Prospectus
Institute for Protein Research
Osaka University

2015

&RQWHQWVGreetings from Director
Concept and Future
History
Organization
Former Directors / Professors Emeriti
Administrative Council
Staff List
Staff & Students /Closing Accounts /Education & Research Activities
Number of Members
Closing Accounts
Periodical Publications
Education Activities
Research Activities
Activities as a Joint-Usage/Research Center, and International Exchange
Joint Researchers and International Collaborative Research
IPR Seminar
International Exchange

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Division of Protein Chemistry
Laboratory of Regulation of Biological Reactions
Laboratory of Extracellular Matrix Biochemistry
Laboratory of Epigenetics
Laboratory of Protein Organic Chemistry

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Division of Protein Structural Biology
Laboratory of Protein Folding
Laboratory of Molecular Biophysics
Laboratory of Protein Crystallography
Laboratory of Membrane Protein Chemistry

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Division of Integrated Protein Functions
Laboratory of Genome and Chromosome Functions
Laboratory of Regulation of Neuronal Development
Laboratory for Molecular and Developmental Biology
Laboratory of Nuclear Network
Laboratory of Homeostatic Integration

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Division of International Collaborative ResearchLaboratory of Guest Professors/Visiting Professors
Tsukihara Laboratory

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Research Center for State-of-the-art Functional Protein Analysis
Laboratory of Protein Profiling and Functional Proteomics
Laboratory of Protein Synthesis and Expression
Laboratory of Supramolecular Crystallography
Laboratory of Protein Informatics
Laboratory of Advanced Protein Characterization
Laboratory of Protein Databases
Open Space laboratory for Advanced Protein Science

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Equipments
Floor Plan
Map / Access

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&RQWHQWVGreetings from Director  
Haruki Nakamura, D.Sci., Director
The machinery of life on the earth has been made of proteins, which are expressed fromthe corresponding genes in genome, and the complicated molecular activities are provided
by huge number of interactions among those proteins. In 1958, Institute for Protein
Research (IPR) was founded by members in Faculty of Science and Medical school of
Osaka University, covering different fields of sciences, such as chemistry, physics, biology
and medicine. Since then, protein research in IPR has made a remarkable progress by
elucidating structures and functions of proteins, and by understanding their biological roles
from the molecular level to the cellular and the higher levels. Through wide and strong
supports from the community, IPR has expanded over time after 57 years. Now, it has four divisions (16 labs) with anattached center, Research Center for State-of-the-Art Functional Protein Analysis (7 labs), which develops its originaltechniques and applies them to reveal protein structures and functions.
IPR had worked as an inter-university joint-use facility attached to Osaka University since its foundation. In April
2010, IPR was qualified as one of the Joint Usage/Research Centers in Japan by MEXT, Ministry of Education,
Culture, Sports, Science and Technology in Japan. In particular, IPR offers the usages of its own synchrotron beamline at SPring-8 and of the Nuclear Magnetic Resonance (NMR) spectrometers with ultra-high sensitivities, todomestic and foreign protein researchers. In addition, IPR has constructed protein structural database (PDB: Protein
Data Bank) as PDBj (PDB Japan), one of the four members of the wwPDB (worldwide PDB), by annotating thedeposited data from structural biologists in Asian and Oceania region and by providing several original services andderived databases. PDBj-BMRB also constructs NMR experimental database, collaborating with BMRB
(BioMagResBank) in U.S.A. IPR has also organized many international collaborative researches with foreign proteinscientists.
Professors and staffs in IPR (about 40 members) work hard for their own researches, as well as for educational
activities to undergraduate students at Faculty of Science and that of Medicine, and Ph. D. students at Graduate
school of Science, Medicine, and Frontier Biosciences. From those Faculties and Graduate schools, nearly 100
students always study at laboratories in IPR, and about 70 postdoctoral fellows make their own original investigations
with various national and international research projects. Those students and postdocs gather from many different
places in the world, and global human interactions are common in IPR.
Paradigm of protein research has been rapidly changed from previous analysis of individual protein molecules tounderstanding of the protein complex that expresses biological activities and to revealing the biological informationfrom protein interactions, still based on the structures and functions of individual proteins. Namely, protein structural
analysis is not a goal as the previous structural biology, but it is a starting point for a novel scientific field, Structural
Life Science, where life science is investigated at multi-scale based on the protein network. IPR is going forward to
this Structural Life Science as the basic science, promoting the principle of Osaka University, "To discover the true
essence of things". In addition, IPR is going to cooperate with the public and the industries through activities at OpenSpace Laboratory for Advanced Protein Science where an industry researcher is invited as a guest professor, andsupport program for industries with database construction and its release to the public. All of those activities willappear on our web page (http://www.protein.osaka-u.ac.jp).

Greetings from Director

1

The processes of life, mediated by a large variety of proteins, are very

Concept and Futurecomplex. Elucidation of these processes through molecular studies of proteinscan be well achieved by efficient cooperation of researchers in various fields of natural sciences. Another requisite is
the establishment of suitable infrastructures that allow close collaborations among scientists across the country. The
Institute for Protein Research (IPR) was founded as a joint-use research organization attached to Osaka University tofulfill these needs, and thus to play a central role in protein science in Japan. Now, IPR works as one of the Joint
Usage/Research Centers authorized by Japanese Government.
Thus, the mission of IPR does not change at all during the long history, and it is summarized as the following two
concepts:
1) With tight collaborations by researchers in IPR having
various backgrounds such as chemistry, physics, biology andmedicine, basic studies are performed on the structure andfunction of proteins and their biological significance at themolecular level, as well as at the cellular level.
2) As the Joint Usage/Research Center, IPR provides theresources for research, databases such as PDB, and scientificcommunication for domestic and international collaborational
studies, promoting protein science for the scientific communityand society.
Based on the above mission, IPR has so far made various
projects on structural biology, neurobiology, proteomics, andmolecular and cellular biology, and produced many excellent results. Although IPR started as a domestic center, it is
now widely recognized as an international center of excellence for protein research. For instance, IPR operates theWorldwide Protein Data Bank (wwPDB) as one of four worldwide centers, mainly covering Asia-Oceania region.
Because of its high level scientific activity, it has attracted many researchers from abroad and will continue to do soin the future. IPR will continue to make essential contributions to revealing the structure and function of proteins andtheir networks for the elucidation of life.

HistoryOsaka University was active in the study of proteins and enzymes since its foundation in 1931,
and it was a long-standing desire of the university authorities to promote further this facet of the university’s activities
by establishing a research institute specialized for protein science. In 1955 an official plan was drafted to establish
such an institute as a part of Osaka University and submitted to the Ministry of Education, Science and Culture. The
Ministry agreed to open a new laboratory for organic chemistry of proteins and amino acids in the Faculty of Science.
The new laboratory was opened in 1956, and Professor Shiro Akabori, who had played a pivotal role in protein
research, was appointed as its supervisor.
In the meantime, among scientists in the relevant fields had continuously grown a strong desire for theestablishment of a central institute for protein research, with the aim of facilitating close cooperation among
researchers from a wide variety of scientific fields. In 1957, the Science Council of Japan urged the Government toConcept and Future / History2

consider the foundation of such an institute somewhere in the country. The Government decided to establish a
research institute for protein science attached to Osaka University. The Institute for Protein Research (IPR) was thus
founded formally on April 1, 1958, as a part of Osaka University, and Professor Shiro Akabori was appointed as its
first director. The IPR has developed significantly in terms of its scientific activity and infrastructures. Now, the IPR
comprises four divisions with 14 laboratories and an attached research center (Research Center for State-of-the-art
Functional Protein Analysis) with 7 laboratories, serving as a joint usage/research institute for the community in thefields of protein and related sciences.

Chronological table
1956

Set up of a new laboratory in Faculty of Science, Osaka University, for organic chemical studies
of proteins and amino acids (the forerunner of Institute for Protein Research) supervised by Prof.
Shiro Akabori.

1958

Establishment of Institute for Protein Research as a Joint-use Research Organization, composed ofthree Divisions (Organic Chemistry, Physical Chemistry and Protein Metabolism). Advisory
Committee on Administration was also founded.

1959

Divisions of Enzymology and Protein Crystallography were added.

1960

Divisions of Protein Chemistry, Physiology and Protein Biosynthesis were added.

1961

The main building (4,130 m2) was completed in the former campus at Nakanoshima.

1962

Set up of the Peptide Center

1964

Division of Molecular Biophysics was added.

1965

Set up of a Branch Division (569 m2) in Torii Memorial Hall

1967

Division of Plasma Proteins was added.

1971

Main building (7,873 m2) of Institute for Protein Research was completed at Suita Campus.

1972

Relocation to new building at Suita campus

1977

Division of Plasma Proteins was renamed to Division of Regulation of Macromlecular Functions

1978

Establishment of Crystallographic Research Center

1979

Buildings of Research Center for Crystal analysis (1,505 m2) and NMR Research Laboratory (267m2) were completed.

1988

Reorganization of the Peptide Center and Crystallographic Research Center as Research Center
for Protein Engineering.

1998

Establishment of Center for Structural Biology

2002

Establishment of Research Center for Structural and Functional Proteomics

2004

Transformed into the Research Institute of Japanese national universities by the National
University Corporation Law

2005

Reorganization of the Research Divisions to 4 Research Divisions with 12 Laboratories.
Laboratory of Foreign Principal Investigators and Endowed Research Division of Molecular
Recognition by Takara Bio Inc. were added.

2006

Set up of Endowed Research Division of Disease Proteomics by Shimadzu

2008

Building of Collaborative Research Facility (1,149 m2) was completed.

2009

Construction of Main building for Earthquake-resistance was completed.

2010

Certified as Joint Usage/Research Center by MEXT.

2012

Establishment of Research Center for State-of-the-Art Functional Protein Analysis.
History / Chronological table

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Organization

Organization4Former Directors_______________________________________________________
1st
2nd
3rd
4th
5th
6th
7th
8th
9th
10th
11th
12th
13th
14th
15th
16th
17th
18th
19th

Shiro AKABORI
Toshizo ISEMURA
Tomoji SUZUKI
Kouzo NARITA
Masao KAKUDO
Yoshiharu IZUMIRyo SATO
Takekazu HORIO
Yukiteru KATSUBE
Hachiro NAKAGAWA
Fumio SAKIYAMA
Yoshimasa KYOGOKU
Yasutsugu SHIMONISHI
Katsuya NAGAI
Hideo AKUTSU
Tomitake TSUKIHARA
Saburo AIMOTO
Toshiharu HASE
Haruki NAKAMURA

1 April 1958
1 December 1961
1 December 1965
15 August 1969
15 August 1971
2 April 1982
1 April 1985
1 April 1987
1 April 1989
1 April 1993
1 April 1995
1 April 1997
1 April 1999
1 April 2000
1 April 2004
1 April 2006
1 April 2008
1 April 2010
1 April 2014

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30 November 1961
30 November 1965
14 August 1969
14 August 1971
1 April 1982
31 March 1985
31 March 1987
31 March 1989
31 March 1993
31 March 1995
31 March 1997
31 March 1999
31 March 2000
31 March 2004
31 March 2006
31 March 2008
31 March 2010
31 March 2014
Present

Professors Emeriti ______________________________________________________
Yoshiharu IZUMI, D. Sci.
Hachiro NAKAGAWA, M. D., D. Med.
Toshio TAKAGI, D. Sci.
Yasutsugu SHIMONISHI, D. Sci.
Hideo AKUTSU, D. Sci.

Yukiteru KATSUBE, D. Sci.
Akira ASANO, D. Sci.
Fumio SAKIYAMA, D. Sci.
Katsuya NAGAI, M. D., D. Med.
Tomitake TSUKIHARA, D. Sci.

