosei Univ., 4Res. Cen. Micro-Nano Tech., Hosei Univ.)
The chemotaxis of Vibrio cholerae, the causative agent of cholera, has been
implicated in pathogenicity and survival in various environments. A classicalbiotype strain of V. cholerae has 44 homologs of methyl-accepting chemotaxis
protein (MCP), which we name MCP-like proteins (MLPs). Our previous
studies showed that Mlp24 and Mlp37, which have two periplasmic PAS-likedomains, are major chemoreceptors that mediate attractant responses to aminoacids. However, the mutant lacking mlp24 and mlp37 (mlp24 mlp37) showed
weak but significant responses to amino acids, suggesting the presence of− S173 −additional amino acid chemoreceptor(s). None of the other MLPs with tandem
PAS-like domains appeared to mediate amino acid taxis. We then turned ourattention to MLPs with a single PAS-like domain. The overproduction of two
members of this family, Mlp2 and Mlp3, in the mlp24 mlp37 strain enhanced
taxis to serine in capillary assays, suggesting that these MLPs mediate attractant
responses to serine. Interestingly, the expression of Mlp3, but not Mlp2,
enhanced taxis to alanine, cysteine, glycine and threonine, implying thedifference in specificity between these MLPs. However, we failed to detect
direct binding of serine to these MLPs in isothermal titration calorimetry
measurements with periplasmic fragments of Mlp2 and Mlp3, as well as
membrane vesicles of an otherwise receptor-less strain of Escherichia coli
expressing Mlp2 or Mlp3. This raises the possibility that these MLPs sense
serine via periplasmic serine-binding protein(s).
3PT189アミノ酸変異による苦味受容体 TAS2R16 機能の多様化

Functional diversity of bitter taste receptor TAS2R16 byamino acid substitution
Hiroo Imai1, Nami Suzuki1, Yoshiro Ishimaru2, Takanobu Sakurai2, Lijie
Yin3, Wenshi Pan3, Keiko Abe2, Takumi Misaka2, Hirohisa Hirai1 (1Primate
Research Institute, Kyoto University, 2Graduate School of Agricultural andLife Sciences, The University of Tokyo, 3School of Life Sciences, Peking
University)
In mammals, bitter taste is mediated by TAS2Rs, which belong to the large familyof seven transmembrane G protein-coupled receptors. Since TAS2Rs are directly
involved in the interaction between mammals and their dietary sources, it is likelythat these genes evolved to reflect species' specific diets during mammalianevolution (1, 2). Here, we investigated the sensitivities of TAS2R16s of various
primates by using a cultured cell expression system in combination withbehavioral tests (3). We found that the sensitivity of each primate species varied
according to the ligand. Especially, the sensitivity of TAS2R16 of Japanesemacaques to salicin, a bitter compound contained in the bark of Salicaceae(willow) plants, was much lower than that of human TAS2R16, which was
supported by behavioral tests. The replacement of amino acid residue at position
86 caused dynamic changes in the sensitivities of TAS2R16s. These results
suggest the possibility that bitter taste sensitivities evolved independently by
replacing specific amino acid residues of TAS2Rs in different primate species toadapt to accessible food items they use. The amino acid residues responsible forthe specificities of each ligand would be discussed in the presentation.
References
1. Suzuki N. et al., Primates 51, 285-289 (2010)
2. Sugawara T. et al., Mol. Biol. Evol. 28, 921-931 (2011)
3. Imai H. et al., Biol. Letters in press (2012)
3PT190弾性ネットワークモデルを利用したβ2アドレナリン受容体のアンサ
ンブルドッキング

Ensemble Docking Simulation for β2 Adrenergic Receptor
Using Elastic Network Models
Tomoyuki Iwamoto1, Hiroshi Wako2, Shigeru Endo3, Haruki Nakamura4(1Department of Biological Sciences, Graduate School of Science, and Facultyof Science Osaka University, 2School of Social Sciences, Waseda
University, 3Department of Physics, Faculty of Science, Kitasato
University, 4Institute for Protein Research, Osaka University)
G protein-coupled receptors (GPCRs) are membrane proteins involved in signaltransduction and participate in many diseases. Therefore, the receptors areimportant targets in the field of drug discovery.
Protein structural information is useful to understand their function and enable
us to develop new therapeutic molecules. However, these membrane proteins
are not easy to crystallize. And it sometimes happens that rigid-body protein
docking simulation for GPCR based on one ligand-receptor complex structure to
discovery new drugs lead to inefficient results with own flexible conformation.
In this study, GPCR’s conformational polymorphism into the virtual screeningof β2 adrenergic receptor (ADRB2) using elastic network model (ENM) andnormal mode analyses (NMA). First, we build the ENM of ADRB2 andperformed NMA. Next, we make virtual models based on ADRB2’s elastic
network normal mode analysis data. And we do docking simulation for thevirtual model group to make up for the above shortcoming.
According to the results, the screening efficiency of virtual model group is
improved by over 20% against that of the initial model.

3PT191

大腸菌走化性受容体 Tar システイン置換シリーズの化学架橋によるクラスター構造推定

Characterization of receptor clustering by cross-linking a series ofcysteine-substituted mutants of the aspartate chemoreceptor Tar
Tsuyoshi Watanabe1, Kosuke Jintori1, Hiroki Irieda2, Ikuro Kawagishi1,3,4(1Dept. Frontier Biosci., Grad. Sch. Eng., Hosei Univ., 2Div. Biol. Sci., Grad.
Sch. Sci., Nagoya Univ., 3Res. Cen. Micro-Nano. Tech., Hosei Univ., 4Dept.
Frontier Biosci., Fac. Biosci. Appl. Chem., Hosei Univ.)
In the chemotaxis of Escherichia coli, the bacterium can respond to a wide
range of attractant concentrations. The chemoreceptors, which exist as
homodimers regardless of ligand binding, form complexes with the adaptor
protein CheW and the histidine kinase CheA. The chemoreceptors form "trimersof dimers" with their cytoplasmic tips in contact with each other, which aresupposed to be organized into a hexagonal array. To account for high sensitivityof the chemotaxis system, it has been proposed that attractant binding to a
receptor dimer affects neighboring receptor dimers. Receptor clustering has also
been implicated in signal gain control by methylation of specific glutamate
residues that is responsible for adaptation to persisting stimuli. However,molecular mechanisms underlying such interactions between receptor dimers
remain to be elucidated. In this study, we carried out in vivo chemical crosslinking assay to probe changes in the receptor array by using divalent maleimide
cross-linkers and a series of site-directed mutant proteins of the aspartate
chemoreceptor Tar with single cysteine residues on the surface of the dimer.
The pattern of cross-linking was generally in agreement with our previous
results of disulfide-crosslinking assay without a chemical cross-linker.
Efficiencies of cross-linking were affected when an attractant or a repellent wasadded. We propose a plausible model of the receptor array, in which threetrimers of dimers are in contact with each other in the periplasmic spacer.
3PT192

コレラ菌走化性解析のためのスウォームアッセイ法の開発

Development of a system for swarm assays to evaluate
chemotaxis of Vibrio cholera
Ryosuke Iwazaki1, Nakagawa Tsubasa2, So-ichiro Nishiyama3, Ikuro
Kawagishi1,2,3,4 (1Dept. Frontier Biosci., Grad. Sci. Eng., Hosei Univ., 2Dept.
Frontier Biosci., Fac. Eng., Hosei Univ., 3Res. Cen. Micro-Nano Tech., Hosei
Univ., 4Dept. Frontier Biosci., Fac. Biosci., Appl. Chem., Hosei Univ.)
Vibrio cholerae, the causative agent of cholera, shows chemotaxis, which has
been implicated in pathogenicity and survival in various environments. A
classical biotype strain of V. cholerae has 44 homologs of methyl-accepting
chemotaxis protein (MCP), which are named MCP-like proteins (MLPs).
Among them, Mlp24 and Mlp37 are major chemoreceptors for amino acid taxis
in V. cholerae. A mutant strain lacking mlp24 and mlp37 ( mlp24  mlp37)
shows significantly weaker attractant responses to amino acids than the wildtype strain in capillary assays. However, in swarm assays, no detectabledifference is observed between the double mutant and the wild-type strains. The
swarm assay is not only easier than the capillary assay but also suited forscreening of mutants. In this study, we aimed at developing a system for swarm
assays to evaluate chemotaxis of V. cholerae. We deleted the mlp32 gene(previously named are-2), which encodes a homolog of the redox sensor Aer for
aerotaxis of Escherichia coli. The resulting triple mutant strain ( mlp24 
mlp37  mlp32) showed significantly slower spreading than the wild-type andthe double mutant strains in tryptone swarm plates. Introduction of a plasmidcarrying the mlp32 gene complemented the defect of the triple mutant strain inswarming. Furthermore, introduction of a plasmid carrying the mlp24 or mlp37
gene significantly enhanced swarming of the triple mutant strain, demonstratingthat this strain can be used as a plasmid host to evaluate amino acid taxis of V.
cholerae by the swarm assay.
3PT201

Seeking molecular and neural mechanisms of temperature
response and resistance in C. elegans
Tomoyo Ujisawa, Satoru Sonoda, Yukie Ohkubo, Hitomi Mizutani, Hiromi
Nagaya, Naoto Kuwahara, Akane Ohta, Atsushi Kuhara (Sch. Sci., Univ.
Konan)
Animals have adaptation mechanisms against environmental temperaturechanges. To understand the molecular mechanism of temperature response, we
are using simple model animal, nematode C. elegans. We recently found that C.
elegans shows cultivation temperature-dependent cold-resistance, which isregulated through neural pathway. After cultivation at 25 degree, wild-type− S174 −animals isolated from U.K. were destroyed by cold stimuli, 2℃ for 48 hours. By
contrast, most of wild-type animals can survive for 48 hours at 2℃, after
cultivation at 15 degree. Detailed temperature-changes experiments revealed
that the cold-resistance is established by temperature memory at adult stage.
We measured cold-resistance in various mutant animals defective insensoryneurons. Abnormal enhancement of cold-resistance is found in the
mutant animals defective in TAX-4 cGMP-gated channel, which is expressed in
10 pairs of sensoryneurons including thermo-sensoyneuron AFD and chemosensoryneurons. Developmental defect of AFD that is caused by mutation in
OTX transcriptional factor did not affect cold-resistance. These results suggestthat cold-resistance is controlled by sensory neurons excepting AFD, and that
sensoryneurons transmit inhibitory signaling for cold resistance. To determine
molecular pathway for cold resistance, we referred previous DNA microarray
analysis (Sugi et al., Nature neurosci., 2011). So far, at least 10 genes such asendonuclease, tubulin kinase and phosphatase PP1 are involved in coldresistance.
3PT202

Inhibitory effect of glutamate-stimulated astrocytes on action
potentials evoked by bradykinin in cultured dorsal root
ganglion neurons
Kazuo Suzuki, Kenji Tonaka, Minehisa Ono (Dept. Biomedical Engr. Tokai
Univ.)The patch-clamp and Ca2+ -imaging techniques have revealed that astrocytes
have dynamic properties including ion channel activity, neurotransmitter
release, such as adenosine triphosphate (ATP) and glutamate. Our previous
study demonstrated that free nerve endings of cultured neuron from dorsal root
ganglion (DRG) are activated by bradykinin (BK). We study whether ATP andglutamate can modulate the BK-response of the neuron cultured with theastrocyte in the mouse DRG, and enunciate the role of the astrocyte in the
nociceptive signal transmission using patch-clamp technique. The astrocytes
were identified using fluorescent antibody, anti-GFAP. The membrane potentialof the astrocyte was about -39 mV. The application of glutamic acid (GA) to thebath evoked the opening of two kinds of Cl- channel of the astrocyte membranewith unit conductance of about 380 pS and 35 pS in the cell-attached mode,
respectively. ATP application evoked the opening of two kinds of K+ channel ofthe astrocyte with unit conductance of about 60 pS and 29 pS, respectively.
Application of BK to the neuron evoked an action potential (spike). The BKapplication of concomitantly with ATP increased the frequency of BK-evoked
spike of the neuron. Stimulation of BK with GA inhibited the BK-evoked spike.
The application of furosemide, a potent cotransporter (Na+ -K+ -Cl-) inhibitor,
prior to the stimulation of BK with GA blocked the inhibition of the spike. It isthought that the inhibition of the spike is related to Cl- movement from theastrocyte.
3PT203

マウス脳ホモジネートにおける一酸化窒素（NO）の測定

Nitric oxide measurement in mice brain homogenate
Yoshiichiro Kitamura, Akira Hirai, Toshihiko Nishio, Yutaka Kirino(Kagawa Sch. Pharm. Sci., Tokushima Bunri Univ.)
It is known that nitric oxide (NO) plays important roles in a number ofphysiological processes over various animal species of vertebrate andinvertebrate, such as neurotransmission, vasodilation and immune response.
Especially in recent years, relationship between nitric oxide (NO) and aging has
been received a lot of attention. However down-regulation of NO signaling
during aging process is strongly suggested, NO dynamics in brain have not beenstudied in detail. Therefore, in this study, NO production in mice brain
homogenate was measured using NO-specific electrode. Brain homogenates(cerebral cortex, cerebellum and hippocampus) were prepared from male wildtype mice (8 and 120 weeks). After oxidation current of the NO electrodestabilized in the saline, each brain homogenate was added for estimation ofbasal NO production. To confirm specificity of the electrode, brain homogenate
was stimulated with calcium ion (Ca2+). When brain homogenate was added,
NO concentration increased and was stabilized at constant level. Ca2+
accelerated the basal NO production in a concentration-dependent manner.
However concentration of basal NO production did not so different in region
and age, Ca2+-induced NO production in aged mice significantly decreased inevery region compared with young mice. From these results, NO production
from mice brain homogenate was successfully measured with the NO-specificelectrode. We also examined that aging induced down-regulation of Ca2+/NO
signaling in mice brain.

3PT204

線虫 AIY 介在神経細胞での局在した神経活動：蛍光イメージングによる研究

Localized neural activities in AIY interneuron of Caenorhabditis elegans: fluorescent imaging study
Hisashi Shidara, Junya Kobayashi, Ryo Tanamoto, Kohji Hotta, Kotaro Oka(Grad. Sch. Sci and Tech., Keio Univ)
The neural activities of subcellular regions are important for understanding therelationship between the cellular morphology and the function in single neurons.
From this view, AIY interneuron of Caenorhabditis elegans has a very uniquefeature of Ca2+ dynamics; Ca2+ change is observed only on the specific regionof neurites, but not on soma during odor and temperature stimulation. To
investigate this dynamics in subcellular regions of AIY in detail, we visualized
the membrane potential and showed that its dynamics is different from the Ca2+one.
For visualization of neural activities on specific neurons in C. elegans, we havedeveloped a specific micro-fluidic device to fix the worm. The worm wasapplied to 10-4 dilution of isoamyl alcohol as the controlled odor stimulation
during 50 sec. The genetic Ca2+ indicator (YC3. 60) expressed in AIY hasrevealed the subcellular neural activities to the external environmental change.
To compare membrane potential change with the dynamics, we also usedvoltage-sensitive fluorescent protein (VSFP) 2. 42 (Akemann et al., 2010).
VSFP2.42 revealed that the odor stimulation depolarized membrane potential
both on the neurite and soma. In addition, the membrane potential showedoscillation with attenuation of the amplitude during odorant stimulation. These
results suggest that AIY has the diversity of biophysical characteristics in the
single cell and it is necessary for understanding its function.
3PT205

コルチコステロンは、シナプス局在の GR-kinase 系を介して、海馬神経シナプスを増やす

Corticosterone induced GR/kinase network-driven rapid
spinogenesis in rat hippocampus
Yoshimasa Komatsuzaki1,2,3, Masatoshi Kasuya2,3, Yasushi Hojo2,3, Suguru
Kawato2,3 (1Dept of Physics, CST, Nihon Univ, 2Grad Sch of Arts and Sci,
Univ of Tokyo, 3Bioinformatics Project, JST)
Modulation of hippocampal synaptic plasticity by corticosterone (CORT) has
been attracting much attention, due to its importance in stress responses. Many
studies have investigated the chronic effect of CORT, but rapid effect of CORT
in hippocampus is unclear. Here, we investigated effect of CORT, a major
glucocorticoid in rat, on density and morphology of dendritic spine which isessential for memory storage processes, in adult male rat hippocampus. The
application of 1 μM CORT induced a rapid increase in the total density of spinesof CA1 pyramidal neurons within 1 h from 0.98 spines/μm to 1.24 spines/μm.
CORT increased the density of large-head (0.5-1.0 μm) and middle-head spines(0.4-0.5 μm), but not small-head (0.2-0.4 μm) spines. Co-presence of RU486, anantagonist of glucocorticoid receptor (GR), completely abolished the effect of
CORT. Blocking ionotropic glutamate receptors with MK-801 inhibited the
CORT effect. This result suggests that Ca2+ homeostasis via NMDA receptors
is necessary to CORT-induced spinogenesis. Blocking various kinases showsthat CORT enhances spinogenesis via MAPK, PKA, PKC, PI3K and calcineurinsignaling pathways. In particular, the contribution of Erk MAPK and PKA isessential. Inhibition of protein synthesis reduced the CORT-induced
spinogenesis but inhibition of transcription. These results indicate that CORT
drives the signaling pathway including synaptic GR and multiple kinase
pathways in hippocampal CA1 neurons. (Komatsuzaki et al., 2012 PLoS-ONE)
3PT206

IP3/Ca2+ シグナリングによる GABA 作動性シナプスの制御

Regulation of GABAergic synapses by IP3/Ca2+ signaling
Hiroko Bannai1, Fumihiro Niwa1, Mark W. Sherwood1, Misa Arizono1,
Akitoshi Miyamoto1, Kotomi Sugiura1, Sabine Levi2, Antoine Triller3,
Katsuhiko Mikoshiba1 (1RIKEN BSI, 2Institut du Fer a Moulin, 3IBENS, ENS
Paris)The number of GABA-A receptors (GABAA R) clustering at the inhibitory
synapse is a crucial determinant of GABAergic synaptic transmission efficacy.
Ca2+ influx in response to excitatory neuronal activity results in the dispersal ofsynaptic GABAAR clusters. In addition to Ca2+ influx, Ca2+ release from theintracellular Ca2+ store also plays various roles in neurons. However, the impact
of Ca2+ release on the inhibitory synapse remains to be elucidated. Here, we− S175 −examined whether the inositol 1,4,5-trisphosphate (IP3)-induced Ca2+ release(IICR) is involved in the regulation of the synaptic clustering of GABAARs. In
hippocampal neurons from mice lacking intracellular Ca2+ releasing channel IP3
receptor type 1 (IP3R1), clusters of GABAAR and those of its scaffold protein
gephyrin were significantly smaller than in wild type neurons. In wild typeneurons, the inhibition of IICR by specific inhibitors resulted in the decrease in
GABAAR and gephyrin cluster size, without reducing the amount of GABAAR
on the cell surface. Single particle tracking with quantum dot (QD-SPT)
revealed that the loss of IP3 R1 and inhibition of IICR enhanced the lateral
diffusion of GABAA Rs. The increase of GABAA R lateral mobility resulting
from the loss of IICR was completely prevented by an inhibitor of calcineurin.
Our results suggest that IP3 /Ca2+ signaling contributes to the stabilization ofsynaptic GABAA R clusters through the regulation of GABAA R lateral
diffusion, possibly through phosphorylation.
3PT207

イ ン ス リ ン を 介 し た 神 経 シ グ ナ ル に よ っ て 制 御 さ れ る 線 虫 C.

elegans の温度適応の解析

Yoshitaka Hasegawa, Hideo Mukai, Makoto Asashima, Yuki Ooishi, Suguru
Kawato (Life sciences, The University of Tokyo)
Activin is known as a sex-hormone in mammalian. We attempt to reveal the roleof activin as a neuromodulator in the adult hippocampus. Activin is a
homodimer of inhibin β, and activin belong to the superfamily of transforming
growth factor-β (TGF-β). We showed endogenous/basal expression of activin in
the hippocampal neurons. Localization of activin receptors in spines wasdemonstrated by immunoelectron microscopy. The incubation of hippocampal
acute slices with activin A altered the density and morphology of spines in CA1
pyramidal neurons. The total spine density was increased by activin treatments.
Activin increased the head diameter of spines. Blocking of MAPK, PKA or
PKC prevented the activin-induced spinogenesis by reducing the density ofspines. We also demonstrated that activin induced the long term potentiation(LTP) of the hippocampal neurons.
カエル神経筋接合部シナプスでの細胞外 Ca2+濃度変化による神経伝達物質放出量増加の二項分布解析

Binomial distribution analysis of increase of transmitter
release depending on [Ca2+]e at the frog neuromuscularjunction
Taisuke Matsuda, Naoya Suzuki (Dept. Phys., Sch. Sci., Univ. Nagoya)
To investigate the action of Ca2+ to induce the transmitter release, we analyzed
the dependency of quantal release parameters on extracellular Ca2+
concentration ([Ca2+]e). Binomial distribution analysis with two parameters,
release probability (p) and number of releasable synaptic vesicles (n) havingthat release probability was applied to the release induced by single stimulations
at frog neuromuscular junction. Endplate potentials (EPPs) and miniature
endplate potentials (MEPPs) were electrically recorded with an intracellularglass microelectrode under five different [Ca2+]e changed from 0.55mM to 0.
95mM at intervals of 0.10mM. The averaged size of EPPs was proportional tothe about 3.0-3.5 power of [Ca2+]e. The binominal analysis of EPPs distribution
with using MEPPs distribution as single unit event gave values of p about 0.1-0.
5 and n about 10-50. Detail analysis of the change of two parameters, p and n,
showed p was approximately proportional to [Ca2+]e and n was proportional tothe about square of that. It suggests that increase of n contributed largely andincrease of p contributed slightly to increase of transmitter release with changeof [Ca2+]e.
3PT2093PT210

海馬でのアクチビンによるシナプス可塑性の急性制御

Acute Modulation of Synaptic Plasticity of Pyramidal Neurons
by Activin in Adult Hippocampus

3PT208

responsible for many of the seed's antioxidant and anti-inflammatory effects,
was used in this study. In every experience, rat cultured embryonic hippocampal
and cortical neurons were treated simultaneously with Aβ1-42 and TQ for 72 h.
The results showed that co-treatment with TQ efficiently attenuated Aβ1-42induced neurotoxicity, as evidenced by the improved cell viability. In addition,
TQ inhibited the mitochondrial membrane potential depolarization and reactive
oxygen species generation caused by Aβ1-42. TQ also restored synaptic vesicle
recycling inhibition, partially reversed the loss of spontaneous activity, andinhibited Aβ1-42 aggregation. These beneficial effects may contribute to theprotection against Aβ-induced neurotoxicity. Together, our results suggest thatthe natural antioxidant TQ has potential for neuroprotection and therefore, may
be a promising candidate for AD treatment.

アミロイドβペプチドが及ぼす神経活動への影響とチモキノンによる保護効果の検討

Thymoquinone, the Nigella sativa Bioactive Compound, Prevents
β amyloid neurotoxicity in cultured rat primary neurons
Amani Alhibshi, Ikuro Suzki, Masao Gotoh (Tokyo University of Technology)
Alzheimer disease (AD) is a neurodegenerative disease characterized byextracellular abnormal accumulation and extensive deposition of amyloid beta
peptide (A β). This accumulation is associated with oxidative damage,inflammatory reactions, synaptic function impairment, synaptic loss and finallyleads to neuronal death, and the use of antioxidants and anti-inflammations
could reduce this risk. Thymoquinone (TQ), the abundant essential oil
compound of Nigella sativa L. seeds, known to be the active principle

Insulin-mediated neural signals negatively regulate
temperature tolerance in C. elegans
Akane Ohta, Tomoyo Ujisawa, Yukari Kinoshita, Hitomi Mizutani, Takurou
Inoue, Naho Inoue, Atsushi Kuhara (Dept. Biol., Facul. Sci. Engin., Konan
Univ.)
Temperature is one of the most critical environmental stimuli and cause
biochemical change in the body. Therefore, animals have adaptation
mechanisms against environmental temperature changes, however, its molecularmechanisms are still poorly understood. Here we show that C. elegans has acultivation temperature-dependent cold resistance, which is regulated byinsulin-mediated pathway. 25℃-grown wild-type animals isolated from U.K.
were destroyed by cold stimuli, 2℃ for 48hr. By contrast, most of 15℃-grown
wild-type animals can survive for 48 hours at 2℃. These facts suggests that C.
elegans has cold resistance with changes in environmental temperature. To
reveal the molecular mechanisms underlying the cold resistance, we measured
cold-resistance in the various mutant animals defective in temperature sensationand temperature-controlled hormonal signaling. We found that the mutants
defective in insulin-like molecule DAF-28, insulin receptor DAF-2 and its
downstream molecules showed enhancement in cold resistance. These results
suggest that insulin-mediated neural signals negatively regulate cold resistance.
In another approach, we focused on the natural variation of wild-type strains
isolated from different areas. We so far found that British strain showed weakerphenotypes in cold resistance than California or Vancouver strains, respectively.
We are also using artificial evolution approaches and forward genetic screening
to isolate the mutation in cold resistance.
3PT211

海馬での年齢依存的な性ホルモン受容体・合成酵素遺伝子の発現変動解析

Age-related changes in the expression of mRNAs encoding forsex steroidogenic enzymes and sex hormone receptors in the
hippocampus
Tetsuya Kimoto, Masahiko Wakabayashi, Suguru Kawato (Dept. Biophys. Life
Sci., Grad. Sch. Art. Sci., Univ. Tokyo)
Although sex steroids play a crucial role in the development/maintenance ofbrain function, the age-related changes in the hippocampal sex steroidogenesis
remain largely unknown. We examined the mRNA expression levels of sex
steroidogenic enzymes and sex steroid receptors in the hippocampus of aged (24month-old) male rats compared with those of young adult (3 month-old) malerats by means of semi-quantitative RT-PCR. The levels of mRNAs forcytochrome P45017 α, 17 β -HSD3 and 5 α -reductase2 reduced in the
hippocampus of aged rats to approx. 45%, 76% and 75% of young adult rats,
respectively. On the other hand, the levels of 17β-HSD1, 5α-reductase1 and
P450aromatase were almost the same between aged and young adulthippocampus. The levels of estrogen receptor β and androgen receptor reducedin aged hippocampus to approx. 84% and 55% of young adult hippocampus,
respectively, while almost no change was observed in the mRNA expression of
estrogen receptor α between aged and young adult hippocampus. These results
indicate that the hippocampal sex steroidogenic properties are substantiallyaltered between aged and young animals.
3PT212− S176 −NMDA 受容体と相互作用することにより CaMKII は分子メモリとして機能する − in vitro 実験系による実証−
CaMKII functions as a molecular memory through interaction
with NMDA receptors - validation in in vitro experiments -Hidetoshi Urakubo1,2, Shin Ishii2, Shinya Kuroda1 (1Dept. Biophys. Biochem.,
Grad. Sch. Sci., U. Tokyo, 2Dept. Syst. Sci., Grad. Sch. Info, Kyoto U.)
In the process of long-term potentiation (LTP) at synapses, calcium-calmodulin(CaM) dependent protein kinase II (CaMKII) is interacted with NMDA
receptors, and the interaction leads to Ca2+ /CaM-induced, but CaMindependent CaMKII activity (Bayer 2001, Nature 411, pp. 801-805). This
sustained activity is considered as a part of mechanisms of LTP maintenance;
however, additional mechanisms are needed to explain reversibility of LTP, andstable basal CaMKII activity even with spontaneous Ca2+ increase at synapses.
Here, in an in vitro experimental system, we found that CaMKII
autophosphorylation at T286 shows hysteresis through interaction of a NMDA
receptors-derived peptide. This indicates that CaMKII activity can work as amolecular memory for LTP maintenance. The hysteresis was regulated byprotein phosphatase 1 (PP1), and canceled by high PP1 application,corresponding to reversal of LTP (Depotentiation). Ca2+ -concentration
dependence of the hysteresis showed that the hysteresis is robust against
spontaneous Ca2+ increase. This can provide a mechanism of reversible LTP
and stable basal CaMKII activity at synapses.
3PT213

新規な神経スパイン解析プログラム

Novel program and its applications for spines in neurons
Hideo Mukai1,2,3, Yuusuke Hatanaka2, Kenji Mitsuhashi2, Gen Murakami2,3,
Yasushi Hojo2,3, Suguru Kawato2,3 (1Dept. Biochem., Fac. Med., Saitama Med.
Univ., 2Dept. Biophys. & Life Sci., Grad. Art & Sci., Univ. Tokyo, 3BIRD, JST)
Dendritic spines in the brain are substrates of synaptic contacts that may be
involved in neuronal computational processes. Visualization and analysis ofdendritic spines is of critical importance to elucidate physiological changes ofmorphological plasticity, as well as effects of various hormones andpharmacological agents. Advent of laser-scanning confocal microscopy / 2-photon
microscopy and fluorescent dye allowed us to collect large body of neuronal image
data, namely tens of dendrites and several tens of thousands of spines residing oneach dendrite of single neurons. Conventional methods using manual tracing
software need time-consuming efforts by researchers and not suitable to handle
large data. Therefore introduction of protocol for a new automatic analysis ofneuronal structure including spines will greatly enhance progress of research in thearea. Here we developed a new software, Spiso-3D, to extract spines as well as
dendrites and in neuronal image based on their geometrical features. Our“geometric method” utilises scale-free, shape-dependent analysis, making lessdependent on brightness in the image. Because Spiso-3D is a Java-based program,it will readily work on a PC with a relatively limited resource.
Using the Spiso, we analyzed the effect of activin, an endogenous sex hormone
in the mammalian brain, on spines of hippocampal neurons. Ref. Mukai et al.,Cerebral Cortex, 21, (12): 2704-2711. (2011)
3PT214

高速ビデオカメラ法および筋電位法によるサル瞬目反射条件付けシ
ステムの開発Evaluation of eyeblink classical conditioning in monkey byusing high-speed video sensing and electromyogram signal
Yasushi Kishimoto1, Shigeyuki Yamamoto2, Kazutaka Suzuki2, Toyoda
Haruyoshi2, Hideo Tsukada2, Yutaka Kirino1 (1Lab. Neurobiophysics, Kagawa
Sch Pharmaceut Sci, Tokushima Bunri Univ., 2Central Research Lab.,
Hamamatsu Photonics Co., Ltd.)Classical eyeblink conditioning is one of the best-characterized behavioral
models of associative learning in mammals. Especially, standard delay
paradigm has been used for assessing the motor learning or cognitive
performance in a variety of mammalian species including, rabbit, rat, humanand mouse. In the present study, we first developed the system for evaluating thedelay eyeblink conditioning in monkey (Macaca mulatta). The eye blinking of amonkey was measured and evaluated by eyelid electromyogram (EMG) method
and a 1khz high-speed image processing (Intelligent Vision Sensor, C8201;
Hamamatsu Photonics K. K.), simultaneously. Within the conditioning
procedure, a 1-kHz tone served as the CS while an air-puff was used as the US.
The EMG-based analysis indicated that monkeys exhibited over 60 % ofconditioned response (CR) frequency during 5-days acquisition session (100trials per day), and rapid extinction of CRs during 2-days extinction session.
Also in the high-speed image processing analysis, the monkeys exhibited
similar results of both in acquisition and extinction of CRs. Hence, we haveconcluded that both methods of the EMG-based and video-based analysis are
effective in measuring eyeblink conditioning in monkey. Our results indicatedthat the conventional delay conditioning procedure can also effective in

conditioning monkeys. The novel multi-measuring system of eyeblink CRs inmonkeys will provide useful tools for elucidating the neural mechanism
underling motor learning in higher mammals.
3PT215視床下部室傍核の吻側部位に存在するヒスタミン H1 受容体発現
ニューロンは摂食抑制作用をもつ

Histamine H1 receptor-expressing neurons in the anterior partof the hypothalamic paraventricular nucleus inhibit foodintakeShuhei Horio (Institute of Health Biosciences, The University of Tokushima
Graduate School)
Food intake in mammals is controlled by neurons in the hypothalamus. To
suppress food intake, the information from the periphery is conveyed to the
hypothalamus via two distinct pathways; one is a humoral pathway that transmit
satiety information to hypothalamic paraventricular nucleus (PVH) via arcuate
nucleus (ARC) that lies adjacent to blood circulation, and the other is a neuronal
pathway that send information to PVH via nucleus solitarius (NTS). Thus
satiety information converges in PVH, which is now considered as a center for
suppressing food intake. Thus far, several neurons in PVH including CRH(corticotropin-releasing hormone) neurons, oxytocin neurons, and melanocortin
neurons are found to suppress food intake.
We studied the role of histamine H1 receptor (H1R)-expressing PVH neurons infood intake. H1R is highly expressed in murine PVH in both the anterior andposterior parts. We developed gene-targeted mice that expressed human IL-2Rα
selectively in H1R-expressing neurons. Injection of immunotoxin to PVH
selectively ablated these neurons (the toxin binds to IL-2Rα and disrupts thecell). The ablation of these neurons caused a 30% increase in food intake. The
body weight was also increased approximately 30% compared to control. The
ablation of the posterior part of H1R-expressing PVH neurons had no effect onfood intake.
These results indicate that H1R-expressing neurons in the anterior PVH have a
crucial role in the regulation of food intake. It is probable that these neurons
constantly suppress food intake.
3PT216

記憶・学習中枢海馬の性差は海馬で合成されるホルモンによるもの
である

Sex difference in profile of hippocampal hormones generates
sex difference in hippocampal function
Yasushi Hojo1,2, Masahiko Wakabayashi1, Kotaro Yoshida1, Tetsuya
Kimoto1,2, Suguru Kawato1,2 (1The University of Tokyo, 2BIRD, JST)Sex difference in the brain is a very attractive problem. For example, thehypothalamus, the brain region responsible for reproductive behavior, exhibits aclear sex difference in the size of nerve nucleus.
In contrast, the hippocampus, a center for learning and memory, does not havesex difference at the anatomical level including the volume and the number ofneurons. Nevertheless, the significant sex difference in the performance ofhippocampus-dependent task such as spatial memory using Morris water mazeor radial arm maze task.
What is the cause of sex difference in the performance of learning and memory
depending on hippocampus? So far, sex difference in the hippocampus had been
attributed to the level of sex hormones in the blood.
However, we revealed that the hippocampal level of sex hormones is muchhigher than the blood level. Surprisingly, hippocampal estradiol (E2), the mostpotent female hormone, in the male is 7-fold higher than female whereas female
E2 in the blood is much higher than male. Moreover, the density of spines(postsynapses) as well as the level of E2 in female hippocampus flucutuateswith a period of 4 days (estrous cycle), whereas those in male are retained at aconstant level.
This clear sex difference in hormonal profile in hippocampus may generate thesex difference in the hippocampal structure at more subtle level, that is, synaptic
level, resulting in the sex difference in the performance of hippocampusdependent task.
3PT217

老化に伴う海馬神経シナプスの密度の減少と記憶の劣化

Age-related changes in spine density and morphology ofhippocampal neurons in relation to memory impairment
Suguru Kawato, Koren Li, Yasushi Hojo (Grad Sch of Arts& Sci, Univ Tokyo)
We have investigated the spine density and morphology of neurons in− S177 −hippocampal CA1 region which regulates spatial memory. Spine is a
postsynapse. We observed a significant decrease in the spine density by goingfrom young rat (3 month old, ~20 years in human) to aged rat (24 month, ~70
years in human). The density of spine as well as the head diameter are analyzed
with Spiso-3D software (mathematical and automated software developed by
Bioinfomatics Project of our group, now commercially available). Spine
morphology is classified into three categories according to the head diameter;(1) small-head spine (0.2-0.4 μm) (2) middle-head spine (0.4-0.5 μm) (3) largehead spine (0.5-1.0 μm). In aged rats, dendrites had significantly less spines (1.7
spines/μm) than young rat (2.3 spines/μm). The density of large-head spine andmiddle-head spine was significantly decreased in aged rats. From many studies,surprisingly, the number of neurons is not decreased and the dendritic atrophy
does not occur upon aging in the hippocampus. Therefore, the observed
decrease in spine density may be essential for loss of memory capacity. It is wellknown that sex-hormone replacement therapy rescues memory loss in humans,
rats and mice, probably by restoration of synaptic density. Therefore, we havebeen investigation a correlation between the decrease in spine density andreduction of sex hormone levels in aged rat hippocampus (not only in blood
circulation), using mass spectrometric determination.
3PT218

オプトジェネティクスを用いた光によるウィスカへの触覚刺激

Whisker photostimulation induces spike and LFP responses inbarrel cortex of ChR2 transgenic rat
Tatsuya Honjoh1,2, Zhi-Gang Ji1,2, Toru Ishizuka1,2, Hiromu Yawo1,2,3(1Tohoku Univ. Grad.Sch. Lif Sci, Sendai, Japan, 2CREST, JST, 3Tohoku Univ.
Center for Neuroscience)
In one of thy1.2-channelrhodopsin 2 (ChR2)-Venus transgenic rat lines, WTChR2V4, the ChR2-Venus conjugate was expressed exclusively in themechanoreceptive, not in the nociceptive subpopulation of trigeminal ganglion
neurons (Ji et al., poster of the 35th annual meeting of the Japan Neuroscience
Society). As the sensory nerve endings are densely innervating each whisker
follicle, its photostimulation is well expected to induce a whisker-related
sensory perception.
The rats were anesthetized with urethane (1.2g/kg body weight). All whiskers ofright side were trimmed to photo-stimulate the trigeminal nerve endings
innervating the hair follicles. For extracellular recording, a 1.4-mm diameter
craniotomy was performed over the barrel cortex of left hemisphere. Glasselectrodes filled with 0.5M Na acetate were inserted into the cortex through an
opening of the dura. Right whisker pad was illuminated broadly with a 50ms
light pulse of blue LED.
We found that the whisker photostimulation could induce the spike and LFP
responses of neurons in somatosensory barrel field. It is suggested that thewhisker photostimulation could activate the whisker-barrel cortex pathway of
mechanoreceptive signaling. The optogenetic actuation of the whisker
mechanoreception would facilitate detailed studies of the rodents’ whiskerbarrel sensory system.
All animal procedures were conducted in accordance with the guiding principlesof Physiological Society of Japan and NIH.
3PT219

軟体動物ナメクジの嗅覚中枢における長期活動ダイナミクス

Dynamics of long-term activities of the olfactory center in theland slug
Yuichi Tanaka1, Kouya Katou1, Minoru Saito3, Yoshimasa Komatsuzaki2(1Grad. Sch. Sci. and Tech., Nihon Univ., 2Dept Phys, CST, Nihon Univ, 3Grad
Sch Integ Basic Sci, Nihon Univ)
Spontaneous oscillation of activity in olfactory system is commonly observed invarious species and is thought to be important for odor-information processing.
However, the functions of the oscillatory activity are not well understood. The
land slug Limax valentianus has a highly developed olfactory center, theprocerebrum (PC), which exhibits oscillation of local field potential (LFP) at
about 1 Hz. Because the application of conditioned odor changes the frequencyof the LFP oscillation in slug PC, odor information is thought to be represented
as the temporal pattern of the oscillation. Here, we investigated the statisticalproperty of spontaneous oscillation in the PC over an extended timescale. To
analyze the autocorrelation of the time series of interspike intervals, we
performed a detrended fluctuation analysis (DFA), which is a scaling analysis
technique used to provide a quantitative parameter (scaling exponent, α). We
found that the fluctuation of spike timing of the PC possessed a long-termcorrelation and can be characterized as 1/f noise at timescales larger than 102
spikes (α~1.10). Furthermore, to investigate the contribution of innervations tospontaneous oscillation, we measured the LFP following the tentacle

amputation. In the scaling region for small n (n < 102 spikes), the scaling
exponent α decreased from 0.73 to 0.58, indicating that the fluctuations of spiketimings are uncorrelated under this experimental condition. These results
suggest that the innervations to PC maintain the intrinsic dynamics.
3PT220ヨーロッパモノアラガイの中枢神経系における神経活動の膜電位イ
メージング

Fluorescent Voltage Imaging of the Neural Activities in theCentral Nervous System of the Pond Snail
Shogo Nakada1, Makoto Hosoi1, Yosimasa Komatuzaki2, Minoru Saito1(1Graduate School of Integrated Basic Sciences, Nihon University, 2College of
Science and Technology, Nihon University)
The brain or ganglia forms a complex network composed of so many neurons
which communicate with one another. We have found that a regulatory neuron(cerebral giant cell; CGC), which is involved in feeding responses, in the cerebral
ganglion of the pond snail Lymnaea stagnalis shows regular beating, regularbursting and irregular bursting discharges depending on the d.c. current throughthe cell membrane. In the present study, we examined how the activities of CGC
affect the neural activities of the buccal ganglion by using fluorescent voltage
imaging technique. After the central nervous system was isolated from the body,it was stained with a voltage-sensitive dye, Di-4-ANEPPS. The stained
preparation was illuminated by a LED (530 nm; LEX2-G, Brain Vision), and the705 nm fluorescent images were acquired through a CCD camera (iXon X3 897,
Andor or ORCA-Flash 4.0, Hamamatsu Photonics). The activities of CGC were
measured simultaneously by intracellular recording using a glass electrode. The d.
c. current was injected into CGC through the electrode to control the activities.
The fluorescent images showed that some neurons in the buccal ganglion fired
coherently, and the firing patterns were affected by the discharge of CGC.
3PT221

マウス海馬スライスの CA1 領域における様々な時空間活動パター
ンのレーザー共焦点イメージング

Laser confocal imaging of various spatiotemporal activity
patterns in the CA1 region of mouse hippocampal slices
Hiromi Osanai1, Akiyoshi Suzuki2, Sachiko Matsumura2, Hideo Mukai3, Minoru
Saito2 (1College of Humanities and Sciences, Nihon University, 2Graduate Schoolof Integrated Sciences, Nihon University, 3Faculty of Medicine, Saitama Medical
University)
The brain forms a complex network composed of so many neurons which
communicate with one another. In addition, even a single neuron shows very
complex activities, and some neurons spontaneously fire due to nonlinear
characteristics. Recently, Ikegaya et al. have developed functional multineuron
calcium imaging which enables us to access brain function with single-neuron
resolution. In the present study, we observed spatiotemporal activity patterns in
the CA1 region of mouse hippocampal slices by a similar technique. The slices(350 μ m) were prepared from 1-week-old male ddY mouse. The slice
preparation was stained with a Ca2+ -sensitive dye, oregon green. The stained
slice was illuminated by an Ar laser (488 nm; 532-BS-A04, Melles Griot), andthe 520 nm fluorescence images were acquired through a Nipkow confocal unit
(CSU-10, Yokogawa) and a CCD camera (iXon X3 897, Andor). As a result,some dozens of neurons fired incoherently in some slices, while they exhibited acompletely coherent activity pattern in other slices. The coherent patternoccurred more frequently under the existence of penicillin and a higher K+
concentration which are often used to induce the epilepsy-like state.
Interestingly, we also observed the irregular switching between the coherent andincoherent activity patterns in other slices. This phenomenon may be explainedby the dynamics of itinerancy among quasi-stable states reported in some neuralnetwork models.
3PT222

QM/MM 法による同化型亜硝酸還元酵素の反応機構についての理論的研究

A QM/MM study on the reaction mechanism of assimilatory
nitrite reductase
Mitsuo Shoji1,2, Kyohei Hanaoka1, Daiki Kondo1, Hiroaki Umeda2, Megumi
Kayanuma2, Katsumasa Kamiya1, Kenji Shiraishi1, Shogo Nakano3, Katsuo
Katayanagi3 (1Grad. Sch. Pure & Appl. Sci., Univ. Tsukuba, 2CCS, Univ.
Tsukuba, 3Grad. Sch. Sci., Hiroshima Univ.)
Assimilatory nitrite reductase (aNiR) catalyzes a multi-electron reduction reactionof nitrite to ammonia, which is a fundamental process for the biological synthesis of− S178 −nitrogen-containing cellular constituents such as plant. Although this multielectron reaction is a novel type of biological catalysis for the reaction specificity
and regulation, the reaction mechanism is not fully understood yet because of thecomplicated reaction process. In this study, the mixed quantum mechanical/
classical mechanical (QM/MM) method was applied to investigate the reaction
mechanism of aNiR. We discuss the reaction efficiency of Nii3, one of the aNiR,
based on the calculated intermediate states and the reaction energy profile.
3PT223

RNase H の RNA 加水分解反応機構の理論的研究

Computational study of the phosphodiester hydrolysis of RNA
by RNase H
Yu Takano, Makoto Kita, Haruki Nakamura (IPR, Osaka University)
The phosphate diester hydrolysis is an important reaction in biology. The
ribonuclease H (RNase H) catalyzes the nonspecific hydrolysis of RNA in
RNA/DNA hybrids, yielding a 3'-hydroxyl and a 5'-phosphate at the hydrolysis
site. This enzyme is found that the Mg2+ ion is essential for the enzymatic
reaction and that aspartate and glutamate are coordinated the Mg2+ ion in theactive site. This catalytic mechanism is ubiquitous among nucleic acidprocessing enzymes and ribozymes. In particular, RNases H has emerged asimportant therapeutic targets because RNase H activity is absolutely requiredfor proliferation of HIV and other retroviruses. Although the X-ray
crystallographic structures of RNase H are determined, there are controversies
about the catalytic mechanism of RNase H, the number of Mg2+ ions, the protontransfer pathway, and the protonation states of the active site residues.
In the present study, we have explored the hydrolysis of the phosphate diester
group of RNA by RNase H, using density functional theory to elucidate how
many Mg2+ ions are required for the catalysis of RNase H. Our computation
demonstrates that both one- and two-metal models show the step-wise hydrolysis
pathway and lower the activation barriers, compared to the non-enzymatichydrolysis of phosphate diester. However, the activation barrier of the two-metalmodel is smaller than that of the one-metal model by about10 kcal mol-1.
3PT224First principles molecular simulation with a generalizedensemble algorithm for studying chemical reactions of
biomolecular systems
Yoshiharu Mori1, Yuko Okamoto2,3,4 (1IMS, 2Dept. of Phys., Nagoya
Univ., 3Structural Biology Research Center, Nagoya Univ., 4Center for
Computational Science, Nagoya Univ.)
It is important to study chemical reactions by molecular simulations because wehave to treat a chemical reaction to understand an interesting chemical systemsuch as biomolecular systems including enzymes. We can perform a firstprinciples molecular simulation to understand these systems. However the timescale of a first principles molecular simulation is limited because its
computational cost is much higher than a simulation with some force fields.
In order to overcome such a difficulty, we need to use generalized-ensemble
algorithms, which enhance conformation sampling in molecular simulations. In
this study, we used a simulated tempering method, Simulated Tempering
Umbrella Sampling (STUS). Molecular simulations with this method canenhance a chemical reaction in reaction coordinate space. As a result, we canobtain the free energy of the chemical reaction accurately.
We performed first principles molecular dynamics simulations with thesimulated tempering method for the system of malonaldehyde in water. The
reaction coordinate fluctuated in large conformational space. As a result, wewere able to calculate the potential of mean force of the reaction coordinate
accurately. We found that molecular simulations with STUS also allow us tounderstand chemical reactions of biomolecular systems.
3PT225

大規模量子化学計算(FMO 法)による HIV-1 糖鎖認識抗体 2G12と糖鎖間の相互作用解析

Interaction Analysis of HIV-1 Antibody 2G12 and Glycans by
Large-Scale Quantum Chemical Calculations based on FMO
methodKaori Noto1, Yuka Koyama2, Keiko Takano2 (1Kitasato University, 2Ochanomizu
University)
Human antibody 2G12 is capable of recognizing the high-mannose glycans on
the HIV-1 surface glycoprotein, gp120. Doores et al. found that Fab-2G12 isbound to D-fructose stronger than the original ligand, D-mannose. Details of theneutral carbohydrate-antibody interaction remain unclear. To elucidate thedifferences in the ligand-binding, we carried out ab initio fragment molecularorbital (FMO) calculations at MP2/6-31G (d) level for Fab-2G12 with Dmannose/D-fructose complexes using their crystal structures (PDB ID: 1OP3,
3OAY). The interaction energy between Fab-2G12 and D-fructose was smaller
than that between Fab-2G12 and D-mannose excluding the effects of water,which was not consistent with the experimental result. We further investigated
the interaction involving water molecules observed in the crystal structures andfound notable pair interaction energies among D-fructose, waters, and severalamino acid residues. In addition, we constructed a model glycan ligand withouta side chain group of the D-fructose and studied its interaction with the antibody
to evaluate the effect of the substituent of the ligand on the interaction. The
obtained results indicated a significant role of C2-OH group of D-fructose in theligand-antibody interaction.
3PT226FTIR 法による光合成酸素発生マンガンクラスター近傍の水素結合
ネットワークの構造解析

FTIR analysis of the hydrogen bonding network around the O2-evolving Mn4CaO5 cluster
Kai Ota, Takumi Noguchi (Grad. Sch. Sci., Univ. Nagoya)Photosynthetic O2 evolution is performed at the Mn4 CaO5 cluster inphotosystem II (PSII) through five intermediates called S states (S0-S4). Recent
X-ray structure of PSII showed that two water molecules are bound to the Ca2+and form a hydrogen-bond network between the Mn4CaO5 cluster and YZ. To
clarify the roles of the water molecules and the hydrogen-bond network aroundthe Ca2+, we have investigated the effect of the replacement of Ca2+ with Ba2+on these structures using Fourier transform infrared (FTIR) difference
spectroscopy. S2 /S1 FTIR difference spectra of moderately hydrated films of
NaCl-washed PSII membranes of spinach were recorded at 283 K in thepresence of either Ca2+ or Ba2+. The weakly hydrogen-bonded OH bands ofwater at 3617/3587 cm-1 observed in Ca2+ -PSII were absent in Ba2+ -PSII. A
broad feature at 2900-2600 cm-1, which arises from strong hydrogen bonds withhigh proton polarizability, also disappeared in Ba2+-PSII. These results strongly
suggest that the above bands of water OH and strong hydrogen bonds originate
from the water molecules bound to Ca2+ and the surrounding hydrogen bond
network, respectively, and the replacement of Ca2+ with Ba2+ with a larger ion
radius changed or destructed these structures. The data support the idea thateither of the water molecules attached to the Ca2+ functions as a substrate andthat the hydrogen-bond network from the Ca2+ to YZ plays an important role inproton transfer in the S2→S3 transition, which is blocked by Ba2+ substitution.

3PT227

QM/Langevin-MD と振動モード解析を用いた光合成初期過程における光化学系 II の理論的研究

A study of QM/Langevin-MD simulation and vibrational
analysis for photosystem II in early process of photosynthesis
Waka Uchida1, Yoshiro Kimura1, Taichi Yuki1, Makoto Hatakeyama1, Koji
Ogata3, Satoshi Yokojima2,3, Shinichiro Nakamura3 (1Graduate School ofTokyo Institute of Technology, 2Tokyo University of Pharmacy and Life
Sciences, 3RIKEN Research Cluster for Innovation)
In the early process of photosynthesis in photosystem II (PSII) protein, theoxidation reaction, evolving one oxygen molecule by oxidizing two watermolecules, occurs in oxygen-evolving complex (OEC) forming oxo-bridgedcluster consisting of four manganese and one calcium ions (Mn4O5Ca). This
reaction goes through the five oxidation states denoted Sn (n=0-4), called Kok
cycle. Here we have performed a QM/Langevin-MD simulation and FTIR
spectrum calculation for OEC and residues surrounding it. The structure of PSII
solved by X-ray crystallography at 1.9 Å was used in this study (PDB entry
3ARC). The structure was optimized using QM/MM calculations. And bychanging Mn oxidation numbers at the same as S2 state ones, the structure
satisfying four Mn oxidation numbers in the S2 state was predicted using
QM/MM calculation from the structure of S1 state. For these optimized
structures, QM/Langevin-MD and vibrational frequency were performed inmany variation of four Mn oxidation numbers. From the experimental
evidences, one Mn ion in the cluster is oxidized during going through S1 to S2states, and meanwhile the spectra of Ala344 and Glu354 show the clear peakshift in IR spectra. We have observed the same peak shift in our vibrationalfrequency calculation. And the trajectory of QM/Langevin-MD simulation
reveals the most stable Mn oxidation numbers of S1 state in the thermal
fluctuation. In addition, based on the trajectory of the S1 state, we will discuss
about four Mn oxidation numbers in the S2 state.

− S179 −3PT228

コケ植物におけるそれぞれ異なる 3 つの消光機構

Three different mechanisms of energy dissipation of adesiccation-tolerant moss to protect reaction centres against
photo-oxidation
Hisanori Yamakawa1, Yoshimasa Fukushima1, Shigeru Itoh1, Ulrich Heber2(1Grad. Sch. Sci., Univ. Nagoya, 2Bio. Sci., Univ. Wurzburg)
Three different types of non-photochemical de-excitation of absorbed lightenergy protect photosystem II of the sun- and desiccation-tolerant moss
Rhytidium rugosum against photo-oxidation. The first mechanism, which islight-induced in hydrated thalli, is sensitive to inhibition by dithiothreitol. It is
controlled by the protonation of a thylakoid protein. Other mechanisms areactivated by desiccation. One of them permits exciton migration towards a farred band in the antenna pigments where fast thermal deactivation takes place.
This mechanism appears to be similar to a mechanism detected before indesiccated lichens. A third mechanism is based on the reversible
photoaccumulation of a radical that acts as a quencher of excitation energy inreaction centres of photosystem II. On the basis of absorption changes around
800 nm, the quencher is suggested to be an oxidized chlorophyll. The data showthat desiccated moss is better protected against photo-oxidative damage thanhydrated moss. Slow drying of moss thalli in the light increases photoprotection more than slow drying in darkness.
3PT229光化学系 II と金ナノ粒子の結合による人工光水分解ナノデバイスの開発

Development of artificial light-driven water splitting
nanodevice using gold nanoparticles and photosystem II
Kousuke Kawahara1, Tatsuya Tomo2,3, Takumi Noguchi1 (1Grad. Sch. Sci.,
Nagoya Univ., 2Faculty of Sci., Tokyo Univ. of Sci., 3JST PRESTO)
Development of efficient artificial photosynthetic systems is crucial to solve theenergy and environmental crisis. We have recently developed a light-driven
water-splitting nanodevice by conjugation of photosystem II (PSII) protein
complexes with gold nanoparticles (GNPs) (Noji et al., J. Phys. Chem. Lett.,2011). However, O2 evolution activity of this PSII-GNP conjugate was only
23% of free PSII. In this study, we have improved the preparation method of the
PSII-GNP conjugates to increase the O2 evolution activity and further detected
light-induced electron transfer in PSII-GNP using attenuated total reflection(ATR)-Fourier transform infrared spectroscopy (FTIR). The previous decreasein the O2 evolution activity in PSII-GNP was found to be caused by a low Ca2+
concentration (30 μM) in the buffer adopted to prevent aggregation of GNP. To
address this problem, the incubation time for binding PSII with GNP in the 30 μ
M Ca2+ buffer was shortened from 120 min to 10 min, and then the resultant
PSII-GNP was suspended in a buffer with 1 mM Ca2+. The PSII-GNP
conjugates were stable in the 1 mM Ca2+ buffer, whereas free GNP wasaggregated in the same buffer. The O2 evolution activity of the PSII-GNP
increased by a factor of two compared with the previous study. A light-induced
ATR-FTIR difference spectrum of the PSII-GNP conjugates in the presence of
DCMU showed typical signals of the semiquinone anion of QA, providing
evidence that light-driven electron transfer took place in this nonodevice.
3PT230光化学系 II 酸素発生中心の暗所安定状態における電子スピン状態とプロトン化状態の理論的研究

Theoretical Study on the Protonation and Electronic Spin
State of the O2-Evolving Complex in Photosystem II at thedark stable S1 state
Makoto Hatakeyama1, Waka Uchida1, Kouji Ogata2, Satoshi Yokojima2,3,
Shinichiro Nakamura2 (1Grad. Sch. Bio., Titech, 2Harima Inst., Riken, 3Sch.
Pharm., Tokyo Univ. Pharm.)
An oxygen evolving complex (OEC) of photosystem II protein has been known
as the catalyst for water splitting and oxygen evolving reactions ofphotosynthesis. The structure and catalytic capability of OEC have been
investigated extensively by both experimental and theoretical studies, in order tolearn how the water splitting proceeds in nature. The latest X-ray diffraction(XRD) analysis has revealed that the OEC contains the multi-nuclear Mn4Ca
cluster with the five oxo bridges. Nonetheless, the XRD analysis has notindicated two indispensable factors for the water splitting mechanism; i.e. (i)coordinate of hydrogen atoms and (ii) electronic spin state of the Mn4Ca cluster.
The coordinate of hydrogen atoms is necessary to analyze the strength of the Mn

ion ligand field and to identify the oxidation Mn site during the water splitting.
Although the Mn4Ca cluster has been elucidated to involve two Mn(III) and two
Mn(IV) ions in the dark stable steady state (S1), the locations of Mn(III)/Mn(IV) ions in the latest crystal structure has been not yet determined inconsidering the coordinate of hydrogen atoms. Taking account on thosearguments, we investigated the electronic spin state of the Mn4Ca cluster at thedark stable state, by respecting the latest crystal structure and by changing theprotonation state of the coordinating O atoms around the Mn4Ca cluster.
3PT231光合成酸素発生の S 状態サイクルにおける NH4+阻害のメカニズム

Mechanism of NH4+ inhibition in the S-state cycle ofphotosynthetic oxygen evolution
Takashi Kobayashi, Takumi Noguchi (Grad.Sch.Sci.,Univ.Nagoya)Photosynthetic O2 evolution by plants and cyanobacteria is performed at the Mncluster in photosystem II (PSII). O2 is released as a result of four-electronoxidation of two water molecules through a light-driven cycle of fiveintermediates called S states (S0-S4). We recently found that NH4+ works as aneffective inhibitor of O2 evolution. However, the detailed mechanism of NH4+
inhibition has not been understood yet. In this study, we investigated themechanism of NH4+ inhibition in the O2 evolving S-state cycle using Fouriertransform infrared (FTIR) spectroscopy. PSII membranes from spinach in thepresence of 200 mM NH4Cl were subjected to12 successive flashes and FTIR
difference spectra upon individual flashes were obtained. It was shown that theamplitude of the caroboxylate stretching bands was significantly reduced at thethird flash, indicating that specific inhibition took place in the S3→S0 transition.
The overall intensity of the FTIR signals was also smaller with NH4Cl than with
NaCl as a control, suggestive of irreversible inactivation of the Mn cluster. TheO2 evolution activity, however, was unchanged when the sample was incubatedwith 1 M NH4Cl in the dark for 1 h and then washed. Flash-induced S2/S1 FTIR
difference spectra repeatedly measured every five minutes showed a signal
decrease as the flash number increases. These results suggest that NH4+
inhibition of O2 evolution is caused by mixed effects of inhibition in the S3→S0transition and irreversible inactivation of the Mn cluster in the S2 state.
3PT232光合成膜蛋白質 PSII の分子動力学シミュレーション

A study of molecular dynamics simulation on Photosystem II protein
Koji Ogata1, Makoto Hatakeyama2, Taichi Yuki2, Waka Uchida2, Shinichiro
Nakamura1 (1RIKEN Research Cluster for Innovation, 2Tokyo Institute ofTechnology)
Photosystem II (PSII) protein, which governs the initial process of thephotosystem in the plant, is transmembrane protein consisting of 20 subunits.
This protein plays an important role of yielding O2 molecules by oxidizing
water molecules. The structure of PSII has been solved by X-ray cystography
techniques and publicly accessed from Protein Data Bank (PDB) and clearlyshows the detail of structure in the active center for photosynthesis and staticalstructure can be observed. Here, to observe the dynamical structure of PSII
protein, we have performed molecular dynamic simulation on a dimer PSII
protein on the thylakoid membrane. The composition of phospholipids andgalactolipids in the modeling thylakoid membrane were built as close as thenative one. The parameters of four lipids (MGDG, DGDG, SQDG and PG) andthe other ligands (Chlorophyll, Plastoquinone, etc.) were assigned in GAFF
atomic types combining the charges obtaining by the quantum chemistrycalculation. A trajectory of 10 ns simulation was analyzed. From the analysis of
the trajectory, water molecules went through into the Mn4CaO5 cluster, which
is important part for the O2 oxidation, using three pathways but not so fast
movements near the Mn4CaO5 cluster. And the two monomer conformations of
PSII showed the correlated motions with each other, the thylakoid membrane
and the other ligands. We will discuss on the influences of the vibrations of PSII
and its surroundings to the mechanism of the oxygen evolution in PSII protein.
3PT233

好熱性紅色硫黄細菌 Thermochromatium tepidum 由来
cytochrome c' における耐熱化メカニズムの検討

Molecular mechanisms for the thermostabilization ofcytochrome c’ from the thermophilic purple sulfur bacterium
Thermochromatium tepidum
Sachiko Kasuga1, Yukihiro Kimura1,2, Kei Furusawa3, Takashi Ohno2, Seiu
Otomo3 (1Grad. Sch. Agri. Sci., Kobe Univ., 2OAST, Kobe Univ., 3Fac. Sci.,
Ibaraki Univ.)
Cytochrome (Cyt) c’ purified from thermophilic purple sulfur photosynthetic− S180 −bacterium, Thermochromatium (Tch.) tepidum, exhibits enhanced thermal
stability as compared with that of the mesophilic counterpart, Allochromatium(Alc.) vinosum despite 82% of sequence homology between two species.
Recently, it was proposed that several factors including numbers of hydrogenbonds, flexibility and compactness of the structure enhance the thermal stabilityof Tch. tepidum Cyt c’. To understand more details of the thermostabilization,
we focused on the amino acid residues at D131 or R129 position in the Cterminal region of Tch. tepidum Cyt c’, and introduced single mutation using theE. coli expression system. The Cyt c’ mutants were examined by differential
scanning calorimetry (DSC) or resonance Raman spectroscopy to reveal theeffects of C-terminal residues on the thermal stability or the heme environmentof Cyt c’. The D131K and D131G mutants in the air-oxidized forms exhibitedthe denaturing temperatures, significantly lower than that of control Cyt c’. In
contrast, R129K and R129A mutants denatured at the temperatures comparable
to that of control Cyt c’. The resonance Raman bands of the control Cyt c’ at1635 and 1625 cm-1 for the intermediate-spin and high-spin states, respectively,
were also affected by the mutations. Based on the results presented here andother finidngs, possible roles of the C-terminal residues of the Tch. tepidum Cyt
c’ are discussed.
3PT234

好熱性紅色硫黄細菌 Thermochromatium tepidum 由来光捕集

1 複合体における BChl-a と Trp 残基間の水素結合相互作用
Hydrogen-bonding interactions between BChl-a and Trp
residues in the light-harvesting1 complexes from Thermochromatium tepidum
Yong Li1, Yukihiro Kimura1,2, Tomoko Numata3, Yuta Inada1, Teruhisa
Arikawa1, Seiu Otomo4, Takashi Ohno1 (1Grad. Sch. Agri. Sco, Kobe
Univ., 2OAST, Kobe Univ., 3HORIBA Co., Ltd., 4Fac. Sci., Ibaraki Univ.)
The light-harvesting 1 reaction center (LH1-RC) from Thermochromatium(Tch.) tepidum exhibits an unusually red-shifted Qy absorption at 915 nm(B915) and the enhanced thermal stability in the presence of Ca ions. To accessfurther details, thermal and spectroscopic properties of the LH1-RCs from wildtype and Sr-substituted Tch. tepidum were investigated by near infrared Raman
spectroscopy and differential scanning calorimetry (DSC). The LH1-RCs
purified from the Sr-substituted Tch. tepidum exhibited the Qy maximum at 888
nm (B888), blue-shifted by 27 nm from the native position. The B915 exhibitedC3-acetyl/C13-keto νC=O bands at 1637/1675 cm-1, which were shifted to~1643/1673 cm-1 when Ca was biosynthetically replaced with Sr. The removalof the original metal cations from B915 and B888 resulted in marked band shiftsof the C3-acetyl/C13-carbonyl νC=O modes to ~1645/~1670 cm-1, supporting
the metal cations to be involved in the fine-tuning of the hydrogen-bonding
between the BChl-a and LH1-polypeptides. The hydrogen-bonding interactions
were almost identical between the Ca2+-depleted B915 and Sr-depleted B888
and between B915 and Ca-substituted B888, despite the significant differencesin their LH1 Qy positions and the DSC denaturing temperatures. Effects of themetal-depletion/-substitution on the hydrogen-bonding interactions strongly
indicate that some factors besides the hydrogen-bondings between BChl-a andLH1-polypeptides are also responsible for the unusual Qy red-shift of the Tch.
tepidum LH1-RC complexes.
3PT235

緑色硫黄細菌のホモダイマ̶光合成反応中心において部位特異的変異が一次電子供与体周辺の構造に与える影響

Local Structural Modifications around the Primary Electron
Donor of the Green Sulfur Bacterial Photosynthetic Reaction
CenterChihiro Azai1, Yuko Sano1, Takumi Noguchi1, Hirozo Oh-oka2 (1Div. Mat. Sci.,
Grad. Sch. Sci., Nagoya Univ., 2Dept. Biol. Sci., Grad. Sch. Sci., Osaka Univ.)
Green sulfur bacteria are obligatory anaerobic photosynthetic bacteria. Their
photosynthetic reaction center (RC) has a unique homodimeric core-protein,which consists of two identical core-polypeptides, while any other kinds of RCs
have heterodimeric core-proteins. The homodimeric structure is considered as aprimitive character from the view of molecular evolution, and the green sulfur
bacterial RC is expected to be a model for the ancestral RC. However, thestructure and the function of green sulfur bacterial RC are barely unknown
because of difficulties in biochemical manipulation arisen from its anaerobic
property. Therefore, molecular biological approach such as analyses using sitedirected mutants is strongly desired to break through the present situation.
This is the first report of site-directed mutants of green sulfur bacterial RC. First,
based on the X-ray structural information of Photosystem I, a homologue ofgreen sulfur bacterial RC, we predicted the amino acid residues nearby the

primary electron donor P840, a special dimer of bacteriochlorophyll a. Then, weconstructed the amino acid substitution mutant RC for each residue, and studied
its effect on the P840 with light-induced FTIR spectroscopy. As a result, thestructural modification was clearly observed as a shift of a keto C=O band ofbacteriochlorophyll a attributable to the P840
3PT236

Light-harvesting Antenna Phosphorylation Enhances Protein
Mobility in Thylakoid Membranes
Masakazu Iwai1,2, Changi Pack2, Yoshiko Takenaka3, Yasushi Sako2, Akihiko
Nakano2,4 (1JST PRESTO, 2RIKEN ASI, 3AIST, 4University of Tokyo)
Phosphorylation of chloroplast light-harvesting antenna complex II (LHCII) is
essential for the energy balance between photosystem I (PSI) and photosystem
II (PSII) in photoadaptation. However, molecular details of how LHCII
phosphorylation causes to switch the energy flow between the two photosystems
are still unclear. Single molecule measurements by using fluorescencecorrelation spectroscopy reveals that LHCII phosphorylation enhances protein
mobility in a certain subfraction of thylakoid membranes in a unicellular green
alga Chlamydomonas reinhardtii. Studies on a mutant of LHCII kinase, stt7,
also revealed that the lack of LHCII phosphorylation slowed protein mobility inthe thylakoid membrane. Our working model illustrates the possible function of
LHCII phosphorylation that affects the molecular crowding in thylakoid
membranes to counterbalance the energy levels between PSI and PSII.
3PT237

Essential role of NADPH oxidase in vitamin D3- and PMAinduced monocytic differentiation of PLB-985 cells
Hiroyuki Kato, Omi Nawa, Asuka Kato, Wakako Hiraoka (Meiji University)
Peripheral cellular differentiation is an important process for keeping
homeostasis especially immune response and development. We have studied therole of reactive oxygen species (ROS) from NADPH oxidase in the process of
monocytic differentiation of myeloid leukemia PLB-985 cells. Exposure to bothor either tumor promoter Phorbol 12-myristate 13-acetate (PMA) and bioactive
secosteroid 1 α, 25-dihydroxyvitamin D3 (vitamin D3) for a few days led thecells to acquire monocytic properties such as phagocytosis and adhesion
activity. Both ESR spin-trapping and chemiluminescence revealed that NADPH
oxidase was activated also in immature state of PLB-985 cells, in which OHand O2- were detected. In order to clarify the role of NADPH oxidase and ROS,
we employed NADPH oxidase gp91phox knockout X-CGD cells, whose
response to PMA and VitD3 were compared to that of normal PLB-985 cell. In
normal cells, VitD3 showed an intense adhesion to dishes, on the other hand
PMA produced the adhesion property of cell-to-cell. These results indicate thatthe function of vitamin D3 was not identical to that of PMA in the process of
monocytic differentiation. The antigen expression of surface molecule upon
phagocytosis was measured with flow cytometry. The degree of expression of
Mac-1α (leukocyte surface antigen :CD11b) and ICAM-1 (CD54) depended on
stimulants and cells. Our result shows that there is some important role of
NADPH oxidase in vitamin D3- and PMA-induced monocytic differentiation of
PLB-985 cells.
3PT238

Effects of inositol hexaphosphate apatite cements on humancells and a biological implication between the cements and ROS

Asuka Kato, Mamoru Aizawa, Wakako Hiraoka (Sch. Sci.&Tec, Univ. Meiji)
Inositol hexaphosphate (IP6), phytic acid, is a potent metal chelator, and is
known as an anti-cancer reagent. Recently, IP6 has received a lot of attention as
ingredient in Hydroxyapatite (Ca10 (PO4)6 (OH)2; HAp) material of artificial
bones and teeth. In this report, we examined the antioxidant effects of IP6 andHAp-IP6 cements, which are created by mixing the HAp powder modified with
IP6, on cell-free and cell culture systems. The enzymatic superoxide (O2 -) from
Hypoxanthine-xanthine oxidase (XO) and hydroxyl radical (HO·) from a fenton
reaction (Fe(II)-H2O2) were employed for antioxidant assay using electron spinresonance (ESR) combined with spin-trapping. In vitro systems, in the case ofIP6, O2- and HO· were suppressed at neutral pH. In contrast, the effect of HApIP6 was so sensitive to pH fluctuation as far as HO· is concerned. For the
biological assay, human leukemia PLB-985 and X-CGD (NADPH oxidase
knockout cell) cultures were utilized. Cells were incubated with 1-10 mM of IP6(pH7.4) for 24 hours. Both the inhibition of cell proliferation and the inductionof apoptosis were detected in the cells treated with a concentration higher than 4
mM of IP6. When the effect of HAp-IP6 to cell survival at pH 7.4 was examined,
there was a distinct difference between PLB-985 and X-CGD. It is possible that
there’ s some biological effect between HAp-IP6 and ROS. These results− S181 −suggested that HAp-IP6 has a potential to activate osteoblastic and osteoclastic
cells in bone formation.
3PT239MHz ultrasound inhibits leukemia cells
Risa Fuji, Yusuke Kobayashi (Grad.Sch. Sci.&Tec, Univ. Meiji)
Recently, ultrasound has been a lot of attention in medical field because of its
widely usage in diagnosis and therapeutic treatment. However, the information
about the therapeutic treatment mechanism and its safety of MHz ultrasound isinsufficient. Therefore we have studied the biological effect of MHz ultrasoundon human lymphoma U937 cells in order to properly evaluate the therapeutic
effect of ultrasound. For an ultrasound apparatus, ceramic resonators of 1.0
MHz and 2.4 MHz were used and placed in the bottom of water tank made ofbrass. A glass tube containing 2. 0 ml cell suspension was set above thetransducer on the water. The frequency of ultrasound was 1.0 MHz, 2.4 MHz, 3.
4 MHz, 5.7 MHz and 7.9 MHz. The intensity of ultrasound was determined withcalorimetric method. The cells were irradiated with ultrasound for 10 seconds.
Because the ultrasonic irradiation to the cells leads to excessive cell death ofnormal cells, we examined the survival rate of cells. The reproductive cell death
was measured for three days after irradiation. In addition, we examined theinduction of apoptosis one day after irradiation by flow cytometer. As a result,the cell growth of U937 was inhibited and the induction of apoptosis wasobserved in 1.0 MHz and 2.4 MHz. On the other hand, the effect of ultrasoundon the cell growth was not seen in 7.9 MHz.