Administrative Council __________________________________________________

 As of 1 April 2015

Outside of Osaka University
Professor Fuyuki ISHIKAWA
Professor Tohru KATAOKA
Professor Akihiko NAKANO
Director Yoichi NABESHIMA
Professor Yoshinori FUJIYOSHI
Professor Kiyoshi KITA
Professor Kiyoko FUKAMI

Inside of Osaka University
Professor㻌 Koichi㻌 FUKASE
Professor Tsuyoshi INOUE
Professor Akira KIKUCHI
Professor Masato OKADA
Professor Kenji NAGAI
Graduate School of Biostudies, Kyoto University
Graduate School of Medicine, Kobe University
Graduate School of Science, The University of Tokyo
Foundation for Biomedical Research and Innovation
Graduate School of Pharmaceutical Sciences㺃Cellular and Structural
physiology Institute, Nagoya University
The Institute of Medical Science, The University of Tokyo
School of Life Science, Tokyo University of Pharmacy and Life Sciences
Graduate School of Science
Graduate School of Engineering
Graduate School of Medicine
Research Institute for Microbial Diseases, Osaka University
Institute of Scientific and Industrial Research

In IPR
Professor Atsushi NAKAGAWA
Professor Toshifumi TAKAO

Institute for Protein ResearchInstitute for Protein Research

Organization

5

Staff List
Director

Professor

Haruki NAKAMURA, D. Sci.

Vice Directors

Professor
Professor

Atsushi NAKAGAWA, D. Sci.
Akira SHINOHARA, D. Sci.

Director of the Research Center

Professor Junichi TAKAGI, D. Sci.

Division of Protein Chemistry
Laboratory of Regulation of Biological ReactionsProfessor
Toshiharu HASE, D. Sci.
Associate Professor
Masato NAKAI, D. Sci.
Assistant Professor
Yoko KIMATA-ARIGA, Ph. D.
Laboratory of Extracellular Matrix Biochemistry Kiyotoshi SEKIGUCHI, D. Sci.
Professor
Masashi YAMADA, Ph. D.
Assistant Professor
Naoko NORIOKA, Ph. D.
Technical Staff
Laboratory of Epigenetics
Professor
Associate Professor
Guest Associate Professor
Assistant Professor
Technical Staff

Shoji TAJIMA, D. Sci.
Isao SUETAKE, Ph. D.
Nobuyasu MAKI, Ph. D.
Hironobu KIMURA, Ph. D.
Naoyuki ABE, M. Sci.

Laboratory of Protein Organic Chemistry
Professor
Associate Professor
Associate Professor
Assistant Professor

Hironobu HOJO, D. Sci.
Toru KAWAKAMI, Ph. D.
Takeshi SATO, Ph. D.
Yuya, ASAHINA, Ph. D.

Division of Protein Structural Biology
Laboratory of Protein FoldingProfessor
Associate professor
Assistant Professor

Yuji GOTO, D. Sci.
Young-Ho LEE, Ph. D.
Masatomo SO, Ph. D.

Laboratory of Molecular Biophysics
Professor
Visiting Professor
Associate Professor
Assistant Professor
Assistant Professor

Toshimichi FUJIWARA, D. Sci.
Akira NAITO, D. Sci.
Chojiro KOJIMA, Ph. D.
Yoh MATSUKI, Ph. D.
Toshihiko SUGIKI, Ph. D

Laboratory of Protein Crystallography
Professor
Associate Professor
Assistant Professor

Genji KURISU, Ph. D.
Hideaki TANAKA, Ph. D.
Risa MUTO, Ph. D.

Laboratory of Membrane Protein Chemistry
Independent Associate Professor
Joji MIMA, Ph. D.

Staff List

6

Division of Integrated Protein Functions
Laboratory of Genome and Chromosome Functions
Professor
Akira SHINOHARA, D. Sci.
Associate Professor
Miki SHINOHARA, Ph. D.
Masahiro TERASAWA, Ph. D.
Laboratory of Regulation of Neuronal Development
Professor
Kazuaki YOSHIKAWA, M.D., D. Med.
Assistant Professor
Koichi HASEGAWA, Ph. D.
Assistant Professor
Kazushiro FUJIWARA, Ph. D.
Laboratory for Molecular and Developmental Biology
Professor
Takahisa FURUKAWA, M.D., D. Med.
Associate Professor
Yoshihiro OMORI, Ph. D.
Assistant Professor
Rikako SANUKI, Ph. D.
Technical Staff
Toshinori TSUJI
Laboratory of Nuclear Network
Independent Associate Professor

Junko KANOH, Ph. D.

Laboratory of Homeostatic Integration
Associate Professor

Nobuaki OKUMURA, Ph. D.

Division of International Collaborative ResearchLaboratory of Foreign PI
Visiting Professor
Visiting Professor
Specially Appointed Associate Professor
Laboratory of Visiting Scientists
Guest Professor

Matthias RÖGNER, Ph. D.
Thomas HAPPE, Ph. D.
Damien R. HALL, Ph.D.
Toshio YAMAZAKI, D. Sci.


Laboratory of Guest Professors/Visiting Professors 
Tsukihara Laboratory
Visiting Professor
Tomitake TSUKIHARA, D. Sci.


Research Center for State-of-the-art Functional Protein Analysis
Laboratory of Protein Profiling and Functional Proteomics
Professor
Specially Appointed Assistant Professor

Toshifumi TAKAO, D. Sci.
Caroline D. REREIRA, Ph. D.

Laboratory of Protein Synthesis and Expression
Junichi TAKAGI, D. Sci.
Professor
Associate Professor
Kenji IWASAKI, Ph. D.
Assistant Professor
Yu KITAGO, Ph. D.
Specially Appointed Assistant Professor
Yukiko MATSUNAGA, Ph. D.
Specially Appointed Assistant Professor
Masataka UMITSU, Ph.D.
Technical Staff
Keiko KAWAKAMI
Laboratory of Supramolecular Crystallography
Atsushi NAKAGAWA, D. Sci.
Professor
Associate Professor
Mamoru SUZUKI, Ph. D.
Assistant Professor
Mayumi AMANO, Ph.D.
Assistant Professor
Eiki YAMASHITA, Ph. D.
Specially Appointed Assistant Professor
Kohei TAKESHITA, Ph. D.
Specially Appointed Assistant Professor
Akifumi HIGASHIURA, Ph. D.

Staff List

7

Laboratory of Protein Informatics
Professor
Associate Professor
Assistant Professor
Visiting Professor
Guest Associate Professor
Guest Associate Professor
Guest Associate Professor
Technical Staff

Haruki NAKAMURA, D. Sci.
Akira KINJO, Ph. D.
Yuko TSUCHIYA, Ph. D.
Junichi HIGO, D. Sci.
Takeshi KAWABATA, Ph.D.
Narutoshi KAMIYA, Ph. D.
Ikuo FUKUDA, Ph.D
Takashi KOSADA, M. Sci.

Laboratory of Advanced Protein Characterization
Atsushi NAKAGAWA, D. Sci.
Professor
Junichi TAKAGI, D. Sci.
Professor
Professor
Toshifumi TAKAO, D. Sci.
Associate Professor
Kenji IWASAKI, Ph. D.
Associate Professor
Takeshi SATO, Ph. D.
Assistant Professor
Eiki YAMASHITA, Ph. D.
Assistant Professor
Naotoshi MIMURA, D. Sci.
Assistant Professor
Toshihiko SUGIKI, Ph.D.
Assistant Professor
Risa MUTO, Ph.D.
Specially Appointed Assistant Professor
Akifumi HIGASHIURA, Ph. D.
Technical Staff
Keiko KAWAKAMI
Naoko NORIOKA, Ph. D.
Technical Staff
Naoyuki ABE, M. Sci.
Technical Staff
Laboratory of Protein Databases
Professor
Professor
Professor
Associate Professor
Associate Professor
Technical Staff
Technical Staff

Haruki NAKAMURA, D. Sci.
Toshimichi FUJIWARA, D. Sci.
Kiyotoshi SEKIGUCHI, D. Sci.
Akira KINJO, Ph. D.
Chojiro KOJIMA, Ph. D.
Takashi KOSADA, M. Sci.
Reiko YAMASHITAOpen Space Laboratory for Advanced Protein Science
Midori UEMURA,
Visiting ProfessorAdministration Office
Head

Ichiro YASUGUCHI

General Affairs Section
Chief
Deputy Chief
Clerk

Shinichi MATSUNAGA
Yoko TANAKA
Noriko YOSHIMURA

Accounting Section
Chief
Deputy Chief
Deputy Chief
Clerk
Clerk

Takashi SHIRAI
Yasuhiro NAKATA
Hiroharu MICHIKI
Yutsuki KITADA
Hiroko NAKATA

Research Cooperation & Equipment Section
Toshizumi MATSUSHITA
Chief
Sadahiro KURIBAYASHI
Deputy Chief
Naoko ARAKI
Deputy Chief
Kazuaki NAGAMI
Clerk

Staff List8

Number of Members                  $VRI$SULO

Staff & Students /Closing Accounts /Education & Research Activities
Staff
Researcher     Student

Professor

Visiting(Guest) ProfessorAssociate Professor

Visiting(Guest) Associate ProfessorAssociate Professor (Lecturer)
Assistant Professor
Specially Appointed Assistant Professor
Technical AssistantAdministrative Staff Technical SupporterAdministrative Assistant

Total



Post Doctoral Fellow
Foreign Post Doctoral Fellow  
JSPS Postdoctoral Fellow

Joint Research Collaborator
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
Total
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
Undergraduate Student

Graduate Student(doctor course)   Graduate Student(master course)
Research Student






Closing Accounts

The Year 2014


Staff & Students /Closing Accounts

9Periodical Publications
1.  Memoirs of the Institute for Protein Research, Osaka University (Annual)
2. Annual Report of the Institute for Protein Research (Annual) 
Education Activities
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Course in Charge
Biological Science ,Graduate School of Science
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Toshiharu HASE
Kiyotoshi SEKIGUCHI
Shoji TAJIMA
Yuji GOTO
Toshimichi FUJIWARA
Genji KURISU
Takahisa FURUKAWA
Akira SHINOHARA
Kazuaki YOSHIKAWA
Toshifumi TAKAO
Junichi TAKAGIAtsushi NAKAGAWA
Haruki NAKAMURA
Hironobu HOJO$VVLVWDQW3URIHVVRU

Toru KAWAKAMI
Masato NAKAI
Isao SUETAKE
Chojiro KOJIMA
Miki SHINOHARA
Yoshihiro OMORI
Nobuaki OKUMURA
Kenji IWASAKI
Mamoru SUZUKI
Akira KINJO
Junko KANOHJoji MIMA
Hideaki TANAKA

Yoko KIMATA-ARIGA
Masashi YAMADA
Hironobu KIMURAYoh MATSUKI
Rikako SANUKI
Koichi HASEGAWA
Kazushiro FUJIWARA
Yu KITAGO
Eiki YAMASHITA

Young-Ho LEE

Akifumi HIGASHIURA

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Takeshi SATO

Chemistry, Graduate School of Science
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Toshimichi FUJIWARA
Toru KAWAKAMI
Toshifumi TAKAOHaruki NAKAMURA
Hironobu HOJO

Chojiro KOJIMA
Akira KINJO  
Takeshi SATO$VVLVWDQW3URIHVVRUYoh MATSUKI
Yuya ASAHINA

Macromolecular Science, Graduate School of Science
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Yuji GOTO
Genji KURISU
Atsushi NAKAGAWA

Mamoru SUZUKI
Hideaki TANAKAYoung-Ho LEE$VVLVWDQW3URIHVVRUEiki YAMASHITA6SHFLDOO\$SSRLQWHG$VVLVWDQW3URIHVVRU
Akifumi HIGASHIURA
Medical Biosignaling, Graduate School of Medicine
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Kazuaki YOSHIKAWA
Takahisa FURUKAWA

Koichi HASEGAWA
Kazushiro FUJIWARA

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Graduate School of Frontier Biosciences
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Toshifumi TAKAO
Junichi TAKAGIAtsushi NAKAGAWA
Haruki NAKAMURA
Takahisa FURUKAWA

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Mamoru SUZUKI
Kenji IWASAKI
Akira KINJO

Education & Research Activities

10Number of Students
Undergraduate

Master Course

Doctor Course

School of Science

7

46

31

School of Medicine

3

1

1

0

5

3

Graduate School of
Frontier Biosciences

Research Activities
2010 㹼2014

Number of Publications
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 Original Paper

132

127

128

128

108

 Review

23

22

27

31

20

 Presentation at Meeting

327

278

279

277

306


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Large Project Researches
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Education & Research Activities

17Activities as a Joint-Usage/Research Center, and International ExchangeIn order to fulfill its aim as a Joint-Usage/Research Center, the Institute carries out the following programs.