− S182 −Name Index (索引)
名字（Family Name）のアルファベット順にソートしています。すべて、オンラインで入力されたデータのまま、表示しています。

2PS053 (S119)
Ando, Toshio (安藤 敏夫)
Aathi, Karunakaran (Aathi Karunakaran)
3PS004 (S146)
Abdul Razak, Nur Wahida (Abdul Razak Nur Wahida)
3PT126 (S162)
Abe, Jun (阿部 淳)
1PT144 (S93)
Abe, Keiko (阿部 啓子)
3PT189 (S174)
Abe, Shuji (阿部 修治)
1PT127 (S90)
Abe, Takahiro (阿部 貴寛)
2PS046 (S118)
Abe, Tetsuya (阿部 哲也)
1PT123 (S89)
Abe, Tetsuyuki (阿部 哲之)
1PT159 (S96)
Abe-Yoshizumi, Rei (吉住 玲)
1B1424 (S21)2F1412 (S47)
2I1448 (S54)
2PT181 (S136)
Accardi, Alessio (アッカーディ アレッシオ)
3PT118 (S160)
Adachi, Kengo (足立 健吾)
1PS031 (S79)
3PS030 (S151)
Adachi, Shin-ichi (足立 伸一)
3PS024 (S150)
Adachi, Shinichi (足立 伸一)
1H1610 (S35)
Adachi, Taiji (安達 泰治)
1PT133 (S91)
1PT148 (S94)
1PT219 (S105)
2PT145 (S129)
Aigaki, Toshiro (敏郎 相垣)
Aihara, Kazuyuki (合原 一幸)
1SC-04 (S4)Aihara, Ryuzo (相原 龍三)
1H1546 (S34)
Aihara, Tomoki (相原 朋樹)
2H1412 (S52)
Aihara, Yusuke (相原 悠介)
2F1510 (S48)
Aikawa, Yoshiki (相川 佳紀)
1PT123 (S89)
Aita, Yusuke (相田 雄亮)
1PT128 (S90)
1YS0930 (S90)
Aizawa, Mamoru (相澤 守)
3PT238 (S181)
Aizawa, Shin-Ichi (相沢 慎一)
2PS031 (S115)
2PS032 (S115)
Aizawa, Sin-Ichi (相沢 慎一)
2PS033 (S115)
Aizawa, Tomoyasu (相沢 智康)
1C1400 (S23)
1PT138 (S92)
2PT177 (S135)
2PT179 (S136)
Akaike, Toshihiro (赤池 敏宏)
1D1400 (S25)
3PS045 (S154)
Akanuma, Satoshi (赤沼 哲史)
1PT135 (S91)
2PT203 (S139)
Akasaka, Kazuyuki (赤坂 一之)
1SB-03 (S2)
1B1400 (S21)
2E1448 (S46)
2E1522 (S46)
Akashi, Satoko (明石 知子)
1PT190 (S101)
Aki, Yayoi (安藝 弥生)
2PT159 (S132)
Akimoto, Takuma (秋元 琢磨)
1PT004 (S84)
3PT155 (S167)
Akimoto, Yuki (秋元 勇輝)
1PT214 (S104)
Akinori, Sarai (皿井 明倫)
2PT102 (S121)
Akitaya, Tatsuo (秋田谷 龍男)
3C0924 (S60)
Akiyama, Hidefumi (秋山 英文)
2PT187 (S137)
Akiyama, Minato (秋山 源)
3PT125 (S162)
Akiyama, Nobuhiko (秋山 信彦)
1PT123 (S89)
Akiyama, Shuji (秋山 修志)
3B1110 (S59)
Akiyama, Yoshinori (秋山 芳展)
3PT122 (S161)
Akiyama, Yutaka (秋山 泰)
2PT104 (S122)
Akiyoshi, Kazunari (秋吉 一成)
3PT125 (S162)
Akutsu, Tatsuya (阿久津 達也)
1PT191 (S101)
Alam, Jahangir Md. (ALAM Jahangir Md.)
3PT173 (S171)
Alam, Jahangir Md. (Alam Jahangir Md.)
2G1424 (S50)
Alhibshi, Amani (あるひぶし あまーに)
3PT209 (S176)
Amano, Aya (天野 亜耶)
2PT164 (S133)
Amano, Ken-ichi (天野 健一)
1PT137 (S92)
3PT116 (S160)
Amano, Marie (天野 万里)
2PT109 (S123)
Amano, Shota (天野 翔太)
1PT221 (S106)
Amemiya, Takayuki (Amemiya Takayuki)
1I1436 (S35)
Amemiya, Takayuki (雨宮 崇之)
1I1424 (S35)
Aminaka, Ryohta (網中 良太)
3PT129 (S162)
Amino, Misako (網野 美紗子)
3PS002 (S146)
Amisaki, Takashi (網崎 孝志)
3PT106 (S158)
Amitani, Ichiro (網谷 一郎)
1PS045 (S82)
Andachi, Tomoyo (安達 知世)
3PT156 (S167)
Ando, Genki (安藤 元気)
1I1510 (S36)
Ando, Hiromune (安藤 弘宗)
2PS024 (S114)
Ando, Joji (安藤 譲二)
2SH-06 (S16)Ando, Koji (安藤 耕司)
3D1046 (S63)

Ando, Shoji (安藤 祥司)
Ando, Toshio (ANDO TOSHIO)
Ando, Toshio (Ando Toshio)
Ando, Toshio (安藤 敏夫)

Ando, Yuriko (安藤 友里子)
Ando, Yuta (安藤 優太)
Aoki, Akihiro (青木 昭宏)
Aoki, Hiroyoshi (青木 弘良)
Aoki, Yoshimitsu (青木 義満)
Aonuma, Hiroka (青沼 宏佳)
Aoyagi, Satoka (青柳 里果)
Aoyama, Kazuhiro (青山 一弘)
Aoyama, Susumu (青山 晋)
Arai, Hidenao (新井 秀直)
Arai, Hidenobu (新井 秀信)
Arai, Munehito (新井 宗仁)

Arai, Noriyoshi (荒井 規允)
Arai, Ritsuko (荒井 律子)
Arai, Ryoichi (新井 亮一)
Arai, Yoshihiro (新井 善博)
Arakawa, Takamasa (荒川 貴将)
Arakawa, Tsutomu (荒川 力)
Araki, Tsutomu (荒木 勉)
Aramaki, Shinji (荒牧 慎二)
Arata, Toshiaki (荒田 敏昭)

Arata, Yukinobu (荒田 幸信)
Arikawa, Keisuke (有川 敬輔)
Arikawa, Kentaro (蟻川 謙太郎)
Arikawa, Teruhisa (有川 曜央)
Arimatsu, Hiroshi (有松 寛)
Arisaka, Fumio (有坂 文雄)

Arita, Mayuno (有田 真優乃)
Ariyama, Hirotaka (有山 弘高)
Arizono, Misa (有薗 美沙)
Asada, Mizue (浅田 瑞枝)
Asai, Yusuke (浅井 佑介)
Asai, Yusuke (浅井 祐介)
Asakawa, Yoshinori (浅川 義範)
Asakura, Tetsuo (朝倉 哲郎)
Asano, Ryutaro (浅野 竜太郎)
Asano, Toshifumi (浅野 豪文)
Asano, Yuka (浅野 友香)
Asano, Yuuki (浅野 裕輝)
Asao, Akihiro (浅尾 晃央)
Asashima, Makoto (浅島 誠)
Asaumi, Akira (浅海 光)
Ashida, Masaaki (芦田 昌明)
Ashizawa, Ryota (芦沢 竜壮)
Ataka, Kenichi (安宅 憲一)
Atomi, Tomoaki (跡見 友章)

Atomi, Yoriko (跡見 順子)

Atsumi, Tatsuo (渥美 龍男)
Awazu, Akinori (粟津 暁紀)

Ayade, Heev (Ayade Heev)
Azai, Chihiro (浅井 智広)
Azechi, Satoe (疇地 里衣)
Azuma, Ryuzo (我妻 竜三)− S183 −1PS018 (S77)
3F1058 (S67)
1H1522 (S34)
2SB-03 (S10)1H1510 (S34)
2G1534 (S51)
1PS046 (S82)
3PS004 (S146)
3PS016 (S148)
3PS017 (S148)
2PT155 (S131)
2PT156 (S131)
2D1448 (S44)
3E0924 (S64)
1PT111 (S87)
1I1610 (S37)
2SH-01 (S16)
1I1546 (S36)
3PS027 (S150)
2A1400 (S38)
2PS043 (S117)
3PS005 (S146)
2PT146 (S130)
3A0936 (S56)
1PT144 (S93)
1PT152 (S94)
1PT158 (S95)
1PT160 (S96)
3PT155 (S167)
1SD-04 (S4)
1PT106 (S86)
1H1534 (S34)
1H1546 (S34)
3PT010 (S157)
1PT005 (S84)
1H1558 (S34)
2A1400 (S38)
2H1412 (S52)
3B1046 (S59)
1PS038 (S80)
1D1424 (S25)
1PT134 (S91)
2PT190 (S138)
1YS1015 (S138)
3PT234 (S181)
2PT240 (S145)
1PS012 (S76)
1PT156 (S95)
2PT144 (S129)
2PT121 (S125)
3PT140 (S165)
3PT206 (S175)
3D1022 (S63)
3F0936 (S66)
2G1610 (S51)
1D1610 (S26)
3E0924 (S64)
1PT111 (S87)
1C1448 (S23)
1PS010 (S75)
1PT211 (S104)
1PT190 (S101)
2PT209 (S140)
3PT207 (S176)
2PS030 (S115)
1H1558 (S34)
3D1034 (S63)
1F1546 (S30)
1SB-04 (S2)
1SB-05 (S2)
1SB-06 (S2)
1SB-04 (S2)
1SB-05 (S2)
1SB-06 (S2)2I1610 (S55)
2PT135 (S128)
2PT141 (S129)
2PT214 (S141)
2PT218 (S141)
1PT125 (S90)
2PT240 (S145)
3PT235 (S181)
3PT149 (S166)
2D1412 (S44)

Azuma, Takachika (東 隆親)Baba, Akinori (馬場 昭典)
Baba, Reisuke (馬場 玲輔)
Baba, Teruhiko (馬場 照彦)
Baba, Toshihiko (馬場 俊彦)
Baigl, Damien (ベイグル ダミアン)
Bannai, Hiroko (坂内 博子)
Banno, Masaki (番野 雅城)
Banno, Miho (阪野 美穂)
Banno, Satomi (坂野 聡美)
Bao, Yulong (包 玉龍)

3B0924 (S58)
2PT226 (S143)
1C1424 (S23)
3PT169 (S170)
3PT123 (S161)
2G1436 (S50)
3PT206 (S175)
2PT006 (S120)
2PT117 (S124)
3PS036 (S152)
1PS011 (S75)
1PS013 (S76)
1PS045 (S82)
Baskin, Ronald (Baskin Ronald)
1PT112 (S87)
Baxter, Nicky (Baxter Nicky)
Berry, Richard (Berry Richard)
1A1534 (S20)
Bertz, Morten (Bertz Morten)
3PT166 (S169)
Bhattacharyya, Bhaswati (Bhattacharyya Bhaswati)
3H1058 (S71)
Biyani, Manish (ビヤニ マニッシュ)
2PT146 (S130)
Blair, D. (Blair David)
2SA-02 (S9)Bloquel, David (Bloquel David)
1H1522 (S34)
Bremerich, Marcel (Bremerich Marcel) 2PT240 (S145)
Caaveiro, Jose (カーベイロ ホセ)
3F0912 (S65)
Caaveiro, Jose M. M. (Caaveiro Jose M. M.)
2PT151 (S131)
Campbell, Kevin L. (Kevin L. Campbell)
1PT101 (S85)
Cao, Wei (曹 巍)
2PT010 (S121)
Carlton, Peter (Carlton Peter)
2SC-05 (S11)
Cervantes-Salguero, Keitel (Cervantes-Salguero Keitel)
3PT109 (S159)
Chadda, Ankita (チャダ アンキタ)
3PT172 (S170)
Chaen, Shigeru (茶圓 茂)
2PS013 (S112)
2PS014 (S112)
Chandak, Mahesh (CHANDAK MAHESH)
1B1412 (S21)
Chang, Le (常 楽)2E1558 (S47)
Chatani, Eri (茶谷 絵理)
1E1424 (S27)
Chaudhuri, Abhishek (Chaudhuri Abhishek)
3H1058 (S71)
Che, Yong-Suk (蔡 栄淑)
2PS001 (S110)
2PS004 (S110)
Chen, Jin (CHEN Jin)
1B1412 (S21)
Chen, Jin (陳 進)1PT143 (S93)
Chiba, Masataka (千葉 雅隆)
3H1110 (S71)
Chiba, Shuntaro (千葉 峻太朗)
2PT185 (S137)
3PT133 (S163)
Chiba, Shuntaro (千葉 峻太郎)
3PT160 (S168)
Chiba, Susumu (千葉 奏)
3PT131 (S163)
Chijenji, Geroge (千見寺 浄慈)
2PT004 (S120)
Chijimatsu, Reiko (千々松 伶子)
2A1424 (S38)
Chikayama, Eisuke (近山 英輔)
2PT237 (S145)
Chikenji, George (千見寺 浄慈)
2PT002 (S119)
2PT003 (S120)
2PT009 (S121)
2PT202 (S139)
Chiwata, Ryohei (千綿 亮平)
3A1022 (S57)
Choi, Seokwoo (崔 錫宇)
2PT175 (S135)
Christie, John M. (Christie John M)
2F1558 (S49)
Crane, Brian R. (Crane Brian R.)
2SA-02 (S9)Dasgupta, Bhaskar (Dasgupta Bhaskar)
2B1436 (S40)David von, Stetten (デヴィッド フォン ステッテン)
2SH-01 (S16)
Davis, Tim (Davis Tim)
1PS040 (S81)
Dedachi, Kenichi (出立 兼一)
2B1400 (S40)
Deguchi, Shinji (出口 真次)
3H1022 (S70)
2PS036 (S116)
Deisseroth, Karl (Deisseroth Karl)
1PT128 (S90)
1YS0930 (S90)
Demura, Makoto (出村 誠)
1SH-01 (S7)1C1400 (S23)
3C0924 (S60)
2PT176 (S135)
2PT177 (S135)
2PT179 (S136)
2PT182 (S136)
Dewa, Takahisa (出羽 毅久)
2G1558 (S51)
Dhara, Koushik (Dhara Koushik)
1C1424 (S23)
Ding, Da-Qiao (丁 大橋)
2SG-06 (S15)
Dombrowski, Christopher (Dombrowski Christopher)
1PS045 (S82)
Dono, Kohei (道野 宏平)
2PS033 (S115)
Dora, Sujit Kumar (Dora Sujit Kumar)
1H1522 (S34)
Dos Remedios, Cristobal G. (dos Remedios Cristobal G.)
1PS016 (S76)Ebihara, Tatsuhiko (海老原 達彦)
Ebina, Teppei (蝦名 鉄平)
Ebisuya, Miki (戎家 美紀)
Echigo, Akinobu (越後 輝敦)
Eiraku, Mototsugu (永楽 元次)
Ejima, Daisuke (江島 大輔)
Endo, Masahiro (遠藤 将弘)
Endo, Masayuki (遠藤 政幸)

3E1022 (S64)
2PT001 (S119)
2SG-04 (S15)
2PT201 (S139)
1PT219 (S105)
2H1534 (S53)
1PT226 (S107)
1PS042 (S81)
3PT111 (S159)
3PT190 (S174)
Endo, Shigeru (猿渡 茂)
3A0924 (S56)
Enoki, Sawako (榎 佐和子)
2PT234 (S144)
Enomoto, Koichiro (榎本 洸一郎)
3F1058 (S67)
Ewald, Maxime (EWALD MAXIME)
Feig, Michael (Feig Michael)
2SD-01 (S12)Feig, Michael (Michael Feig)
1PT201 (S102)
1YS1100 (S102)
Ferreon, Josephine C. (Ferreon Josephine C.)
1PT158 (S95)
Fuchigami, Sotaro (渕上 壮太郎)
1PT190 (S101)
2PT134 (S127)
Fuji, Kana (冨士 香奈)
1PT189 (S101)
3PT239 (S182)
Fuji, Risa (藤 里砂)
3D0936 (S62)
Fujihashi, Yuta (藤橋 裕太)
Fujii, Hiroshi (藤井 浩)
3I0924 (S71)
3I1046 (S73)
Fujii, Masaaki (藤井 正明)
3PS038 (S152)
Fujii, Masashi (藤井 雅史)
2PT141 (S129)
2PT218 (S141)
Fujii, Satoshi (藤井 聡)
1C1534 (S24)
2PT106 (S122)
3PT108 (S158)
Fujii, Takashi (藤井 高志)
2SB-05 (S10)
2SE-01 (S12)
Fujii, Takasi (藤井 高志)
3PT184 (S173)
Fujii, Takeshi (藤井 毅)
Fujii, Tetsuya (藤井 徹矢)
1PT133 (S91)
Fujii, Yasuhiro (藤井 康裕)
3PT154 (S167)
Fujimoto, Eriko (藤本 恵理子)
2I1610 (S55)
Fujimura, Shoko (藤村 章子)
3PS030 (S151)
Fujino, Masayuki (藤野 真之)
1PT102 (S85)
Fujisaki, Hiroshi (藤崎 弘士)
2B1412 (S40)
1PT170 (S98)
1PT189 (S101)
2PT137 (S128)
Fujisawa, Masao (藤澤 雅夫)
2B1510 (S41)
Fujisawa, Takuya (藤澤 拓也)
3G1010 (S68)
Fujita, Eri (藤田 恵理)
1SB-04 (S2)
1SB-06 (S2)
Fujita, Hideaki (藤田 英明)
3PS037 (S152)
Fujita, Katsumasa (藤田 克昌)
3PS037 (S152)
Fujita, Keisuke (藤田 恵介)
1PS049 (S82)
1YS0900 (S82)
1PT006 (S84)
Fujita, Masaya (藤田 昌也)
1PT163 (S96)
Fujita, Shuji (藤田 嵩治)
3PS004 (S146)
Fujita, Yutaro (Fujita Yutaro)
1PT114 (S87)
Fujita-Yanagibayashi, Sari (柳林(藤田) 彩理)
1F1436 (S29)
Fujitani, Hideaki (藤谷 秀章)
1PT173 (S98)
Fujiwara, Hitomi (藤原 瞳)
1PT163 (S96)
Fujiwara, Kei (藤原 慶)
1E1448 (S27)
1PT226 (S107)
1PT227 (S107)
1PT228 (S107)
Fujiwara, Kuniyo (藤原 邦代)
2PT182 (S136)
Fujiwara, Satoru (藤原 悟)
2H1400 (S51)
1PT003 (S83)
Fujiwara, Shin-ichi (藤原 伸一)
3PT106 (S158)
Fujiwara, Takahiro (藤原 敬宏)
1H1448 (S33)
1PT209 (S103)
2PS041 (S117)
Fujiwara, Takahiro K. (藤原 敬宏)
1G1412 (S31)
3PT172 (S170)
Fujiyoshi, Satoru (藤芳 暁)
2SI-03 (S17)
Fujiyoshi, Yoshinori (藤吉 好則)
3B0900 (S57)
3F0924 (S66)
Fukada, Harumi (深田 はるみ)
2E1522 (S46)
Fukagawa, Akihiro (深川 暁宏)
3PT104 (S158)
Fukaminato, Tuyoshi (深港 豪)3PT102 (S157)
Fukano, Hiroko (深野 弘子)
1PT238 (S109)
Fukuchi, Satoshi (Fukuchi Satoshi)
1I1436 (S35)
Fukuchi, Satoshi (福地 佐斗志)
1I1424 (S35)
Fukuda, Ikuo (福田 育夫)
3PT010 (S157)
Fukuda, Mao (福田 真生)
3PS019 (S149)
Fukuda, Masaki (福田 真己)
1PT135 (S91)
Fukuda, Norio (福田 紀男)
2H1510 (S52)
2H1546 (S53)
1PS009 (S75)
1PS016 (S76)
Fukuda, Shingo (福田 真悟)
1H1510 (S34)
Fukuda, Takafumi (福田 隆文)
1PT151 (S94)
Fukuda, Tetsuya (福田 哲也)
2G1610 (S51)
3F0936 (S66)
Fukuda, Toshio (福田 敏男)
1PT229 (S107)

1PT233 (S108)
1PT106 (S86)
2PS045 (S118)
2SE-02 (S13)2PS005 (S110)
1PT169 (S97)
2PT122 (S125)
2I1546 (S55)
2I1558 (S55)
2PS003 (S110)
1PT139 (S92)
Fukuoka, Mayuko (福岡 万佑子)
3PS027 (S150)
Fukushima, Kazuhiko (福島 和彦)
3PS036 (S152)
Fukushima, Mitsuyasu (福島 光鎮)
Fukushima, Yoshimasa (福島 佳優)
3PT228 (S180)
Funatsu, Takashi (船津 高志)
3C1058 (S61)
1PT208 (S103)
1PT234 (S108)
1PT236 (S108)
3PS010 (S147)
1YS0915 (S147)
3PS029 (S151)
3PS040 (S153)
1G1510 (S31)
Funayama, Tomoo (舟山 知夫)
Furuike, Shou (古池 晶)
3A1022 (S57)
1PS035 (S80)
Furukawa, Ayako (古川 亜矢子)
3C1034 (S61)
3PT003 (S155)
Furukawa, Eiji (古川 英嗣)
2PT110 (S123)
Furukawa, Taichi (古川 太一)
1H1558 (S34)
Furukawa, Yoshiaki (古川 良明)
2B1534 (S41)
2B1546 (S41)
2B1558 (S41)
2B1610 (S41)
3C0900 (S59)
Furukawa, Yukio (古川 進郎)
2PS005 (S110)
Furuki, Takao (古木 隆夫)
1PT162 (S96)
Furuki, Takao (古木 隆生)
3G0912 (S67)
Furuno Tadahide (古野 忠秀)
1PT206 (S103)
Furuno, Tadahide (古野 忠秀)
2D1558 (S45)
3PT176 (S171)
Furuno, Taiji (古野 泰二)
3PS018 (S149)
Furusawa, Chikara (古澤 力)
1PT218 (S105)
Furusawa, Hiroyuki (古澤 宏幸)
1PT162 (S96)
Furusawa, Kei (古澤 敬)
3PT233 (S180)
Furuta, Akane (古田 茜)
3PS002 (S146)
Furuta, Ken'ya (古田 健也)
1SI-04 (S8)2PS019 (S113)
3PS002 (S146)
Furuta, Masakazu (古田 雅一)
2I1610 (S55)
Furuta, Miyuki (古田 美由紀)
3PT135 (S164)
Furuta, Tadaomi (古田 忠臣)
2PT125 (S126)
2PT137 (S128)
3PT133 (S163)
3PT160 (S168)
Furutani, Yuji (古谷 祐詞)
1C1546 (S24)
1C1558 (S24)1F1400 (S29)
2G1610 (S51)
3F0936 (S66)
2PT182 (S136)
2PT183 (S137)
3PT168 (S170)
Furutani-Umezu, Nozomi (梅津 のぞみ)
1PS051 (S83)
Furuya, Kishio (古家 喜四夫)
1PT203 (S102)
Furuya, Sonoko (古家 園子)
1PT203 (S102)
Futaki, Shiro (二木 史朗)
2SB-02 (S10)Ge, Zhenyi (葛 臻翼)
2PT010 (S121)
Geetha, Hiremath (Geetha Hiremath)
2PS049 (S118)
Gekko, Kunihiko (月向 邦彦)
1PT108 (S86)
Gennis, Robert (Gennis Robert)
2PT155 (S131)
2PT156 (S131)
Getzoff, Elizabeth (Getzoff Elizabeth)
2F1546 (S49)
Getzoff, Elizabeth D. (Getzoff Elizabeth D)
2F1558 (S49)
Getzoff, Elizabeth D. (ゲトゾフ エリザベス)
2F1534 (S49)
Go, Mitiko (郷 通子)
2PT127 (S126)
Go, Nobuhiro (郷 信広)
3PS026 (S150)
Gohara, Kazutoshi (郷原 一寿)
1D1522 (S26)
Gohara, Mizuki (郷原 瑞樹)
1B1424 (S21)
2I1448 (S54)
Golestanian, Ramin (Golestanian Ramin)
1SF-03 (S6)
Gomibuchi, Yuki (五味渕 由貴)
1PS001 (S74)
Gonzalez-Bonet, G. (Gonzalez-Bonet Gabriela)
2SA-02 (S9)Goto, Masaki (後藤 優樹)
3G0936 (S68)
3G0948 (S68)
Goto, Susumu (五斗 進)
2PT107 (S122)
Goto, Takafumi (後藤 孝文)
2PS034 (S115)
Goto, Takashi (後藤 孝)
2G1522 (S50)
3PT158 (S168)
Goto, Touichiro (後藤 東一郎)
3PT148 (S166)
Goto, Yuji (後藤 祐児)
1E1436 (S27)
Fukui, Sadaharu (福井 貞晴)
Fukujin, Fumihito (福神 史仁)
Fukumori, Yoshihiro (福森 義宏)
Fukumura, Takuma (福村 拓真)
Fukunaga, Kentaro (福永 謙太郎)
Fukunaga, Yukihiro (福永 幸裕)
Fukuoka, Hajime (福岡 創)− S184 −1PT145 (S93)
1PT220 (S105)
1PT221 (S106)
3PS019 (S149)
3PT209 (S176)
Gouaux, Eric (Gouaux Eric)
1PT103 (S85)
1YS1030 (S85)
Gowrishankar, Kripa (Gowrishankar Kripa)
3H1058 (S71)
1H1522 (S34)
Gruet, Antoine (Gruet Antoine)
2SH-01 (S16)
Guerin, Laurent (ゲラン ロラン)
2PT210 (S140)
Gunji, Yukio (郡司 幸夫)
2PT234 (S144)
2PT235 (S144)
Gunji, Yukio-Pegio (郡司 ペギオ-幸夫)
2PT236 (S144)
Guo, Hao (Guo Hao)
1C1546 (S24)
Gustafsson, Nils (Gustafsson Nils)
1PS030 (S79)
Habchi, Johnny (Habchi Johnny)
1H1522 (S34)
Hada, Takuya (秦 拓也)
3PT119 (S160)
Haga, Hisashi (芳賀 永)
1PT230 (S107)
Hagiwara, Yohsuke (萩原 陽介)
1B1610 (S22)
3PT133 (S163)
Hagiya, Tomoka (萩谷 朋佳)
1PT206 (S103)
Hagiyama Man (萩山 満)
Hagiyama, Man (萩山 満)
2D1558 (S45)
Hakamada, Kazumi (袴田 和巳)
2PT228 (S143)
3PS048 (S154)
Hakoshima, Toshio (箱嶋 敏雄)
1B1448 (S21)
Hamachi, Itaru (浜地 格)
2PT151 (S131)
Hamada, Daizo (浜田 大三)
1PT159 (S96)
Hamada, Hiroshi (濱田 博司)
1SF-02 (S6)
Hamada, Shogo (浜田 省吾)
1PT227 (S107)
3PT109 (S159)
3PT112 (S159)
3PT113 (S159)
Hamada, Syougo (浜田 省吾)
1PT226 (S107)
Hamada, Tomoyo (浜田 智代)
2D1510 (S44)
3PS020 (S149)
3PS022 (S149)
Hamada, Tsutomu (濱田 勉)
3PT153 (S167)
1YS1045 (S167)
Hamada, Tsutomu (濵田 勉)
3G1022 (S68)
Hamaguchi, Hiro-o (浜口 宏夫)
2SH-03 (S16)
Hamajima, Yuki (濱島 裕輝)
1PT113 (S87)
Hamasaki, Naotaka (濱崎 直孝)
3PT117 (S160)
Hamasaki, Yuuta (浜崎 雄太)
2PT233 (S144)
Han, Sung-Woong (韓 成雄)
1PT148 (S94)
Han, Yong-Woon (韓 龍雲)
1SD-01 (S4)
Hanaoka, Kyohei (花岡 恭平)
3PT222 (S178)
Hanashima, Akira (花島 章)
1PS011 (S75)
1PS013 (S76)
Hanazono, Yuya (花園 祐矢)
1PT123 (S89)
Handa, Daisuke (半田 大祐)
3PT169 (S170)
Handa, Naofumi (半田 直史)
1PS046 (S82)
Haneda, Takahiro (羽子田 貴広)
2PS047 (S118)
Hara, Chiho (原 千穂)
3PT180 (S172)
Hara, Masayuki (原 雅行)
3I1010 (S72)Hara, Noritaka (原 典孝)
2I1424 (S54)Hara, Yukari (原 ゆかり)
3C0936 (S60)
Harada, Kouji (原田 耕治)
2C1558 (S43)
Harada, Ryuhei (原田 隆平)
1PT201 (S102)
1YS1100 (S102)
Harada, Tomoaki (原田 知明)
1PT237 (S109)
Harada, Yoshie (原田 慶恵)
1SD-01 (S4)3C1046 (S61)
1PS042 (S81)
3PS010 (S147)
1YS0915 (S147)
3PS031 (S151)
3PT111 (S159)
Haraguchi, Tokuko (原口 徳子)
2SG-06 (S15)Harata, Masahiko (原田 昌彦)
3PS033 (S151)
3PS034 (S152)
Haruna, Taichi (春名 太一)
2PT217 (S141)
Haruyoshi, Toyoda (豊田 晴義)
3PT214 (S177)
Haseda, Keisuke (長谷田 圭亮)
3PS041 (S153)
Hasegawa, Katsuya (長谷川 克也)
1SB-04 (S2)
1SB-06 (S2)Hasegawa, Koji (長谷川 浩司)
1H1610 (S35)
Hasegawa, Masashi (長谷川 正)
1PT113 (S87)
Hasegawa, Yoshitaka (長谷川 賢卓)
3PT207 (S176)
Hasemi, Takatoshi (長谷見 崇俊)
2PT177 (S135)
Hashi, Yurika (橋 友理香)
2PS035 (S116)
Hashikami, Kentaro (階上 健太郎)
1C1448 (S23)
Hashimoto, Kazuhito (橋本 和仁)
1I1558 (S36)
3D1058 (S63)
Hashimoto, Ken (橋本 健)
3G1010 (S68)
Hashimoto, Mamoru (橋本 守)
1H1558 (S34)
Hasimoto, Yuh (橋本 優)
3PS030 (S151)
Hata, Hiroaki (畑 宏明)
3C0912 (S60)
Hata, Tomonori (秦 智紀)
2A1448 (S38)
Hatae, Hinako (波多江 日成子)
3PT117 (S160)
Hatakeyama, Makoto (畠山 允)
3PT227 (S179)
3PT230 (S180)
Gotoh, Masao (後藤 正男)

Hatakeyama, Tetsuhiro (畠山 哲央)
Hatanaka, Rie (畑中 理恵)
Hatanaka, Yuusuke (畑中 悠介)
Hatano, Ken-ichi (秦野 賢一)
Hatano, Tatsurou (波多野 達朗)
Hatori, Kuniyuki (羽鳥 晋由)
Hatta, Ichiro (八田 一郎)
Hattori, Akihiro (服部 明弘)

Hattori, Junko (服部 純子)
Hattori, Koji (服部 浩二)
Hattori, Motoyuki (服部 素之)
Hattori, Yoshikazu (服部 良一)
Hayakawa, Kimihide (早川 公英)
Hayakawa, Masayuki (早川 雅之)
Hayakawa, Yoshinori (早川 慶紀)
Hayakawa, Yoshinori (早川 美徳)
Hayano, Yosuke (早野 陽介)
Hayashi, Fumio (林 史夫)

Hayashi, Fumio (林 文夫)
Hayashi, Katsuhiko (林 克彦)
Hayashi, Kumiko (林 久美子)

Hayashi, Nobuhiro (林 宣宏)
Hayashi, Ryunosuke (林 龍之介)
Hayashi, Shigehiko (林 重彦)

Hayashi, Takashi (林 高史)
Hayashi, Tomohiko (林 智彦)
Hayashi, Yukiko (林 由起子)
Hayashi, Yuuki (林 勇樹)
Hayashida, Takuya (林田 拓也)
Hazemoto, Norio (櫨本 紀夫)
Head, David (Head David)
Heber, Ulrich (Heber Ulrich)Heberle, Joachim (Heberle Joachim)
Hegemann, Peter (Hegemann Peter)
Hellingwerf, Klaas (Hellingwerf Klaas)
Hexig, Bayar (賀喜 白乙)Hibino, Katsutoshi (日比野 勝俊)
Hibino, Kayo (日比野 佳代)
Hibino, Masahiro (日比野 政裕)
Hida, Akira (肥田 哲)
Hidaka, Tetsurou (日高 徹郎)
Higa, Seiji (比嘉 世滋)
Higo, Junichi (肥後 順一)
Higuchi, Hideo (樋口 秀男)

Higuchi, Yoshiki (樋口 芳樹)
Hiiragi, Takashi (柊 卓志)
Hijikata, Hiroko (土方 博子)
Hikima, Takaaki (引間 孝明)
Himeno, Hiroki (姫野 泰輝)
Hira, Riichiro (平 理一郎)
Hirai, Akira (平井 彰)
Hirai, Hirohisa (平井 啓久)
Hirai, Mitsuhiro (平井 光博)

Hirai, Teruhisa (平井 照久)
Hiraiwa, Tetsuya (平岩 徹也)
Hiramatsu, Hirotsugu (平松 弘嗣)
Hiramitsu, Yuuki (平光 佑基)
Hirano, Atsushi (平野 篤)
Hirano, Ken (平野 研)
Hirano, Minako (平野 美奈子)

3PT232 (S180)
2PT231 (S144)
1PT162 (S96)
3PT213 (S177)
2PT114 (S124)
1PT192 (S102)
1PS048 (S82)
2PS012 (S112)
3G1110 (S69)
3PT151 (S167)
3PS023 (S149)
3PS046 (S154)
3PS049 (S154)
1PT102 (S85)
3PS044 (S153)
3PS051 (S155)
1PT103 (S85)
1YS1030 (S85)
1B1424 (S21)
2B1448 (S40)
2PS034 (S115)
1PT224 (S106)
2C1436 (S42)1SC-02 (S3)
1PT155 (S95)
2PS030 (S115)
2PT110 (S123)
2PT119 (S124)
2PT120 (S125)
2PT180 (S136)
3PT161 (S168)
1PT117 (S88)
2SI-04 (S17)
1PS028 (S78)
1PS029 (S79)
3PT124 (S161)
1PS029 (S79)
3B1034 (S59)
1PT128 (S90)
1YS0930 (S90)
2PT178 (S136)
2PT162 (S133)
2PT164 (S133)
2F1400 (S47)
3PT002 (S155)
1C1436 (S23)
1PT160 (S96)
3PT105 (S158)
3C0924 (S60)
1PS018 (S77)
3PT228 (S180)
1F1546 (S30)
1PT128 (S90)
1YS0930 (S90)
2PT175 (S135)
1D1400 (S25)
3PS045 (S154)
2PT011 (S121)
1G1558 (S32)
2PS044 (S117)
3G1010 (S68)
2PS007 (S111)
1F1558 (S30)
2PT149 (S130)
1PT168 (S97)
1SI-01 (S8)1H1412 (S33)
2A1522 (S39)
2A1558 (S39)
3B0912 (S57)
2SF-05 (S14)
3PT172 (S170)
2D1522 (S45)
3PT146 (S166)
3G1022 (S68)
2SH-05 (S16)
3PT203 (S175)
3PT189 (S174)
1C1610 (S25)
1F1412 (S29)
2PT129 (S126)
3PT137 (S164)
3PT117 (S160)
3H0948 (S70)
2PT226 (S143)
2E1424 (S46)
1PT108 (S86)
3PT158 (S168)
1PT005 (S84)
3PT101 (S157)
2G1546 (S51)
3PT163 (S169)
3PT167 (S169)

Hirano, Yoshinori (平野 秀典)
Hirano, Yuuji (平野 祐司)
Hiraoka, Wakako (平岡 和佳子)

Hiraoka, Yasushi (平岡 泰)
Hirashima Naohide (平嶋 尚英)
Hirashima, Naohide (平嶋 尚英)

Hirashima, Tsuyoshi (平島 剛志)
Hirata, Hiroaki (平田 宏聡)
Hirata, Kunio (平田 邦生)
Hiratsuka, Yuichi (平塚 祐一)
Hirayama, Takuma (平山 卓磨)
Hiroaki, Hidekazu (廣明 秀一)
Hirokawa, Erisa (広川 恵里沙)
Hirokawa, Nobutaka (廣川 信隆)
Hirokawa, Takatsugu (広川 貴次)
Hiromasa, Ohta (大田 浩正)
Hironaka, Ken-ichi (廣中 謙一)
Hirosaki, Ken (廣崎 賢)
Hirosawa, Koichiro M. (廣澤 幸一朗)
Hirose, Mika (廣瀬 未果)
Hirose, Noboru (廣瀬 昇)

Hiroshi, Kihara (木原 裕)
Hiroshima, Akinori (広島 明宣)
Hiroshima, Michio (廣島 通夫)

Hirota, Hiroshi (廣田 洋)
Hirota, Shun (廣田 俊)
Hirotsune, Shinji (広常 真治)
Hisamoto, Naoki (久本 直毅)
Hisatome, Ichiro (久留 一郎)
Hisatomi, Osamu (久冨 修)
Hisatomi, Osamu (久富 修)
Hitomi, Kenichi (人見 健一)
Hitomi, Kenichi (人見 研一)
Hiyama, Miyabi (樋山 みやび)
Hizukuri, Yohei (檜作 洋平)
Ho, Chien (Chien Ho)
Hoerning, Marcel (Hoerning Marcel)
Hojo, Eri (北條 江里)
Hojo, Yasushi (北条 泰嗣)
Hojo, Yasushi (北條 泰嗣)

Homma, Michio (本間 道夫)

Honda, Hajime (本多 元)

Honda, Kazufumi (本田 一文)
Hongo, Kunihiro (本郷 邦広)
Honjoh, Tatsuya (本城 達也)
Honma, Michio (本間 道夫)
Hori, Naoto (堀 直人)

Hori, Yuichiro (堀 雄一郎)
Horigome, Miyako (堀籠 美也子)
Horio, Shuhei (堀尾 修平)
Hoshi, Takahiko (星 貴彦)
Hoshida, Masayuki (星田 政行)
Hoshino, Kazuki (星野 一樹)
Hoshino, Masato (星野 真人)
Hoshino, Rintaro (星野 倫太朗)
Hoshino, Tatsuya (星野 達也)
Hosoda, Kazufumi (細田 一史)
Hosoda, Kazuo (細田 和男)
Hosoi, Makoto (細井 誠)
Hosokawa, Chie (細川 千絵)
Hosokawa, Yoichiroh (細川 陽一郎)

− S185 −1PT004 (S84)
3PS038 (S152)
1PT140 (S92)
3PT237 (S181)
3PT238 (S181)
2SG-06 (S15)
1PT206 (S103)
3PT139 (S164)
3PT176 (S171)
3PT177 (S171)
1G1400 (S31)
3H1046 (S71)
1PT128 (S90)
1YS0930 (S90)
3PS005 (S146)
1PT164 (S97)
2SB-02 (S10)1I1424 (S35)
1PS009 (S75)
3H0900 (S69)
2PT104 (S122)
2PT123 (S125)
2PT220 (S142)
3PT124 (S161)
1G1412 (S31)
1PT116 (S88)
2PT116 (S124)
1SB-04 (S2)
1SB-05 (S2)
1SB-06 (S2)
1PT115 (S88)
1PT175 (S98)
1D1424 (S25)
1G1610 (S32)
3PT104 (S158)
3PT179 (S172)
3E0936 (S64)
1SE-04 (S5)2A1436 (S38)
1PT233 (S108)
1PT104 (S86)
2PT192 (S138)
2F1436 (S48)
2F1534 (S49)
2F1546 (S49)
2F1558 (S49)
2PT187 (S137)
3PT122 (S161)
1PT101 (S85)
1SB-07 (S3)2C1534 (S43)
1C1400 (S23)
3PT217 (S177)
3PT205 (S175)
3PT213 (S177)
3PT216 (S177)
1A1546 (S20)
1A1610 (S20)
1B1424 (S21)
2I1448 (S54)
2I1510 (S54)
2I1522 (S55)
2I1534 (S55)
1PT118 (S88)
1PT229 (S107)
1PT233 (S108)
2PT160 (S132)
1SB-08 (S3)
1PS014 (S76)
1PS015 (S76)
2PS019 (S113)
2PT117 (S124)
1PT164 (S97)
3PT218 (S178)
2I1400 (S53)
3C0948 (S60)
1PT171 (S98)
1PT172 (S98)
1C1424 (S23)3F1046 (S67)
3PT215 (S177)
2D1522 (S45)
1PS014 (S76)
1PT117 (S88)
3PS024 (S150)
3PS050 (S155)
3PT146 (S166)
2PT209 (S140)
1I1424 (S35)
3PT220 (S178)
1D1546 (S26)
1G1400 (S31)
2D1558 (S45)

Hosokawa-Muto, Junji (武藤 淳二)
Hosoya, Satoshi (細谷 悟史)
Hotta, Kohji (堀田 耕司)
Houseman, Grant (Houseman Grant)
Hsu, Wei-Lin (許 維麟)
Hua, Yue-jin (Hua Yue-jin)
Hughes, Kelly T. (Hughes Kelly T.)
Husimi, Yuzuru (伏見 譲)
Ichihashi, Norikazu (市橋 伯一)
Ichii, Momoko (一居 桃子)
Ichikawa, Mai (市川 麻衣)
Ichikawa, Masatoshi (市川 正敏)
Ichikawa, Muneyoshi (市川 宗厳)
Ichikawa, Satoshi (市川 論)
Ichikawa, Satoshi (市川 諭)
Ichikawa, Takaaki (市川 貴章)
Ichiki, Takanori (一木 隆範)
Ichimiya, Masayoshi (一宮 正義)
Ichimura, Kaoru (市村 薫)
Ichimura, Taro (市村 垂生)
Ichinomiya, Katsuo (一宮 勝雄)
Ichiyanagi, Kouhei (一柳 光平)
Ide, Takahiro (井手 隆広)
Ide, Toru (井出 徹)

Igarashi, Kiyohiko (五十嵐 圭日子)
Igarashi, Noriyuki (五十嵐 教之)
Ihara, Kunio (井原 邦夫)

Iida, Yoshihiro (飯田 禎弘)
Iijima, Giichi (飯島 義市)
Iino, Ryota (飯野 亮太)

Iino, Takanori (飯野 敬矩)
Iizuka, Kojiro (飯塚 浩二郎)
Iizuka, Ryo (飯塚 怜)

Ikebe, Emi (池辺 詠美)
Ikebe, Mitsuo (池辺 光男)
Ikeda, Kanami (池田 佳奈美)
Ikeda, Kyohei (池田 京平)
Ikeda, Satoshi (池田 諭史)
Ikeda, Yohei (池田 洋平)
Ikegami, Koji (池上 浩司)
Ikegami, Takahisa (池上 貴久)

Ikeguchi, Masamichi (池口 雅道)
Ikeguchi, Mitsunori (池口 満徳)

Ikeo, Masahito (池尾 眞人)
Ikeuchi, Akinori (池内 暁紀)
Ikezaki, Keigo (池崎 圭吾)
Ikura, Teikichi (伊倉 貞吉)
Imachi, Masayoshi (井町 昌義)
Imada, Katsumi (今田 勝己)
Imada, Katsumi (今田 勝巳)

Imafuku, Yasuhiro (今福 泰浩)
Imai, Hiroo (今井 啓雄)

Imai, Kiyohiro (今井 清博)

1PT155 (S95)
3PT124 (S161)
1PT216 (S105)
3PT204 (S175)
3E0948 (S64)
2PT125 (S126)
1PT129 (S90)
1YS1000 (S90)
2PS028 (S114)
2PT146 (S130)
2PT204 (S139)
1G1534 (S32)
2PT205 (S139)
2PT154 (S131)
1PT235 (S108)
3PT101 (S157)
2A1448 (S38)
2A1546 (S39)
3PS015 (S148)
2D1546 (S45)
2G1522 (S50)
3PT158 (S168)
1PT236 (S108)
2PT147 (S130)
1H1558 (S34)
1PT115 (S88)
1PT157 (S95)
3PS037 (S152)
3PS033 (S151)
2PT121 (S125)
3PS024 (S150)
3PS006 (S146)
3PS007 (S147)
2G1546 (S51)
3PT163 (S169)
3PT167 (S169)
1PT236 (S108)
2PT123 (S125)
1F1522 (S30)
2I1400 (S53)
2PT201 (S139)
1PT165 (S97)
1H1546 (S34)
1A1436 (S19)
3A0924 (S56)
1PS033 (S79)
1YS1115 (S79)
3PT103 (S157)
1G1400 (S31)
2D1558 (S45)
2PT234 (S144)
3C1058 (S61)
1PT234 (S108)
1PT236 (S108)
3PS029 (S151)
1PS018 (S77)
2PS053 (S119)
2PT110 (S123)
1PT159 (S96)
3PS035 (S152)
3PT117 (S160)
3A0912 (S56)
1C1412 (S23)
1PT145 (S93)
3PT178 (S172)
1PT120 (S89)
1PT142 (S93)
1F1534 (S30)
1PS034 (S80)
1PT190 (S101)
2PT134 (S127)
2PT136 (S128)
2PS010 (S111)
1C1510 (S24)
2PS011 (S111)
1C1522 (S24)
1PT146 (S93)
3PT114 (S159)
2SE-05 (S13)
1SA-07 (S2)2I1424 (S54)
1PT105 (S86)
1PT121 (S89)
2PS005 (S110)
2PT109 (S123)
2PT118 (S124)
2PT124 (S125)
1PS030 (S79)
1F1400 (S29)
2PT189 (S138)
3PT189 (S174)
2PT154 (S131)
2PT159 (S132)

Imai, Masayuki (今井 正幸)
Imaizumi, Ryo (今泉 亮)
Imaki, Takahito (今木 貴仁)
Imamoto, Akira (今本 公)
Imamoto, Yasushi (今元 泰)
Imanishi, Miki (今西 未来)
Imao, Asato (今尾 麻人)

Imoto, Daisuke (井元 大輔)
Imoto, Seiya (井元 清哉)
Inaba, Naomichi (稲葉 直道)
Inaba, Satomi (稲葉 理美)
Inaba, Satoshi (稲葉 敏)
Inaba, Takehiko (稲葉 岳彦)
Inaba, Takehio (稲葉 岳彦)
Inada, Noriko (稲田 のりこ)
Inada, Yuta (稲田 勇太)
Inagaki, Kenji (稲垣 賢二)
Inagaki, Kouya (稲垣 宏弥)
Inaguma, Asumi (稲熊 あすみ)
Inai, Koji (稲井 公二)
Inanami, Takashi (稲波 崇)
Inoh, Yoshikazu (伊納 義和)
Inomata, Kohsuke (猪股 晃介)
Inomata, Naoki (猪股 直生)
Inoue, Keiichi (井上 圭一)
Inoue, Masayo (井上 雅世)
Inoue, Masayoshi (井上 正義)
Inoue, Naho (井上 奈穗)
Inoue, Rintaro (井上 倫太郎)
Inoue, Takurou (井上 琢朗)
Inoue, Tsuyoshi (井上 豪)
Inoue, Yasuhiro (井上 康博)
Inoue, Yuichi (井上 裕一)

Inui, Takaya (乾 貴矢)
Irie, Katsumasa (入江 克雅)
Irieda, Hiroki (入枝 泰樹)
Iritani, Yuka (入谷 有香)
Iseki, Mineo (伊関 峰生)
Ishibashi, Munenori (石橋 宗則)
Ishida, Hanako (石田 英子)
Ishida, Hideharu (石田 秀治)
Ishida, Hisashi (石田 恒)
Ishida, Nobuhiro (石田 亘広)
Ishida, Noriyoshi (石田 誠宜)Ishida, Takeshi (石田 武志)
Ishidate, Fumiyoshi (石館 文善)
Ishido, Satoshi (石戸 聡)
Ishigami, Izumi (石上 泉)
Ishihama, Yasushi (石濱 泰)

Ishihara, Jun-ichi (石原 潤一)
Ishihara, Shuji (石原 秀至)

Ishii, Jun (石井 純)
Ishii, Kentaro (石井 健太郎)
Ishii, Shin (石井 信)
Ishii, Shuya (石井 秀弥)
Ishii, Takeshi (石井 毅)
Ishijima, Akihiko (石島 秋彦)

Ishikawa, Kumiko (石川 久美子)

Ishikawa, Takuji (石川 拓司)
Ishikawa, Yoshiro (石川 慶郎)
Ishikita, Hiroshi (石北 央)
Ishikura, Takakazu (石倉 孝一)

2PT161 (S133)
2PT163 (S133)
3PT142 (S165)
1PT120 (S89)
1PT140 (S92)
2PT140 (S128)
1F1424 (S29)
3B0936 (S58)
1SE-03 (S5)2H1424 (S52)
1PS002 (S74)
1PS003 (S74)
1PS004 (S74)
2PT239 (S145)
2SD-04 (S12)3PS034 (S152)
2E1522 (S46)
2PS032 (S115)
3PS032 (S151)
3PS036 (S152)
3PS010 (S147)
1YS0915 (S147)
3PT234 (S181)
2PT109 (S123)
1B1436 (S21)
2PT183 (S137)
2PT208 (S140)
1PT167 (S97)
3PT176 (S171)
2SB-02 (S10)
3PS013 (S148)
1F1448 (S29)
2PT181 (S136)
2C1424 (S42)
1PT142 (S93)
3PT210 (S176)
1E1424 (S27)
3PT210 (S176)
1PT116 (S88)
1PT133 (S91)
1PT219 (S105)
1A1412 (S19)2I1546 (S55)
2I1558 (S55)
1PS041 (S81)
2PS003 (S110)
3PS013 (S148)
3PT186 (S173)
3B0900 (S57)
3PT191 (S174)
3PS036 (S152)
1H1610 (S35)
3D0924 (S62)
1G1412 (S31)
1PT116 (S88)
2PT116 (S124)
2PS024 (S114)
3PT008 (S156)
1C1510 (S24)
2H1424 (S52)
1PS002 (S74)
1PS003 (S74)
1PS004 (S74)
1PT182 (S100)
2PT222 (S142)
1PT209 (S103)
3B1022 (S58)
3I1022 (S72)
2G1448 (S50)
2G1510 (S50)
3PT162 (S169)
2C1510 (S43)
1SF-04 (S6)
1SI-06 (S9)1D1436 (S25)
1PT234 (S108)
3B1046 (S59)
3PT212 (S177)
3B0948 (S58)
3F1034 (S67)
1SA-04 (S1)
1A1412 (S19)
2I1558 (S55)
1PS041 (S81)
2PS003 (S110)
3PS013 (S148)
1PS043 (S81)
1PS044 (S81)
2PS038 (S116)
1SF-02 (S6)
3PT172 (S170)
3D0912 (S62)
1PT192 (S102)

Ishikura, Zen (石倉 禅)
Ishimaru, Yoshiro (石丸 喜朗)
Ishimori, Kazuki (石杜 和希)
Ishitani, Ryuichiro (石谷 隆一郎)

2D1546 (S45)
3PT189 (S174)
1PT010 (S85)
1SH-02 (S7)
1PT128 (S90)
1YS0930 (S90)
Ishiura, Masahiro (石浦 正寛)
3B1046 (S59)
1PT122 (S89)
2PT229 (S143)
2PT230 (S143)
2SG-05 (S15)Ishiwata, Shin'ichi (石渡 信一)
2H1546 (S53)
3B0948 (S58)
3H0912 (S69)
3H1110 (S71)
1PS047 (S82)
2PS009 (S111)
Ishiwata, Shin ichi (石渡 信一)
2H1510 (S52)
1PS009 (S75)
1PS016 (S76)
1PS017 (S77)
1PS010 (S75)
Ishizuka, Toru (石塚 徹)
2PT183 (S137)
3PT218 (S178)
Isjijima, Akihiko (石島 秋彦)
2I1546 (S55)
Islam, Mohammad M (Islam Mohammad M)
2E1546 (S47)
Islam, Mohammad, M (Islam Mohammad, M)
2E1534 (S47)
Isojima, Hiroshi (磯島 広)
1A1436 (S19)
Isomura, Yoshikazu (礒村 宜和)
2SH-05 (S16)
Isono, Kunio (磯野 邦夫)
2PT190 (S138)
1YS1015 (S138)
1PT107 (S86)
Isozumi, Noriyoshi (五十棲 規嘉)
Itaba, Takeshi (板場 武史)
1PS051 (S83)
1PS052 (S83)
Itabashi, Takeshi (板橋 岳志)
3H0912 (S69)
Itagaki, Tetsuhiko (板垣 哲彦)
2PT166 (S133)
Ito Akihiko (伊藤 彰彦)
1PT206 (S103)
Ito, Akihiko (伊藤 彰彦)
2D1558 (S45)
Ito, Ayumi (伊藤 亜由美)
1PS024 (S78)
Ito, Daisuke (伊東 大輔)
1D1522 (S26)
Ito, Etsuro (伊藤 悦朗)
1D1534 (S26)
Ito, Hiroshi (伊藤 浩史)
2PT232 (S144)
Ito, Jotaro (伊藤 丈太郎)
1PS024 (S78)
Ito, Jumpei (伊藤 順平)
1PT128 (S90)
1YS0930 (S90)
Ito, Jun-ichi (伊東 純一)
2PT005 (S120)
Ito, Kenji (伊藤 健二)
2PT123 (S125)
Ito, Kentaro (伊藤 賢太郎)
1SF-06 (S7)Ito, Masahiro (伊藤 政博)
2PS002 (S110)
2PS006 (S111)
Ito, Mashiho (伊藤 真志保)
2PT143 (S129)
Ito, Nobutoshi (伊藤 暢聡)
1C1522 (S24)
1PT146 (S93)
Ito, Shota (伊藤 奨太)
2F1436 (S48)
3D0924 (S62)
Ito, Yoshihiro (伊藤 嘉浩)
2PT145 (S129)
Ito, Yuko (伊藤 祐子)
1PS034 (S80)
Ito, Yuma (伊藤 由馬)
1H1424 (S33)
3PS034 (S152)
3PS035 (S152)
3PT181 (S172)
Ito, Yutaka (伊藤 隆)
2SB-02 (S10)
1B1448 (S21)
Itoga, Kazuyoshi (糸賀 和義)
2PS047 (S118)
Itoh, Hideki (伊藤 秀城)
2H1510 (S52)
Itoh, Koji (伊藤 光二)
1PT006 (S84)
Itoh, Mai (伊藤 真衣)
3PT129 (S162)
Itoh, Nobuya (伊藤 伸哉)
2PT148 (S130)
Itoh, Satoru (伊藤 暁)
3E1058 (S65)
Itoh, Satoru G. (伊藤 暁)
1PT131 (S91)
Itoh, Shigeru (伊藤 繁)
3PT228 (S180)
Itoh, Toshiki (伊藤 俊樹)
3PT141 (S165)
Itoh, Toshiyuki (伊藤 俊幸)
1H1534 (S34)
Itou, Takashi (伊藤 隆司)
3PS038 (S152)
Iwadate, Yoshiaki (岩楯 好昭)
2PS048 (S118)
2PS052 (S119)
Iwai, Hidenao (岩井 秀直)
2D1424 (S44)
Iwai, Masakazu (岩井 優和)
3PT236 (S181)
Iwai, Shigenori (岩井 成憲)
2F1534 (S49)
2F1546 (S49)
Iwai, Sosuke (岩井 草介)
2PS014 (S112)
2PT221 (S142)
Iwaki, Masayo (岩城 雅代)
3F0936 (S66)
Iwaki, Mitsuhiro (岩城 光宏)
1PS049 (S82)
1YS0900 (S82)
1PT006 (S84)
Iwaki, Takafumi (岩城 貴史)
3C1110 (S61)
Iwama, Kazuya (岩間 和也)
1PT140 (S92)
Iwamoto, Hiroyuki (岩本 裕之)
2H1610 (S53)
Iwamoto, Kazunari (岩本 一成)
2PT215 (S141)
Iwamoto, Masayuki (岩本 真幸)
2SH-01 (S16)− S186 −2G1558 (S51)
3PT164 (S169)
2PT111 (S123)
Iwamoto, Shigeto (岩本 成人)
Iwamoto, Tomoyuki (岩本 知之)
3PT190 (S174)
Iwamoto, Yui (岩本 唯)
2PT155 (S131)
2PT156 (S131)
Iwane, Atsuko (岩根 敦子)
1PS049 (S82)
1YS0900 (S82)
1PT006 (S84)2PS043 (S117)
Iwane, Atsuko H. (岩根 敦子)
2PT176 (S135)
Iwano, Katsunori (岩野 亮宗)
2B1400 (S40)
Iwaoka, Michio (岩岡 道夫)
3PT003 (S155)
Iwaoka, Ryo (岩岡 諒)
Iwasa, Takuya (岩佐 拓也)
3D1034 (S63)
Iwasa, Tatsuo (岩佐 達郎)
1F1412 (S29)
3F1046 (S67)
Iwasa, Yoh (巌佐 庸)
2PT220 (S142)
Iwasaki, Hideo (岩崎 秀雄)
2C1510 (S43)
2PT232 (S144)
Iwasaki, Kenji (岩崎 憲治)
1H1610 (S35)
Iwase, Kouhei (岩瀬 航平)
1PS015 (S76)
2PT230 (S143)
Iwata, Keita (岩田 慶太)
3PT182 (S172)
Iwata, Seigo (岩田 聖悟)
Iwata, Tatsuya (岩田 達也)
2F1436 (S48)
2F1448 (S48)
2F1546 (S49)
3D0924 (S62)
Iwatani, Yasumasa (岩谷 靖雅)
1PT102 (S85)
3PT003 (S155)
Iwayama, Masashi (岩山 将士)
1E1400 (S27)
3F1010 (S66)
Iwazaki, Ryosuke (岩崎 良祐)
3PT192 (S174)
1PT156 (S95)
Iwura, Takafumi (井浦 貴文)
3PS031 (S151)
Izawa, Kazushi (井澤 和司)
J. McBride, Mark (J. McBride Mark)
2SE-05 (S13)Javkhlantugs, Namsrai (ジャブクラントゥクス ナムスライ)
3PT127 (S162)
Ji, Zhi-Gang (姫 志剛)
3PT218 (S178)
Jikihara, Kazunori (直原 一徳)
2PT169 (S134)
Jin, Mingyue (金 明月)
2A1412 (S38)
2A1424 (S38)
1PT186 (S100)
Jinguji, Masaaki (神宮司 将晃)
3PT185 (S173)
Jintori, Kosuke (陣取 孝輔)
3PT191 (S174)
Johmura, Masae (城村 雅恵)
2SB-01 (S10)
Jolley, Craig (Jolley Craig)
2C1522 (S43)
Joti, Yasumasa (城地 保昌)
3PS026 (S150)
Ju, Jungmyoung (朱 正明)
1I1610 (S37)
Jung, Kwang-Hwan (Jung Kwang-Hwan)
1F1510 (S30)
2PT177 (S135)
2PT179 (S136)
Kagemoto, Tatsuya (影本 達也)
1PS016 (S76)
Kageyama, Takeshi (景山 健志)
2PT186 (S137)
Kai, Noriko (甲斐 憲子)
1H1534 (S34)
Kainosho, Masatsune (甲斐荘 正恒)
1B1424 (S21)
Kaizu, Kazunari (海津 一成)
2SI-04 (S17)2C1412 (S42)
2PT215 (S141)
3PT183 (S172)
Kajiwara, Kentaro (梶原 健太郎)
3PS024 (S150)
Kakibe, Sayuri (柿部 小百合)
3G0936 (S68)
Kalay, Ziya (Kalay Ziya)
2C1400 (S42)
Kamada, Mayumi (鎌田 真由美)
1PT191 (S101)
Kamagata, Kiyoto (鎌形 清人)
2E1412 (S45)
1PT152 (S94)
1PT222 (S106)
Kamatari, Yuji O. (鎌足 雄司)
1PT139 (S92)
1PT155 (S95)
Kambara, Taketoshi (神原 丈敏)
2A1522 (S39)
Kameda, Tomoshi (亀田 倫史)
1PT005 (S84)
1PT112 (S87)
3PT107 (S158)
Kamei, Takashi (亀井 敬)
3PT102 (S157)
Kamekura, Masahiro (亀倉 正博)
2PT201 (S139)
Kami-ike, Nobunori (上池 伸徳)
2I1436 (S54)
2PS006 (S111)
2PS029 (S115)
Kamikubo, Hironari (上久保 裕成)
2PT173 (S135)
Kamikubo, Hironari (上久保 裕生)
2F1424 (S48)
1PT124 (S89)
1PT138 (S92)
1PT153 (S95)
2PT170 (S134)
2PT172 (S134)
2PT174 (S135)
Kamikubo, Hironori (上久保 裕生)
2PT171 (S134)
Kamimura, Atsushi (上村 淳)
2SF-02 (S14)
2PT216 (S141)
Kamino, Keita (神野 圭太)
2PS045 (S118)
Kamio, Yoshiyuki (神尾 好是)
3F0948 (S66)
Kamioka, Tetsuya (上岡 哲矢)
1C1412 (S23)
Kamitani, Yasushi (紙谷 泰史)
3PT150 (S166)

Kamiya, Katsumasa (神谷 克政)
Kamiya, Kenshu (神谷 健秀)
Kamiya, Koki (神谷 厚輝)

Kamiya, Masakatsu (神谷 昌克)

Kamiya, Motoshi (神谷 基司)
Kamiya, Narutoshi (Kamiya Narutoshi)
Kamiya, Narutoshi (神谷 成敏)

Kamiya, Ritsu (神谷 律)

Kamiya, Yukiko (神谷 由紀子)
Kamo, Naoki (加茂 直樹)

Kan, Yumi (菅 悠美)
Kanaba, Teppei (金場 哲平)
Kanada, Ryo (金田 亮)
Kanada, Soun (金田 創運)
Kanamaru, Shuji (金丸 周司)
Kanamori, Toshiyuki (金森 敏幸)

Kanaori, Kenji (金折 賢二)
Kanaya, Toshiji (金谷 利治)
Kanayama, Kenji (金山 賢治)
Kanba, Keisuke (神庭 圭祐)
Kanbe, Toshio (神戸 俊夫)
Kandori, Hideki (KANDORI HIDEKI)
Kandori, Hideki (神取 秀樹)

Kanehisa, Minoru (金久 實)
Kaneko, Jun (金子 淳)
Kaneko, Kunihiko (金子 邦彦)

Kaneko, Tomoyuki (金子 智行)

Kanematsu, Keita (兼松 啓太)
Kaneta, Yusuke (金田 祐輔)
Kang, Jiyoung (Kang Jiyoung)

Kano, Hideaki (加納 英明)
Kano, Masanobu (狩野 方伸)
Kanuka, Hirotaka (嘉糠 洋陸)
Karino, Yasuhito (狩野 康人)
Kariya, Ryusho (刈谷 龍昇)
Kasahara, Kota (笠原 浩太)
Kasai, Haruo (河西 春郎)
Kasai, Rinshi (笠井 倫志)
Kasai, Rinshi S. (笠井 倫志)
Kasai, Taishi (笠井 大司)
Kasai, Takuma (葛西 卓磨)
Kashibe, Masayoshi (樫部 順美)Kashima, Risa (鹿島 理沙)
Kashiwazaki, Jun (柏崎 隼)
Kasuga, Sachiko (春日 草千子)
Kasuya, Daisuke (糟屋 大介)

3PT222 (S178)
1PT176 (S99)
3PT136 (S164)
3PT143 (S165)
3PT163 (S169)
1C1400 (S23)
2PT177 (S135)
2PT179 (S136)
2PT178 (S136)
2B1436 (S40)
3B1010 (S58)
1PT130 (S90)
3PT010 (S157)
2A1412 (S38)
2PS050 (S118)
3PS003 (S146)
3PS006 (S146)
3PS007 (S147)
1B1436 (S21)
1F1558 (S30)
2PT176 (S135)
2PT177 (S135)
2PT179 (S136)
3PT142 (S165)
1B1448 (S21)
1PS037 (S80)
1F1522 (S30)
1PS012 (S76)
2PT144 (S129)
3PS044 (S153)
3PS051 (S155)
3PT123 (S161)
3PT169 (S170)
1C1522 (S24)
2PT153 (S131)
1E1424 (S27)
3PT123 (S161)
3PT003 (S155)
3C0924 (S60)
3F1058 (S67)
1F1400 (S29)
1F1448 (S29)
1F1510 (S30)
2F1412 (S47)
2F1436 (S48)
2F1448 (S48)
2F1534 (S49)
2F1546 (S49)
2G1610 (S51)
3D0912 (S62)
3D0924 (S62)
3F0936 (S66)
2PT181 (S136)
2PT182 (S136)
2PT189 (S138)
2PT107 (S122)
3F0948 (S66)
1SC-05 (S4)
1PT218 (S105)
2PT216 (S141)
2PT224 (S142)
2PT231 (S144)
2D1510 (S44)
3PS020 (S149)
3PS022 (S149)
3PS023 (S149)
3PS046 (S154)
3PS049 (S154)
3PS041 (S153)
3PT160 (S168)
1B1610 (S22)
1I1448 (S35)
2PT142 (S129)
2PT166 (S133)
2PT167 (S133)
2SH-03 (S16)
2SH-05 (S16)
1I1546 (S36)
1E1522 (S28)
2PT150 (S130)
2PT105 (S122)
3PT131 (S163)
2SH-05 (S16)
1H1448 (S33)
1G1412 (S31)
3PT172 (S170)
2PS024 (S114)
2PT140 (S128)
3PT134 (S163)
2E1412 (S45)
2PS016 (S112)
3PT233 (S180)
3E1010 (S64)

Kasuya, Masatoshi (粕谷 昌寿)
Kasuya, Yuzo (粕谷 有造)
Katagiri, Toyomasa (片桐 豊雅)
Katahira, Masato (片平 正人)

Kataoka, Hironao (片岡 博尚)
Kataoka, Maachi (片岡 真亜知)
Kataoka, Mikio (片岡 幹雄)

Kataoka, Yuto (片岡 悠人)
Katayama, Kazuhiko (片山 和彦)
Katayama, Kota (片山 耕大)
Katayama, Yukie (片山 幸江)
Katayanagi, Katsuo (片柳 克夫)
Kato, Asuka (加藤 あす香)
Kato, Chiaki (加藤 千明)
Kato, Hideaki E. (加藤 英明)
Kato, Hiroyuki (加藤 博之)
Kato, Koichi (KATO Koichi)
Kato, Koichi (加藤 晃一)
Kato, Mikio (加藤 幹男)
Kato, Minoru (加藤 稔)
Kato, Noritaka (加藤 徳剛)
Kato, Satoru (加藤 知)
Kato, Shoichi (加藤 翔一)
Kato, Takashi (加藤 尚志)
Kato, Takayuki (加藤 貴之)

Kato, Yoshitaka (加藤 善隆)
Kato, Yumiko (加藤 弓子)
Katoh, Kaoru (加藤 薫)
Katou, Kouya (加藤 功弥)
Katsura, Hitomi (桂 ひとみ)
Katsurada, Yuki (桂田 悠基)
Kawabata, Kazushige (川端 和重)
Kawabata, Takeshi (川端 猛)
Kawagishi, Ikuro (川岸 郁朗)

Kawaguchi, Kazutomo (川口 一朋)

Kawaguchi, Tatsuya (川口 辰也)
Kawaguchi, Tetsuya (川口 徹也)
Kawahara, Kousuke (河原 弘典)
Kawai, Takeaki (河合 武揚)
Kawai, Yuka (河合 優香)
Kawai-Noma, Shigeko (河合 繁子)
Kawakami, Keisuke (川上 恵典)
Kawakami, Masaru (川上 勝)
Kawakami, Tomohiro (河上 知広)
Kawakami, Tomoya (川上 智也)
Kawakami, Yuji (川上 祐司)
Kawakita, Yoshito (川北 祥人)
Kawamoto, Akihiro (川本 晃大)
Kawamoto, Shuhei (川本 周平)
Kawamura, Izuru (川村 出)

Kawamura, Takashi (河村 高志)− S187 −1PT180 (S99)
3PT205 (S175)
3A1046 (S57)
1PS027 (S78)
3F0912 (S65)
3C0936 (S60)
3C1034 (S61)
3PT003 (S155)
2F1436 (S48)
2PT192 (S138)
3G0900 (S67)
1E1534 (S28)2F1424 (S48)
1PT124 (S89)
1PT138 (S92)
1PT153 (S95)
2PT170 (S134)
2PT171 (S134)
2PT172 (S134)
2PT173 (S135)
2PT174 (S135)
2SD-04 (S12)3E0948 (S64)1F1400 (S29)
2PT189 (S138)
3I1046 (S73)
3PT129 (S162)
3PT222 (S178)
3PT237 (S181)
3PT238 (S181)
1PT113 (S87)
1PT128 (S90)
1YS0930 (S90)
3PT237 (S181)
1B1412 (S21)
1B1436 (S21)
1PT112 (S87)
2I1610 (S55)
2B1510 (S41)
1PT141 (S92)
3PT126 (S162)
3G1110 (S69)
3PT149 (S166)
2D1522 (S45)
1PT208 (S103)
2SE-01 (S12)
2PS028 (S114)
3PS012 (S147)
3PT001 (S155)
1F1510 (S30)
3PT119 (S160)
2PS042 (S117)
3PT219 (S178)
2PT188 (S138)
1SB-06 (S2)
1PT230 (S107)
2PT103 (S121)
1PT121 (S89)
2PS049 (S118)
3PS032 (S151)
3PS036 (S152)
3PT180 (S172)
3PT184 (S173)
3PT185 (S173)
3PT186 (S173)
3PT187 (S173)
3PT188 (S173)
3PT191 (S174)
3PT192 (S174)
1E1400 (S27)
3F1010 (S66)
2PT131 (S127)
2PT109 (S123)
3PT188 (S173)
3PT229 (S180)
3F0912 (S65)
3F0948 (S66)
1PT151 (S94)
3D0948 (S62)
2E1400 (S45)
1PT136 (S91)
1PS022 (S77)
3A1022 (S57)
2PT106 (S122)
2PS022 (S113)
2PS028 (S114)
2PS043 (S117)
2G1400 (S49)
1F1558 (S30)
3F1046 (S67)
2PT168 (S134)
3PT114 (S159)
1PT113 (S87)

Kawanabe, Akira (川鍋 陽)
Kawano, Keiichi (河野 敬一)

Kawano, Ryuji (川野 竜司)

Kawasaki, Kazunori (川崎 一則)
Kawashima, Yoshiaki (川島 嘉明)
Kawashima, Yurie (川島 有里恵)
Kawata, Daiki (川田 大輝)
Kawata, Masaaki (川田 正晃)
Kawata, Yasushi (河田 康志)
Kawato, Suguru (川戸 佳)

Kaya, Motoshi (茅 元司)

Kayama, Yoko (香山 容子)
Kayano, Isao (茅野 功)
Kayanuma, Megumi (栢沼 愛)
Kazama, Toshiya (風間 俊哉)
Kazumi, Tanaka (田中 一巳)
Kazuta, Yasuaki (数田 恭章)
Ken'ya, Furuta (古田 健也)
Kenmoku, Hiromichi (兼目 裕充)
Kenri, Tsuyoshi (見理 剛)
Kenzaki, Hiroo (検崎 博生)
Khan, Alam, M (Khan Alam, M)
Khan, Monsur A (Khan Monsur A)
Ki, Jyunan (紀 十男)
Kidera, Akinori (木寺 詔紀)

Kidoaki, Satoru (木戸秋 悟)
Kidokoro, Shun-Ichi (城所 俊一)
Kidokoro, Shun-ichi (城所 俊一)
Kihara, Hiroshi (木原 裕)
Kihara, Takanori (木原 隆典)
Kihara, Tatsuya (木原 達也)
Kijima, Saku (貴嶋 紗久)
Kikawada, Takahiro (黄川田 隆洋)
Kikuchi, Hiroto (菊地 浩人)
Kikuchi, Katsuya (菊地 克也)
Kikuchi, Kazuya (菊地 和也)

Kikuchi, Macoto (菊池 誠)
Kikuchi, Satoru (菊池 覚)
Kikuchi, Takeshi (菊地 武司)

Kikuchi, Yohsuke (菊池 洋輔)
Kikukawa, Takashi (菊川 峰志)Kikushima, Kenji (菊島 健児)
Kim, Do-myoung (金 渡明)
Kim, Hyonchol (金 賢徹)
Kimoto, Tetsuya (木本 哲也)
Kimura, Akatsuki (木村 暁)
Kimura, Akihiro (木村 明洋)
Kimura, Hiroshi (木村 宏)
Kimura, Kazuya (木村 和哉)
Kimura, Kenjiro (木村 建次郎)
Kimura, Masako (木村 雅子)
Kimura, Ryota (木村 亮太)
Kimura, Shota (木村 彰太)
Kimura, Sumiko (木村 澄子)

Kimura, Tetsunari (木村 哲就)