Joint Researchers and International Collaborative Research ___________________________
A joint research program has been established to provide visiting scientists, who are engaged in studies onproteins, from outside the Institute with an opportunity to perform coordinated research at the Institute for up to 6
months. More than 50 scientists are selected yearly from applications of various domestic institutions.
The program covers the research and travel expense, and the Institute provides the facilities for accommodations.
This program started in 1959, and total 2,217 researchers were so far admitted during the past 55 years. Researchers,
who want to use big instruments such as the X-ray analysis facilities, the superconducting magnet NMR, and mass
spectrometers, should apply for the visiting scientist program.

Number of Joint ResearchersRegular(Year)

NumberMonth

Number

2000

29

28

15

18

2001

31

55

21

30

2002

24

20

27

35

2003

25

21

43

52

2004

33

27

24

33

2005

29

24

29

37

2006

29

18

37

45

2007

33

20

35

45

2008

38

25

39

48

2009

44

30

44

51

2010

57

21

48

2011

51

35

2012

59

2013
2014

Beamline

NMR
Number

Theme

53

15

15

48

52

15

15

20

52

59

12

12

69

22

58

63

14

14

79

30

65

65

12

12

Joint-Usage/Research Center

18

Theme

Year
In addition, in 2005, the Institute started an international collaborative
research program, inviting researchers widely from overseas countries. The
research should be conducted in the form of a collaboration including atleast one of the Principal Investigators at IPR, or it should use particularexperimental facilities of IPR. So far, the researchers came from USA, UK,
Sweden, Spain, New Zealand, Hungary, Poland, Bangladesh, India, Taiwan,
China, Korea, France, Russia, Cuba, Germany, Malaysia, Indonesia,
Netherlands, Singapore, Viet Nam and Italy using this program.
In the fiscal year of 2014, 17 overseas researchers visited the Institutefrom 12 countries, Germany, Korea, Thailand, USA, India, Malaysia,
Taiwan, Australia, Hungary, Indonesia, UK and Sweden, ,and madecollaborative researches with the Principal Investigators at the Institute.

Number

Days

2005

6

305

2006

8

213

2007

4

44

2008

6

140

2009

8

252

2010

9

152

2011

9

431

2012

15

323

2013

13

224

2014

18

382

IPR Seminar _________________The Institute holds annually around 16 seminars on various topics. Applied topics are selected by the Advisory
Committee on Research Programs. Speakers are supplied with travel expenses incurred in attending the seminar.

IPR Seminars from April 2014 to March 2015
㻌
1

2

3

4

5

6

7

8

9

10

11

12

Title / Organizer
Workshop on the Beamline for Biological Macromolecule Assemblies
Atsushi NAKAGAWA (IPR, Osaka Univ.), Eiki YAMASHITA (IPR, Osaka Univ.), Akifumi
HIGASHIURA (IPR, Osaka Univ.)
Practical Aspects of Non-uniform Sampling in Multi-dimensional NMR Spectroscopy andApplication for Biological Systems
Takahisa IKEGAMI (Yokohama City University), Koh TAKEUCHI (AIST)
Molecular mechanism of chromosome transmission: From replication to partitioning
Toshiki TSURIMOTO (Kyusyu Univ.), Hisao Masukata (School of Science, Osaka Univ.)
Hiroshi MASUMOTO (Kazusa DNA Res. Inst.)
Regulation and Environmental Adaptation of Photosynthesis: An Attractive Theme for Structural
Life Science
Toshiharu HASE (IPR, Osaka Univ.), Akira SUZUKI 㸦Institut Jean- Pierre Bourgin,INRA Centre
de Versailles), Genji KURISU 㸦IPR, Osaka Univ.)
Genome stability and exchange of DNAs

Date
May 12-14, 2014㻌 㻌June 18-19, 2014㻌 㻌
September 25-26, 2014㻌 㻌
October 24, 2014㻌 㻌
November 14, 2014㻌

Susan GASSER (FMI), Akira SHINOHARA (IPR, Osaka Univ.) 㻌

Mechanism and regulation of aberrant protein aggregation
Yuji GOTO (IPR, Osaka Univ.), Jozef KARDOS 䠄Eotvos Larand Univ.)
Eri CHATANI (Kobe Univ.), Hisashi YAGI (Tottori Univ.)
7th Retina Research Meeting
Akira MURAKAMI (Juntendo Univ.), Takeshi IWATA (Tokyo Medical Center)
Atsushi MIZOTA (Tokyo Univ.), Sumiko WATANABE (The Univ. of Tokyo)
Neural basis of information integration for decision making-Neural circuitry mechanism and its formational developmentMakoto SATO (Faculty of Medicine, Osaka Univ.), Kazuaki YOSHIKAWA (IPR, Osaka Univ.)
The 5th symposium on “Fusion of neuronal science and structural biology”
Michihiro IGARASHI (Niigata Univ.), Nobuyuki SHIINA (National Institute for Basic Biology)
Tomoo NISHIOKA (Nagoya Univ.), Kozo KAIBUCHI (Nagoya Univ.)
Junichi TAKAGI (IPR,Osaka Univ.), Atsushi NAKAGAWA (IPR, Osaka Univ.)
Looking to the future of Notch signaling
Kenji MATSUNO (School of Science, Osaka Univ.), Junichi TAKAGI (IPR, Osaka Univ.)
Yumiko SAGA (National Institute of Genetics)-The 11th Japan-Korea Bilateral Symposium on Biological NMR-International NMR Symposium on Pharmaceutical NMRToshimichi FUJIWARA (IPR, Osaka Univ.), Chojiro KOJIMA (IPR, Osaka Univ.)
Bong-Jin LEE䠄Seoul National Univ.)
Molecular Crowding and Macromalecular Association

November 17-21, 2014㻌

Yuji GOTO (IPR, Osaka Univ.), Damien HALL (Australian National Univ.)

 㻌

 㻌
November 22, 2014㻌 㻌
November 27-28, 2014㻌 㻌
December 4-5, 2014㻌 㻌
December 18, 2014㻌 㻌
December 19-20, 2014㻌 㻌
February 5, 2015㻌

Joint-Research Center, and International Exchange

19

13

14

15

Joint workshop by PDBj & Platform for Drug Discovery, Informatics, and Structural Life Science

February 20, 2015㻌

Haruki NAKAMURA (IPR, Osaka Univ.) 㻌

Cutting edge of Structural biology and biochemistry of anaerobic proteins

March 5, 2015㻌

HGenji KURISU (IPR, Osaka Univ.), Yuichi FUJITA (Nagoya Univ.) 㻌

Analysis and prediction of protein assembly structures by bioinformatics
Haruki NAKAMURA (IPR, Osaka Univ.), Kei YURA (Ochanomizu Univ.)

16

Photomovement responses, photosensor proteins, optgenetics.

March 5, 2015

~ Watanabe In Memoriam~

Kenji IWASAKI (IPR, Osaka Univ.), Mineo ISEKI (Toho Univ.)

March 6, 2015㻌

International ExchangeThe post of Visiting Professor from foreign countries or regions is provided at the Research Center for State-of-the-art
Functional Protein Analysis. The Institute also accepts foreign scientists through programs sponsored by the Japan Society
for the Promotion of Science, or through other programs when financing is guaranteed from outside the Institute. More than
two hundred visiting foreign scientists have participated in research activities since the establishment of the Institute. In
2005, the Institute has started a new program, the International Collaborative Research, for overseas researchers who
perform coordinated research at the Institute and use particular experimental facilities of IPR, including a synchrotron beamline for biological macromolecular assemblies at SPring-8, as well as in a joint research program for Japanese researchers.
The Institute has also accepted the international exchange students through the program called FrontierLab@OsakaU. To
promote further cooperation in research, the Institute has concluded Inter-Faculty Academic Exchange Agreements asfollows.
Peking University, Institute of Physical Chemistry (China) from 1987
Center for Genetic Engineering and Biotechnology (Cuba) from 2003
National Synchrotron Radiation Research Center (Taiwan) from 2007
Indian Institute of Chemical Biology (India) from 2009
Seoul National University, College of Pharmacy (Korea) from 2009
Airlangga University, Institute of Tropical Disease (Indonesia) from 2014
National Center for Protein Science, Shanghai (China) from 2014
Peking University, Center of Protein Science (China) from 2014
The Australian National University, College of Physical and Mathematical Sciences (Australia) from 2014

Collaborations with Foreign Institutions㻌㻌㻝㻌

Representative/Institution/Coutry/Project Title
Prof. Zengyi Chang, Center of Protein Science, Peking University, China
㺃Promotion of Protein Science㻌㻞㻌 Prof. Ming Lei, National Center for Protein Science Shanghai
㺃Promotion of Structural Biology
㻟㻌 Dr. Akira Suzuki, INRA, Versailles, France               㺃Molecular physiology of plant amino acid synthesis
㻠㻌 Dr. Guy T. Hanke, Osnab䡎ueck University, Germany㻌 㻌 㻌 㻌 㻌 㻌 㻌 㻌 㻌 㻌 㻌䞉Redulation of redox metabolisms in chloroplast㻡㻌 Prof. Peter F. Stadler, University Lipzig, Department of Computer Science, Bioinformatics, Lipzig, Germany
㺃Bioinformatics of DNA methylation
㻢㻌 Dr.Norimasa Iwanami, Max Planck Institute of Immunobiology and Epigenetics, Freiburg, Germany
㺃Analysis of Dnmt1 mutation in zebrafish
㻣㻌 Dr. Jorge Fernandez-de-Cossio, Center for Genetic Engineering and Biotechnology, Cuba㺃Development of Analytical Software for Interpretation of Mass Spectral Data㻤㻌 Prof. Luca Tamagnone, University of Torino, Italy
㺃Development of function-modulating antibodies against human plexin D1.
㻥㻌 Prof. Gregory A. Petsko,Cornell University, USA㺃Development of pharmacological chaperone for sorLA.
㻝㻜㻌 Director, Prof. Shangjr Gwo, Taiwan
㺃Structure biology research using synchrotron radiation
㻝㻝㻌 Prof. Chun-Jung Chen, National Cheng Kung University
㺃Crystal structures of key proteins and complexes involved in two-component regulatory systems in Pseudomonas
aeruginosa for the regulatory mechanism

Joint-Research Center, and International Exchange

20㻝㻞㻌 Prof. Janos Hajdu, Uppsala University, Sweden
㺃Studies of coherent X-ray imaginf for virus particles
㻝㻟㻌 Prof. Thomas Happe, Ruhr University Bochum, Germany
㺃X-ray structural analysis of [FeFe]-hydrogenase from green alga㻌㻝㻠㻌 Prof. Matthias Rögner, Ruhr University Bochum, Germany
㺃Crystallization of NDH1 from Thermophilic cyanobacterium㻌㻝㻡㻌 Prof. Michael Hippler, University of Münster, Germany
㺃Structural dynamics of calredoxin, a novel EF-hand and thioredoxin domain protein from Chlamydomonas
reinhardtii㻌
㻝㻢㻌 Prof. James R. Ketudat-Cairns, Suranaree University of Technology, Thailand
㺃Structural studies of a glycoside hydrolase family 116 beta-glycosidase from a thermophilic bactera㻌㻝㻣㻌 Prof. Jozsef Kardos, Etovos Lorand University, Hungary
䡡Understanding the mechanism of protein abberant aggregation and amyloid formation
㻝㻤㻌 Prof, Gennaro Esposito, University of Udine, Italy
䡡Amyloid fibril formation of β2-microglobulin
㻝㻥㻌 Prof. Alexey Merz, University of Washington School of Medicine
㺃Functions of yeast Sec1/Munc18 (SM) -family proteins in SNARE-mediated membrane docking and fusion
㻞㻜㻌 Prof. William Wickner, Dartmouth Medical School
㺃Membrane tethering mediated by the HOPS complex in 㺃SNARE-dependent membrane fusion
㻞㻝㻌 Prof. András Per㹡zel, Etovos Lorand University, Hungary
㺃Protein misfolding and early stage aggregates㻞㻞㻌 Prof. Masayori Inouye, Robert Wood Johnson Medical School, University of Medicine and Dentistry of New
Jersey, USA
䞉Isotope labeling by single protein production systems
㻞㻟㻌 Prof. John L. Markley, University of Wisconsin-Madison, USA㻌 㻌 㻌 㻌䞉Databank for biological NMR
㻞㻠㻌 Prof. Praveen Ballabh, New York Medical College, USA      㺃Mechanisms of neurogenesis in human fetal brain
㻞㻡㻌 Prof. Zhengang Yang, Fudan University, China            㺃Molecular mechanism of cortical interneuron development
㻞㻢㻌 Prof. Neil Hunter, University of California Davis, USA       㺃Study on mechanisms of meiotic recombination
㻞㻣㻌 Prof. Susan Gasser, Friedrich Mericier Institute, Switzerland       㺃Molecular mechanism of chromosome dynamics

International Exchange

21

Division of Protein Chemistry

Division of Protein Chemistry Laboratory of Regulation of Biological Reactions  Professor
Associate Professor
Assistant Professor

Toshiharu HASE
Masato NAKAI
Yoko KIMATA-ARIGA

Correspondence Tel: +81-6-6879-8611; Fax: +81-6-6879-8613; E-mail:enzyme@protein.osaka-u.ac.jp
The plant organelles collectively referred as plastids play adiverse set of physiological functions represented byphotosynthesis, and soluble and membrane-bound proteins,
localized in certain subplastidal compartments, areinvolved in the organelle functions. We have been
studying the function and biogenesis of plastids andplastidal proteins with techniques of biochemistry, genetics
and cell biology using higher plants and cyanobacteria.
Current projects are as following. i) Reducing power
necessary for carbon, nitrogen and sulfur assimilation are
utilized by combination of an electron carrier protein,ferredoxin and ferredoxin-dependent enzymes, enabling
plants to assimilate inorganic raw materials to organic
compounds such as sugar and amino acid. The structure ofelectron transfer complex and catalytic mechanisms offerredoxin and partner redox enzymes are studied. ii)
Cytosolically synthesized polypeptides are transported into
chloroplasts and converted to functional mature proteins.
The mechanisms of protein translocation across theenvelope membranes of chloroplasts and involvements ofmolecular chaperones are studied. Recently, a new multiprotein complex with a size of one-megadalton was
discovered, and its structure and function are underinvestigation. iii) Malaria cells contain an organelle calledapicoplast, in which redox metabolisms for parasite vitality
take place. We are studying malaria ferredoxin and its
partner redox enzymes to explore how redox cascade isoperative in apicoplasts.

Fig.2. A novel one-megadalton protein translocation
machinery at the inner envelope of chloroplasts.

࠙Referencesࠚ
1. Unconvering the Protein Translocon at the Chloroplast Inner
Envelope Membrane. Kikuchi S, Nakai M, et al. (2013)
Science 339, 571-574.
2. Concentration-dependent oligomerization of cross-linked
complexes between ferredoxin and ferredoxin-NADP+
reductase. Kimata-Ariga Y, Kubota-Kawai H, Lee Y-H,
Muraki N, Ikegami T, Kurisu G, and Hase T. (2013) Biochem.
Biophys. Res. Commun. 434, 867-872.
3. N-terminal structure of maize ferredoxin:NADP+ reductase
determines recruitment into different thylakoid membranecomplexes. Altmann B, Twachtmann M, Muraki N, Voss I,
Okutani S, Kurisu G, Hase T, and Hanke GT. (2012) Plant
Cell 24, 2979-2991
4. Electron Transfer of Site-specifically Cross-linked Complexes
between Ferredoxin and Ferredoxin-NADP+ Reductase.
Kimata-Ariga Y, Sakakibara Y, Ikegami T, and Hase
T. (2010) Biochemistry 49, 10013-10023.
5. A 1-megadalton translocation complex containing Tic20 andTic21 mediates chloroplast protein import at the innerenvelope membrane. Kikuchi S, Oishi M, Hirabayashi Y,
Nakai M, et al. (2009) Plant Cell 21, 1781-1797.
6. Molecular interaction of Ferredoxin and Ferredoxin-NADP+
reductase from human malaria parasite. Kimata-Ariga Y, Hase
T, et al. (2007) J. Biochem. 142, 715-720.

࠙Current Research Programsࠚ
1) Electron partitioning in redox metabolisms ofphotosynthetic and non-photosynthetic plastids2) Reaction mechanism of ferredoxin-dependent enzymes3) Molecular mechanism of chloroplast biogenesis
4) Structure and function of chloroplast protein translocons5) Structure and reaction mechanism of ferredoxin and its
oxidoreductase of malaria apicoplast

Fig.1. Electron transfer complex of ferredoxin
and ferredoxin-dependent enzymes in chloroplasts and root plastids.

Laboratory of Regulation of Biological Reactions

23

Division of Protein Chemistry Laboratory of Extracellular Matrix Biochemistry
Professor
Assistant Professor
Senior Technical Staff

Kiyotoshi SEKIGUCHI
Masashi YAMADANaoko NORIOKA

Correspondence㻌Tel: +81-6-6879-8617; Fax: 06-+81-6-6879-8619; E-mail: sekiguch@protein.osaka-u.ac.jp

The goal of our laboratory is to understand themolecular basis of tissue architecture and cellularfunctions in multi-cellular organisms based on thecell-extracellular matrix (ECM) interaction. ECM is nota mere scaffold between cells but rather an
information-rich supra-molecular structure that provides
cells with signals that regulate cell growth,differentiation, and apoptosis. Cells read thebarcode-like signals written in the ECM with a varietyof cell surface receptors and determine whether theyshould grow or differentiate. The composition of the
ECM is spatiotemporally regulated during embryonicdevelopment and differs from one cell type to another.
We performed a comprehensive immunohistochemical
survey of more than 40 basement membrane proteins inmouse embryos. The immunohistochemical data wascompiled into a high-resolution digital image database(“Mouse Basement Membrane Bodymap”), which isavailableontheinternetat
http://www.matrixome.com/bm/.

Fig. 2. Localization of laminin alpha 5 (left; green) and alpha 1 (right;
green) chains in E7 mouse embryos, double-stained with anti-Oct-3/4(red). Laminin alpha 5 was detected along the entire basement membrane,
while laminin alpha 1 was localized at the extraembryonic basement
membrane.

࠙Current Research Projectsࠚ
1) Purification and characterization of laminins andother basement membrane proteins.
2) Studies on spatiotemporal customization of
basement
membranecomposition
duringdevelopment.
3) Mechanisms of integrin-mediated cell-substratum
adhesion and signal transduction.
4) Regulation of cell-cell and cell-substrateinteractions by tetraspanin CD151.
5) Regulation of stem cell proliferation anddifferentiation
throughengineeringofextracellular environment.

Fig. 3. Mouse Basement Membrane Bodymap database. Morethan 90% of basement membrane components have been
localized within E16.5 mouse embryos by means of
immunohistochemistry.

࠙Referencesࠚ
1. Transcriptome-based systematic identification of extracellularmatrix proteins. Manabe, R. et al. (2008) Proc. Natl. Acad. Sci.
USA 105, 12849-12854.
2. Polydom/SVEP1 is a ligand for integrin D9E1. Sato-Nishiuchi,
R. et al. (2012) J. Biol. Chem. 287, 25615-25630.
3. Basement membrane assembly of the integrin D8E1 ligand
nephronectin requires Fraser syndrome-associated proteins.
Kiyozumi, D. et al. (2012) J. Cell Biol. 197, 677-689.
4. Laminin E8 fragments support efficient adhesion andexpansion of dissociated human pluripotent stem cells.
Miyazaki, T. et al. (2012) Nature Commun. 3, 1236;
doi :10.1038/ncomms2231.
5. A novel efficient feeder-free culture system for the derivationof human induced pluripotent stem cells. Nakagawa, M. et al.
(2014) Sci. Rep. 4, 3594; doi:10.1038/srep03594.

Fig. 1. Regulation of cell growth, differentiation, and survival by
extracellular matrix. Cells have their own customized
extracellular matrix.

Laboratory of Extracellular Matrix Biochemistry24Division of Protein Chemistry

Laboratory of Epigenetics
Professor
Associate Professor
Assistant Professor
Technical Assistant

Shoji TAJIMA
Isao SUETAKE
Hironobu KIMURA
Naoyuki ABECorrespondenceTel: +81-6-6879-8628; Fax: +81-6-6879-8629; E-mail: tajima@protein.osaka-u.ac.jp
In vertebrates, genomic DNA is packaged into chromatin,of which fundamental structure is a nucleosome. The
methylation modification of cytosine in the sequence of
CpG and the posttranslational modifications of core
histones affect packaging of chromatin. These
modifications are crucial for gene expression. We are
specially focusing on DNA, histones H3 lysine 9 and 27,
and H4 lysine 20 methylations, which are known tocontribute to gene silencing via forming heterochromatin.
The methyl groups are transferred from S-adenosyl-Lmethionine by methyltransferases. Our final goal is toelucidate the mechanisms how DNA methylation state areregulated, and the silencing marks on the lysine residues inhistone H3 and H4 are affecting the DNA methylation
state.

Fig.3. Recognition of histone H3K9me3 in nucleosome
structure by HP1Į. The selective binding to histone H3K9me3 innucleosomes via the chromodomain (CD) is cooperatively
enhanced by the balance of net positive charge [Ȉ(+)] of thehinge (HR) and the suppressive effect of the chromoshadow
domain (CSD). The delicate charge balance of the HR and CSD
allows the selective binding of HP1Į to histone H3K9me3 innucleosomes. (ref. 4)

࠙Current Research Programsࠚ
1) Effect of methylation and histone modification on
chromatin structure2) Mechanism of creation and inheritance of the DNA
methylation patterns3) Identifying the factors interacting with DNA
methyltransferases

Fig. 4. The RTS domain plugging the catalytic pocket of Dnmt1
is removed by direct interaction with Uhrf1 (SRA), and thus the
DNA can access to the catalytic center. (ref. 6)
ިReferencesީ
1. Mouse Dnmt3a preferentially methylates linker DNA and isinhibited by histone H1. Takeshima H, Suetake I, Tajima S(2008) J. Mol. Biol. 383, 810-821.
2. Structural insight into maintenance methylation by mouse
Dnmt1. Takeshita K, Suetake I, Yamashita E, Suga M, Narita
H, Nakagawa A, Tajima S (2011) Proc. Natl. Acad. Sci. USA
108, 9055-9059.
3. Characterization of DNA-binding activity in the N-terminal
domain of the DNA methyltransferase Dnmt3a. Suetake I,
Mishima Y, Kimura H, Lee YH, Goto Y, Takeshima H,
Ikegami T, Tajima S (2011) Biochem. J. 437, 141-148.
4. Hinge and chromoshadow of HP1Į participate in recognitionof K9 methylated histone H3 in nucleosomes. Mishima Y,
Watanabe M, Kawakami T, Jayasinghe CD, Otani J,
Kikugawa Y, Shirakawa M, Kimura H, Nishimura O, Aimoto
S, Tajima S, Suetake I (2013) J. Mol. Biol. 425, 54-70.
5. Cell cycle-dependent turnover of 5-hydroxymethyl cytosine
in mouse embryonic stem cells. Otani J, Kimura H, Sharif J,
Endo TA, Mishima Y, Kawakami T, Koseki H, Shirakawa M,
Suetake I, Tajima S. (2013) PLoS ONE 8, e82961.
6. The DNA methyltransferase Dnmt1 directly interacts with the
SET and RING finger associated (SRA) domain of themultifunctional protein Uhrf1 to facilitate accession of thecatalytic center to hemi-methylated DNA. Berkyurek AC,
Suetake I, Arita K, Takeshita K, Nakagawa A, Shirakawa M,
Tajima S (2014) J. Biol. Chem. 289, 379-386.

Fig.1. Schematic illustration of the members of DNA
methyltransferase family Dnmts. Up to present, 4 genes, Dnmt1,
Dnmt3a, Dnmt3b, and Dnmt3L, are identified.
Fig. 2. Multi-domain structure ofmouse Dnmt1. Figure shows
ribbon model of mouse Dnmt1. The
C-terminal catalytic domain are
surrounded by the RFTS, CXXC
motif, and two BAH domains(BAH1 and BAH2). Four zinc ions
are shown in red spheres. All of thezinc ions are in a motif similar to
Zn-finger motif. The KG-repeat
linker connecting the N-terminal
region and the C-terminal catalytic
domain is in a flexible structure.