1F1510 (S30)
1C1400 (S23)
2PT177 (S135)
2PT179 (S136)
2G1412 (S49)
3PT136 (S164)
3PT143 (S165)
3PT163 (S169)
3G0900 (S67)
1PT184 (S100)
3PT139 (S164)
2PT119 (S124)
2PT189 (S138)
1PT183 (S100)
1PT164 (S97)
3PT205 (S175)
3PT207 (S176)
3PT211 (S176)
3PT213 (S177)
3PT216 (S177)
3PT217 (S177)
1SI-01 (S8)2A1522 (S39)
2A1558 (S39)
1H1534 (S34)
3PS027 (S150)
3PT222 (S178)
1SF-06 (S7)
1PS035 (S80)
2PT205 (S139)
2A1546 (S39)
1D1610 (S26)
2PS022 (S113)
3PT007 (S156)
3PT009 (S157)
2E1534 (S47)
2E1546 (S47)
3F0900 (S65)
1F1534 (S30)
2B1412 (S40)
1PT147 (S93)
2PT134 (S127)
1PT231 (S107)
1PT232 (S108)
1PT107 (S86)
1PT149 (S94)
1PT179 (S99)
1PT157 (S95)
3H1034 (S71)
1PT223 (S106)
2SE-03 (S13)
1PT162 (S96)
2PT137 (S128)
3PS038 (S152)
1C1424 (S23)
3PS039 (S153)
3PS042 (S153)
3PS043 (S153)
3PT134 (S163)
1B1534 (S22)
1PT161 (S96)
1PS048 (S82)
1I1400 (S35)
3E1034 (S65)
1PT127 (S90)
1PS023 (S78)
1C1400 (S23)
2PT176 (S135)
2PT177 (S135)
2PT179 (S136)
2PT182 (S136)
3PT123 (S161)
3PT128 (S162)
1H1412 (S33)
1C1510 (S24)
3PS046 (S154)
3PS047 (S154)
3PT211 (S176)
3PT216 (S177)
1SD-04 (S4)
1SI-05 (S9)3D0936 (S62)
2SG-01 (S15)
1PS027 (S78)
2SI-05 (S18)2H1558 (S53)
1C1610 (S25)
3PT137 (S164)
3PT138 (S164)
1PS011 (S75)
1PS012 (S76)
1PS013 (S76)
1C1546 (S24)1C1558 (S24)
2PT182 (S136)
2PT183 (S137)
Kimura, Toru (木村 徹)
1PT166 (S97)
Kimura, Yoshiro (木村 嘉朗)
3PT227 (S179)
Kimura, Yuki (木村 雄貴)
1PT102 (S85)
Kimura, Yukihiro (木村 行宏)
3PT233 (S180)
3PT234 (S181)
2I1546 (S55)
Kinbara, Kazushi (金原 数)
2B1436 (S40)
Kinjo, Akira (Kinjo Akira)2PT103 (S121)
Kinjo, Akira (金城 玲)
3PT005 (S156)
Kinjo, Masataka (Kinjo Masataka)
1SD-05 (S5)Kinjo, Masataka (金城 政孝)
1G1546 (S32)
1H1400 (S33)
1PT205 (S103)
3PT004 (S156)
Kinoshita, Kengo (木下 賢吾)
3E1046 (S65)
2PT105 (S122)
3PT131 (S163)
Kinoshita, Masahiro (木下 正弘)
1PS034 (S80)
1PT137 (S92)
1PT154 (S95)
3PT116 (S160)
Kinoshita, Michiyo (木下 充代)
2PT190 (S138)
1YS1015 (S138)
Kinoshita, Miki (木下 実紀)
2I1424 (S54)
Kinoshita, Syunsuke (木下 俊祐)
1PS015 (S76)
Kinoshita, Yukari (木下 ゆかり)3PT210 (S176)
Kinosita Jr, Kazuhiko (木下 一彦)
1PS035 (S80)
Kinosita, Jr., Kazuhiko (木下 一彦)
1PS031 (S79)Kinosita, Kazuhiko (木下 一彦)
3A1046 (S57)
1PS026 (S78)
1PS027 (S78)
3PT166 (S169)
Kinosita, Kazuhiko Jr. (木下 一彦)
3A1022 (S57)
Kinosita, Yoshiaki (木下 佳昭)
3A1058 (S57)
Kira, Atsushi (吉良 敦史)
3PT114 (S159)
Kirino, Yutaka (桐野 豊)
3PT203 (S175)
3PT214 (S177)
Kishi, Takaaki (岸 孝亮)
2I1522 (S55)
Kishida, Naoko (岸田 直子)
1PT170 (S98)
Kishimoto, Ayaka (岸本 彩花)
2PT162 (S133)
2PT164 (S133)
Kishimoto, Yasushi (岸本 泰司)
3PT214 (S177)
Kiso, Makoto (木曽 真)
2PS024 (S114)
Kita, Makoto (喜多 真琴)
3PT223 (S179)
Kita, Sayaka (喜多 清)1H1412 (S33)
Kitagawa, Daiju (北川 大樹)
2SB-04 (S10)
Kitagawa, Teizo (北川 禎三)
3I0924 (S71)
2PT159 (S132)
Kitahara, Ryo (北原 亮)
1PT112 (S87)
Kitahata, Hiroyuki (北畑 裕之)
2C1448 (S42)
Kitajima-Ihara, Tomomi (北島ー井原 智美) 2PT201 (S139)
Kitamoto, Dai (北本 大)
3PT176 (S171)
Kitamura, Kazuo (喜多村 和郎)
2SH-05 (S16)
Kitamura, Natsumi (北村 奈津美)
2PT164 (S133)
Kitamura, Shingo (北村 紳悟)
3PT003 (S155)
Kitamura, Yoshiichiro (北村 美一郎)
3PT203 (S175)
Kitano, Takahiro (北野 貴寛)
2PT188 (S138)
Kitao, Akio (北尾 彰朗)
1A1522 (S20)
1PS019 (S77)
1PT174 (S98)
2PT132 (S127)
Kitaoka, Maya (北岡 麻耶)
2I1400 (S53)
Kitayama, Hiroki (北山 寛貴)
1E1436 (S27)
Kitazawa, Soichiro (北沢 創一郎)
1PT112 (S87)
Kitoh-Nishioka, Hirotaka (鬼頭ー西岡 宏任)
3D1046 (S63)
Klumpp, Stefan (クランプ ステファン)
2I1412 (S54)Ko, Jong Soo (Ko Jong Soo)
1I1610 (S37)
Kobatake, Takahiro (小畑 貴広)
2PS013 (S112)
Kobayashi, Asako (小林 麻子)
1I1522 (S36)
Kobayashi, Chigusa (小林 千草)
2PT130 (S127)
2PT139 (S128)
Kobayashi, Emiko (小林 恵美子)
1PT184 (S100)
2PS042 (S117)
Kobayashi, Junya (小林 純也)
3E0900 (S63)
3PT204 (S175)
Kobayashi, Masaru (小林 大)
1PS025 (S78)
Kobayashi, Naoya (小林 直也)
1PT106 (S86)
Kobayashi, Ryo (小林 亮)
1SF-06 (S7)
Kobayashi, Ryota (小林 亮太)
3PT145 (S165)
Kobayashi, Shiori (小林 詩織)
1B1424 (S21)
2I1448 (S54)
Kobayashi, Takashi (小林 聖)
3PT231 (S180)
Kobayashi, Takuya (小林 琢也)
2A1558 (S39)
Kobayashi, Tetsuya (小林 徹也)
1D1424 (S25)
Kobayashi, Tetsuya J. (小林 徹也)
2SF-02 (S14)
Kobayashi, Toshihide (Kobayashi Toshihide)
2G1424 (S50)
Kobayashi, Toshihide (小林 俊秀)
3PT146 (S166)
Kobayashi, Yasuhiko (小林 泰彦)
1G1510 (S31)

3PT239 (S182)
Kobayashi, Yusuke (小林 裕亮)
1PT159 (S96)
Kobayashi, Yuta (小林 祐大)
Kobirumaki-Shimozawa, Fuyu (小比類巻 生)
1PS009 (S75)
Kodama, Mieko (兒玉 美恵子)
1PT211 (S104)
Kodama, Toru (児玉 亮)
3PS050 (S155)
Kodani, Noriko (小谷 紀子)
3PS027 (S150)
2SB-03 (S10)
Kodera, Noriyuki (古寺 哲幸)
1PS046 (S82)
2PS053 (S119)
3PS017 (S148)
2PT187 (S137)
Koga, Nobuaki (古賀 伸明)
2F1412 (S47)
Kogure, Kazuhiro (木暮 一啓)
2PT181 (S136)
Kohchi, Takayuki (河内 孝之)
2PT188 (S138)
Kohno, Toshiyuki (河野 俊之)
2PT111 (S123)
3PT178 (S172)
Kohori, Ayako (小堀 綾子)
3A1022 (S57)
Koike, Masafumi J. (小池 雅文)
1PT118 (S88)
Koike, Ryotaro (小池 亮太郎)
1B1546 (S22)
1I1424 (S35)
2PT130 (S127)
1B1424 (S21)
Kojima, Chojiro (児嶋 長次郎)
Kojima, Hiroaki (小嶋 寛明)
1PS050 (S82)
2PS019 (S113)
3PS001 (S146)
3PS002 (S146)
3PS009 (S147)
Kojima, Keiichi (小島 慧一)
1F1424 (S29)
Kojima, Masaru (小嶋 勝)
1PT229 (S107)
1PT233 (S108)
Kojima, Seiji (小嶋 誠司)
1A1546 (S20)
1A1610 (S20)
2I1400 (S53)
2I1448 (S54)
2I1510 (S54)
2I1522 (S55)
2I1534 (S55)
2PT160 (S132)
Komatsu, Daiki (小松 大貴)
3H1022 (S70)
1PT228 (S107)
2PS036 (S116)
Komatsu, Hideyuki (小松 英幸)
1PT011 (S85)
Komatsu, Yasuo (小松 康雄)
2PT158 (S132)
Komatsu, Yu (小松 勇)
1PT135 (S91)
Komatsuzaki, Yoshimasa (小松崎 良将)
2PT233 (S144)
3PT205 (S175)
3PT219 (S178)
Komatuzaki, Yosimasa (小松崎 良将)
3PT220 (S178)
Komeda, Tadahiro (米田 忠弘)
2E1412 (S45)
Komichi, Nobutaka (小道 信孝)
1PT130 (S90)
Komiya, Ken (小宮 健)
1I1522 (S36)
Komiyama, Ryosuke (小見山 亮祐)
1PS036 (S80)
Komori, Tomotaka (小森 智貴)
2PS011 (S111)
Komuro, Yasuaki (小室 靖明)
2PT139 (S128)
Kon, Takahide (昆 隆英)
1SI-02 (S8)3B1010 (S58)
Kondo, Akihiko (近藤 昭彦)
1PT234 (S108)
Kondo, Daiki (近藤 大生)
3PT222 (S178)
Kondo, Hiroko (近藤 寛子)
1PT188 (S101)
Kondo, Jiro (近藤 次郎)
3C1010 (S60)
Kondo, Takao (近藤 孝男)
3B1058 (S59)
3B1110 (S59)
2PT232 (S144)
Kondo, Yoshito (近藤 嘉人)
1PT008 (S84)
Konno, Asahi (昆野 朝陽)
1PT169 (S97)
Kono, Hidetoshi (河野 秀俊)
1SD-03 (S4)1B1522 (S22)
3PS026 (S150)
3PT108 (S158)
Kono, Ryohei (河野 良平)
2B1510 (S41)
1PT114 (S87)
Kori, Hiroshi (郡 宏)
2PT232 (S144)
Koshii, Takaya (越井 貴也)
1PT217 (S105)
Kosuge, Takashi (小菅 隆)
2PT123 (S125)
Kosugi, Masayuki (小杉 正幸)
3E0912 (S63)
Kotani, Susumu (小谷 享)
2PS035 (S116)
Kotera, Masaaki (小寺 正明)
2PT107 (S122)
Koua, Faisal Hammad Mekky(Koua Faisal Hammad Mekky)
3D1022 (S63)
Kouduki, Naoharu (上月 尚治)
1PT138 (S92)
Kouyama, Mizuno (神山 勉)
2PT184 (S137)
Kouyama, Tsutomu (神山 勉)
1SH-06 (S8)1F1522 (S30)
Kowalczykowski, Stephen (Kowalczykowski Stephen)
1PS045 (S82)
Koyama, Masako (小山 昌子)
2SB-06 (S10)
Koyama, Tsubasa (小山 翼)
2PS014 (S112)
Koyama, Yuka (小山 裕佳)
3PT225 (S179)
Koyanagi, Mitsumasa (小柳 光正)
2PT190 (S138)
1YS1015 (S138)
Koyasako, Kotaro (小屋迫 光太郎)
2A1412 (S38)
2A1436 (S38)− S188 −Koyasu, Kazuma (小安 司馬)
Kubo, Koji (久保 康児)
Kubo, Minoru (久保 稔)
Kubo, Tomohiro (久保 智広)
Kubo, Yoko (久保 葉子)
Kubo, Yoshihiro (久保 義弘)
Kubori, Tomoko (久堀 智子)

3A1010 (S56)
3C0924 (S60)
3I1034 (S72)
3PS003 (S146)
2PT191 (S138)
3PT168 (S170)
1PT105 (S86)
2PT124 (S125)
1I1412 (S35)
Kubota, Chihiro (久保田 千尋)
3B0948 (S58)
Kubota, Hiroaki (久保田 寛顕)
2F1522 (S48)
Kubota, Kenshuke (久保田 健介)
2SI-01 (S17)Kudo, Seishi (工藤 成史)
2SI-02 (S17)
1PS022 (S77)
1PS029 (S79)
2PS029 (S115)
2PS030 (S115)
2PS046 (S118)
Kudo, Shogo (工藤 翔吾)
3PT113 (S159)
Kudo, Yukihiko (工藤 恭彦)
2PS041 (S117)
Kudoh, Suguru N. (工藤 卓)
1D1546 (S26)
3PT201 (S174)
Kuhara, Atsushi (久原 篤)
3PT210 (S176)
Kuma, Hiroyuki (隈 博幸)
3PT117 (S160)
Kumachi, Shigefumi (熊地 重文)
2PT204 (S139)
Kumagai, Izumi (熊谷 泉)
1C1448 (S23)1C1510 (S24)Kumagai, Yumiko (熊谷 裕美子)
3PT112 (S159)
Kumaki, Yasuhiro (熊木 康裕)
1C1400 (S23)
Kumashiro, Yoshikazu (熊代 善一)
2PS047 (S118)
Kumemoto, Ryusei (久米本 龍生)
2PS012 (S112)
Kuramochi, Maiko (倉持 麻衣子)
2PS042 (S117)
2PS035 (S116)
Kuramoto, Ayumu (倉本 歩)
1PS013 (S76)
Kurasawa, Masayoshi (倉沢 正義)
Kuribayashi, Kaori (栗林 香織)
3PT143 (S165)
Kuribayashi-Shigetomi, Kaori (栗林(繁富) 香織)
1I1546 (S36)
Kurihara, Kazue (栗原 和枝)
1PT163 (S96)
Kurihara, Kazuo (栗原 和男)
2PT170 (S134)
Kurihara, Kensuke (栗原 顕輔)
3PT142 (S165)
Kurikawa, Tomoki (栗川 知己)
2PT224 (S142)
Kurisaki, Ikuo (栗崎 以久男)
2E1436 (S46)
2PT165 (S133)
Kurisu, Genji (栗栖 源嗣)
3B1010 (S58)
Kurita, Yuki (栗田 祐輝)
3PS050 (S155)
Kuroda, Katsushi (黒田 克史)
3PS027 (S150)
Kuroda, Shinya (黒田 真也)
2SF-03 (S14)
1PT214 (S104)
2PT219 (S142)
3PT212 (S177)
Kuroda, Takuya (黒田 琢弥)
1PT105 (S86)
2PT124 (S125)
Kuroda, Yutaka (Kuroda Yutaka)
2E1546 (S47)
Kuroda, Yutaka (黒田 裕)
1C1412 (S23)2E1534 (S47)
2PT001 (S119)
Kuroi, Kunisato (黒井 邦巧)
2PT169 (S134)
Kuroki, Ryota (黒木 良太)
3E0912 (S63)
Kurosawa, Masaru (黒沢 大)2B1558 (S41)
Kuroyanagi, Shigehide (畔柳 成秀)
1B1610 (S22)
Kurumizaka, Hitoshi (胡桃坂 仁志)
2SG-03 (S15)
1PT190 (S101)
Kusakabe, Hitoshi (日下部 均)
2PT109 (S123)
Kusumi, Akihiro (楠見 明弘)
1G1412 (S31)
1H1448 (S33)
1PT209 (S103)
2PS041 (S117)
3PT172 (S170)
Kusunoki, Masami (楠 正美)
3D1010 (S62)
Kuwabara, Yumiko (桑原 由美子)
3PT128 (S162)
Kuwahara, Naoto (桑原 直人)
3PT201 (S174)
Kuwajima, Kunihiro (KUWAJIMA Kunihiro)
1B1412 (S21)
Kuwajima, Kunihiro (桑島 邦博)
1PT143 (S93)
1PT160 (S96)
2PT150 (S130)
Kuwata, Kazuo (桑田 一夫)
1PT139 (S92)
1PT155 (S95)
Lee, Sang Min (Lee Sang Min)
1I1610 (S37)
Lee, Young-Ho (李 映昊)
1E1436 (S27)
2PT126 (S126)
Leppla, Stephen (Leppla Stephen)
2PS041 (S117)
Levadny, Victor (LEVADNY Victor)
3PT173 (S171)
Levadny, Victor (Levadny Victor)
3PT174 (S171)
Levi, Sabine (LEVI Sabine)
3PT206 (S175)
Li, Hua (李 華)
1PT106 (S86)
Li, Junhua (Li Junhua)
3PT170 (S170)
Li, Koren (李 香蓮)
3PT217 (S177)
Li, Na (李 な)
1A1546 (S20)
Li, Pai-Chi (Li Pai-Chi)
3B1022 (S58)
Li, Wenfei (Li Wenfei)
1PT178 (S99)
Li, Yong (永 麗)3PT234 (S181)
Liang-da, Chiu (Liang-da Chiu)
3PS037 (S152)

Libchaber, Albert (リップシャーバー アルバート)
2SC-06 (S11)
1PT209 (S103)
Limin, Chen (陳 莉敏)Lin, Szu-Ning (Lin Szu-Ning)
2PS025 (S114)
Lin, Tsai-Shun (Lin Tsai-Shun)
2PS008 (S111)
Lin, Yuxi (林 雨㬢)
1E1436 (S27)
Liu, An-An (刘 安安)
2PS041 (S117)
1SE-02 (S5)
Liu, Feng (Liu Feng)
1I1558 (S36)
Liu, Huan (劉 歓)
2PS041 (S117)
Liu, Shihui (Liu Shihui)
2PS008 (S111)
Lo, Chien-Jung (Lo Chien-Jung)
2PS025 (S114)
1A1534 (S20)
Lo, Chien-Jung (羅 健榮)Longhi, Sonia (Longhi Sonia)
1H1522 (S34)
Lu, Yue (魯 ゆえ)
3D1058 (S63)
MIzuno, Daisuke (水野 大介)
2PT240 (S145)
Mabuchi, Issei (馬渕 一誠)
2PS016 (S112)
Machiyama, Hiroaki (町山 裕亮)
3H1046 (S71)
Maeda, Kohji (前田 耕治)
3PS021 (S149)
3PT159 (S168)
Maeda, Miki (前田 美紀)
2PT008 (S120)
1G1510 (S31)
Maeda, Munetoshi (前田 宗利)
1F1424 (S29)
Maeda, Ryo (前田 亮)
Maeda, Tomoko (前田 友子)
2PT157 (S132)
Maeda, Toshinori (前田 俊徳)
2PT191 (S138)
Maeda, Yuichiro (前田 雄一郎)
1B1546 (S22)
1PT118 (S88)
Maeda, Yusuke (前多 裕介)
2SC-06 (S11)
Maejima, Masami (前島 雅美)
1PT102 (S85)
Maekawa, Takuma (前川 拓摩)
1PT104 (S86)
Maeno, Akihiro (前野 覚大)2E1448 (S46)
2E1522 (S46)
1B1448 (S21)
Maesaki, Ryoko (前崎 綾子)
1SA-05 (S1)
Maeshima, Kazuhiro (前島 一博)
Makabe, Koki (MAKABE KOKI)
1B1412 (S21)
Makabe, Koki (真壁 幸樹)
1PT160 (S96)
Maki, Koichiro (牧 功一郎)
1PT148 (S94)
Maki, Kosuke (槇 亙介)
1PT150 (S94)
Maki-Yonekura, Saori (眞木 さおり)
2D1522 (S45)
Makino, Fumiaki (牧野 文信)
3PS012 (S147)
Makino, Tsukasa (牧野 司)
1PS037 (S80)
Makino, Yoshihide (牧野 祥嗣)
2PT148 (S130)
Makino, Yoshiteru (槙野 義輝)
1F1558 (S30)
Makishi, Kou (牧志 洸)
2PT213 (S141)
Manandhar, Yasodha (Manandhar Yasodha)
2PT145 (S129)
Marathe, Rahul (マーラー ラウール)
2I1412 (S54)
Marcucci, Lorenzo (マルクッチ ロレンツォ)
1PS049 (S82)
1YS0900 (S82)
Maruko, Yuuto (丸子 勇人)
1PS014 (S76)
Marunaka, Yoshinori (丸中 良典)
2C1546 (S43)
Maruta, Shinsaku (丸田 晋策)
1PS038 (S80)
1PS051 (S83)
1PS052 (S83)
1PT213 (S104)
2PS038 (S116)
3PT182 (S172)
Maruta, Takahiro (丸田 隆広)
2PS013 (S112)
2PS014 (S112)
Maruyama, Atsushi (丸山 篤史)
1D1400 (S25)
Maruyama, Atushi (丸山 篤史)
3PS045 (S154)
Maruyama, Ichiro (丸山 一郎)
3PT121 (S161)
Maruyama, Yuusuke (丸山 雄介)
3E1022 (S64)
Masai, Ryouta (政井 良太)
2PT108 (S122)
Masaike, Tomoko (政池 知子)
2H1448 (S52)
3A0912 (S56)
3A1034 (S57)
1PS025 (S78)
3PS030 (S151)
Masaki, Noritaka (正木 紀隆)
3PS028 (S150)
Masaoka, Takashi (正岡 崇志)
1PT102 (S85)
Mashima, Tsukasa (真嶋 司)
3C0936 (S60)
Mashimo, Tadaaki (真下 忠彰)
3B1010 (S58)
Mashio, Yuka (眞塩 由佳)
2PT147 (S130)
Masubuchi, Takeya (増渕 岳也)
3PT111 (S159)
Masuda, Shinji (増田 真二)
2F1522 (S48)
Masuda, Tadashi (増田 正)
2PS015 (S112)
Masumoto, Hiroshi (増本 博)
2G1522 (S50)
3PT158 (S168)
Matsubara, Tomo (松原 倫)
2PT141 (S129)
Matsubara, Yuki (松原 佑記)
3PT110 (S159)
Matsubayashi, Nobuyuki (松林 伸幸)
1PS034 (S80)
Matsubayasi, Nobuyuki (松林 伸幸)
2PT185 (S137)
Matsuda, Chie (松田 知栄)
3PT002 (S155)
Matsuda, Shoichi (松田 翔一)
1I1558 (S36)
Matsuda, Syoichi (松田 翔一)
3D1058 (S63)
Matsuda, Taisuke (松田 泰祐)
3PT208 (S176)
Matsuda, Tomoki (松田 知己)
1C1412 (S23)
Matsudaira, Paul T. (Matsudaira Paul T.)
2SA-03 (S9)Matsueda, Ryohei (松枝 遼平)
2D1448 (S44)
2G1522 (S50)
Matsui, Takashi (松井 貴志)
1PS025 (S78)

Matsui, Tsubasa (松井 翼)
Matsui, Tsubasa S. (松井 翼)
Matsui, Tsutomu (松井 つとむ)
Matsuki, Hitoshi (松木 均)

3H1022 (S70)
2PS036 (S116)
1PT157 (S95)
3G0936 (S68)
3G0948 (S68)
Matsumoto, Atsushi (松本 淳)
3PT008 (S156)
Matsumoto, Hideki (松本 英樹)
1G1510 (S31)
1PT110 (S87)
Matsumoto, Takashi (松本 崇)
1C1400 (S23)
Matsumoto, Takashi (松本 貴之)
3PT221 (S178)
Matsumura, Sachiko (松村 佐智子)
1PT115 (S88)
Matsumura, Yoshitaka (松村 義隆)
1PT157 (S95)
2D1522 (S45)
Matsunaga, Sachihiro (松永 幸大)
Matsunaga, Yasuhiro (松永 康佑)
2B1412 (S40)
Matsunami, Hideyuki (松波 秀行)
2SE-01 (S12)
Matsuo, Koichi (松尾 光一)
1PT108 (S86)
Matsuo, Lisa (松尾 里紗)
2PS022 (S113)
Matsuo, Takatoshi (松尾 高稔)
2PT161 (S133)
Matsuo, Tatsuhito (松尾 龍人)
2H1400 (S51)
Matsuoka, Daisuke (松岳 大輔)
2PT116 (S124)
Matsuoka, Masanari (松岡 雅成)
1I1400 (S35)
1PT116 (S88)
Matsuoka, Shigeru (松岡 茂)
2PT116 (S124)
Matsuoka, Toru (枩岡 亨)
2PT167 (S133)
Matsushita, Hisashi (松下 尚嗣)
3PS042 (S153)
Matsushita, Katsuyoshi (松下 勝義)
1PT161 (S96)
Matsushita, Mitsugu (松下 貢)
1SC-03 (S3)Matsutaka, Hiroshi (松鷹 宏)
1A1412 (S19)
Matsuura, Azuma (松浦 東)
2F1400 (S47)
Matsuura, Koji (松浦 宏治)
1PT211 (S104)
Matsuura, Tomoaki (松浦 友亮)
1C1534 (S24)
2PT205 (S139)
2SB-06 (S10)
Matsuura, Yoshiyuki (松浦 能行)
3E0900 (S63)
Matsuyama, Atsushi (松山 淳)
2PT176 (S135)
Matsuyama, Take (松山 武)
3B0936 (S58)
Matsuzaki, Masanori (松崎 政紀)
2SH-05 (S16)
Matsuzaki, Yuri (松崎 由理)
2PT104 (S122)
Matsuzawa, Tomohiro (松澤 朋寛)
3F0924 (S66)
Matubayasi, Nobuyuki (松林 伸幸)
1E1522 (S28)
Matuda, Yui (松田 唯)
3PT135 (S164)
Maturana, Andres D. (Maturana Andres D.)
1PT128 (S90)
1YS0930 (S90)
Matuta, Shinsaku (丸田 晋策)
1PS043 (S81)
Matuyama, Takashi (松山 崇)
1C1510 (S24)
Mayama, Shigeki (真山 茂樹)
2PS047 (S118)
Mayanagi, Kota (真柳 浩太)
2A1400 (S38)
2A1412 (S38)
Mayor, Satyajit (Mayor Satyajit)
3H1058 (S71)
McBride, Mark J. (McBride Mark J.)
2PS026 (S114)
Michiue, Tatsuo (道上 達男)
1D1400 (S25)Mie, Yasuhiro (三重 安弘)
2PT158 (S132)
Migita, Taiko (右田 たい子)
3I0948 (S72)
Mikhailenko, Sergey V. (Mikhailenko Sergey V.)
2PS009 (S111)
Miki, Kunio (三木 邦夫)
3E0912 (S63)
1PT123 (S89)
Miki, Masao (三木 正雄)
2H1412 (S52)
Miki, Norihisa (三木 典尚)3PT163 (S169)
Mikoshiba, Katsuhiko (御子柴 克彦)
3PT206 (S175)
Mikuni, Shintaro (Mikuni Shintaro)
3PT005 (S156)
Mikuni, Shintaro (三國 新太郎)
1PT205 (S103)
3PT004 (S156)
Mimatsu, Saori (三松 さおり)
2PS042 (S117)
Mimura, Yasumasa (三村 泰昌)
1SC-01 (S3)Minagawa, Jun (皆川 純)
2PS050 (S118)
Minakata, Shiho (南方 志帆)
1PT118 (S88)Minami, Kazuyuki (南 和幸)
2PS048 (S118)
Minami, Shintaro (南 慎太朗)
2PT004 (S120)
2PT009 (S121)
2PT202 (S139)
Minami, Shintaro (南 慎太郎)
2PT002 (S119)
2PT003 (S120)
Minamino, Tohru (南野 徹)
1SA-07 (S2)
2SE-01 (S12)2I1424 (S54)
2I1436 (S54)
2PS005 (S110)
2PS029 (S115)
Mine, Shouhei (峯 昇平)
1PT184 (S100)
Minezaki, Toshiya (峯崎 俊哉)
2B1400 (S40)
Mino, Hiroyuki (三野 広幸)
3D0900 (S61)
3D1022 (S63)
1PT122 (S89)
Mio, Kazuhiro (三尾 和弘)
2G1534 (S51)
3E1022 (S64)
3PT002 (S155)
Mio, Muneyo (三尾 宗代)
2G1534 (S51)
3PT002 (S155)
Misaka, Takumi (三坂 巧)
3PT189 (S174)
Mishima, Hirokazu (三嶋 浩和)
3PT116 (S160)
Mishima, Masaki (三島 正規)
1B1448 (S21)− S189 −Mishima, Masanori (Mishima Masanori)
Mitaku, Shigeki (美宅 成樹)

Mitaku, Sigeki (美宅 成樹)
Mitarai, Namiko (御手洗 菜美子)
Mitomi, Yasushi (三冨 康司)
Mitsuhashi, Kenji (三橋 賢司)
Mitsui, Hiromasa (三井 広大)
Mitsui, Toshiyuki (三井 敏之)

Mitsuoka, Kaoru (光岡 薫)
Mitsutake, Ayori (光武 亜代理)
Miura, Nami (三浦 奈美)
Miura, Takashi (三浦 岳)
Miwa, Yoshihiro (三輪 佳宏)
Miyagi, Hiraku (宮城 拓)
Miyaji, Reiji (宮地 礼司)
Miyakawa, Makiyo (宮川 真紀代)
Miyakawa, Takeshi (宮川 毅)
Miyakawa, Takuya (宮川 拓也)
Miyake, Jun (三宅 淳)

Miyamori, Takaaki (宮森 一彰)
Miyamoto, Akitoshi (宮本 章歳)
Miyamoto, Keita (宮本 恵太)
Miyamoto, Mariko (宮本 万理子)
Miyamoto, Tatsuya (宮本 達也)
Miyanaga, Akihiko (宮永 晃彦)
Miyano, Satoru (宮野 悟)
Miyanoiri, Yohei (宮ノ入 洋平)
Miyasa, Ryota (宮佐 亮太)
Miyashita, Naoyuki (宮下 尚之)

Miyata, Makoto (Miyata Makoto)
Miyata, Makoto (宮田 真人)

Miyata, Tomoko (宮田 知子)

Miyawaki, Atsushi (宮脇 敦史)
Miyazaki, Makito (宮崎 牧人)
Miyazaki, Naoyuki (宮崎 直幸)
Miyazaki, Naoyuli (宮崎 直幸)
Miyazawa, Sanzo (宮澤 三造)
Miyazono, Yuya (宮薗 侑也)
Miyoshi, Hiromi (三好 洋美)
Miyoshi, Yohei (三吉 洋平)
Mizobata, Tomohiro (溝端 知宏)
Mizuguchi, Kenji (水口 賢司)
Mizuhara, Yukinobu (水原 志暢)Mizukami, Shin (水上 進)

Mizukami, Taku (水上 卓)
Mizukami, Takuya (水上 琢也)
Mizuno, Akari (水野 紅理)
Mizuno, Daisuke (水野 大介)

Mizuno, Katsuhide (水野 克秀)
Mizuno, Takafumi (水野 敬文)
Mizushima, Ryota (水島 良太)
Mizushima, Tsunehiro (水島 恒裕)
Mizuta, Toshifumi (水田 敏史)
Mizutani, Hitomi (水谷 仁美)
Mizutani, Kana (水谷 佳奈)
Mizutani, Takeomi (水谷 武臣)
Mizutani, Yusuke (水谷 祐輔)
Mochiji, Shota (持地 翔太)
Mochizuki, Atsushi (望月 敦史)

Mochizuki, Masao (望月 正雄)

1PS040 (S81)
2PT011 (S121)
2PT108 (S122)
3PT120 (S161)
1A1400 (S19)2C1424 (S42)
2B1558 (S41)
3PT213 (S177)
2PT191 (S138)
1G1448 (S31)
1I1510 (S36)
2D1436 (S44)
3H1010 (S70)
3E1010 (S64)
1PT180 (S99)
2B1424 (S40)
2PT117 (S124)
2C1448 (S42)
1G1412 (S31)
1PT209 (S103)
3PT121 (S161)
1PT139 (S92)
3PT153 (S167)
1YS1045 (S167)
1PT135 (S91)
2PT131 (S127)
2PT114 (S124)
3H1034 (S71)
2PT228 (S143)
3PS048 (S154)
2F1558 (S49)
3PT206 (S175)
2H1522 (S52)
2PT214 (S141)
1PT229 (S107)
2PT117 (S124)
2SD-04 (S12)1B1424 (S21)
2PT168 (S134)
3B1022 (S58)
3E1110 (S65)
1PT212 (S104)
2PS027 (S114)
2SE-04 (S13)3A1058 (S57)
3E1022 (S64)
2PS021 (S113)
2PS022 (S113)
2PS023 (S114)
2PS024 (S114)
2SE-01 (S12)2I1436 (S54)
3PS012 (S147)
3PT001 (S155)
2SH-07 (S17)3H1110 (S71)
3E0948 (S64)
1H1610 (S35)
2PT007 (S120)
1PS042 (S81)
1I1610 (S37)
3PT011 (S157)
1PT164 (S97)
1PT177 (S99)
1PS039 (S81)
3PS039 (S153)
3PS042 (S153)
3PS043 (S153)
3PT134 (S163)
1PT001 (S83)
1PT150 (S94)
2H1510 (S52)
1PS009 (S75)2SC-03 (S11)
1PS018 (S77)
3PT170 (S170)
2PT184 (S137)
2PS048 (S118)
2PT126 (S126)
2PT162 (S133)
2PT164 (S133)
1PT164 (S97)
3PT201 (S174)
3PT210 (S176)
3A1058 (S57)
1PT230 (S107)
2D1546 (S45)
3PS014 (S148)
2PS050 (S118)
1G1436 (S31)
2C1436 (S42)2C1510 (S43)
3I1034 (S72)

Mochizuki, Seiichi (望月 精一)
Moffat, Keith (Moffat Keith)
Mogami, George (最上 譲二)

3PS027 (S150)
3I0936 (S72)
1E1558 (S28)
2H1424 (S52)
1PS002 (S74)
1PS003 (S74)
1PS004 (S74)
1PS005 (S74)
1PT010 (S85)
1PT182 (S100)
3PT157 (S168)
Mori, Takaharu (森 貴治)
2PT123 (S125)
Mori, Takeharu (森 丈晴)
1B1448 (S21)
Mori, Tomoyuki (森 智行)
Mori, Toshiaki (森 利明)
3C1110 (S61)
Mori, Yoshiharu (森 義治)
3PT224 (S179)
Mori, Yuki (森 勇樹)
3PS042 (S153)
Morigaki, Kenichi (森垣 憲一)
3G0924 (S68)
3PT161 (S168)
Moriguchi, Yusuke (森口 雄介)
1PS046 (S82)
Morii, Hisayuki (森井 尚之)
2E1522 (S46)
Morikawa, Mika (森川 美佳)
1D1534 (S26)
1PT135 (S91)
Morikawa, Ryota (森河 良太)
2PT131 (S127)
Morikawa, Takamitsu (森川 高光)
2PT118 (S124)
Morimatsu, Fumiki (森松 文毅)
1C1400 (S23)
Morimoto, Jumpei (森本 淳平)
1C1436 (S23)
Morimoto, Nobuyuki (森本 展行)
1E1558 (S28)
1PT010 (S85)
1PT182 (S100)
Morimoto, Wakako (森本 和加子)
1A1610 (S20)
Morimoto, Yukio (森本 幸生)
1PT104 (S86)
1PT110 (S87)
2SE-01 (S12)Morimoto, Yusuke (森本 雄祐)
2I1436 (S54)
Morimoto, Yusuke V. (森本 雄祐)
2PS028 (S114)
Morino, Kai (森野 佳生)
1SC-04 (S4)Morishita, Naoki (森下 直樹)
1C1400 (S23)Morishita, Ryo (森下 了)
3C1034 (S61)
Morishita, Yoshihiro (森下 喜弘)
2PT220 (S142)
Morita, Masamune (森田 雅宗)
3PT153 (S167)
1YS1045 (S167)
Morita, Mizuki (森田 瑞樹)
2PT101 (S121)
Morita, Shin-ichi (盛田 伸一)
1D1412 (S25)
Morita, Takami (森田 貴己)
1PS019 (S77)
Morita, Yoshiro (森田 恭朗)
3PS021 (S149)
Morita, Yuya (森田 有哉)
1PT110 (S87)
Moritsugu, Kei (森次 圭)
1PT147 (S93)
Moriwaki, Yoshitaka (森脇 由隆)
2PT151 (S131)
Moriya, Kazuki (守谷 和騎)
2PT121 (S125)
Moriya, Yuki (守屋 勇樹)
2PT107 (S122)Moriyama, Toru (森山 徹)
2PT234 (S144)
Morotomi-Yano, Keiko (諸冨 桂子)
1G1424 (S31)
Morris, Roger J. (モリス ロジャー)
3PT172 (S170)
Motojima, Fumihiro (元島 史尋)1E1412 (S27)
Motojima-Miyazaki, Yuko (元島(宮崎) 優子)
1E1412 (S27)
Motokawa, Tatsuo (本川 達雄)
2PS040 (S117)
Mouri, Kazunari (毛利 一成)
2SI-04 (S17)
1PT204 (S102)
2PS044 (S117)
Mukai, Hideo (向井 秀夫)
1PT235 (S108)
3PT207 (S176)
3PT213 (S177)
3PT221 (S178)
Mukai, Hiroaki (向井 大陽)
3PS043 (S153)
Mukai, Tatsuo (向 辰朗)
2PT009 (S121)
Mukai, Yuri (向井 有理)
3PT126 (S162)
Mukaiyama, Atsushi (向山 厚)
3B1110 (S59)
Muneyuki, Eiro (宗行 英朗)
2SI-02 (S17)
1PS022 (S77)
1PS024 (S78)
1PS032 (S79)
2PT139 (S128)
Muneyuki, Eiroh (宗行 英朗)
1PS023 (S78)
Murakami, Gen (村上 元)
3PT213 (S177)
Murakami, Hisashi (村上 久)
2PT234 (S144)
Murakami, Kosuke (村上 耕介)
3E0948 (S64)
Murakami, Midori (村上 緑)
1SH-06 (S8)1F1522 (S30)
Murakami, Satoshi (村上 聡)
1PS021 (S77)
Murakami, Seiko D. (村上 聖子)
1I1424 (S35)
Murakami, Shinya (村上 真也)
1G1448 (S31)
2D1436 (S44)
3H1010 (S70)
Murakoshi, Hideji (村越 秀治)
1PT207 (S103)
Muramatsu, Chikako (村松 知可子)
3F0936 (S66)
Muramoto, Kazumasa (村本 和優)
2PT155 (S131)
2PT156 (S131)
2PT157 (S132)
2PT160 (S132)
Muraoka, Takahiro (村岡 貴博)
2I1546 (S55)
Murata, Kazuyoshi (村田 和義)
1H1534 (S34)
3E0948 (S64)

Murata, Michio (村田 道雄)
Murata, Satoshi (村田 智)

Murata, Shizuaki (村田 静昭)
Murayama, Koichi (村山 幸市)
Murayama, Shuhei (村山 秀平)
Murayama, Takashi (村山 尚)
Murayama, Yoriko (村山 依子)
Muto, Ai (武藤 愛)
Mutoh, Risa (武藤 梨沙)

Nagadoi, Aritaka (長土居 有隆)
Nagae, Takayuki (永江 峰幸)Nagai, Hiroki (永井 宏樹)
Nagai, Ken (永井 健)
Nagai, Ken H. (永井 健)
Nagai, Masako (長井 雅子)
Nagai, Rina (永井 里奈)
Nagai, Takeharu (永井 健治)
Nagai, Tetsuro (永井 哲郎)
Nagai, Toshiki (長井 俊樹)
Nagai, Yukifumi (長井 幸史)
Nagai, Yumiko (永井 弓子)
Naganawa, Tatsuya (永縄 達也)
Nagao, Chioko (長尾 知生子)
Nagao, Hidemi (長尾 秀実)

Nagaoka, Masataka (長岡 正隆)

Nagasaki, Akira (長崎 晃)
Nagasaki, Masao (長崎 正朗)
Nagase, Shinobu (長瀬 忍)
Nagashima, Hiroki (長嶋 宏樹)
Nagashima, Yasuto (長嶋 泰人)
Nagata, Chikahiro (永田 親弘)
Nagata, Kumiko (永田 久美子)
Nagata, Mitsunori (永田 光範)
Nagata, Taashi (永田 崇)
Nagata, Takashi (永田 崇)

Nagata, Tomohiro (永田 智啓)
Nagatani, Akira (長谷 あきら)
Nagatani, Yasuko (永谷 康子)
Nagatani, Yukinori (永谷 幸則)
Nagatomo, Shigenori (長友 重紀)
Nagaya, Hiromi (長屋 ひろみ)
Nagayama, Kuniaki (永山 國昭)

Nagayama, Masaharu (長山 雅晴)
Nagayoshi, Wataru (永吉 航)
Naiki, Hironobu (内木 宏延)
Naito, Akira (内藤 晶)

Naito, Hiroshi (内藤 洋)
Naito, Tatsuya (内藤 達也)
Naka, Yasuhiro (仲 泰嘉)
Nakabayashi, Jun (中林 潤)
Nakada, Chieko (中田 千枝子)
Nakada, Hinako (中田 比奈子)
Nakada, Kyoko (中田 恭子)
Nakada, Shogo (仲田 正吾)
Nakagaki, Toshiyuki (中垣 俊之)
Nakagawa, Ayumi (中川 あゆみ)
Nakagawa, Hiroshi (中川 洋)
Nakagawa, Kanako (中川 香奈子)
Nakagawa, Takeshi (中川 健)
Nakagawa, Takuya (中川 卓哉)− S190 −1PT116 (S88)
2PT116 (S124)
1PT226 (S107)
1PT227 (S107)
3PT109 (S159)
3PT112 (S159)
3PT113 (S159)
3C0924 (S60)
1PT187 (S101)
2SB-02 (S10)2A1610 (S40)
2PT232 (S144)
2PT107 (S122)
1PT122 (S89)
2PT229 (S143)
2PT230 (S143)
1PT190 (S101)
1PT113 (S87)
1PT105 (S86)
2PT124 (S125)
2SC-02 (S11)
1PT224 (S106)
2PT154 (S131)
2PT159 (S132)
2PS043 (S117)
1SA-03 (S1)
2PT118 (S124)
3PT144 (S165)
2B1424 (S40)
2PT154 (S131)
2PT159 (S132)
3PT177 (S171)
2H1448 (S52)
1PT177 (S99)
1E1400 (S27)
3F1010 (S66)
1PT001 (S83)
2PT131 (S127)
1E1510 (S28)
2E1436 (S46)
2PT165 (S133)
2SE-03 (S13)
2SD-04 (S12)3PS038 (S152)
3D0900 (S61)
3D1022 (S63)
3PS017 (S148)
1PT117 (S88)
1F1448 (S29)
1A1412 (S19)
1PS041 (S81)
3C1034 (S61)
3C0936 (S60)
2PT190 (S138)
1YS1015 (S138)
3PT003 (S155)
1H1558 (S34)
2F1510 (S48)
2PT123 (S125)
1H1534 (S34)
1H1546 (S34)
2PT152 (S131)
2PT159 (S132)
3PT201 (S174)
1H1534 (S34)
1H1546 (S34)
3E0948 (S64)
1G1400 (S31)
2C1448 (S42)
3PS050 (S155)
1E1436 (S27)
1PT145 (S93)
1F1558 (S30)
3E0936 (S64)
3F1046 (S67)
2PT168 (S134)
3PT114 (S159)
3PT127 (S162)
2PT112 (S123)
3A1034 (S57)
3PT159 (S168)
2PT211 (S140)
3PT172 (S170)
2PT148 (S130)
1E1510 (S28)
3PT220 (S178)
1SF-06 (S7)
2PT121 (S125)
1E1534 (S28)
1PT142 (S93)
1A1424 (S19)
1PS028 (S78)
1PS005 (S74)

Nakagawa, Taro (中川 太郎)
Nakagawa, Yuki (中川 裕規)
Nakagawa, Zen-ichi (中川 善一)
Nakahara, Asahi (中原 旭)
Nakahara, Naoya (中原 直哉)
Nakajima, Akihiko (中島 昭彦)
Nakajima, Masahiro (中島 正博)

2PT161 (S133)
2PT003 (S120)
2PT107 (S122)
3PS031 (S151)
2H1558 (S53)
3H0936 (S70)
1PT229 (S107)
1PT233 (S108)
3F1034 (S67)
Nakajima, Masato (中島 正人)
2PS003 (S110)
Nakajima, Mizuki (中嶋 瑞樹)
2PT203 (S139)
Nakajima, Yoshiki (中島 慶樹)
3PT168 (S170)
Nakajo, Koichi (中條 浩一)
1I1448 (S35)
Nakaki, Ryo (仲木 竜)
Nakamasu, Akiko (中益 朗子)
1PS018 (S77)
Nakamichi, Katsuhisa (中道 勝久)
3PT101 (S157)
Nakamori, Kota (中森 弘太)
2PT002 (S119)
Nakamrua, Tatsuya (中村 竜也)
3PS039 (S153)
Nakamura, Atsushi (中村 淳志)
2PT142 (S129)
Nakamura, Daigo (中村 大吾)
3PS036 (S152)
Nakamura, Haruki (Nakamura Haruki)
2B1436 (S40)
Nakamura, Haruki (中村 春木)
1C1412 (S23)
3B1010 (S58)
1PT119 (S88)
1PT126 (S90)
1PT130 (S90)
1PT168 (S97)
2PT103 (S121)
3PT010 (S157)
3PT190 (S174)
3PT223 (S179)
Nakamura, Mitsuru (中村 允)
1PT216 (S105)
Nakamura, Shigeyoshi (中村 成芳)
1PT107 (S86)
1PT149 (S94)
1PT179 (S99)
Nakamura, Shinichiro (中村 振一郎)
3PT227 (S179)
3PT230 (S180)
3PT232 (S180)
Nakamura, Shogo (中村 昇吾)
1D1400 (S25)
Nakamura, Shotaro (中村 祥太郎)
3PS021 (S149)
Nakamura, Shougo (中村 昇吾)
3PS045 (S154)
Nakamura, Shugo (中村 周吾)
2PT010 (S121)
2PT101 (S121)
Nakamura, Shuichi (中村 修一)
2SE-01 (S12)
2PS029 (S115)
2PS030 (S115)
2PS046 (S118)
Nakamura, Syuichi (中村 修一)
1PS029 (S79)
2PS006 (S111)
Nakamura, Takashi (NAKAMURA Takashi)
1B1412 (S21)
Nakamura, Takashi (中村 敬)
1PT143 (S93)
1PT160 (S96)
2PT150 (S130)
Nakamura, Teruya (中村 照也)
1PT129 (S90)
1YS1000 (S90)
Nakamura, Tomoyuki (中村 知幸)
2PS049 (S118)
Nakamura, Yuki (中村 由樹)
1H1436 (S33)
Nakane, Daisuke (中根 大介)
2SE-05 (S13)3A1058 (S57)
3E1022 (S64)
2PS022 (S113)
2PS023 (S114)
2PS024 (S114)
2PS026 (S114)
Nakanishi Mamoru (中西 守)
1PT206 (S103)
Nakanishi, Mamoru (中西 守)
3PT176 (S171)
Nakanishi, Naoya (中西 直哉)
1PT233 (S108)
Nakanishi, Shuji (中西 周次)
1I1558 (S36)
3D1058 (S63)
Nakanishi, Taichi (中西 太市)
1F1522 (S30)
Nakanishi, Takeshi (中西 猛)
1C1448 (S23)Nakanishi, Yosuke (中西 陽介)
3PS043 (S153)
Nakano, Akihiko (中野 明彦)
3PT236 (S181)
Nakano, Kentaro (中野 賢太郎)
2PS037 (S116)
Nakano, Masahiro (中野 雅裕)
3A0948 (S56)
Nakano, Minoru (中野 実)
2G1448 (S50)
2G1510 (S50)
3PT162 (S169)
Nakano, Shogo (中野 祥吾)
3PT222 (S178)
Nakao, Hiroyuki (中尾 裕之)
3PT162 (S169)
Nakao, Ryota (中尾 亮太)
1PT185 (S100)
Nakasako, Masayoshi (中迫 雅由)
2D1522 (S45)
2D1534 (S45)
Nakashima, Hiromichi (中嶋 寛倫)
2PT122 (S125)
Nakashima, Hitomi (中嶋 仁珠)
2PS048 (S118)
Nakashima, Ryosuke (中島 良介)
1PT117 (S88)
3PT115 (S160)
Nakashima, Satoru (中島 聡)
3I1022 (S72)
3I1034 (S72)
Nakashima, Yuta (中島 雄太)
2PS048 (S118)
Nakasone, Yusuke (中曽根 祐介)
2F1510 (S48)
2F1558 (S49)
2PT169 (S134)

2PT175 (S135)
2PT109 (S123)
3PT135 (S164)
2PT192 (S138)
1PS013 (S76)
2SE-05 (S13)
2PS026 (S114)
3A0912 (S56)
Nakayama, Rinako (中山 莉奈子)
3PS030 (S151)
1D1424 (S25)Nakazato, Kenichi (中里 研一)
1C1510 (S24)Nakazawa, Hikaru (中澤 光)
3G1110 (S69)
Nakazawa, Hiromitsu (中沢 寛光)
3PT149 (S166)
Nakazumi, Taichi (中住 太一)
1C1400 (S23)
Namba, Keiichi (難波 啓一)
2SE-01 (S12)2I1424 (S54)
2I1436 (S54)
2PS005 (S110)
2PS006 (S111)
2PS028 (S114)
2PS029 (S115)
3PS012 (S147)
3PT001 (S155)
Namba, Toshinori (難波 利典)
2PT225 (S143)
Namsrai, Javkhlantugs (ナムスライ ジャブクラントゥス)
3E0936 (S64)
Namsrai, Javkhlantugs (ナムズライ ジャフカラントゥクス)
3PT114 (S159)
Nara, Toshifumi (奈良 敏文)
2PT176 (S135)
Narihara, Itaru (成原 格)
3PT007 (S156)
Narita, Akihiro (成田 哲博)
1H1610 (S35)
2H1436 (S52)
1PT118 (S88)
1PS039 (S81)
Narita, Jun (成田 純)
1PT169 (S97)
Narumi, Masatoshi (鳴海 正寿)
2PT174 (S135)
Naruse, Keiji (成瀬 恵治)
1PT209 (S103)
1PT211 (S104)
Natali, Francesca (Natali Francesca)
2H1400 (S51)
Natsume, Yuno (夏目 ゆうの)3G1034 (S69)
Nawa, Omi (名和 臣)
3PT237 (S181)
Negami, Tatsuki (根上 樹)
2PT133 (S127)
Negishi, Lumi (根岸 瑠美)
3PT124 (S161)
Nemoto, Naoto (根本 直人)
1PT234 (S108)
2PT147 (S130)
2PT204 (S139)
Nemoto, Yuri L. (根本 悠宇里)
1G1412 (S31)
3PT172 (S170)
Neya, Saburo (根矢 三郎)
3I0924 (S71)
Nie, Qing-Miao (Nie Qing-Miao)
2PS017 (S112)
Nigorikawa, Shinpei (濁川 慎平)
3PT152 (S167)
Nii, Daisuke (二井 大輔)
3PT105 (S158)
Niida, Atsushi (新井田 厚司)
2SD-04 (S12)
Niioka, Hirohiko (新岡 宏彦)
1H1558 (S34)
Niisato, Naomi (新里 直美)
2C1546 (S43)
Niitani, Yamato (新谷 大和)
1A1448 (S19)
Niiyama, Mayumi (新山 真由美)
1PT116 (S88)
2PT116 (S124)
Niizato, Takayuki (新里 高行)
2PT210 (S140)
2PT234 (S144)
Nishi, Shunsuke (西 俊介)
3D0948 (S62)
Nishida, Koji (西田 幸次)
1E1424 (S27)
Nishigaki, Koichi (西垣 功一)
2PT204 (S139)
Nishigaki, Takehiko (西垣 岳彦)
2I1400 (S53)
Nishigaki, Takeshi (西垣 武)
3I1022 (S72)
Nishihara, Yasutaka (西原 泰孝)
1A1522 (S20)
Nishijo, Kaname (西條 要)
2PT144 (S129)
Nishikata, Koro (西方 公郎)
1F1534 (S30)
Nishikawa, Hirohide (西川 大英)3F1046 (S67)
Nishikawa, Kouki (西川 幸希)
3F0924 (S66)
Nishikawa, Takehiro (西川 雄大)
2PT205 (S139)
Nishikawa, Yoshihiro (中村 真梨子)
3A0948 (S56)
Nishikawa, Yusuke (西川 雄亮)
2SE-03 (S13)
2PS039 (S116)
Nishikomori, Ryuta (西小森 隆太)
3PS031 (S151)
Nishima, Wataru (二島 渉)
1PT212 (S104)
Nishimori, Hiraku (西森 拓)
2PT141 (S129)
2PT214 (S141)
2PT218 (S141)
Nishimoto, Etsuko (西本 悦子)
2E1610 (S47)
2PT122 (S125)
Nishimura, Atsuhito (西村 篤人)
2PT229 (S143)
Nishimura, Erika (西村 恵里夏)
2PT112 (S123)
Nishimura, Katsuyuki (西村 勝之)
2PT115 (S124)
3PT178 (S172)
Nishimura, Kohei (西村 航平)
1PT168 (S97)
Nishimura, Megumi (西村 めぐみ)
1E1400 (S27)
3F1010 (S66)
Nishimura, Shin I. (西村 信一郎)
2PT227 (S143)
Nishimura, Takahiro (西村 隆宏)
1I1534 (S36)
Nishimura, Yoshifumi (西村 善文)
1PT190 (S101)
Nishino, Kunihiko (西野 邦彦)
1PT117 (S88)
3PT115 (S160)
Nakata, Haruka (中田 春香)
Nakata, Satoshi (中田 聡)
Nakatani, Youichi (中谷 陽一)
Nakauchi, Yuni (中内 祐二)
Nakayama, Koji (中山 浩次)

Nishino, Takeshi (西野 武士)
Nishino, Tomohide (西野 伴英)
Nishio, Izumi (西尾 泉)
Nishio, Kazuya (西尾 和也)
Nishio, Toshihiko (西尾 俊彦)
Nishioka, Noriko (西岡 典子)
Nishiyama, Hidetoshi (西山 英利)
Nishiyama, Masayoshi (西山 雅祥)

2PT137 (S128)
2E1424 (S46)
3PT154 (S167)
1PT104 (S86)
3PT203 (S175)
2I1400 (S53)
3E1022 (S64)
1A1558 (S20)
3H0924 (S70)
1PT121 (S89)
Nishiyama, So-ichiro (西山 宗一郎)
3PT185 (S173)
3PT188 (S173)
3PT192 (S174)
Nishiyama, Yuta (西山 雄太)
2PT234 (S144)
Nishizaka, Takayuki (西坂 崇之)
2H1448 (S52)
3A0912 (S56)
3A1034 (S57)
3A1058 (S57)
3E1022 (S64)
1PS025 (S78)
2PS023 (S114)
3PS030 (S151)
1PT128 (S90)
Nishizawa, Tomohiro (西澤 知宏)
1YS0930 (S90)
Nitta, Ryo (仁田 亮)
3H0900 (S69)
Niwa, Fumihiro (丹羽 史尋)
3PT206 (S175)
Niwa, Ken (丹羽 健)
1PT113 (S87)
Niwa, Tatsuya (丹羽 達也)
1B1510 (S21)Nobe, Yukiko (野辺 由紀子)
1I1424 (S35)
Noda, Naoki (野田 直紀)
2PS020 (S113)
1YS0945 (S113)
Noda, Sigeho (野田 繁穂)
2SD-03 (S12)
1PT101 (S85)
Noguchi, Hiroki (野口 大貴)
3I0936 (S72)
Noguchi, Masato (野口 正人)
Noguchi, Takumi (野口 巧)
3D1034 (S63)
3PT226 (S179)
3PT229 (S180)
3PT231 (S180)
3PT235 (S181)
Noguchi, Taro Q.P. (野口 太郎)
2SE-03 (S13)
2PS035 (S116)
Noguchi, Yukina (野口 有希奈)
2PS006 (S111)
Noji, Hiroyuki (野地 博之)
3A0936 (S56)
Noji, Hiroyuki (野地 博行)
1SA-02 (S1)
1A1436 (S19)
3A0924 (S56)
3A0948 (S56)
3A1010 (S56)
3F0900 (S65)
1PS029 (S79)
1PS033 (S79)
1YS1115 (S79)
2PT207 (S140)
3PT103 (S157)
Noji, Sumihare (野地 澄晴)
1F1436 (S29)Nomiya, Yui (野宮 唯)
1PS012 (S76)
Nomura, Fumimasa (野村 典正)
2D1510 (S44)
3PS020 (S149)
3PS022 (S149)
3PS023 (S149)
3PS046 (S154)
3PS049 (S154)
Nomura, Keiji (野村 圭史)
3G0900 (S67)
Nomura, Shin-ichiro (野村 慎一郎)
3PT112 (S159)
3PT113 (S159)
Nomura, Shin-ichiro M. (野村 M. 慎一郎)
1E1448 (S27)
1PT228 (S107)
Nomura, Shin-ichiro. M (野村 慎一郎)
1PT226 (S107)
Nomura, Sin-ichiro (野村 慎一郎)
1PT227 (S107)
Nomura, Takao (野村 尚生)
2B1558 (S41)
Nonaka, Takamasa (野中 孝昌)
1PT179 (S99)
Noriyuki, Kodera (Noriyuki Kodera)
1H1522 (S34)
Noto, Kaori (能登 香)
3PT225 (S179)
Nozawa, Shunsuke (野澤 俊介)
3PS024 (S150)
Nozoe, Takashi (野添 嵩)
2C1610 (S43)
Nukina, Nobuyuki (貫名 信行)
2B1558 (S41)
Numata, Osamu (沼田 治)
2PS037 (S116)
Numata, Tomoko (沼田 朋子)
3PT234 (S181)
Nunomura, Wataru (布村 渉)
1PT107 (S86)
Nureki, Osamu (濡木 理)
1SH-02 (S7)
1PT128 (S90)
1YS0930 (S90)
Oasa, Sho (大浅 翔)
1PT205 (S103)
Oba, Tetsuya (大羽 哲也)
2I1510 (S54)
Obana, Eriko (尾華 絵里子)
3PT119 (S160)
Oda, Masayuki (織田 昌幸)
1C1522 (S24)
2E1522 (S46)
3B0924 (S58)
1PT130 (S90)
Oda, Toshiro (小田 俊郎)
1PT003 (S83)
Odawara, Aoi (小田原 あおい)
1PT220 (S105)
Ode, Hirotaka (大出 裕高)
1PT102 (S85)
Ogata, Aya (緒方 彩)
1PT231 (S107)− S191 −Ogata, Kazuhiro (緒方 一博)
Ogata, Koji (緒方 浩二)

3PT010 (S157)
3PT227 (S179)
3PT232 (S180)
Ogata, Kouji (緒方 浩二)
3PT230 (S180)
Ogawa, Naoki (小川 直輝)
3PT101 (S157)
Ogawa, Ruriko (小川 瑠里子)
2PS043 (S117)
Ogawa, Yoko (小川 洋子)
1PT138 (S92)
2SB-01 (S10)
Ogawa, Yutaka (小川 泰)
1E1436 (S27)
Ogi, Hirotsugu (荻 博次)
3I0924 (S71)
Ogura, Takashi (小倉 尚志)
3I1010 (S72)
3I1022 (S72)
3I1034 (S72)
3I1046 (S73)
2PT159 (S132)
Ogura, Toshihiko (小椋 俊彦)
3H0900 (S69)
3PS011 (S147)
Ogura, Yusuke (小倉 裕介)
1I1534 (S36)
Oh-oka, Hirozo (大岡 宏造)
3PT235 (S181)
Ohara, Masahiro (小原 正裕)
1H1534 (S34)
Ohara, Osamu (小原 收)
1PT202 (S102)
3PS031 (S151)
3PT145 (S165)
Ohba, Tetsuhiko (大場 哲彦)
Ohba, Yasunori (大庭 裕範)
1PT144 (S93)
Ohhashi, Yumiko (大橋 祐美子)
2PT113 (S123)
Ohkawa-Nishiwaki, Taeko (大川(西脇) 妙子)
3B1058 (S59)
Ohki, Shinya (大木 進野)
1PT107 (S86)
Ohki, Takashi (大木 高志)
3B0948 (S58)
3H1110 (S71)
1PS047 (S82)
2PS009 (S111)
2SH-05 (S16)
Ohkubo, Fuki (大久保 文貴)
2PT212 (S141)
Ohkubo, Jun (大久保 潤)
Ohkubo, Yukie (大久保 幸恵)
3PT201 (S174)
Ohmae, Eiji (大前 英司)
2E1448 (S46)
Ohnishi, Norikazu (大西 紀和)
2PS050 (S118)
Ohno, Shin-ya (大野 真也)
2PT186 (S137)
Ohno, Takashi (大野 隆)
3PT233 (S180)
3PT234 (S181)
Ohno, Tetsuo (大野 哲生)
2H1558 (S53)
Ohnuki, Jun (大貫 隼)
1PS020 (S77)
1PT002 (S83)
Ohnuma, Kiyoshhi (大沼 清)
3PS045 (S154)
Ohnuma, Kiyoshi (大沼 清)
1D1400 (S25)
3PS051 (S155)
Ohonuma, Kiyoshi (大沼 清)
3PS044 (S153)
Ohta, Akane (太田 茜)
3PT201 (S174)
3PT210 (S176)
Ohta, Makoto (太田 信)
3F0948 (S66)
Ohta, Noboru (太田 昇)
1C1610 (S25)
Ohta, Yoshihiro (太田 善浩)
3PS041 (S153)
3PT175 (S171)
Ohtomo, Hideaki (大友 秀明)
1PT120 (S89)
1PT142 (S93)
Ohtsuki, Ryuji (大槻 隆司)
1C1400 (S23)
Ohuchi, Hideyo (大内 淑代)
1F1436 (S29)
Ohuchi, Shokichi (大内 将吉)
2PT149 (S130)
3PS050 (S155)
Ohue, Masahito (大上 雅史)
2PT104 (S122)
Oikawa, Hiroyuki (小井川 浩之)
1PT152 (S94)
1PT222 (S106)
Oiki, Shigetoshi (老木 成稔)
2SH-01 (S16)
2G1558 (S51)
2G1610 (S51)
3PT164 (S169)
3PT165 (S169)
Oiwa, Kazuhiro (大岩 和弘)
2SC-02 (S11)
1PS031 (S79)
3PS001 (S146)
3PS009 (S147)
Oka, Kotaro (岡 浩太郎)
1PT216 (S105)
3PT204 (S175)
Oka, Masahito (岡 勝仁)
2PT208 (S140)
Oka, Tatsuya (岡 辰也)
3PT169 (S170)
Oka, Toshihiko (岡 俊彦)
3G1058 (S69)
1PT123 (S89)
Okabe, Hiroki (岡部 紘輝)
2PS049 (S118)
Okabe, Kohki (岡部 弘基)
3PS010 (S147)
1YS0915 (S147)
3PS040 (S153)
Okada, Fumiko (岡田 文子)
3G0924 (S68)
Okada, Kaoru (岡田 薫)
3A1034 (S57)
Okada, Keisuke (岡田 佳祐)
3PT161 (S168)
Okada, Mariko (岡田 眞理子)
2PT140 (S128)
3PT179 (S172)
Okada, Norihiro (岡田 昇大)3I1058 (S73)
Okada, Ryuichi (岡田 龍一)
1D1534 (S26)
Okada, Seiji (岡田 誠治)
2PT150 (S130)
Okada, Takeru (岡田 健)
1A1412 (S19)
Okada, Taketo (岡田 岳人)
1D1610 (S26)
Okada, Yasushi (岡田 康志)
2SI-04 (S17)3H0900 (S69)Okada-Hatakeyama, Mariko (岡田 眞理子)
Okahata, Yoshio (岡畑 恵雄)
Okajima, Koji (岡島 公司)
Okajima, Takaharu (岡嶋 孝治)

Okamoto Keisuke (岡本 恵佑)
Okamoto, Ken (岡本 研)
Okamoto, Kenji (岡本 憲二)
Okamoto, Tetsuaki (岡本 哲明)
Okamoto, Yoh (岡本 洋)
Okamoto, Yuko (岡本 祐幸)

Okamura, Hideyasu (岡村 英保)
Okano, Keiko (岡野 恵子)
Okano, Teruo (岡野 光夫)
Okano, Toshiyuki (岡野 俊行)
Okazaki, Fumihiro (岡崎 文洋)
Okazaki, Yuichiro (岡崎 裕一郎)

Oki, Keisuke (沖 佳祐)
Okimoto, Noriaki (沖本 憲明)
Okimura, Chika (沖村 千夏)
Okitsu, Takashi (沖津 貴志)
Okubo, Hiroyuki (大久保 宏之)
Okuda, Satoru (奥田 覚)
Okuma, Yasuo (大熊 康夫)
Okumura, Hisashi (奥村 久士)
Okuno, Daichi (奥野 大地)

Okuno, Masanari (奥野 将成)
Okura, Reiko (大倉 玲子)
Okushita, Keiko (奥下 慶子)
Okuta, Akiko (奥田 明子)
Omagari, Katsumi (尾曲 克己)
Onai, Kiyoshi (小内 清)
Onishi, Yukiko (大西 幸子)
Ono, Koji (小野 晃司)
Ono, Minehisa (小野 巌久)
Ono, Sumire (小野 すみれ)
Ono, Takahito (小野 崇人)
Ono, Takao (小野 尭生)
Ono, Yukiko (小野 友紀子)
Onodera, Yoshikuni (小野寺 宜郷)
Onoe, Hiroaki (尾上 弘晃)

Onoue, Yasuhiro (尾上 靖宏)
Ooishi, Yuki (大石 悠貴)
Oosawa, Kenji (大澤 研二)

Oosawa, Tsuyoshi (大澤 剛)
Oroguchi, Tomotaka (苙口 友隆)
Osaki, Toshihisa (大崎 寿久)

Osako, Masato (大迫 政人)
Osanai, Hiromi (小山内 裕美)
Oshima, Atsunori (大嶋 篤典)
Oshima, Hiraku (尾嶋 拓)

Osoegawa, Hiroya (小副川 博也)
Ota, Hiroki (大田 啓貴)
Ota, Kai (大田 該)
Ota, Motonori (Ota Motonori)Ota, Motonori (太田 元規)

Otani, Nao (大谷 直)
Otomo, Seiu (大友 征宇)
Otsuka, Kensuke (大塚 健介)
Otsuka, Yo (大塚 陽)

Owa, Mikito (大和 幹人)

1G1610 (S32)
1PT162 (S96)
2F1510 (S48)
2D1546 (S45)
3PS014 (S148)
3PS015 (S148)
1PT206 (S103)
2PT137 (S128)
2PT128 (S126)
2SI-02 (S17)
1PS006 (S74)
1PT132 (S91)
3PT130 (S163)
3PT144 (S165)
3PT224 (S179)
3PT003 (S155)
2PT191 (S138)
2PS047 (S118)
2PT191 (S138)
3PT150 (S166)
1PS002 (S74)
1PS003 (S74)
1PS004 (S74)
3PT151 (S167)
1PT188 (S101)
2PT140 (S128)
2PS048 (S118)
1F1558 (S30)
3F1046 (S67)
2PS013 (S112)
1PT219 (S105)
3E1010 (S64)
3E1058 (S65)
1PT131 (S91)
2G1546 (S51)
3A0948 (S56)
3PT167 (S169)
2SH-03 (S16)2C1610 (S43)
1PT215 (S105)
3E0924 (S64)
1D1534 (S26)
2PT138 (S128)
2PT229 (S143)
2PT230 (S143)
2G1546 (S51)
3PT167 (S169)
1PT172 (S98)
3PT202 (S175)
1PT215 (S105)
3PS013 (S148)
1PT236 (S108)
2F1448 (S48)
1PT117 (S88)
1I1546 (S36)
1PT224 (S106)
1PT225 (S106)
3B1110 (S59)
3PT207 (S176)
2PS030 (S115)
2PT110 (S123)
2PT119 (S124)
2PT120 (S125)
3PT124 (S161)
2D1522 (S45)
2D1534 (S45)
2G1412 (S49)
3PT136 (S164)
3PT143 (S165)
3PT163 (S169)
3B1110 (S59)
3PT221 (S178)
3F0924 (S66)
1PT137 (S92)
1PT154 (S95)
3PT116 (S160)
2PT149 (S130)
2A1610 (S40)
3PT226 (S179)
1I1436 (S35)
1B1546 (S22)
1I1424 (S35)
2PT130 (S127)
3PT110 (S159)
3PT233 (S180)
3PT234 (S181)
1G1510 (S31)
1G1448 (S31)
2D1436 (S44)
3H1010 (S70)
3PS006 (S146)
3PS007 (S147)

Oyama, Hirofumi (大山 浩史)

1H1424 (S33)
3PS035 (S152)
2H1510 (S52)
Oyama, Kotaro (大山 廣太郎)
2H1546 (S53)
1PS009 (S75)
Ozaki, Yu-ichi (尾崎 裕一)
2PT219 (S142)
Ozaki, Yukihiro (尾崎 幸洋)
1D1412 (S25)
2F1412 (S47)
Ozaki, Yuya (尾崎 裕哉)
2SH-05 (S16)
Ozawa, Katsuya (小澤 克也)
2PT143 (S129)
Ozawa, Takeaki (小澤 岳昌)
1PT166 (S97)
P. Terada, Tomoki (寺田 智樹)
2E1448 (S46)
P.N., Sunilkumar (P.N. Sunilkumar)
1PT151 (S94)
Pack, Chan-Gi (白 燦基)
Pack, Chan-gi (白 燦基)
1D1424 (S25)
Pack, Chang-Gi (白 燦基)
3PT179 (S172)
Pack, Chang-gi (白 燦基)
2SI-04 (S17)Pack, Changi (白 燦基)
3PT236 (S181)
Pan, Wenshi (Pan Wenshi)
3PT189 (S174)
2PS041 (S117)
Pang, Dai-Wen (庞 代文)Park, Sam-Yong (朴 三用)
3I0912 (S71)
1PT101 (S85)
2SA-02 (S9)Paul, Koushik (Paul Koushik)
3PT124 (S161)
Paxton, Thanai (Paxton Thanai)
Picollo, Alessandra (ピッコロ アレサンドラ)
3PT118 (S160)
Pilizota, Teuta (Pilizota Teuta)
1A1534 (S20)
Plazanet, Marie (Plazanet Marie)
1PT003 (S83)
Ramakrishnan, Charu (Ramakrishnan Charu)
1PT128 (S90)
1YS0930 (S90)
Rao, Madan (Rao Madan)
3H1058 (S71)
Re, Suyong (李 秀栄)
3E1110 (S65)
3D0948 (S62)
Renger, Thomas (レンガー トーマス)
1PT209 (S103)
Rie, Nagai (理恵 永井)
Roberts, Thomas M. (Roberts Thomas M.)
2PS020 (S113)
1YS0945 (S113)
Roder, Heinrich (Roder Heinrich)
1PT150 (S94)
Ronald D., Vale (Ronald D. Vale)
3PS004 (S146)
Royant, Antoine (ロヤン アントン)
2SH-01 (S16)Ryo, Akihide (梁 明秀)
3C1034 (S61)
Sadakane, Kei (貞包 慧)
2PS038 (S116)
Sadamoto, Hisayo (定本 久世)
1D1610 (S26)
Saeki, Shinjiro (佐伯 真二郎)
2PT190 (S138)
1YS1015 (S138)
Saeki, Yasushi (佐伯 泰)
3E1010 (S64)
1PT104 (S86)
1PT180 (S99)
Saeki, Yuko (佐伯 夕子)
1G1610 (S32)
3PT179 (S172)
Sagawa, Takashi (佐川 貴志)
2I1546 (S55)
2I1558 (S55)
Saido, Takaomi (西道 隆臣)
2PT111 (S123)
Saijo-Hamano, Yumiko (西條 由見子)
2PS005 (S110)
Saikusa, Kazumi (七種 和美)
1PT190 (S101)
Sainoo, Yasuyuki (道祖尾 恭之)
2E1412 (S45)
Saita, Eiichiro (税田 英一郎)
1PS026 (S78)
1PS035 (S80)
Saito, Ayumu (斉藤 あゆむ)
2SD-04 (S12)
Saito, Hiroaki (齋藤 大明)1E1400 (S27)
3F1010 (S66)
2PT131 (S127)
Saito, Hiroyuki (斎藤 博幸)
3PT169 (S170)
Saito, Kazuya (齋藤 一弥)
2PT152 (S131)
Saito, Kei (斎藤 慧)
2A1448 (S38)
Saito, Keisuke (斉藤 圭亮)
3D0912 (S62)
Saito, Mami (齋藤 真美)
1PT181 (S100)
Saito, Minoru (斎藤 稔)
1PT235 (S108)
2PT233 (S144)
3PT219 (S178)
3PT220 (S178)
3PT221 (S178)
Saito, Shinji (斉藤 真司)
3PT165 (S169)
Saito, Shoma (齊藤 翔馬)
2PS035 (S116)
Saito, Takashi (斉藤 貴志)
2PT111 (S123)
Saito, Takeshi (斎藤 健)
2SF-03 (S14)
Saito, Takeshi (齊藤 健)
2PT219 (S142)
Saito, Tomofumi (齋藤 智文)
3PS017 (S148)
Saitoh, Sho (斉藤 翔)
1PT222 (S106)
Saitoh, Zenpei (斎藤 善平)
1H1534 (S34)
Sakae, Yoshitake (榮 慶丈)
1PT132 (S91)
Sakaguchi, Miyuki (坂口 美幸)
3I1046 (S73)
Sakai, Chihiro (坂井 ちひろ)
1C1400 (S23)
Sakai, Eri (坂井 絵厘)
1PT153 (S95)
Sakai, Kazumi (酒井 佳寿美)
1F1436 (S29)
Sakai, Kazunari (坂井 一成)
3PT159 (S168)
Sakai, Makoto (酒井 誠)
2SH-02 (S16)
3PS038 (S152)
Sakai, Shin-ichi (坂井 慎一)
1D1400 (S25)
3PS045 (S154)
Sakakibara, Hitoshi (榊原 斉)
3PS001 (S146)
3PS009 (S147)
Sakakibara, Manabu (榊原 学)
1D1534 (S26)− S192 −Sakakibara, Shotaro (榊原 正太郎)
Sakamoto, Akihiko (坂本 明彦)
Sakamoto, Hiroshi (坂本 寛)
Sakamoto, Shigetaka (坂本 盛宇)
Sakata-Sogawa, Kumiko (十川 久美子)