Laboratory of Epigenetics

25Division of Protein Chemistry

Laboratory of Protein Organic Chemistry
Professor
Associate Professor
Associate Professor
Assistant Professor

Hironobu HOJO
Toru KAWAKAMI
Takeshi SATO
Yuya ASAHINA


CorrespondenceTel: +81-6-6879-8601; Fax: +81-6-6879-8603; E-mail: hojo@protein.osaka-u.ac.jp
Chemical methods enable the synthesis of proteins, which
can not be prepared by the recombinant method, such assite-specifically labeled, glycosylated and phosphorylated

proteins. Laboratory of Protein Organic Chemistry isaiming to promote new protein researches using thesesynthetic proteins. Thus, our laboratory is developing
facile methods for protein synthesis based on ligation
chemistries. In addition, the synthetic method is applied
for the preparation of membrane proteins and their partial
sequences to elucidate the signal transduction mechanism
by solid state NMR and IR. Modified histones and their
partial sequences, glycosylated proteins are also being
synthesized for the functional analyses.
Fig. 2. Concept for our research on activation mechanism ofreceptor tyrosine kinase. Our research focuses on structure of
the transmembrane and the juxtamembrane regions tounderstand how these regions involve in the activation
mechanism.

࠙Current Research Programsࠚ
1) General studies on a chemical protein synthesis
2) Development of methods for peptide ligation
3) Development of methods for site-specificmodification of peptides and proteins
4) Synthetic studies of membrane proteins
5) Structural and functional studies of membraneproteins
6) Synthetic studies of modified histones, andelucidation of the role of modifies

࠙Referencesࠚ
1. Peptidyl N-alkylcysteine as a peptide thioester surrogate in thenative chemical ligation. Asahina Y, Nabeshima K, Hojo H(2015) Tetrahedron Lett., 56, 1370-1373.
2.Fast preparation of an N-acetylglucosaminylated peptide
segment for the chemoenzymatic synthesis of a glycoprotein.
Asahina Y, Kanda M, Suzuki A, Katayama H, Nakahara Y,
Hojo H (2013) Org. Biomol. Chem., 11, 7199–7207.
3. Chemoenzymatic synthesis of immunoglobulin domain of
Tim-3 carrying the complex type N-glycan using the one-pot
ligation method. Asahina Y, Kamitori S, Takao T, Nishi N, Hojo
H (2013) Ang. Chem. Int. Ed., 52, 9733-9737.
4. Model study using designed selenopeptides on the importanceof the catalytic triad for the antioxidative functions ofglutathione peroxidase. Takei T, Urabe Y, Asahina Y, Hojo H,
Nomura T, Dedachi K, Arai K, Iwaoka M (2014) J. Phys. Chem.
B, 118, 492-500.
5. Enhancement in the Rate of Conversion of Peptide Cys-Pro
esters to Peptide Thioesters by Structural Modification.
Kawakami T, Kamauchi A, Harada E, Aimoto S (2014)
Tetrahedron Lett., 55, 79–81.
6.Sequential Peptide Ligation by Combining the Cys-Pro Ester(CPE) and Thioester Methods and its Application to theSynthesis of Histone H3 Containing a Trimethyl Lysine
Residue. Kawakami T, Akai Y, Fujimoto H, Kita C, Aoki Y,
Konishi T, Waseda M, Takemura L, Aimoto S (2013) Bull.
Chem. Soc. Jpn., 86, 690-697.
7.Coupling of transmembrane helix orientation to membranerelease of the juxtamembrane region in FGFR3. Tamagaki H,
Furukawa Y, Yamaguchi R, Hojo H, Aimoto A, Smith SO,
Sato T (2014) Biochemistry, 53, 5000-5007.

Fig. 1. Synthetic route of human interleukin-2 as a example ofchemical protein synthesis. The key compound of the method is a
peptide thioester which is prepared by the solid-phase method.
The thioester group is then chemoselectively reacted with theterminal amino group of the other segment to give a peptide bond.
The reaction is repeated until the entire sequence of the desired
protein is assembled. After deprotection and folding, the correctly
folded functional protein is obtained.
Laboratory of Protein Organic Chemistry26

Division of Protein Structural Biology

Division of Protein Structural Biology

Laboratory of Protein Folding
ᴾ

Professor
Associate Professor
Assistant Professo

Yuji GOTO
Young-Ho LEE
Masatomo SO

ᴾ
CorrespondenceᴾTel: +81-6-6879-8614; Fax: +81-6-6879-8616; E-mail: ygoto@protein.osaka-u.ac.jp
ᴾ
Protein folding is a process in which an extended
polypeptide chain acquires a unique folded conformation
with biological activity. Clarifying the mechanism ofprotein folding is essential to improve our understandingof the structure and function of proteins. It is also
important because many critical biological processes anddisease states involve protein misfolding and aggregation
reactions. We study the conformational stability and themechanisms of protein folding and misfolding withvarious approaches including spectroscopies (NMR,
fluorescence, CD), physicochemical measurements(calorimetry,
analyticalultracentrifugation)
andfluorescence microscopy.

ẔCurrent Research Programsẕ
1) Observation of folding processes and clarification of
the mechanism of protein folding2) Structural stability and formation of amyloid fibrils
3) Comprehensive understanding of protein folding andmisfolding on the basis of solubility andsupersaturation

Fig. 3. Overview (A) and schematic illustration (B) of the a
HANdai Amyloid Burst Inducer (HANABI) system. HANABI
performs a high-throughput analysis of ultrasonication-forced
amyloid fibrillation. (Ref.3)

Fig. 1. Schematic presentation of folding pathway of bovine
E-lactoglobulin with non-native ǂ-helical intermediate.
Fig. 4. Comparison of crystallization of sodium acetate andamyloid fibrillation of amyloid E. (Ref.4)ިReferencesީ
1. Kinetic intermediates of amyloid fibrillation studied byhydrogen exchange methods with nuclear magnetic
resonance. Lee Y-H, Goto Y (2012) Biochim. Biophys. Acta
1824, 1307-1323.
2. High-throughput analysis of the ultrasonication-forced
amyloid fibrillation reveals the mechanism underlying thelarge fluctuation in the lag time. Umemoto A, Yagi H, So M,
Goto Y. (2014) J. Biol. Chem. 289, 27290-27299.
3. Supersaturation-limited amyloid fibrillation of insulin
revealed by ultrasonication. Muta H, Lee YH, Kardos J, LinY, Yagi H, Goto Y. (2014) J. Biol. Chem. 289,18228-18238.
4. Heat of supersaturation-limited amyloid burst directly
monitored by isothermal titration calorimetry. Ikenoue T, Lee,
YH, Kardos J, Yagi H, Ikegami T, Naiki H, Goto, Y. (2014)
Proc. Natl. Acad. Sci. USA 111, 6654-6659.

Fig. 2. Real time observation of the growth of amyloid-E fibril
using total internal reflection fluorescence microscopy.
Laboratory of Protein Folding27

Division of Protein Structural BiologyLaboratory of Molecular Biophysics
Professor
AssociateProfessor
Assistant Professor

Toshimichi FUJIWARA
Chojiro KOJIMAYoh MATSUKI

CorrespondenceTel: +81-6-6879-8598; Fax: +81-6-6879-8699; E-mail: tfjwr@protein.osaka-u.ac.jp
Laboratory of molecular biophysics is engaged in studying
the biological macromolecular structure and theirintermolecular interactions mainly by using nuclear
magnetic resonance (NMR). NMR provides informationon the protein structure at work with atomic resolution
even in cells. Taking this advantage, we can understand thebiological activities for signal transduction and energy
conversion from structures. Structures of Fo c-ring,
Fig. 2 1H–13C HSQC spectra of methylated ubiquitin andlight-harvesting Bchl c complex, E2-microglobulin
methylated
ubiquitin interacting with protein YUH1 shown on theamyloid,florigen,interacting
ubiquitin,andleft. Ubiquitin and YUH1 are shown in the complex (1CMX) bymembrane-bound mastoparan-X have been elucidated.
the ribbon and surface representation, respectively. Larger andSince supramolecular systems such as membrane protein
smaller chemical shift changes are colored red and gray,complexes play important roles in biological systems, we
respectively. This simple post-methylation method gives strong
are also developing new methodologies in NMR to analyze
CH3 NMR signals for detecting the protein interactions. (Ref. 2)
those challenging structures. One of our programs forsolid-state NMR features high-field dynamic nuclear
polarization (DNP) for a 1000-fold signal enhancement byusing high-intensity terahertz light source, gyrotron. Thesedevelopments aim to contribute to not only academic but
also industrial NMR applications such as drug discovery.
࠙Current Research Programsࠚ
1) NMR analysis of proteins and their interactions
2) Sensitivity enhancement of high-resolution NMR byhyperpolarization
3) New methodologies in biological NMR including
isotope-labeled sample preparation and data analysis

Fig. 3 Automatic solid-state NMR structural analysis of protein.
13
C-NMR spectra of proteins in solids often show unresolved
signals. Our spectral fitting softwane RESPLS enables chemical
shift assignments and secondary structure prediction based on
the databases PDBj and BMRB. This method simplifies the
structural analysis by providing reliable information even forlyophilized states. (Ref. 3)

ᇻReferencesᇼ
1. Utilization of paramagnetic relaxation enhancements forhigh-resolution NMR structure determination of a soluble
loop-rich protein with sparse NOE distance restraints. Furuita
K, Kataoka S, Sugiki T, Hattori Y, Kobayashi N, Ikegami T.,
Shiozaki K, Fujiwara T, Kojima C (2015) J. Biomol. NMR, 61,
55-64.
2. Utilization of lysine 13C-methylation NMR for protein-protein
interaction studies. Hattori Y, Furuita K, Ohki I, Ikegami T,
Fukada H, Shirakawa M, Fujiwara T, Kojima C (2013) J.
Biomol. NMR 55, 19-31.
3. Secondary Structural Analysis of proteins based on 13C
chemical shift assignments in unresolved solid-state NMR
spectra enhanced by fragmented structure database, Ikeda K,
Egawa A, Fujiwara T (2013) J. Biomol. NMR 55, 189-200.
4. Helium-cooling and -spinning dynamic nuclear polarization for
sensitivity-enhanced solid-state NMR at 14 T and 30 K,
Matsuki Y, Ueda K, Idehara T, Ikeda R, Ogawa I, Nakamura S,
Toda M, Anai T, Fujiwara T (2012) J. Magn. Reson. 225, 1-9.

Fig. 1. NMR magnet for 600-MHz solid-state NMR on the leftand 395-GHz gyrotron for high-intensity light source ofterahertz-wave on the right. Hyperpolarization generated withthese instruments increases the NMR sensitivity of proteins. This
DNP-NMR spectrometer was developed in Institute for Protein
Research. (Ref. 4)

Laboratory of Molecular Biophysics

28

Division of Protein Structural Biology

Laboratory of Protein Crystallography
Professor
Associate Professor
Assistant Professor

Genji KURISU
Hideaki TANAKA
Risa MUTOH

CorrespondenceTel: +81-6-6879-8605; Fax: +81-6-6879-8606; E-mail: gkurisu@protein.osaka-u.ac.jp
Protein crystallography is the best method to determine
atomic structure of protein molecules, in order to elucidate
the molecular mechanism of the highly organizedbiological systems. The main aim of our group is the X-ray
structure determination of the biological macromolecularassemblies including membrane protein complexes. We arefocusing on macromolecular assemblies aroundphotosystem I, and dynein motor including the possible
cargo such as huge vault complex.
࠙Current Research Programsࠚ
1㸧Structural studies of photosynthetic energy-transducing
membrane protein complex and related redox enzymes
2㸧Crystal structure analyses of dynein motors
3㸧High resolution structural analysis of rat liver vault

Fig. 1. Crystal structure of the hetero-tetrameric catalytic
component
NB-proteinofDPOR(Dark-operative
Protochlorophyllide OxidoReductase). The [4Fe-4S] clusters areshown in CPK model, and the Pchlide molecules in stick model.
The BchN and BchB subunits in one dimer are colored in green
and blue.