Sakiyama, Hiroki (崎山 裕紀)
Sakiyama, Yusuke (崎山 雄介)
Sako, Yasushi (佐甲 靖志)

Sakonji, Mitsuhiro (左近司 充洋)
Sakumura, Yuichi (作村 諭一)
Sakuraba, Shun (櫻庭 俊)
Sakurai, Kazumasa (櫻井 一正)
Sakurai, Keisuke (櫻井 啓介)

Sakurai, Minoru (櫻井 実)

Sakurai, Takanobu (桜井 敬展)
Sakurai, Takashi (櫻井 孝司)
Sakurai, Takashi (櫻井 貴志)
Sakurai, Takashi (櫻井 隆)
Sakurai, Takeshi (櫻井 武)
Sakurai, Yasuyuki (櫻井 靖之)
Sakurai, Yu (櫻井 佑)
Samejima, Masahiro (鮫島 正浩)
Sannomiya, Takumi (三宮 工)
Sano, Masaki (佐野 雅己)
Sano, Tadahisa (佐野 宰久)
Sano, Takahiko (佐野 貴彦)
Sano, Yuko (佐野 裕子)
Sarai, Akinori (皿井 明倫)
Saruyama, Yasuo (猿山 靖夫)
Sasahara, Kenji (笹原 健二)
Sasai, Masaki (笹井 理生)

Sasai, Yoshiki (笹井 芳樹)
Sasaki, Akira (佐々木 章)
Sasaki, Akira (佐々木 顕)
Sasaki, Kazuo (佐々木 一夫)

Sasaki, Takanori (佐々木 貴規)
Sasaki, Yuji (佐々木 裕次)
Sasaki, Yuji C. (佐々木 裕次)
Sasamoto, Kouhei (笹本 浩平)
Sasano, Kouhei (佐々野 耕平)
Sato, Akihiko (佐藤 秋彦)
Sato, Chikara (佐藤 主税)

Sato, Daisuke (佐藤 大輔)
Sato, Francielle (佐藤 フランシエーレ)
Sato, Fuminori (佐藤 文憲)
Sato, Hiroyuki (佐藤 博之)
Sato, Kaori (佐藤 香)
Sato, Katsuya (佐藤 克也)
Sato, Keiko (佐藤 啓子)
Sato, Koji (佐藤 幸治)

2B1448 (S40)
1PT208 (S103)
3PS040 (S153)
1PT185 (S100)
1I1424 (S35)
2SG-02 (S15)1H1424 (S33)
3PS033 (S151)
3PS034 (S152)
3PS035 (S152)
3PT181 (S172)
3PT177 (S171)
2PS053 (S119)
1D1412 (S25)
1D1424 (S25)
1G1558 (S32)
1G1610 (S32)
1PT151 (S94)
1PT204 (S102)
1PT210 (S104)
2PS044 (S117)
2PT128 (S126)
3PT179 (S172)
3PT236 (S181)
2PT172 (S134)
2PS052 (S119)
2PT185 (S137)
1E1436 (S27)
1PT145 (S93)
1PT117 (S88)
2PT162 (S133)
2PT164 (S133)
3PT115 (S160)
2F1400 (S47)
3G0912 (S67)
1PT162 (S96)
2PT125 (S126)
2PT185 (S137)
3PT133 (S163)
3PT160 (S168)
3PT189 (S174)
2D1424 (S44)
1PT234 (S108)
2A1610 (S40)
2PT112 (S123)
2B1546 (S41)
3C0936 (S60)
1PT236 (S108)
1H1546 (S34)
1SF-01 (S6)
2PT109 (S123)
3PT158 (S168)
3PT235 (S181)
2PT106 (S122)
3PT108 (S158)
3PT150 (S166)
3PT151 (S167)
2B1522 (S41)
1SE-01 (S5)1G1522 (S32)
1PT166 (S97)
1PT167 (S97)
1PT217 (S105)
2PS010 (S111)
2PS017 (S112)
2PT213 (S141)
1PT219 (S105)
1H1400 (S33)
1PT205 (S103)
2PT211 (S140)
1A1424 (S19)2H1522 (S52)
1PS028 (S78)
1PS029 (S79)
3PT126 (S162)
2PT121 (S125)
3PS024 (S150)
3B0924 (S58)
2C1546 (S43)
1PS013 (S76)
1PS040 (S81)
3E1022 (S64)
3H0900 (S69)
1PT183 (S100)
1PT142 (S93)
2PT169 (S134)
2PT116 (S124)
2F1400 (S47)
1PS017 (S77)
2PS048 (S118)
2SE-05 (S13)
2PS026 (S114)
2G1412 (S49)Sato, Masaaki (佐藤 政秋)

2SI-04 (S17)3PS013 (S148)
3H1022 (S70)
Sato, Masaaki (佐藤 正明)
2PS036 (S116)
Sato, Masayuki J. (佐藤 雅之)
2PS051 (S119)
Sato, Nozomi (佐藤 希美)
3I1058 (S73)
Sato, Takaki (佐藤 孝樹)
3PS038 (S152)
1PS020 (S77)
Sato, Takato (佐藤 昂人)
1PT002 (S83)
3PS024 (S150)
Sato, Tokushi (佐藤 篤志)
1PT227 (S107)
Sato, Yusuke (佐藤 佑介)
2PS039 (S116)
Satoh, Ayumi (佐藤 歩美)
1B1436 (S21)
Satoh, Tadashi (佐藤 匡史)
Satoshi, Fujii (藤井 聡)
2PT102 (S121)
Satoshi, Komori (小森 慧)
2PT102 (S121)
Satou, Osamu (佐藤 治)
2PS053 (S119)
Sawabu, Yusuke (澤武 裕輔)
2C1448 (S42)
Sawada, Kengo (澤田 賢吾)
2PT002 (S119)
2PT003 (S120)
2PT004 (S120)
2PT009 (S121)
2PT202 (S139)
3PT125 (S162)
Sawada, Shin-ichi (澤田 晋一)
Sawada, Yasuhiro (澤田 泰宏)
3H1046 (S71)
Sawada, Yasuyuki (澤田 康之)
3PT132 (S163)
Sawai, Satoshi (澤井 哲)
1SF-04 (S6)3H0936 (S70)2PS045 (S118)
2PT239 (S145)
3PT171 (S170)
Sawaki, Hiroyuki (澤木 浩之)
3PS036 (S152)
Sawano, Yoriko (澤野 頼子)
2PT114 (S124)
2SH-03 (S16)
Segawa, Hiroki (瀬川 尋貴)
1PS028 (S78)
Seino, Akihiko (清野 晶彦)
Seki, Kazuha (関 一葉)
2PT161 (S133)
Seki, Yasutaka (関 安孝)
1PT009 (S84)
1PT179 (S99)
Sekiguchi, Hiroshi (関口 博史)
3B0924 (S58)
2PT121 (S125)
Sekiguchi, Junichi (関口 順一)
1PT106 (S86)
Sekiguchi, Tetsushi (関口 哲志)
1PT234 (S108)
Sekiguchi, Yuki (関口 優希)
2D1522 (S45)
Sekiguichi, Hiroshi (関口 博史)
3PS024 (S150)
Sekijima, Masakazu (関嶋 政和)
1PT189 (S101)
Sekimura Miho (関村 美穂)
1PT206 (S103)
Sekine, Rui (関根 瑠威)
1PT234 (S108)
Senda, Miki (千田 美紀)
3E1022 (S64)
Senda, Toshiya (千田 俊哉)
3E1022 (S64)
Seno, Keiji (妹尾 圭司)
2PT180 (S136)
Seo, Daisuke (瀬尾 悌介)
2PT112 (S123)
Seo, Hideo (瀬尾 英雄)
1PS043 (S81)
1PS044 (S81)
2PS038 (S116)
Serizawa, Takahiro (芹澤 隆博)
2H1510 (S52)
Setou, Mitsutoshi (瀬藤 光利)
2SH-04 (S16)
3A0912 (S56)
3PS028 (S150)
Sezutsu, Hideki (瀬筒 秀樹)
2PT206 (S140)
Shaevitz, Joshua W. (Shaevitz Joshua W.)
2SE-06 (S13)
Shaji, Divya (Shaji Divya)
1I1436 (S35)
Shen, Jian-Ren (沈 建仁)
3D0948 (S62)
3D1022 (S63)
Shen, Nan (沈 楠)
1PT186 (S100)
Sherwood, Mark W. (SHERWOOD Mark W.)
3PT206 (S175)
Shiba, Rumi (芝 るみ)
1PT124 (S89)
Shibafuji, Yusuke (柴藤 佑介)
1F1558 (S30)
Shibata, Akihiro (柴田 明裕)
1PT209 (S103)
Shibata, Akihiro C. E. (柴田 明裕)
3PT172 (S170)
Shibata, Keitaro (柴田 桂太朗)
2A1448 (S38)
2A1510 (S39)
Shibata, Mikihiro (SHIBATA MIKIHIRO)
3F1058 (S67)
Shibata, Mikihiro (柴田 幹大)
3PS016 (S148)
Shibata, Tatsuo (柴田 達夫)
2SF-04 (S14)1D1424 (S25)
3H0948 (S70)
2PT225 (S143)
2PT226 (S143)
Shibata, Yutaka (柴田 穣)
3D0948 (S62)
Shibayama, Naoya (柴山 修哉)
3I0912 (S71)
Shichida, Yoshinori (七田 芳則)
1F1424 (S29)
1F1436 (S29)
3B0936 (S58)
3F1022 (S66)
2PT190 (S138)
1YS1015 (S138)
Shidara, Hisashi (設樂 久志)
3PT204 (S175)
Shigeri, Yasushi (茂里 康)
3G0900 (S67)
Shigeta, Yasutami (繁田 泰民)
3PT149 (S166)
Shiina, Masaaki (椎名 政昭)
3PT010 (S157)
Shima, Tomohiro (島 知弘)
2A1522 (S39)
Shimabukuro, Katsuya (島袋 勝弥)
2PS020 (S113)

1YS0945 (S113)
2F1424 (S48)
3I1046 (S73)
2PT156 (S131)
2PT162 (S133)
2PT164 (S133)
3PT129 (S162)
2PT203 (S139)
Shimada, Masami (島田 真実)
2H1534 (S53)
Shimada, Masato (島田 万里)
2PT173 (S135)
Shimada, Mayu (島田 麻由)
3PT171 (S170)
Shimada, Nao (島田 奈央)
2SC-01 (S11)
Shimamoto, Yuta (島本 勇太)
2SD-04 (S12)
Shimamura, Teppei (島村 徹平)
Shimazaki, Megumi (嶋崎 恵)
3PT159 (S168)
Shimazaki, Ryo (島崎 亮)
1PT235 (S108)
Shimbo, Yudai (新保 雄大)
1PT009 (S84)
Shimizu, Hirofumi (清水 啓史)
2SH-01 (S16)
2G1610 (S51)
Shimizu, Kana (清水 佳奈)
2PT005 (S120)
Shimizu, Kentaro (清水 謙多郎)
2PT006 (S120)
2PT010 (S121)
2PT101 (S121)
2PT133 (S127)
2PT151 (S131)
Shimizu, Miho (清水 美穂)
1SB-04 (S2)
1SB-05 (S2)
1SB-06 (S2)
Shimizu, Nobutaka (清水 伸隆)
2PT123 (S125)
Shimizu, Norihiro (清水 則宏)
1PS008 (S75)
Shimizu, Youské (清水 洋輔)
3PS009 (S147)
Shimobayashi, Shunsuke (下林 俊典)
3C1110 (S61)
Shimokata, Kunitoshi (下方 国稔)
3PT129 (S162)
2PT233 (S144)
Shimokawa, Tomoya (下川 智也)
3B0900 (S57)
Shimomura, Takushi (下村 拓史)
Shimono, Kazumi (下野 和実)
2PT176 (S135)
Shimosato, Taku (下里 卓)
2B1400 (S40)
Shimozawa, Masahiro (下沢 将啓)
3PT181 (S172)
Shimozawa, Togo (下澤 東吾)
2H1448 (S52)
Shimura, Nanako (志村 七子)
1PT202 (S102)
3PS031 (S151)
Shindo, Yuki (新土 優樹)
2PS044 (S117)
Shinjio, Masaji (新庄 正路)
1PT115 (S88)
Shinjo, Masaji (新庄 正路)
1PT157 (S95)
Shinoda, Keiko (篠田 恵子)
1PT173 (S98)
Shinoda, Wataru (篠田 渉)
2G1400 (S49)
Shinohara, Kaoru (Shinohara Kaoru)
1PT114 (S87)
Shinohara, Kyosuke (篠原 恭介)
1SF-02 (S6)Shinohara, Satoko (篠原 聡子)
3H1034 (S71)
Shinohara, Yasuo (篠原 康雄)
3PT119 (S160)
Shinozaki, Daisuke (篠﨑 大将)
1PT005 (S84)
Shinozaki, Kozue (篠崎 梢)
2PT203 (S139)
Shintani, Seine (新谷 正嶺)
2H1510 (S52)
2H1546 (S53)
Shinyashiki, Kengo (新屋敷 健悟)
2PT109 (S123)
Shinzawa-Itoh, Kyoko (伊藤・新澤 恭子)
2PT157 (S132)
Shinzawa-Itoh, Kyoko (新澤-伊藤 恭子)
3I1034 (S72)
Shinzawa-Itoh, Kyoko (新澤伊藤 恭子)
3I1022 (S72)
Shionyu, Masafumi (塩生 真史)
2PT127 (S126)
Shirai, Natsuki (白井 菜月)
2SB-06 (S10)
Shirai, Nobu C. (白井 伸宙)
1B1534 (S22)
Shirai, Tadao (白井 忠雄)
1H1546 (S34)
Shiraishi, Arata (白石 新)
3PS050 (S155)
Shiraishi, Kenji (白石 賢二)
3PT222 (S178)
Shirakawa, Masahiro (白川 昌宏)
2SB-02 (S10)
3PS042 (S153)
Shiraki, Kentaro (白木 賢太郎)
2H1534 (S53)
1PT005 (S84)
Shirakihara, Yasuo (白木原 康雄)
1PS021 (S77)
Shirasaki, Yoshitaka (白崎 善隆)
1PT202 (S102)
3PS031 (S151)
Shiro, Yoshitsugu (城 城宜嗣)
2PT160 (S132)
Shiro, Yoshitsugu (城 宜嗣)
3I1010 (S72)
3I1058 (S73)
2PT155 (S131)
2PT156 (S131)
Shiro, Yoshitugu (城 宜嗣)
2PT157 (S132)
Shiroguchi, Katsuyuki (城口 克之)
3A1022 (S57)
Shiroi, Tetsuya (城井 哲也)
3PT187 (S173)
Shirota, Matsuyuki (城田 松之)3E1046 (S65)
3PT131 (S163)
Shohda, Koh-ichiroh (庄田 耕一郎)
3G1046 (S69)
Shoji, Mitsuo (庄司 光男)
3PT222 (S178)
Shoji, Shuichi (庄子 習一)
1PT234 (S108)
Shoji, Toru (庄司 亨)
3PS023 (S149)
Shomura, Yasuhito (庄村 康人)
3B0912 (S57)
Shouji, Shuichi (庄子 習一)
3PS031 (S151)
Siato, Hiroaki (齋藤 大明)
1PT001 (S83)
Simozawa, Togo (下澤 東吾)
1PS009 (S75)
Sircar, R. (Sircar Ria)
2SA-02 (S9)Sneppen, Kim (Sneppen Kim)
2C1424 (S42)
So, Masatomo (宗 正智)
1E1436 (S27)
Soda, Kunitsugu (曽田 邦嗣)
1PT009 (S84)
1PT179 (S99)
Shimad, Mayu (島田 麻由)
Shimada, Hideo (島田 秀夫)− S193 −Sodeyama, Fumiaki (袖山 文彰)
Sodeyama, Kohei (袖山 浩平)
Soga, Haruka (曽我 遥)
Soga, Naoki (曽我 直樹)

1PS011 (S75)
1PT160 (S96)
1C1534 (S24)
3A1046 (S57)
1PS027 (S78)
Sogo, Takao (十河 孝夫)
2PT207 (S140)
Sohma, Yoshiro (相馬 義郎)
2G1534 (S51)
3PT133 (S163)
1C1412 (S23)
Sohya, Shihori (惣谷 志保里)
2B1448 (S40)
Sokabe, Masahiro (曽我部 正博)
1PT203 (S102)
2PS034 (S115)
3PT132 (S163)
Somei, Yusaku (染井 悠作)
3PS048 (S154)
Somiya, Takayasu (宗宮 孝安)
2H1412 (S52)
Song, Jianxing (Song Jianxing)
1PT115 (S88)
1PT157 (S95)
Sonoda, Satoru (園田 悟)
3PT201 (S174)
Sonoyama, Masashi (園山 正史)
3PT123 (S161)
3PT128 (S162)
Sowa, Yoshiyuki (Sowa Yoshiyuki)
1A1534 (S20)
1A1558 (S20)
Sowa, Yoshiyuki (曽和 義幸)
2PS001 (S110)
2PS004 (S110)
3PS032 (S151)
3PT180 (S172)
Sowa, Yosiyuki (曽和 義幸)
3PT186 (S173)
Stasevich, Timothy J. (スタセビッチ ティモシー)2SG-01 (S15)
Stewart, Murray (Stewart Murray)
2PS020 (S113)
1YS0945 (S113)
Sudo, Yuki (須藤 雄気)
1F1558 (S30)
1C1436 (S23)
Suga, Hiroaki (菅 裕明)
3E1022 (S64)
Suga, Mitsuo (須賀 三雄)
Sugase, Kenji (菅瀬 謙治)
3C1034 (S61)
1PT112 (S87)
Sugase, Kenjji (菅瀬 謙治)
2E1510 (S46)
Sugawa, Mitsuhiro (須河 光弘)
1PS025 (S78)
Sugawara, Hideaki (菅原 英明)
3PT158 (S168)
Sugawara, Ko (菅原 皓)
3PS040 (S153)
Sugawara, Tadashi (菅原 正)3G1046 (S69)
3PT142 (S165)
Sugawara, Takeshi (菅原 武志)
1SD-04 (S4)Sugihara, Fuminori (杉原 文徳)
3PS042 (S153)
3PS043 (S153)
Sugihara, Minoru (杉原 稔)
1SH-06 (S8)
Sugiki, Toshihiko (杉木 俊彦)
3PT002 (S155)
Sugimori, Kimikazu (杉森 公一)
2PT131 (S127)
Sugimoto, Hiroshi (杉本 宏)
3I1010 (S72)
3I1058 (S73)
Sugimoto, Ikuyo (杉本 育代)
1PT211 (S104)
Sugimoto, Manabu (杉本 学)
1G1448 (S31)
2D1436 (S44)
3H1010 (S70)
Sugimoto, Yasunobu (杉本 泰伸)
3H1034 (S71)
Sugimura, Kaoru (杉村 薫)
1SI-06 (S9)1D1436 (S25)
Sugishima, Masakazu (杉島 正一)
3I0936 (S72)
Sugita, Masatake (杉田 昌岳)
3E1034 (S65)
1PT127 (S90)
Sugita, Yuji (杉田 有治)
3B1022 (S58)
3E1110 (S65)
1PT201 (S102)
1YS1100 (S102)
1PT212 (S104)
2PT130 (S127)
2PT139 (S128)
3PT157 (S168)
Sugiura, Haruka (杉浦 晴香)
2PT238 (S145)
Sugiura, Kotomi (杉浦 琴美)
3PT206 (S175)
Sugiura, Miwa (杉浦 美羽)
3D1034 (S63)
Sugiura, Shinji (杉浦 慎治)
3PS044 (S153)
3PS051 (S155)
Sugiura, Wataru (杉浦 亙)
1PT102 (S85)
3PT003 (S155)
Sugiyama, Hiroshi (杉山 弘)
2SI-06 (S18)
1PS042 (S81)
3PT111 (S159)
Sugiyama, Shigeru (杉山 成)
1PT116 (S88)
2PT116 (S124)
Sugiyama, Sho (杉山 将)
2PT161 (S133)
Sumi, Tomonari (墨 智成)
2I1412 (S54)
Sumikama, Takashi (炭竈 享司)
2G1558 (S51)
3PT165 (S169)
Sumikoshi, Kazuya (角越 和也)
2PT010 (S121)
Sumino, Ayumi (角野 歩)
2G1558 (S51)
Sumino, Yutaka (住野 豊)
2SC-02 (S11)
Sumita, Kazumasa (住田 一真)
1PT121 (S89)
Sumitomo, Koji (住友 弘二)
3PT148 (S166)
Sunada, Kai (砂田 海)
1PS006 (S74)
Sunaga, Yasuhiro (須永 泰弘)
2SD-03 (S12)Sunami, Takeshi (角南 武志)
2PT205 (S139)
Sunami, Tomoko (角南 智子)
1B1522 (S22)Sunato, Ayumi (砂戸 歩美)
Sutoh, Kazuo (須藤 和夫)
Suwa, Makiko (諏訪 牧子)
Suyama, Akira (陶山 明)

Suyong, Re (李 秀栄)
Suzki, Ikuro (鈴木 郁朗)
Suzuki Ryo (鈴木 亮)
Suzuki, Akihiro (鈴木 明大)
Suzuki, Akiyoshi (鈴木 章義)
Suzuki, Atsuo (鈴木 淳巨)
Suzuki, Haruo (鈴木 春男)
Suzuki, Hiroaki (鈴木 宏明)
Suzuki, Hirofumi (鈴木 博文)
Suzuki, Hiroki (鈴木 博己)
Suzuki, Hiromi (鈴木 博実)
Suzuki, Ikuro (鈴木 郁郎)
Suzuki, Ikurou (鈴木 郁郎)
Suzuki, Kazuo (鈴木 和夫)
Suzuki, Kazutaka (鈴木 一隆)
Suzuki, Kazuya (鈴木 和也)
Suzuki, Kenichi (鈴木 健一)
Suzuki, Kenichi G. N. (鈴木 健一)
Suzuki, Koji (鈴木 康二)
Suzuki, Madoka (鈴木 団)
Suzuki, Makoto (鈴木 誠)

Suzuki, Miho (鈴木 美穂)
Suzuki, Nami (鈴木 南美)
Suzuki, Naoya (鈴木 直哉)
Suzuki, Nobutake (鈴木 信勇)
Suzuki, Shingo (鈴木 真吾)
Suzuki, Takao K. (鈴木 誉保)Suzuki, Toshiharu (鈴木 俊治)

Suzuki, Yo (鈴木 陽)
Suzuki, Yu (鈴木 悠)
Suzuki, Yuta (鈴木 雄太)
Tabata, Kazuhito (田端 和仁)
Tabata, Kazuhito V. (田端 和仁)
Tabei, Yasuo (田部井 靖生)
Tachibana, Hideki (橘 秀樹)
Tachikawa, Masashi (立川 正志)

Tachiwana, Hiroaki (立和名 博昭)
Tadakuma, Hisashi (多田隈 向史)
Tadakuma, Hisashi (多田隈 尚史)
Tadokoro, Satoshi (田所 哲)
Taga, Shun (多賀 瞬)
Tagaki, Seiji (高木 清二)
Tagawa, Miho (田川 美穂)
Taguchi, Hideki (田口 英樹)
Taguchi, Takahisa (田口 隆久)
Tahara, Kentaro (田原 健太朗)
Tahara, Kohei (田原 耕平)
Taiji, Makoto (泰地 真弘人)

Tajika, Ryosuke (田鹿 良介)
Tajima, Hirotaka (田島 寛隆)

Tajima, Kana (田島 可奈)
Tajima, Tomoki (田島 朋樹)
Tajiri, Yusuke (田尻 祐介)
Takaba, Kiyofumi (高場 圭章)
Takada, Sayuri (高田 さゆり)
Takada, Shoji (Takada Shoji)
Takada, Shoji (高田 彰二)

1PT142 (S93)
3A1022 (S57)
1SH-06 (S8)3C0912 (S60)
3G1046 (S69)
3PT110 (S159)
1PT212 (S104)
3PT209 (S176)
1PT206 (S103)
2D1522 (S45)
3PT221 (S178)
1PT102 (S85)
1PT175 (S98)
2PT205 (S139)
1PT119 (S88)
2G1522 (S50)
1PT109 (S87)
1PT220 (S105)
1PT221 (S106)
3PS019 (S149)
3PT202 (S175)
3PT214 (S177)
3H0912 (S69)
2PS041 (S117)
1G1412 (S31)
1PT102 (S85)
2H1510 (S52)
1E1558 (S28)
2H1424 (S52)
1PS002 (S74)
1PS003 (S74)
1PS004 (S74)
1PS005 (S74)
1PT010 (S85)
1PT182 (S100)
2PT204 (S139)
3PT189 (S174)
3PT208 (S176)
1PT202 (S102)
3PS031 (S151)
2PT209 (S140)
2PT206 (S140)
3A1046 (S57)
1PS027 (S78)
1PS035 (S80)
3C0900 (S59)
1PT111 (S87)
1PT152 (S94)
3F0900 (S65)
2PT207 (S140)
1PS033 (S79)
1YS1115 (S79)
2PT005 (S120)
2B1510 (S41)
1PT114 (S87)
1D1424 (S25)
1G1436 (S31)2C1510 (S43)
1PT190 (S101)
3PT111 (S159)
3F0912 (S65)
1PS042 (S81)
3PT139 (S164)
3PT177 (S171)
2PS030 (S115)
2C1534 (S43)
3PT110 (S159)
1B1510 (S21)
1PT151 (S94)
1D1546 (S26)
1PT236 (S108)
3PT139 (S164)
1PT009 (S84)
1PT188 (S101)
2PT140 (S128)
1PT122 (S89)
1PT233 (S108)
3PT186 (S173)
3PT187 (S173)
3F1046 (S67)
3PS038 (S152)
2PT153 (S131)
3E0912 (S63)
3I0948 (S72)
1PT178 (S99)
1SE-05 (S5)2E1558 (S47)
3C0948 (S60)
1PS037 (S80)
1PT172 (S98)
1PT181 (S100)
2PT143 (S129)
3PT006 (S156)

3PT007 (S156)
3PT009 (S157)
3PT011 (S157)
Takada, Syoji (高田 彰二)
1PT171 (S98)
Takagi, Daisuke (高木 大輔)
2PT228 (S143)
Takagi, Hiroaki (高木 拓明)
2PS051 (S119)
Takagi, Hiroyuki (高木 啓行)
1E1400 (S27)
3F1010 (S66)
3H0912 (S69)
Takagi, Jun (高木 潤)
2PT162 (S133)
Takagi, Kenji (高木 賢治)
2PT164 (S133)
3G1022 (S68)
Takagi, Masahiro (高木 昌宏)
3PT153 (S167)
1YS1045 (S167)
Takagi, Toshiyuki (高木 俊之)
3PT123 (S161)
3PT169 (S170)
Takahashi, Chisato (高橋 知里)
2G1510 (S50)
Takahashi, Fumio (高橋 文雄)
2F1436 (S48)
2PT192 (S138)
Takahashi, Hironobu (高橋 宏暢)
1D1610 (S26)
Takahashi, Hiroshi (高橋 浩)
3PT123 (S161)
3PT146 (S166)
3PT147 (S166)
Takahashi, Hiroto (高橋 泰人)
2I1546 (S55)
2I1558 (S55)
2PS003 (S110)
Takahashi, Kei (髙橋 慧)
3PT171 (S170)
Takahashi, Ken-ichi (高橋 健一)
2PT127 (S126)
Takahashi, Koichi (高橋 恒一)
2SD-02 (S12)2C1412 (S42)
2PS044 (S117)
2PT215 (S141)
3PT183 (S172)
2SI-04 (S17)
Takahashi, Kouichi (高橋 恒一)
Takahashi, Kyohei (高橋 恭平)
1PT190 (S101)
Takahashi, Layla (高橋 玲羅)
3F1034 (S67)
Takahashi, Masahiro (高橋 正裕)
1PT210 (S104)
Takahashi, Ryosuke (高橋 亮輔)
3PS015 (S148)
Takahashi, Satoshi (高橋 聡)
2E1412 (S45)
1PT144 (S93)
1PT152 (S94)
1PT222 (S106)
Takahashi, Seiya (高橋 聖弥)
2E1412 (S45)
Takahashi, Takuya (高橋 卓也)
1PT008 (S84)
Takahashi, Takuya (髙橋 卓也)
1PT007 (S84)
Takahashi, Tomoki (高橋 智樹)
3G1058 (S69)
3PT173 (S171)
Takahashi, Yuka (高橋 優嘉)
2PS002 (S110)
Takahashi, Yukio (高橋 幸生)
2D1522 (S45)
Takai, Eisuke (高井 英輔)
2H1534 (S53)
Takai, Shinji (高井 進二)
1PS038 (S80)
Takai, Tomoyo (高井 朋代)
2PT126 (S126)
Takaine, Masak (高稲 正勝)
2PS037 (S116)
Takakuwa, Yuichi (高桑 雄一)
1PT107 (S86)
Takamatsu, Atsuko (高松 敦子)
1SF-02 (S6)
Takanezawa, Sota (高根沢 聡太)
1D1412 (S25)
Takano, Hiroshi (高野 宏)
2B1424 (S40)
Takano, Keiko (鷹野 景子)
3PT225 (S179)
Takano, Mitsunori (高野 光則)
1PS020 (S77)
1PS039 (S81)
1PT002 (S83)
1PT168 (S97)
1PT169 (S97)
Takano, Yu (鷹野 優)
3B1010 (S58)
3PT223 (S179)
Takaoka, Yousuke (高岡 洋輔)
2PT151 (S131)
Takaori, Akifumi (高折 晃史)
3C1034 (S61)
Takasu, Masako (高須 昌子)
1PT135 (S91)
2PT131 (S127)
Takata, Masaki (高田 昌樹)
3PT146 (S166)
Takayama, Yuki (高山 裕貴)
2D1522 (S45)
Takayanagi, Masayoshi (高柳 昌芳)
2E1436 (S46)
2PT165 (S133)
Takayuki, Uchihashi (内橋 貴之)
3PS004 (S146)
Takeda, Kazuki (竹田 一旗)
3E0912 (S63)
1PT123 (S89)
Takeda, Mahoshi (武田 真星)
1PT145 (S93)
Takeda, Shigeki (武田 茂樹)
3F1034 (S67)
Takeda, Shuichi (武田 修一)
1B1546 (S22)
Takehana, Yasuhiro (竹花 康弘)
2PS040 (S117)
Takei, Hiroyuki (竹井 弘之)
3PS047 (S154)
Takei, Takaya (竹井 敬哉)
2PT149 (S130)
Takei, Yodai (武井 洋大)
3PS029 (S151)
Takekawa, Norihiro (竹川 宜宏)
2I1534 (S55)
Takemori, Shigeru (竹森 重)
1SB-09 (S3)2H1558 (S53)
Takemura, Kazuhiro (竹村 和浩)
1PS019 (S77)
1PT174 (S98)
2PT132 (S127)
Takenaka, Toshio (TAKENAKA Toshio)
1B1412 (S21)
Takenaka, Toshio (竹中 健朗)
1PT143 (S93)
Takenaka, Yoshiko (武仲 能子)
1SB-02 (S2)
3PT236 (S181)− S194 −Takeshita, Daijiro (竹下 大二郎)
Taketani, Hiroshi (竹谷 寛司)
Takeuchi, Hideo (竹内 英夫)
Takeuchi, Kazuki (竹内 一樹)

3C1022 (S61)
2SI-02 (S17)2E1424 (S46)
1C1610 (S25)
2PT129 (S126)
Takeuchi, Ken (竹内 健)
3E0936 (S64)
2PT192 (S138)
1I1546 (S36)
Takeuchi, Shoji (竹内 昌治)
2G1412 (S49)
1PT225 (S106)
2PT207 (S140)
3PT143 (S165)
3PT163 (S169)
Takeuchi, Syoji (竹内 昌治)
3PT136 (S164)
Takigami, Satoshi (滝上 慧)
1D1534 (S26)
Takiguchi, Kingo (滝口 金吾)
3PT141 (S165)
Takiguchi, Yohko (滝口 陽子)
3PT141 (S165)
Takinoue, Masahiro (瀧ノ上 正浩)
1PT224 (S106)
2PT238 (S145)
Takuo, Yasunaga (安永 卓生)
3E1010 (S64)
Tamada, Taro (玉田 太郎)
3E0912 (S63)
2PT170 (S134)
Tamada, Tarou (玉田 太郎)
2SD-04 (S12)
Tamada, Yoshinori (玉田 嘉紀)
Tamai, Nobutake (玉井 伸岳)
3G0936 (S68)
3G0948 (S68)
Tamaoki, Nobuyuki (玉置 信之)
3PT102 (S157)
Tamashiro, Tomonari (玉城 智成)
1C1522 (S24)
Tamba, Yukihiro (丹波 之宏)
3PT148 (S166)
Tame, Jeremy R.H. (Jeremy R.H. Tame)
1PT101 (S85)
Tamogami, Jun (田母神 淳)
2PT176 (S135)
Tamori, Masaki (田守 正樹)
2PS040 (S117)
Tamura, Atsuo (田村 厚夫)
1E1546 (S28)
1PT165 (S97)
3PT156 (S167)
Tamura, Hiroyuki (田村 浩之)
2PT185 (S137)
Tamura, Koichi (田村 康一)
3B1034 (S59)
Tamura, Takashi (田村 隆)
2PT109 (S123)
Tanabe, Yoichi (田邊 陽一)
1C1522 (S24)
Tanaka, Akihiro (田中 晃弘)
2PS023 (S114)
Tanaka, Aya (田中 あや)
3PT148 (S166)
Tanaka, Gouhei (田中 剛平)
1SC-04 (S4)
Tanaka, Hiroto (田中 裕人)
1PS050 (S82)
Tanaka, Isao (田中 勲)
3F0948 (S66)
Tanaka, Kaori (田中 香織)
2PT149 (S130)
Tanaka, Keiji (田中 啓二)
1PT104 (S86)
Tanaka, Keiko (田中 啓子)
1PT213 (S104)
Tanaka, Masatoshi (田中 正俊)
2PT186 (S137)
Tanaka, Motomasa (田中 元雅)
2PT113 (S123)
Tanaka, Ryosuke (田中 良昌)
1PT230 (S107)
Tanaka, Saeko (田中 佐江子)
3G0936 (S68)
Tanaka, Tomohiro (田中 智大)
1PT171 (S98)
Tanaka, Yoshikazu (田中 良和)
3F0948 (S66)
Tanaka, Yoshimi (田中 良巳)
1SF-06 (S7)
Tanaka, Yuichi (田中 裕一)
3PT219 (S178)
Tanaka, Yusuke (田中 裕介)
3B0924 (S58)
Tanamoto, Ryo (棚元 亮)
3PT204 (S175)
Tang, Chao (Tang Chao)
2SA-04 (S9)Tani, Iori (谷 伊織)
2PT236 (S144)
Tani, Yoshiaki (谷 芳明)
2A1522 (S39)
Tanida, Jun (谷田 純)
1I1534 (S36)
Tanigaki, Yasuyuki (谷垣 保幸)
1PT234 (S108)
Tanigawara, Mizue (谷川原 瑞恵)
3A1010 (S56)
Taniguchi, Daisuke (谷口 大相)
1SF-04 (S6)
Taniguchi, Mieko (谷口 美恵子)
1A1400 (S19)
Taniguchi, Yuichi (谷口 雄一)
2SB-01 (S10)
Tanikawa, Hiromi (谷川 ひろみ)
1PS021 (S77)
Tanio, Michikazu (谷生 道一)
2PT115 (S124)
3PT178 (S172)
Tanizawa, Hideki (谷沢 英樹)
1A1400 (S19)
Tanokura, Masaru (田之倉 優)
2PT114 (S124)
Taoka, Azuma (田岡 東)
2SE-02 (S13)
Tardin, Catherine (Tardin Catherine)
3PT170 (S170)
Tashiro, Yuki (田代 裕己)
1PT159 (S96)
Tate, Shin-ichi (楯 真一)
2E1448 (S46)
Tateno, Masaru (舘野 賢)
1B1610 (S22)
1I1448 (S35)
2PT142 (S129)
2PT166 (S133)
2PT167 (S133)
Tateyama, Emi (館山 絵美)
2PT158 (S132)
Tatsumi, Hitoshi (辰巳 仁史)
2B1448 (S40)
2PS034 (S115)
Taylor, David (Taylor David)
3E0948 (S64)
Terada, Tohru (寺田 透)
1PT147 (S93)
2PT133 (S127)
2PT151 (S131)
Terada, Tomoki (寺田 智樹)
1PT167 (S97)
Terada, Tomoki P. (寺田 智樹)
1PT217 (S105)
2PS010 (S111)
2PT213 (S141)
Terahara, Naoya (寺原 直矢)
2PS006 (S111)
Terakawa, Tsuyoshi (Terakawa Tsuyoshi)
1PT178 (S99)Terakawa, Tsuyoshi (寺川 剛)