Fig. 3. Overall structure of the vault shell. (Left) The whole vault
shell comprises a 78-mer of MVP molecules. One molecule of
MVP is colored in red, and the others are colored in blue.
(Right) The MVP monomer is folded into nine structural repeat
domains, a shoulder domain, a cap-helix domain, and a cap-ringdomain. Each domain is depicted in a different color.

crystal structure
20 ns

㻥㻜㼻

30 ns
40 ns
10 ns

࠙Referencesࠚ

50 ns

1. The 2.8 Å crystal structure of the dynein motor domain. Kon etal., (2012) Nature, 484, 345-350
2. X-ray
crystalstructureofthelight-independent
protochlorophyllide reductase. Muraki et al. (2010) Nature,
465, 110-116.
3. The Structure of Rat Liver Vault at 3.5 Angstrom Resolution.
Tanaka et al. (2009) Science, 323, 384-388.
4. Structural Basis of Equisetum arvense Ferredoxin Isoform II
Producing an Alternative Electron Transfer withFerredoxin-NADP+ Reductase. Kurisu et al. (2005) J. Biol.
Chem., 280, 2275-2281.
5. Structure of the Cytochrome b6f complex of Oxygenic
Photosynthesis: Tuning the cavity. Kurisu et al. (2003)
Science 302, 1009-1014.
6. Structure of the electron transfer complex between ferredoxin
and ferredoxin-NADP(+) reductase. Kurisu et al. (2001)
Nature Struct. Biol., 8, 117-121.

Fig. 2. Overall structure of the dynein motor domain (upper
panel) and the swing motion of the stalk region (lower panel).
Dynein is a microtubule-based motor protein, whose motor
activity is located in the motor domain of the heavy chain.
(Upper) A crystal structure of the motor domain is drawn as aribbon model showing linker, six AAA modules constituting ring,
stalk–strut coiled-coils and C-sequence at the outside of the ring.
(Lower) Crystal structure is superimposed with the models of fivesimulated structures derived from the molecular dynamics
simulation. The orientation of the swing is parallel to the AAA
ring and is consistent with a diffusive search-like motion for the
next step along a microtubule.

Laboratory of Protein Crystallography

29

Division of Protein Structural Biology

Laboratory of Membrane Protein Chemistry
Associate Professor

Joji MIMA

Correspondence Tel: +81-6-6879-4326; Fax: +81-6-6879-4329
E-mail: Joji.Mima@protein.osaka-u.ac.jp

Laboratory of Membrane Protein Chemistry is working on
in vitro reconstituted proteoliposomal studies to understandthe molecular machineries of membrane tethering, docking,and fusion processes in the endomembrane organelle
systems of eukaryotic cells (from yeast to human).
Intracellular membrane tethering, docking, and fusion
events are fundamental and conserved biological reactions,which are vital for vesicle trafficking between subcellularorganelle membrane compartments and plasma membranes,
organelle morphology, hormone secretion, and also
synaptic neurotransmission. Earlier seminal studies haverevealed that membrane tethering, docking, and fusion aretemporally and spatially regulated in cells by the diverse
sets of key protein components, which include
SNARE-family proteins, SNARE-interacting chaperone
proteins such as Sec1/Munc18-family proteins, Rab-familysmall GTPases, Rab-interacting effector proteins, andtethering multisubunit complexes. However, it has still
remained enigmatic how those essential protein factors
cooperate to specifically and efficiently mediate membrane
tethering, docking, and fusion events. In our group, we
explored the vital tethering/docking/fusion machineries byin vitro reconstitution with purified recombinant proteins(SNAREs, Rabs, and so on) and synthetic lipid bilayerswith defined lipid compositions. Using homotypic yeast
vacuole membrane fusion as a model, we found that thefunctional synergy of SNARE chaperones (Sec17p,
Sec18p, and the HOPS complex) and phosphoinositides isessential to trigger rapid SNARE-dependent membranefusion. Next, by comprehensively studying 14 purified
SNAREs in yeast, which localize at not only vacuoles but
also endosomes, Golgi, and endoplasmic reticulum (ER),for their capacity to assemble into QabcR-SNARE
complexes and initiate reconstituted proteoliposomal
fusion, we uncover the novel concept that SNAREs
employ multiple and distinct strategies to confer the
specificity of membrane fusion. Moreover, we recently
have developed the in vitro assays to quantitatively analyze
membrane tethering of synthetic liposomes in the presenceof the protein factors related to physiological tethering
processes. Our reconstitution studies now establish that
membrane-anchored human Rab GTPases, including
ER/Golgi Rab2a, endosomal Rab5a, and lysosomal Rab7a,directly and specifically catalyze membrane tethering in a
guanine nucleotide-independent manner. This leads us tofurther study how exactly Rab GTPases and their effectors
or tethering factors work together on membranes tomediate membrane tethering for ensuring the directionalityof intracellular membrane trafficking.

࠙Current Research Programsࠚ
1) Understanding the molecular machinery to catalyze
membrane tethering, docking, and fusion events ineukaryotic endomembrane systems
2) Mechanisms by which miscellaneous sets of
SNAREs, Rab GTPases, and their interacting
proteins control the directionality of intracellular
membrane trafficking














Fig. 1. Reconstituted proteoliposomes with purified membraneproteins and synthetic liposomes with defined lipid compositions.
ᇻReferencesᇼ1. Membrane-anchored human Rab GTPases directly mediate
membrane tethering in vitro. Tamura N, Mima J (2014) Biol
Open 3, 1108-1115.
2. Multiple and distinct strategies of yeast SNAREs to confer
the specificity of membrane fusion. Furukawa N, Mima J(2014) Sci Rep 4, 4277.
3. Distinct contributions of vacuolar Qabc- and R-SNARE
proteins to membrane fusion specificity. Izawa R, Onoue T,
Furukawa N, Mima J (2012) J Biol Chem 287, 3445-3453.
4. Minimal membrane docking requirements revealed byreconstitution of Rab GTPase-dependent membrane fusionfrom purified components. Stroupe C, Hickey CM, Mima J,
Burfeind A, Wickner W (2009) Proc Natl Acad Sci USA 106,
17626-17633.
5. Phosphoinositides and SNARE chaperones synergisticallyassemble and remodel SNARE complexes for membranefusion. Mima J, Wickner W (2009) Proc Natl Acad Sci USA
106, 16191-16196.
6. Complex lipid requirements for SNARE- and SNARE
chaperone- dependent membrane fusion. Mima J, Wickner W(2009) J Biol Chem 284, 27114-2712.
7. Reconstituted membrane fusion requires regulatory lipids,
SNAREs and synergistic SNARE chaperones. Mima J,
Hickey CM, Xu H, Jun Y, Wickner W (2008) EMBO J 27,
2031-2042.

Laboratory of Membrane Protein Chemistry30

Division of Integrated Protein Functions

Division of Integrated Protein Functions

Laboratory of Genome and Chromosome Functions
Professor
AssociateProfessor
AsistantProfessor

Akira SHINOHARA
Miki SHINOHARA
Masahiro TERASAWACorrespondenceTel: +81-6-6879-8624; Fax: +81-6-6879-8626; E-mail: ashino@protein.osaka-u.ac.jp
Laboratory of Genome-Chromosome Functions is engaged
in the following chromosome and genome stability
research. 1. Molecular mechanisms of recombination;
homologous
recombination
andnon-homologous
end-joining. 2. Mechanism of genome stability andgenome instability associated with cancer. 3. Mechanismof meiotic recombination which is essential for productionof genome diversity and chromosome segregation. 4.
Control of chromosome morphogenesis such as
synaptonemal complex formation and chromosome
Fig. 2. Immunostaining of chromosome spreads identified adynamics in meiosis.
meiosis-specific chromosome structure, the synaptonemal
Errors in the recombination lead chromosome instability
complex (SC), in which paternal and maternal homologous
which results in turmorigenesis in somatic cells andchromosomes are synapsed along the chromosomes. Zip1 (green)is a component of the central element of the SC while Rec8 (red)miscarriage and aneuploidy diseases such as Down
is a cohesion component for the axial elements of the SC.
syndrome. The biological relevance to these diseases isalso explored.
Fig. 3. Anaphase bridge formation in
mitotic mammalian cells. When DNA
double-strand breaks are introducedon mitotic chromosomes, anaphasebridges are formed, which, unlessrepaired, leads to chromosome
instability.

࠙Current Research Programsࠚ
1) Analysis of proteins working with RecA homologues
in recombination
2) Studies on chromosome morphogenesis
3) Analysis of the roles of chromatin modification inmeiotic recombination
4) Mechanisms of choice of DSB repair pathways
5) Analysis of recombination in human cells

࠙Referencesࠚ
1. DNA damage response clamp contributes to chromosomal assemblyof ZMM-SIC pro-crossover factors during meiosis. Shinohara, M,
Hayashihara, K., Grubb, J.T., Bishop, D.K., and Shinohara, A. (2015)
J. Cell. Sci. 128, 1494-506.
2. Canonical non-homologous end joining in mitosis induces genome
instability and is suppressed by M-phase specific phosphorylation of
XRCC4 via CDKs. Terasawa, M., Shinohara A., and Shinohara, M.
(2014) PLoS Genetics, 10, e1004563, 2014.
3. Dot1-dependent histone H3K79 methylation promotes the formationof meiotic double-strand breaks in the absence of histone H3K4
methylation in budding yeast. Bani Ismail, M., Shinohara M. and A.
Shinohara. (2014) PLoS One. 9, e96648.
4. A new protein complex promoting the assembly of Rad51 filaments.
Sasanuma, H., Tawaramoto, M.S., Lao, J, Hosaka, H., Sanda, E.,
Suzuki, M. Yamashita, E., Shinohara, M. Hunter, N., Nakagawa A.
and Shinohara, A. (2013) Nature Comms. 4, 1676-1688.
5. Saccharomyces cerevisiae Srs2 helicase disassembles Rad51 frommeiotic chromosomes. Sasanuma, H., Furihata Y., Shinohara, M. andShinohara, A. (2013) Genetics, 194, 859-872.
6. CDK-dependent phosphorylation of Lif1 and Sae2 control imprecise
non-homologous end joining accompanied with DSB resection.

Fig. 1. Promotion of Rad51 filament formation by Psy3-Csm2
dimer. We identified a new protein complex involved inhomologous recombination. This complex containing Psy3, Csm2,
Shu1 and Shu2 promote the assembly of Rad51. X-ray structure
analysis revealed that a core complex of Psy3-Csm2 is
structurally similar to Rad51 dimer. Based on the structure of thecomplex, we propose a model in which Psy3-Csm2 (blue and red)
dimer bound to single-stranded DNAs promotes the assembly of
Rad51 filament (green) for homology search

Matsuzaki K., Terasawa, M., Iwasaki, D., Higashide, M. andShinohara, M. (2012) Genes-to-Cells, 17, 473-493.
7. Crossover assurance and crossover interference are distinctly
regulated by the ZMM proteins during yeast meiosis. Shinohara, M.,
Oh, S., Hunter, N., and Shinohara, A. (2008) Nature Genet. 40,
299-309.
8. A protein complex containing Mei5 and Sae3 promotes the assemblyof the meiosis-specific RecA homolog Dmc1. Hayase, A., Takagi, M.,
Miyazaki, T., Oshiumi, H., Shinohara, M. and Shinohara, A. (2004)
Cell 119, 927-940.
Laboratory of Genome and Chromosome Functions

31

Division of Integrated Protein Functions

Laboratory of Regulation of Neuronal Development
Professor
Assistant Professor
Assistant Professor

Kazuaki YOSHIKAWA
Koichi HASEGAWA
Kazushiro FUJIWARA

Correspondence Tel: +81-6-6879-8621; Fax: +81-6-6879-8623; E-mail: yoshikaw@protein.osaka-u.ac.jp
Laboratory of Regulation of Neuronal Development isengaged in studying the protein-protein interaction (PPI)
networks that are involved in neuronal differentiation andsurvival. In 1991, we discovered the novel protein necdin(neurally differentiated embryonal carcinoma-derived
protein) that was induced in neurally differentiatedpluripotent stem cells. The necdin gene (NDN) is
expressed only from the paternal allele through genomic
imprinting, a placental mammal-specific epigenetic controlof gene expression. Necdin is a member of the MAGE
(melanoma antigen) family proteins that share a highly
conserved domain known as the MAGE homology domain(MHD)(Fig. 1). Necdin binds to many regulatory proteins
involved in the proliferation, differentiation, survival anddeath of mammalian neurons and neural stem/precursor
cells. Thus, necdin serves as a hub of PPI networks forneuronal development (Fig. 2). We are also studying thepossibilities that necdin deficiency causes abnormal brain
development and neurodenerative disorders in humans.

Fig. 2. Necdin as a hub of the protein-protein interaction(PPI) network for neuronal development. Necdin interacts withmany proteins and forms PPI networks that are operative inmammalian neurons and neural stem/precursor cells. Necdin
binds to nuclear proteins (p53, E2F, Sirt1, FoxO, HIF, Bmi1,etc.) and transmembrane receptor proteins (Trk, p75, etc.).
These proteins also serve as hubs of the PPI networks involvedin cell survival (death), cell proliferation, and energy
metabolism. Thus, necdin is likely to integrate these PPI
networks in neurons and neural stem/precursor cells. Presented
are functionally relevant PPIs reported by us (light blue
elements) and others (yellowish green elements). Necdin
interacts physically with dozens of proteins other than these
proteins.

࠙Current Research Programsࠚ
1) Molecular mechanisms of neuronal differentiation
2) Molecular mechanisms of neuronal survival࠙Referencesࠚ
1. Antagonistic interplay between necdin and Bmi1 controls
proliferation of neural precursor cells in the embryonicmouse neocortex. Minamide R, et al. (2014) PLoS One 9,e84460.
2. Necdin controls proliferation and apoptosis of embryonicneural stem cells in an oxygen tension-dependent manner.
Huang Z, et al. (2013) J Neurosci 33, 10362-10373.
3. Non-epithelial stem cells and cortical interneuron productionin the human ganglionic eminences. Hansen DV, et al. (2013)
Nature Neuroscience 16, 1576-1587.
4. ERK inhibition rescues defects in fate specification of
Nf1-deficient neural progenitors and brain abnormalities.
Wang Y, et al. (2012) Cell 150, 816-830.
5. Necdin controls Foxo1 acetylation in hypothalamic arcuate
neurons to modulate the thyroid axis. Hasegawa K, et al.
(2012) J Neurosci 32, 5562-5572.
6. Thyroid hormone signaling acts as a neurogenic switch by
repressing Sox2 in the adult neural stem cell niche.
Lopez-Juarez A, et al. (2012) Cell Stem Cell 10, 531-543.
7. Necdin controls proliferation of white adipocyte progenitor
cells. Fujiwara K, et al. (2012) PLoS One 7, e30948.

Fig. 1. A model structure of necdin MAGE homology domain.
A structural model of necdin MAGE homology domain (MHD)
was predicted on the basis of structural data of necdin-like 2(PDB 3NW0) using the Spanner program developed in our
institute. Necdin MHD has a goblet-like appearance and consistsof two winged helix motifs (WH-A, WH-B), which are often foundin nuclear proteins that interact with DNA and proteins to form
supramolecular complexes. Studies using necdin MHD mutants
suggest that the hydrophobic pocket region (right, HP) including
helices 4 and 5 (left, H4-H5) interacts with various regulatory
proteins.

Laboratory of Regulation of Neuronal Development

32

Division of Integrated Protein Functions

ᴾ

Laboratory for Molecular and Developmental Biology
Professor
Associate Professor
Assistant Professor
Technical Assistant

Takahisa FURUKAWA
Yoshihiro OMORI
Rikako SANUKI
Toshinori TSUJIIᴾ
Correspondence Tel: +81-6-6879-8631; Fax: +81-6-6879-8633;E-mail:takahisa.furukawa@protein.osaka-u.ac.jp
ᴾ

Our laboratory studies molecular mechanisms
underlying the development and function of thevertebrate central nervous system (CNS) using
various research methods of molecular biology,
mouse genetics, biochemistry, cell biology and neuralphysiology. Our brain consists of more than 100
billions of neurons. To function as a brain, numerous
numbers of neurons must be generated at right places
and they must be interconnected each other. We use
the retina as a model system to understand how DNA
encodes programs to generate various neurons andglial cells, form precise neuronal circuits, and enable
complicated neuronal function. We also focus on how
abnormality of biological processes in development
and maturation leads to human diseases. We are eager
to contribute to development of diagnosis and cure ofhuman diseases. Together, our lab aims to elucidate
mechanisms and principles underlying the CNS
development from DNA programs to physiological
function and human diseases.
ẔCurrent Research Programsẕ
1) Molecular analysis of synapse formation in the
CNS.
2) Elucidation of functional roles of microRNAs(miRNAs) in CNS development.
3) Analysis of molecular mechanisms underlying
neuronal differentiation.
4) Functional analysis of cilium, an antenna of a cell,in the CNS.







Fig. 1. Electron tomography of rod photoreceptor synapse
terminals using ultrahigh-voltage electron microscopy. In
the WT mouse retina, photoreceptor axonal terminus forms
invagination to appose dendritic terminals of horizontal cells
and bipolar cells to form the ribbon synapse. On the other
hand, Pikachurin KO mice showed improper apposition of
the bipolar cell dendritic tips to the photoreceptor ribbon
synapses.

Fig. 2. Aberrant sprouting of mossy fibers in themiR-124a KO mouse. The mossy giver terminals werevisualized by Timm staining with Nissl counterstaining at
postnatal day 10. Scale bars represent 500 um.
ᇻReferencesᇼ
1. Protein-4.1G-Mediated Membrane Trafficking Is Essential
for Correct Rod Synaptic Location in the Retina and forNormal Visual Function. Sanuki R et al. (2015) Cell Rep 10,
796–808.
2. ICK is essential for cell type-specific ciliogenesis and theregulation of ciliary transport. Chaya T, Omori Y, Kuwahara
R, Furukawa T (2014) EMBO J 33, 1227-1242.
3. Presynaptic Dystroglycan-Pikachurin Complex Regulates theProper Synaptic Connection between Retinal Photoreceptor
and Bipolar Cells. Omori et al. (2012) J of Neuroscience 2,6126-6137.
4. miR-124a is required for hippocampal axogenesis and retinal
cone survival through Lhx2 suppression. Sanuki et al. (2011)
Nature Neuroscience 14, 1125-1134.
5. Negative regulation of ciliary length by ciliary male germcell-associated kinase (Mak) is required for retinal
photoreceptor survival. Omori et al. (2010) PNAS 107,
22671-22676.
6. Blimp1 suppresses Chx10 expression in differentiating retinal
photoreceptor precursors to ensure proper photoreceptor
development. Katoh et al. (2010) J of Neuroscience 12,6515-6526.
7. Pikachurin, a dystroglycan ligand, is essential forphotoreceptor ribbon synapse formation. Sato et al. (2008)
Nature Neuroscience 11, 923-931.

Laboratory for Molecular and Developmental Biology

33

Division of Integrated Protein Functions

Laboratory of Nuclear Network
Associate Professor Junko KANOH

Correspondence Tel: +81-6-6879-4328; Fax: +81-6-6879-4329
E-mail: jkanoh@protein.osaka-u.ac.jp
Organisms promptly and appropriately respond to various
environmental changes for their survival. In order to                           
respond to various forms of stress, cells have developed
signal transduction cascades for each stress. We will
clarify the molecular bases for networks of proteins orcellular signals, focusing on the key proteins that arecommonly involved in several distinct signal transductions.
Our current interests are crosstalk of signal transductions
for chromosome maintenance and those for nutrient
recognition. Our study will contribute to understanding themechanisms of chromosome disease, cancer or diabetes.
࠙Current Research Programsࠚ
1) Analysis of molecular functions of telomere-binding
proteins.
2) Analysis of functional networks among thechromosomal non-coding regions (including telomeres).
3) Analysis of molecular bases for Tel2-PIKK network.

Fig. 3. Tel2-PIKK network. Tel2 protein interacts with all thePIKK (phosphoinositide 3-kinase-related kinase) family proteins
and with Tti1 and Tti2 proteins. ATR/ATM proteins are involved
in DNA damage/replication checkpoint. TOR proteins regulate
uptake of nutrients and cell growth. TRRAP proteins are thecommon components of histone acetyltransferase, and regulate
variety of cellular processes. How Tel2 regulates PIKK proteinsis a big issue.

࠙ReferenceࠚFig. 1. Movement of telomeres in mitosis. A telomere-binding
protein Taz1 (red), the nuclear envelope (green), and microtubule(green) were observed using the live cell-imaging microscope.
Telomeres are transiently released from the nuclear envelope
during mitosis.

Recruitment of non-telomeric proteinsto telomeres

Genome-wide Networks
via Telomeres
Chromosome

Telomere

Recruitment of telomeric proteins
to non-telomeric regionsFig. 2. Telomere-binding proteins are involved in various cellular
phenomena. We focus on analyses of the functional networks
among telomeres, nuclear architectures, and other chromosomal
non-coding regions.

Laboratory of Nuclear Network34

1. Release of chromosomes from the nuclear envelope: auniversal mechanism for eukaryotic mitosis? Kanoh J (2013)
Nucleus 4, 100-104.
2. Identification of the functional domains of the telomere protein
Rap1 in Schizosaccharomyces pombe. Fujita I et al. (2012)
PLoS ONE 7, e49151.
3. Telomere-nuclear envelope dissociation promoted by Rap1
phosphorylation ensures faithful chromosome segregation.
Fujita I et al. (2012) Curr. Biol. 22, 1932-1937.
4. A conserved motif within RAP1 plays diversified roles intelomere protection and regulation in different organisms.
Chen Y et al. (2011) Nat. Struc. Mol. Biol. 18, 213-221.
5. Fission yeast Ku protein is required for recovery from DNA
replication stress. Miyoshi T et al. (2009) Genes Cells 14,1091-1103.
6. Fission yeast Pot1-Tpp1 protects telomeres and regulates
telomere length. Miyoshi T et al. (2008) Science 320,
1341-1344.
7. Rapamycin-sensitivity of the S. pombe tor2 mutant andorganization of two highly phosphorylated TOR complexes byspecific and common subunits. Hayashi T et al. (2007) Genes
Cells 12, 1357-1370.
8. Tel2 is required for the activation of Mrc1-mediated DNA
replication checkpoint. Shikata M et al. (2007) J. Biol. Chem.
282, 5346-5355.
9. Telomere-binding
proteinTaz1
establishes
Swi6
heterochromatin independently of RNAi at telomeres. Kanoh J
et al. (2005) Curr. Biol. 15, 1808-1819.

Division of Integrated Protein Functions

Laboratory of Homeostatic Integration
AssociateProfessor
Nobuaki OKUMURA

Correspondence Tel: +81-6-6879-4312; Fax: +81-6-6879-4332
 E-mail: nokumura@protein.osaka-u.ac.jp

Laboratory of Homeostatic Integration is engaged instudying animal metabolism and physiology based onprotein sciences. We especially focus on the enzymes thatare involved in dipeptide metabolism to elucidate thefunctional roles of dipeptides and their components.
Recently we are studying non-covalent complexes of a
dipeptidase, CN2, using ESI-TOF MS to analyze its
reaction mechanism, metal affinity and substrate
recognition. We are also trying to develop new proteomic
techniques for researches in physiology, pathology anddiagnosis.㹱

࠙Current Research Programsࠚ

1) Studies on molecular mechanisms of biosynthesis
and degradation of histidine dipeptides.
2) Analysis of non-covalent complexes of proteinsusing mass spectrometry.
3) Development of proteomic techniques and its
application to physiology and pathology.
4) Development of procedures for evaluation of protein
preparations for structural analysis and its
application.

Fig. 1. Synthesis and degradation of carnosine Carnosine is anaturally occurring dipeptide present at high concentrations inthe skeletal muscles and the brain in mammals. This issynthesized from beta-alanine and histidine by carnosine
synthetase, while degraded into these amino acids by carnosine
dipeptidases. We found a Mn2+-dependent cytosolic dipeptidase,
CN2, can hydrolyze carnosime and analyzed its primary andtertially structures (Ref. 6).

Fig. 2 Localization of carnosinase CN2 in the hypothalamus.
The carnosine-hydrolyzing dipeptidase, CN2, is expressed in avariety of tissues in mice, but its expression levels are highlyvariable depending on cell types. In the brain, CN2 (B, E) ishighly expressed in histaminergic neurons in the
tuberomammillary nucleus of the hypothalamus (TMN), where it
is colocalized with the histamine-synthesizing enzyme, histidine
decarboxylase (A, D)(Ref. 7).