1SD-02 (S4)
3PT006 (S156)
3PT007 (S156)
3PT011 (S157)
Terakita, Akihisa (寺北 明久)
1SH-05 (S7)
2PT190 (S138)
1YS1015 (S138)
2PT111 (S123)
Terasawa, Hiroaki (寺沢 宏明)
3PT118 (S160)
Terashima, Hiroyuki (寺島 浩行)
2D1448 (S44)
Terashita, Masahiro (寺下 匡大)2F1510 (S48)
Terazima, Masahide (寺嶋 正秀)
2F1558 (S49)
2PT169 (S134)
2PT175 (S135)
Terazono, Hideyuki (寺薗 英之)
3PS046 (S154)
3PS047 (S154)
Tereda, Tomoki P. (寺田 智樹)
2PS017 (S112)
Terui, Takako (照井 貴子)
1PS009 (S75)Teshima, Tetsuhiko (手島 哲彦)
1I1546 (S36)
Thomas, Neil (Thomas Neil)
1PS030 (S79)
Tirtom, Naciye Esma (Tirtom Naciye Esma)
3A0948 (S56)
3PT005 (S156)
Tiwari, Manisha (Tiwari Manisha)Toba, Shiori (鳥羽 栞)
2A1436 (S38)
Tochio, Hidehito (杤尾 豪人)
2SB-02 (S10)Toda, Masashi (戸田 真志)
2PT234 (S144)
Toda, Masaya (戸田 雅也)
3PS013 (S148)
Toda, Mikito (戸田 幹人)
1PT170 (S98)
1PT189 (S101)
1PT191 (S101)
Toda, Osamu (戸田 収)
3PT124 (S161)
Togashi, Yuichi (冨樫 祐一)
1SA-01 (S1)
2B1610 (S41)
Toichi, Keisuke (東一 圭祐)
2PT107 (S122)
Tokimatsu, Toshiaki (時松 敏明)
Tokino, Takashi (時野 隆至)
2E1412 (S45)
Tokita, Masayuki (鴇田 昌之)
3PT135 (S164)
3PT152 (S167)
Tokuda, Naoko (徳田 直子)
1G1522 (S32)
Tokuda, Naomi (徳田 尚美)
3PT178 (S172)
Tokuda, Syougo (徳田 昇悟)
1PS006 (S74)
Tokuhisa, Atsushi (徳久 淳師)
3PS026 (S150)
Tokunaga, Fumio (徳永 史夫)
2PT190 (S138)
1YS1015 (S138)
Tokunaga, Makio (徳永 万喜洋)
2SG-02 (S15)1H1424 (S33)
3PS033 (S151)
3PS034 (S152)
3PS035 (S152)
3PT104 (S158)
3PT181 (S172)
Tokuraku, Kiyotaka (徳楽 清孝)
2SE-03 (S13)
2PS035 (S116)
2PS039 (S116)
Tokutomi, Satoru (徳富 哲)
2F1510 (S48)
2PT169 (S134)
2PT188 (S138)
Tomaru, Hidetoshi (都丸 英敏)
2PT110 (S123)
Tomaru, Takenori (都丸 武宜)
2PT235 (S144)
Tomii, Kentaro (富井 健太郎)
2PT005 (S120)
Tominaga, Keisuke (富永 圭介)
1E1546 (S28)
3PT156 (S167)
Tominaga, Takashi (冨永 貴志)
1D1558 (S26)
Tominaga, Yoko (冨永 洋子)
1D1558 (S26)
Tomisawa, Satoshi (冨澤 聡)
1C1400 (S23)
Tomishige, Michio (富重 道雄)
1A1436 (S19)
1A1448 (S19)
1PS036 (S80)
1PS037 (S80)
Tomita, Kozo (富田 耕造)
3C1022 (S61)
Tomita, Masahiro (冨田 昌弘)
3PT138 (S164)
Tomita, Masanori (冨田 雅典)
1G1510 (S31)
Tomita, Masaru (冨田 勝)
2PS044 (S117)
Tomita, Noriko (冨田 典子)
3F0948 (S66)
Tomo, Tatsuya (鞆 達也)
3PT229 (S180)
Tomoko, Masaike (政池 知子)
1PS023 (S78)
Tomomi, Ishido (石堂 智美)
3PT101 (S157)
Tomonaga, Yuya (友永 雄也)
1F1558 (S30)
Tomonari, Sayuri (友成 さゆり)
1F1436 (S29)
Tonaka, Kenji (渡仲 健仁)
3PT202 (S175)
Tonooka, Taishi (外岡 大志)
2G1412 (S49)
Torii, Jumpei (鳥井 淳平)
2E1610 (S47)
Torimitsu, Keiichi (鳥光 慶一)
3PT148 (S166)
Torisawa, Takayuki (鳥澤 嵩征)
2A1448 (S38)
2A1510 (S39)
2A1546 (S39)
Tosha, Takehiko (當舎 武彦)
3I1058 (S73)
Touyama, Kanako (當山 奏子)
1PS043 (S81)
1PS044 (S81)
Toyabe, Shoichi (鳥谷部 祥一)
2SI-02 (S17)
1PS023 (S78)
1PS032 (S79)
Toyabe, Shoici (鳥谷部 祥一)
1PS022 (S77)
Toyabe, Syoichi (鳥谷部 祥一)
1PS024 (S78)

Toyama, Yusuke (遠山 祐典)
Toyoshima, Yoko (豊島 陽子)

1D1448 (S25)
1A1510 (S19)2A1448 (S38)
2A1510 (S39)
2A1534 (S39)
2A1546 (S39)
Toyota, Taro (豊田 太郎)
3G1034 (S69)
3PT142 (S165)
3PT171 (S170)
3PT206 (S175)
Triller, Antoine (TRILLER Antoine)
2C1424 (S42)
Trusina, Ala (Trusina Ala)
3PT192 (S174)
Tsubasa, Nakagawa (中川 翼)
3G1058 (S69)
Tsuboi, Taka-aki (坪井 駿明)
3PT174 (S171)
Tsuchiya, Masahiro (土屋 雅博)
3PS014 (S148)
Tsuda, Koji (津田 宏治)
2PT005 (S120)
Tsuji, Yotaro (辻 祐太郎)
3PT163 (S169)
Tsuji, Yusuke (辻 悠佑)
2PT191 (S138)
Tsujikawa, Hiroto (辻川 寛人)
2PT101 (S121)
Tsujiuchi, Yutaka (辻内 裕)
2D1448 (S44)
2G1522 (S50)
3PT158 (S168)
3PT214 (S177)
Tsukada, Hideo (塚田 秀夫)
Tsukada, Yoshihiro (塚田 祥弘)
1PS008 (S75)
Tsukamoto, Hisao (塚本 寿夫)
2PT183 (S137)
2PT190 (S138)
1YS1015 (S138)
Tsukamoto, Takashi (塚本 卓)
2PT179 (S136)
Tsukaoto, Hisao (塚本 寿夫)
3PT168 (S170)
Tsukazaki, Tomoya (塚崎 智也)
1PT128 (S90)
1YS0930 (S90)
Tsukihara, Tomitake (月原 冨武)
2PT157 (S132)
3PT129 (S162)
2F1522 (S48)
Tsukiji, Yuuki (築地 祐樹)
Tsumoto, Kanta (湊元 幹太)
3PT138 (S164)
Tsumoto, Kouhei (津本 浩平)
2E1510 (S46)
3F0912 (S65)
2PT151 (S131)
Tsuneshige, Antonio (常重 アントニオ)
2PT153 (S131)
Tukamoto, Seiichi (塚本 精一)
1PT142 (S93)
Tulum, Isil (Tulum Isil)
2PS027 (S114)
Tunnacliffe, Alan (Tunnacliffe Alan)
1PT162 (S96)
Tuzi, Satoru (辻 暁)
3F1046 (S67)
2PT168 (S134)
3PT178 (S172)
Uchibori, Soyomi (内堀 そよみ)
2PS042 (S117)
Uchida, Junya (内田 潤也)
3PS021 (S149)
Uchida, Kaoru (内田 薫)
2PS031 (S115)
2PS033 (S115)
Uchida, Nariya (内田 就也)
1SF-03 (S6)
Uchida, Waka (打田 和香)
3PT227 (S179)
3PT230 (S180)
3PT232 (S180)
Uchida, Yumiko (内田 裕美子)
1PT105 (S86)
Uchihashi, Takayuki (UCHIHASHI TAKAYUKI)
3F1058 (S67)
Uchihashi, Takayuki (内橋 貴之)
2SB-03 (S10)1H1510 (S34)
2G1534 (S51)
3PS016 (S148)
Uchikoga, Nobuyuki (内古閑 伸之)
2PT104 (S122)
Uchiyama, Seiichi (内山 聖一)
3PS010 (S147)
1YS0915 (S147)
Uda, Shinsuke (宇田 新介)
2SF-03 (S14)
1PT214 (S104)
2PT219 (S142)
Udin, Bahrudin (Udin Bahrudin)
1PT104 (S86)
Ueda, Asami (植田 朝美)
1C1448 (S23)
Ueda, Hiroki (上田 泰己)
1SA-06 (S1)
2C1522 (S43)
Ueda, Kazuyoshi (上田 一義)
3PT127 (S162)
Ueda, Keisuke (植田 啓介)
2H1412 (S52)
Ueda, Masahiro (上田 昌宏)
1G1558 (S32)
2PS051 (S119)
Ueda, Takuya (上田 卓也)
1B1510 (S21)
3F0912 (S65)
1PS042 (S81)
3PT111 (S159)
Ueda, Taro Q.P. (上田 太郎)
2PS039 (S116)
Ueda, Yusuke (植田 悠介)
1D1546 (S26)
Uehara, Wataru (上原 航)
2PT163 (S133)
Ueki, Shoji (植木 正二)
2H1412 (S52)
Uekusa, Yoshinori (植草 義徳)
1B1436 (S21)
Uemura, Tatsuya (植村 達也)
1PT164 (S97)
Ueno, Hiroshi (上野 博史)
2SI-02 (S17)
1PS022 (S77)
1PS023 (S78)
1PS024 (S78)
1PS032 (S79)
Ueno, Shingo (上野 真吾)
2PT147 (S130)
Ueno, Takamasa (上野 貴将)
2E1510 (S46)
Ueno, Taro (上野 太郎)
3PS029 (S151)
Ueno, Yutaka (上野 豊)
1PT184 (S100)− S195 −Uenoyama, Atsuko (Uenoyama Atsuko)
Ueta, Masami (上田 雅美)
Ujisawa, Tomoyo (宇治澤 知代)

2PS027 (S114)
3PT001 (S155)
3PT201 (S174)
3PT210 (S176)
Umeda, Hiroaki (梅田 宏明)
3PT222 (S178)
Umeda, Yusuke (梅田 祐介)
2PT101 (S121)
Umeki, Nobuhisa (梅木 伸久)
2SE-03 (S13)
2PS018 (S113)
2PS047 (S118)
Umemura, Kazuo (梅村 和夫)
3PT105 (S158)
1PT163 (S96)
Umemura, Taizo (梅村 太三)
3PT180 (S172)
Umemura, Tohru (梅村 徹)
1C1448 (S23)
Umetsu, Mitsuo (梅津 光央)
1C1510 (S24)
Umezawa, Koji (梅澤 公二)
1PS020 (S77)
1PT002 (S83)
1PT168 (S97)
1PT169 (S97)
Umezawa, Yuki (梅澤 祐貴)
2PT001 (S119)
Unno, Masashi (海野 雅司)
2F1522 (S48)
Unzai, Satoru (雲財 悟)
1PT101 (S85)
3PT212 (S177)
Urakubo, Hidetoshi (浦久保 秀俊)
3PT130 (S163)
Urano, Ryo (浦野 諒)
Urisu, Tsuneo (宇理須 恒雄)
1SH-03 (S7)
Usukura, Jiro (臼倉 治郎)
1PT118 (S88)
Utida, Yumiko (内田 裕美子)
1PT121 (S89)
Utsumi, Ayaka (内海 彩香)
1PT232 (S108)
Utsumi, Yui (内海 唯)
2A1510 (S39)
Uyeda, Taro (上田 太郎)
1PS001 (S74)
2PS018 (S113)
Uyeda, Taro Q.P. (上田 太郎)
2SE-03 (S13)
Uzawa, Takanori (鵜澤 尊規)
2PT145 (S129)
3PT170 (S170)
Villaruz, Lara (Villaruz Lara)
1F1558 (S30)
Wada, Akimori (和田 昭盛)
3F1046 (S67)
Wada, Akira (和田 明)
3PT001 (S155)
Wada, Hirofumi (和田 浩史)
2SC-04 (S11)
2SE-05 (S13)
2PS026 (S114)
Wada, Naohisa (和田 直久)
2PT186 (S137)
Wada, Yuji (和田 侑士)
2E1448 (S46)
Wakabayashi, Chiaki (若林 千晃)
1PS035 (S80)
Wakabayashi, Ken-ichi (若林 憲一)
2PS050 (S118)
3PS007 (S147)
Wakabayashi, Kenichi (若林 憲一)
3PS006 (S146)
Wakabayashi, Masahiko (若林 正彦)
3PT211 (S176)
3PT216 (S177)
Wakabayashi, Masaki (若林 真樹)
2G1448 (S50)
2G1510 (S50)
3PT162 (S169)
Wakabayashi, Takeyuki (若林 健之)
1PS001 (S74)
Wakai, Nobuhiko (若井 信彦)
1PS019 (S77)
Wakamatsu, Kaori (若松 馨)
3F1034 (S67)
Wakamoto, Yuichi (若本 祐一)
2SF-01 (S14)1D1400 (S25)
2C1610 (S43)
1PT215 (S105)
3PS045 (S154)
Wako, Hiroshi (輪湖 博)
3PT190 (S174)
Wang, Wei (Wang Wei)
1SE-02 (S5)
1PT178 (S99)
2PT145 (S129)
Wang, Yangtian (王 楊天)
1E1558 (S28)
Wang, Yaolai (Wang Yaolai)
1SE-02 (S5)
Wang, Zhuo (王 卓)3E1010 (S64)
Watabe, Masaki (渡部 匡己)
3PT183 (S172)
Watanabe, Aya (渡辺 綾)
2PT120 (S125)
Watanabe, Hajime (渡邉 肇)
1C1534 (S24)
Watanabe, Hikari (渡邊 ひかり)
3E0936 (S64)
Watanabe, Hiroki (渡辺 大輝)
3PS016 (S148)
Watanabe, Keiichi (渡邉 啓一)
2PT122 (S125)
Watanabe, Kunihiko (渡部 邦彦)
3G0900 (S67)
Watanabe, Masakatsu (渡辺 正勝)
1H1610 (S35)
3D0924 (S62)
Watanabe, Nobuhisa (渡邉 信久)
1PT102 (S85)
1PT113 (S87)
Watanabe, Rikiya (渡邉 力也)
3A1010 (S56)
3F0900 (S65)
1PS033 (S79)
1YS1115 (S79)
3PT103 (S157)
Watanabe, Rikiya (渡邊 力也)
3A0936 (S56)
Watanabe, Satoru (渡部 暁)
1PT106 (S86)
Watanabe, Shingo (渡辺 真悟)
1F1412 (S29)
Watanabe, Tomonobu (渡邉 朋信)
2PT118 (S124)
3PS037 (S152)
Watanabe, Toshiyuki (渡辺 敏行)
1SB-06 (S2)
Watanabe, Tsuyoshi (渡邊 剛)
3PT191 (S174)
Watanabe, Yurika (渡辺 百合香)
2PT125 (S126)
Watanabe, Yuta (渡辺 裕多)
2A1448 (S38)
Watanabe-Nakayama, Takahiro (中山 隆宏)
2SI-02 (S17)
Wazawa, Tetsuichi (和沢 鉄一)
1E1558 (S28)

2H1424 (S52)
1PS002 (S74)
1PS003 (S74)
1PS004 (S74)
1PS005 (S74)
1PT182 (S100)
Wijaya, I Made Mahaputra (ウィジャヤ イ マディ マハプトラ)
2F1534 (S49)
Williamson, Michael P. (Williamson Michael P.)
1PT112 (S87)
1PT108 (S86)
Woody, Robert W. (Woody Robert)
1PT158 (S95)
Wright, Peter E. (Wright Peter E.)
1C1412 (S23)
Wu, Nan (呉 楠)
Wulff, Michael (ウルフ ミカエル)
2SH-01 (S16)Xu, Ming (Xu Ming)
1PT150 (S94)
Yagi, Hisashi (八木 寿梓)
1E1436 (S27)
Yagi, Maho (矢木 真穂)
1PT112 (S87)
Yagi, Masahiro (八木 正浩)
1PT140 (S92)
Yagi, Naoto (八木 直人)
2H1610 (S53)
2PT121 (S125)
3PS024 (S150)
2SF-06 (S14)
Yagi, Takeshi (八木 健)
3H0924 (S70)
Yagi, Toshiki (八木 俊樹)
3PS003 (S146)
Yajima, Hiroaki (矢島 孔明)
3H0900 (S69)
Yajima, Junichiro (矢島 潤一郎)
1SI-03 (S8)1A1510 (S19)2A1534 (S39)
1PS040 (S81)
Yamada, Akira (山田 章)
2PS040 (S117)
Yamada, Daichi (山田 大智)2F1546 (S49)
Yamada, Kazuteru (山田 一輝)
1I1510 (S36)
1I1534 (S36)
Yamada, Kenji (山田 憲嗣)
2PT187 (S137)
Yamada, Kenta (山田 健太)
Yamada, Kosuke (山田 浩輔)
3PT120 (S161)
Yamada, Makiko (山田 麻紀子)
2G1510 (S50)
Yamada, Masafumi D. (山田 正文)
1PS038 (S80)
Yamada, Ryotaro (山田 遼太郎)
3PS051 (S155)
Yamada, Tesshi (山田 哲司)
2PT117 (S124)
Yamada, Yoshiteru (山田 好輝)
1PT142 (S93)
Yamagata, Yuriko (山縣 ゆり子)
1PT129 (S90)
1YS1000 (S90)
Yamagata, Yutaka (山形 豊)
1I1610 (S37)
Yamagishi, Akihiko (山岸 明彦)
1PT135 (S91)
2PT203 (S139)
Yamagishi, Junya (山岸 純也)
2PT140 (S128)
Yamagishi, Mai (山岸 舞)
1PT202 (S102)
3PS031 (S151)
Yamagishi, Masahiko (山岸 雅彦)
1A1510 (S19)
Yamaguchi, Akihito (山口 明人)
1PT117 (S88)
3PT115 (S160)
Yamaguchi, Chiaki (山口 千秋)
2PT191 (S138)
Yamaguchi, Hideaki (山口 秀明)
3C0924 (S60)
Yamaguchi, Hiroshi (山口 宏)
1PT104 (S86)
Yamaguchi, Keiichi (山口 圭一)
1PT139 (S92)
1PT155 (S95)
Yamaguchi, Maki (山口 眞紀)
2H1558 (S53)
Yamaguchi, Mariko (山口 真理子)
2F1424 (S48)
1PT124 (S89)
1PT138 (S92)
1PT153 (S95)
2PT170 (S134)
2PT171 (S134)
2PT172 (S134)
2PT174 (S135)
Yamaguchi, Rui (山口 類)
2SD-04 (S12)
Yamaguchi, Shigeo (山口 繁生)
2PT170 (S134)
Yamaguchi, Shin (山口 真)
2A1534 (S39)
1PS040 (S81)
Yamaguchi, Yoshiki (山口 芳樹)
1PT212 (S104)
Yamai, Toshihide (山井 俊英)
3F1034 (S67)
Yamakawa, Akiko (山川 明来子)
3PS036 (S152)
Yamakawa, Hisanori (山川 壽伯)
3PT228 (S180)
Yamakawa, Kentaro (山川 賢太郎)
1PT162 (S96)
Yamamori, Yu (山守 優)
1PT174 (S98)
Yamamoto, Akie (山本 晃衣)
2H1412 (S52)
Yamamoto, Eiji (山本 詠士)
1PT004 (S84)
3PT155 (S167)
Yamamoto, Hiroki (山本 泰生)
2PS021 (S113)
Yamamoto, Hirotsugu (山本 裕紹)
1I1534 (S36)
Yamamoto, Johtaro (山本 条太郎)
1G1546 (S32)
Yamamoto, Junpei (山元 淳平)
2F1534 (S49)
2F1546 (S49)
Yamamoto, Kazuyuki (山本 和志)
2PS019 (S113)
Yamamoto, Kentaro (山本 健太郎)
3PS032 (S151)
Yamamoto, Kimiko (山元 季実子)
3PT184 (S173)
3PT188 (S173)
Yamamoto, Kimiko (山本 希美子)
2SH-06 (S16)
Yamamoto, Kyohei (山本 恭平)
1PS039 (S81)
Yamamoto, Masahide (山本 雅英)
1E1424 (S27)
Yamamoto, Masaki (山本 雅貴)
2D1522 (S45)
Yamamoto, Naoki (山本 直樹)
1E1546 (S28)
3PT156 (S167)

Yamamoto, Shigeyuki (山本 茂幸)
Yamamoto, Syou (山本 匠)
Yamamoto, Takenori (山本 武範)
Yamamoto, Tsubasa (山本 翼)
Yamamoto, Yohei (山本 陽平)
Yamamura, Masayuki (山村 雅幸)
Yamamura, Yasuhisa (山村 泰久)
Yamanaka, Akihiro (山中 章弘)
Yamane, Mitsunori (山根 光智)
Yamane, Tsutomu (山根 努)
Yamano, Akihito (山野 昭人)
Yamanobe, Takanobu (山野辺 貴信)
Yamanoha, Banri (山之端 万里)
Yamaoki, Yudai (山置 佑大)
Yamasaki, Hideki (山崎 秀樹)
Yamasaki, Seiji (山崎 聖司)

3PT214 (S177)
1PT125 (S90)
3PT119 (S160)
1PT141 (S92)
2PT121 (S125)
1I1522 (S36)
2PT152 (S131)
1SH-04 (S7)
1PS016 (S76)
2PT136 (S128)
1PT110 (S87)
2PT223 (S142)
2PS038 (S116)
3C0936 (S60)
1PT126 (S90)
1PT117 (S88)
3PT115 (S160)
Yamasaki, Tatsuya (山崎 達哉)
2PS031 (S115)
Yamasaki, Tsuyoshi (山崎 剛)
2PS035 (S116)
Yamashita, Daichi (山下 大智)3F0948 (S66)
Yamashita, Eiki (山下 栄樹)
2PT157 (S132)
2G1534 (S51)
Yamashita, Hayato (山下 隼人)
3F0948 (S66)
Yamashita, Keitaro (山下 恵太郎)
Yamashita, Naoki (山下 直樹)
3F1034 (S67)
Yamashita, Risa (山下 理沙)
1PT185 (S100)
Yamashita, Shoji (山下 昭二)
2E1610 (S47)
2PT122 (S125)
Yamashita, Takahiro (山下 高廣)
1F1424 (S29)
1F1436 (S29)
3F1022 (S66)
Yamashita, Takefumi (山下 雄史)
1B1558 (S22)
Yamashita, Yutaka (山下 豊)
2D1424 (S44)
1PT192 (S102)
Yamato, Takahisa (倭 剛久)
2PT111 (S123)
Yamauchi, Hitomi (山口 瞳)
Yamauchi, Seigo (山内 清語)
1PT144 (S93)
Yamauchi, Toyohiko (山内 豊彦)
2D1424 (S44)
Yamazaki, Kaoru (山崎 薫)
1PT235 (S108)
Yamazaki, Masahito (Yamazaki Masahito)
2G1424 (S50)
Yamazaki, Masahito (山崎 昌一)
3G1058 (S69)
3PT140 (S165)
3PT173 (S171)
3PT174 (S171)
Yamazaki, Naoshi (山﨑 尚志)
3PT119 (S160)
Yamazaki, Toshimasa (山崎 俊正)
1PT111 (S87)
Yamazaki, Yoichi (山崎 洋一)
2F1424 (S48)
1PT124 (S89)
1PT153 (S95)
2PT170 (S134)
2PT171 (S134)
2PT172 (S134)
2PT173 (S135)
2PT174 (S135)
Yanagawa, Masataka (柳川 正隆)
3F1022 (S66)
Yanagida, Toshio (柳田 敏雄)
1PS049 (S82)
1YS0900 (S82)
1PT006 (S84)
1PT210 (S104)
2PS011 (S111)
2PS028 (S114)
2PT118 (S124)
3PT167 (S169)
Yanagisawa, Haru-aki (柳澤 春明)
2A1412 (S38)
Yanagisawa, Miho (柳澤 実穂)
2G1436 (S50)
3PT135 (S164)
3PT152 (S167)
Yanagisawa, Sachiko (柳澤 幸子)
3I0924 (S71)
3I1010 (S72)
Yanagisawa, Yuki (柳沢 祐樹)
2PT186 (S137)
Yanaka, Saeko (谷中 冴子)
2E1510 (S46)
Yang, Wei (Yang Wei)
1PT129 (S90)
1YS1000 (S90)
Yang, Zhuohao (楊 倬皓)
3C1058 (S61)
Yano, Akira (谷野 章)
3PS027 (S150)
Yano, Ken-ichi (矢野 憲一)
1G1424 (S31)
Yano, Naomine (矢野 直峰)
2PT157 (S132)
2PT160 (S132)
Yao, Haruhiko (八尾 晴彦)
3PT150 (S166)
3PT151 (S167)
Yao, Min (姚 閔)
3F0948 (S66)
Yashima, Kenta (八島 健太)
2PT211 (S140)
Yasuda, Kenji (安田 賢二)
2D1510 (S44)
3PS020 (S149)
3PS022 (S149)
3PS023 (S149)
3PS046 (S154)
3PS047 (S154)
3PS049 (S154)
Yasuda, Ryohei (安田 涼平)
1PT207 (S103)
3PS016 (S148)
Yasuda, Satoshi (安田 哲)
1PS038 (S80)
Yasuda, Satoshi (安田 賢司)
1PT154 (S95)
3PT116 (S160)− S196 −Yasui, Masato (安井 正人)

2G1534 (S51)
1PT004 (S84)
3PT155 (S167)
Yasui, Shin-ichiro (安居 慎一郎)
1PS005 (S74)
Yasui, So (安井 聡)
2PT229 (S143)
Yasunaga, Takuo (安永 卓生)
2A1400 (S38)
2A1412 (S38)
2A1424 (S38)
2A1436 (S38)
2D1412 (S44)
1PT180 (S99)
1PT185 (S100)
1PT186 (S100)
1PT223 (S106)
Yasunaga, Yakuo (安永 卓生)
1PS008 (S75)
Yasuoka, Jun-ichi (安岡 潤一)
3PT125 (S162)
Yasuoka, Kenji (泰岡 顕治)
1PT004 (S84)
3PT155 (S167)
Yawo, Hiromu (八尾 寛)
1PS010 (S75)
3PT218 (S178)
Yawo, Hiromu (八尾 寛)
2PT183 (S137)
3E0924 (S64)
Yazawa, Koji (矢澤 宏次)
1PS007 (S75)
Yazawa, Yoichi (矢沢 洋一)
Yildiz, Ahmet (YILDIZ Ahmet)
2SA-01 (S9)Yin, Lijie (Yin Lijie)
3PT189 (S174)
Yizhar, Ofer (Yizhar Ofer)
1PT128 (S90)
1YS0930 (S90)
Yohda, Masafumi (養王田 正文)
1PT123 (S89)
2PT121 (S125)
Yoichi Yamazaki, Yoichi Yamazaki (山崎 洋一)
1PT138 (S92)
Yokogawa, Daisuke (横川 大輔)
3PT178 (S172)
3PT227 (S179)
Yokojima, Satoshi (横島 智)
3PT230 (S180)
Yokomaku, Yoshiyuki (横幕 能行)
1PT102 (S85)
Yokomizo, Shunnya (横溝 駿矢)
2H1558 (S53)
Yokota, Hideo (横田 秀夫)
2SD-03 (S12)
Yokota, Hiroaki (横田 浩章)
3C1046 (S61)
Yokota, Yuichiro (横田 裕一郎)
1G1510 (S31)
Yokoyama, Hirokazu (横山 弘和)
2G1448 (S50)
Yokoyama, Keiko (横山 慶子)
1D1522 (S26)
Yokoyama, Ken (横山 謙)
3A0948 (S56)
Yokoyama, Yasunori (横山 泰範)
2PT011 (S121)
2PT108 (S122)
3PT120 (S161)
Yomo, Tetsuya (四方 哲也)
1C1534 (S24)
1G1534 (S32)
2PT205 (S139)
2PT209 (S140)
Yoneda, Shigetaka (米田 茂隆)
1PT175 (S98)
Yoneda, Takuro (米田 拓郎)
1A1610 (S20)
Yonekita, Taro (米北 太郎)
1C1400 (S23)
Yonekura, Koji (米倉 功治)
2D1522 (S45)
Yonetani, Takashi (米谷 隆)
2PT153 (S131)
Yonetani, Yoshiteru (米谷 佳晃)
1SD-03 (S4)Yonezawa, Kento (米澤 健人)
2PT170 (S134)
2PT171 (S134)
Yoon, Dong H. (Yoon Dong H.)
1PT234 (S108)
Yoshida, Aya (吉田 文)2E1400 (S45)
Yoshida, Hideji (吉田 秀司)
3PT001 (S155)
Yoshida, Hikaru (吉田 光)
1PS024 (S78)
Yoshida, Hironori (吉田 浩典)
3PT149 (S166)
Yoshida, Hiroshi (吉田 寛)
1D1510 (S26)
Yoshida, Kaoru (吉田 薫)
3PS007 (S147)
Yoshida, Kazunari (吉田 一也)
3PT154 (S167)
Yoshida, Keito (吉田 桂人)
2PT170 (S134)
2PT171 (S134)
Yoshida, Kotaro (吉田 広太郎)
3PT216 (S177)
Yoshida, Manabu (吉田 学)
3PS007 (S147)
Yoshida, Masasuke (吉田 賢右)1E1412 (S27)
3A1022 (S57)
3A1046 (S57)
1PS026 (S78)
1PS027 (S78)
1PS031 (S79)
1PS035 (S80)
2PS020 (S113)
1YS0945 (S113)
Yoshida, Muneki (吉田 宗生)
3PS037 (S152)
Yoshida, Ryo (吉田 亮)
2SD-05 (S12)
Yoshida, Taka-aki (吉田 貴亮)
3PT135 (S164)
Yoshida, Yumi (吉田 裕美)
3PS021 (S149)
3PT159 (S168)
Yoshidome, Takashi (吉留 崇)
1PS034 (S80)
Yoshihara, Shizue (吉原 静恵)
2PT188 (S138)
Yoshikawa, Kenichi (吉川 研一)
2C1534 (S43)
2G1436 (S50)
3C0924 (S60)
3C1110 (S61)
3PT101 (S157)
Yoshikawa, Shinya (吉川 信也)
3I1022 (S72)
3I1034 (S72)
2PT157 (S132)

Yoshimatsu, Daiki (吉松 大輝)
Yoshimitsu, Ryosuke (吉満 亮介)
Yoshimoto, Jun-ichi (吉本 順一)
Yoshimura, Hideyuki (吉村 英恭)

Yoshimura, Takeo (吉村 武朗)
Yoshimura, Yuichi (吉村 優一)
Yoshinaga, Natsuhiko (義永 那津人)
Yoshino, Masaru (吉野 賢)
Yoshioka, Yoshichika (吉岡 芳親)

3PT129 (S162)
3PT175 (S171)
3PS044 (S153)
2PS009 (S111)
1PT237 (S109)
1PT238 (S109)
3PS025 (S150)
2PT149 (S130)
3PS050 (S155)
1E1436 (S27)
1SF-05 (S6)
3PT123 (S161)
3PS042 (S153)

Yoshizawa, Keiko (慶澤 景子)
Yoshizawa, Susumu (吉澤 晋)
You, Huijuan (游 慧娟)Yu, Isseki (優 乙石)
Yugi, Katsuyuki (柚木 克之)
Yuki, Taichi (由木 太一)
Yura, Kei (由良 敬)
Zaccai, Giuseppe (Zaccai Giuseppe)
Zhang, Feng (Zhang Feng)
Zhang, Jin (張 進)− S197 −2PT118 (S124)
2F1412 (S47)
2PT181 (S136)
3PT103 (S157)
1E1510 (S28)
1PT214 (S104)
3PT227 (S179)
3PT232 (S180)
1I1412 (S35)
2H1400 (S51)
1PT128 (S90)
1YS0930 (S90)
2PT184 (S137)

Zhang, Peijuan (Zhang Peijuan)

1PS018 (S77)
2PT240 (S145)
2F1436 (S48)
Zhang, You (張 宇)Zhang, Yu (張 宇)
2F1534 (S49)
2F1546 (S49)
Zhao, Chenchao (Zhao Chenchao)
2H1412 (S52)
Zhao, Ye (Zhao Ye)
1PT129 (S90)
1YS1000 (S90)
1A1546 (S20)
Zhu, Shiwei (朱 世偉)
2PT188 (S138)
Zikihara, Kazunori (直原 一徳)
Zinchenko, Anatoly (ジンチェンコ アナトーリ)
3C0924 (S60)

S198

S199

S200

S201

S202

S203

S204

S205

本学会の連絡先は下記の通りです．
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編集発行
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生物物理Vol. 52㈱ニコン

インステックSupplement 1

広告

INDEX

S1-1

インフォコム㈱S1-14

日本エフイー・アイ㈱S1-2㈱菱化システム

S1-15

オリンパス㈱

S1-3

大陽日酸㈱

S1-16

共立出版

S1-4

シュレーディンガー㈱S1-17

エルゼビア・ジャパン㈱S1-5

ブレインビジョン㈱S1-18, 19

WDB ㈱

S1-6

バイオリサーチセンター㈱

S1-20

遠藤科学㈱

S1-7

アンドール・テクノロジー PLC

S1-21

理科研㈱

S1-8

フォトニックインストゥルメンツ㈱

S1-22

㈱木下理化

S1-9

和光純薬工業㈱S1-23

キコーテック㈱S1-10

橋本電子工業㈱S1-24㈱島津製作所S1-11

ブルカー・オプティクス㈱S1-25㈱ JCC ギミック

S1-12

ブルカー・バイオスピン㈱S1-26㈱日立製作所S1-13※ 直接連絡される場合には，必ず「生物物理を見て」とお付け加え下さい．

＜資料請求番号 S1-4＞

＜資料請求番号 S1-5＞＜資料請求番号 S1-6＞

＜資料請求番号 S1-7＞

＜資料請求番号 S1-8＞＜資料請求番号 S1-9＞

＜資料請求番号 S1-10＞＜資料請求番号 S1-11＞

＜資料請求番号 S1-12＞＜資料請求番号 S1-13＞

＜資料請求番号 S1-14＞

＜資料請求番号 S1-15＞

＜資料請求番号 S1-16＞

＜資料請求番号 S1-17＞

＜資料請求番号 S1-18＞

＜資料請求番号 S1-19＞

＜資料請求番号 S1-20＞

＜資料請求番号 S1-21＞

＜資料請求番号 S1-22＞

＜資料請求番号 S1-23＞

＜資料請求番号 S1-24＞

＜資料請求番号 S1-25＞

＜資料請求番号 S1-26＞

＜資料請求番号 S1-1＞＜資料請求番号 S1-2＞

SUPPLEMENT 1

SEIBUTSU BUTSURI

生物物理

Vol. 52

2012 年 8 月（増刊号）

ISSN 0582-4052 CODEN : SEBUAL
2012 年 8 月（増刊号）

SUPPLEMENT 1
Vol.52

日本生物物理学会第2012

回年会講演予稿集

50＜資料請求番号 S1-3＞

昭和42年8月1日 国鉄大局特別扱承認雑誌第216号
平成24年8月15日
増刊号

2012 年 Vol.52
SUPPLEMENT 1

定価 3,500円

第 50回年会講演予稿集
2012.9.22 (土) ～ 24 (月)名古屋大学・東山キャンパス

主催

日本生物物理学会