࠙Referencesࠚ
1. Carnosine and the control of blood glucose. Okumura N. et al.
(2015) in Food and Nutritional Components in Focus, RSC
publishing.
2. Carnosine dipeptidase II. Okumura N (2013) in Handbook ofproteolytic enzymes, 3rd ed., 1596-1600, Elsevier.
3. Diversity in protein profiles of individual calcium oxalatekidney stones. Okumura N, et al. (2013) PLos One 8, e68624.
4. Identification of cargo proteins specific for importin-beta with
importin-alpha applying a stable isotope labeling by aminoacids in cell culture (SILAC)-based in vitro transport system.
Kimura M, et al. (2013) J. Biol. Chem. 288, 24540-24549.
5. Role of L-carnosine in the control of blood glucose, bloodpressure, thermogenesis, and lipolysis by autonomic nerves in
rats: involvement of the circadian clock and histamine. Nagai
K, et al. (2012) Amino Acids. 43, 97-109.
6. Structural basis for substrate recognition and hydrolysis bymouse carnosinase CN2. Unno H, et al. (2008) J. Biol. Chem.
283, 27289-27299.
7. Colocalization of a carnosine-splitting enzyme, tissuecarnosinase (CN2)/cytosolic non-specific dipeptidase 2(CNDP2),withhistidine
decarboxylaseinthetuberomammllary nucleus of the hypothalamus. Otani H, et al.
(2008) Neurosci Lett. 445, 166-169.

Laboratory of Homeostatic Integration

35

Division ofInternational Collaborative Research

Division of International Collaborative Research  Laboratory of Foreign PI 
Visiting Professor
Visiting Professor

Matthias RÖGNER
Thomas HAPPE(Lehrstuhl für Biochemie der Pflanzen, Fakultät für Biologie &Prof. RÖGNER
Biotechnologie, Ruhr-Universitäet Bochum)
CorrespondenceE-mail: matthias.roegner@rub.de & thomas.happe@rub.de

Prof. Happe4) Structure-function relationships of natural andsemiartifical Fe-Fe hydrogenases, ferredoxins andmaturases (Ref. 5+6).

Research in the laboratory of Matthias Rögner focuses on
the structure, function, regulation and biogenesis of energy
transducing membrane proteins from cyanobacteria. Topics
involve dynamics and adaptations of bioenergetic
processes in the thylakoid membrane in response toenvironmental signals both on the level of individual
proteins, their (transient) interaction partners and on thelevel of membrane composition. First, inter-molecularinteractions of the electron transfer complex between
PhotosystemI and Fd from Thermosynechococcus
elongatus were analyzed by the transferred cross-saturation
method of NMR and X-ray crystallography. Most recently,asubtype
of
the
NDH-1
complexfrom
Thermosynechococcus elongatus, NDH-S, which is
involved in a unique inorganic C-concentration mechanism,
was crystallized in collab. with the Kurisu group (IPR).
Additionally, the structure of recombinant CupS was
solved by NMR in collaboration with T. Ikegami (IPR),
including a model for its interaction with thetransmembrane subunits of the complex (1, 2). Also, thesolution structure of the Cyt. b6f-complex subunit PetP,
which is specific for cyanobacteria and red alga andregulates linear vs. cyclic photosynthetic electron transport,
was determined for the first time by NMR.
The Happe group analyzes the anaerobic metabolism of thegreen alga Chlamydomonas reinhardtii in all its aspects.
They also study structure-function relationships of Fe-Fehydrogenases including a detailed characterization of theactive center (H-cluster) and the catalytic turnover process.
A novel in vitro maturation assay was established leading
to semi-artificial hydrogenase with high catalytic activity(4-6). The structure of these novel biocatalysts wasrecently solved together with the Kurisu group (Fig. 1).
Both groups cooperate in the creation of a cyanobacterial
design cell which combines the mechanism ofphotosynthetic water-splitting with hydrogen production
via imported hydrogenase at the expense of CO2-fixation(Fig. 2). Prerequsite is the re-routing of photosynthetic
electrons by modifying interactions between Ferredoxin(Fd) and FNR on the one side and Fd with hydrogenase(H2ase) on the other. For this purpose, various
FNR-mutants have been designed in coop. with T. Hase
(IPR) and characterized by in vitro activity assays andinteraction studies (ITC, with the Ikegami-group, and SPR).
The most promising mutants have recently beentransformed into Synechocystis PCC 6803 and are now
under evaluation.
࠙Current Research Projectsࠚ
1) Structural dynamics of cyanobacterial thermophilic
NDH-1 complexes (Ref. 1+2) & Cyt. b6f-complex.
2) Strategies for designing H2-producing cyanobacterial
model cells (Ref. 3).
3) Photobiological H2 production in green algae, cell
metabolism and signaling under anaerobiosis (Ref. 4).

Fig.1. Crystal structures of the catalytic center of active and inactive
semiartifical [FeFe]-hydrogenase maturated in vitro with different
synthetic 2Fe complexes of the kind Fe2[μ-(SCH2)2X](CN)2(CO)42-. The
central group of the dithiolate bridge is indicated next to the respective
structure.

Fig. 2. Cyanobacterial design cell for hydrogen production from water.
Key elements are the water-splitting complex PS2 as source of electrons
and the distribution of electrons at the acceptor side of PS1 between
CO2-fixation and H2-production, guided by affinity design of Fd vs. FNR
and hydrogenase, respectively. (3)࠙Referencesࠚ
1. Korste A, Wulfhorst H, Ikegami T, Nowaczyk MM, Stoll R(2015) Solution structure of the NDH-1 complex subunit CupS
from Thermosynechococcus elongatus BBA Bioenergetics,
accepted doi:10.1016/j.bbabio.2015.05.003
2. Korste A,
Wulfhorst H, Ikegami T, Nowaczyk MM, Stoll R(2015) 1H, 13C and 15N chemical shift assignments of the
NDH-1 complex subunit CupS. Biomol NMR Assign
9:169-171
3. Rögner, M. (2013) Metabolic engineering of cyanobacteria forthe production of hydrogen from water; Biochemical Society
Transactions 41, 1254-1259
4. Hemschemeier A., Düner M., Casero D., Merchant S.S.,
Winkler M. and Happe T. (2013) Hypoxic survival requires a2-on-2 hemoglobin in a process involving nitric oxide. Proc.
Natl. Acad. Sci. USA. 110: 10854-10859
5. Esselborn J., Lambertz C., Adamska A., Simmons T., BerggrenG., Noth J., Siebel J., Hemschemeier A., Artero V., Reijerse E.,
Fontecave M., Lubitz W. & Happe T. (2013) Spontaneous
activation of [FeFe]-hydrogenases by an inorganic [2Fe] active
site mimic. Nature Chem Bio 9, 607-609.
6. Rumpel S., Siebel J.F., Fares C., Duan J., Reijerse E., Happe T.,
Lubitz W. and Winkler M. (2014) Enhancing hydrogenproduction of microalgae by redirecting electrons fromphotosystem I to hydrogenase. Energy & Environmental
Science 7: 3296–3301

Laboratory of Foreign PI

37

Division of International Collaborative ResearchLaboratory of Foreign PI    Specially Appointed Associate Professor   Damien HALL

(Research School of Chemistry, The Australian National University.
Building 137, Acton, 2601. Canberra, ACT. AUSTRALIA).
CorrespondenceE-mail:damien.hall@anu.edu.auResearch in the Hall laboratory is concerned with thephysical study of important biochemical processes related
to disease states. One example of this coupledscience/medicine research program is the study of amyloid
formation and its relationship to the spectrum ofamyloidosis diseases [1,2]. Amyloid fibers are linear
protein aggregates capable of being formed from various
peptides and proteins [2,3]. In their most basic form,
amyloid fibers have dimensions characterized by a widthof 4-20 nm and a length in the nanometer to sub-millimeter
range [2,3]. Peptide/protein monomeric units are joined byinter-peptide beta-sheets formed along the long axis of thefiber. The designation of amyloid is conferred upon aprotein aggregate if, in addition to displaying fiber likestructure, it possesses specific dye binding capabilities anda cross-beta X-ray fiber diffraction pattern [1,2]. Although
amyloid formation is coming to be thought of as apotentially common property of most, if not all proteins, inmedicine there are only twenty-seven distinct peptides thatare known to be associated with an amyloidosis diseasestate [1,4]. These twenty-seven basic forms of amyloidosis(and their mutational variants) display a huge diversity inbehaviors with respect to the pathophysiology of diseaseprogression, with some showing tissue/organ specificity,while others tend towards non-specific dispersal ofamyloid throughout the body. In general much of what weknow (or think we know) about amyloid and its
relationship to disease is based on qualitative experimental
observation [1,5]. This is partly due to the generalcomplexity of the systems studied, the difficulties of
performing quantitative measurement in patients, and thelimitations associated with the methodological approaches
employed to study these processes. Work in the Hall
laboratory is focused on sharpening up some of thesefoundations by approaching the topic from a biophysicalperspective i.e. experiments interpreted through the lens ofa constraining mathematical model [1-8]. Using a
combination of experimental/computational approaches wewould like to, in combination with Japanese colleagues at
the IPR, explore the following topics,

6) Elucidation of peptide structural precursor states ofamyloid using NMR measurements.
7) Construction of an arc-length distance matrix data basefor all proteins in the PDBj [1,8].
8) Systematic application of a classification scheme fordefining the surface chemical properties of amyloid [1].
࠙Referencesࠚ
1. D Hall, H Edskes (2012) Computational modeling of therelationship between amyloid and disease. Biophysical reviews
4: 205-222.
2. D Hall. (2012) Semi-automated methods for simulation andmeasurement of amyloid fiber distributions obtained fromtransmission electron microscopy experiments. Analyticalbiochemistry 421: 262-277
3. D Hall, L Huang (2012) On the use of size exclusion
chromatography for the resolution of mixed amyloid
aggregate distributions: I. equilibrium partition models.
Analytical biochemistry 426: 69-85.
(C)

Fig. 1. (A,B) Theoretical and experimental modeling of amyloid
migration through a size-exclusion chromatography column.[3] (C)Semi-automated analysis of electron microscopy measurements of insulin
derived amyloid fibers using the ADM software [2].

Fig. 2. Description of a new protein distance matrix based on surface
arc-length that is relevant to the interaction of non-structured polymers(such as intrinsically disordered proteins) with globular proteins [8]࠙Current Research Programsࠚ

4. K Sasahara, D Hall, D Hamada (2010) Effect of lipid type on
the binding of lipid vesicles to islet amyloid polypeptide
amyloid fibrils. Biochemistry 49: 3040-3048.
5. D Hall, H Edskes. (2009) A model of amyloid's role in disease
based on fibril fracture. Biophysical chemistry 145: 17-28
6. D Hall, N Hirota. (2009) Multi-scale modelling of amyloid
formation from unfolded proteins using a set of theory derived
rate constants. Biophysical chemistry 140: 122-1285.
7. D Hall, J Kardos, H Edskes, JA Carver, Y Goto (2015) A
multi-pathway
perspectiveonproteinaggregation:
Implications for control of the rate and extent of amyloid
formation FEBS letters 589: 672-679.
8. D Hall, S Li, K Yamashita, R Azuma, JA Carver, DM Standley(2014) A novel protein distance matrix based on the minimumarc-length between two amino-acid residues on the surface of a
globular protein. Biophysical chemistry 190, 50-55.

1) Efficient means for fractionating and purifying
heterogeneous amyloid fiber samples [2,3]2) Non-subjective methods for characterizing grossamyloid structural properties using AFM and TEM [3]3) Examine amyloid fiber breakage position effects asdeterminants of amyloid fiber behavior [5,6]4) Placement effects of peptides within larger polypeptide
chains as determinants of amyloid growth [5,6].
5) Spatial modeling of amyloidosis disease progression
using computation and in vivo imaging data [1,6,7].
Laboratory of Foreign PI

38

Division of International Collaborative ResearchLaboratory of Visiting Scientist
Guest Professor

Toshio YamazakiCorrespondenceTel: +81-45-503-9262; Fax: +81-45-503-9641;
           E-mail: toshio.yamazaki@riken.jp        We are investigating biomolecules using liquid-state andsolid-state nuclear magnetic resonance (NMR) to
understand their structure and function. We are also
developing experimental methods for those purposes.

Soluble parts of membrane proteins are often important forfunction and binding of drugs. In the Aquaporin Z case, we
can observe chemical shift perturbation by binding of drug.
The determination of the binding site of drug is one of the
purpose after the assignment.

࠙Current Research Programsࠚ
1) Solid-state NMR of biomolecules.
2) Solution-state NMR for detecting motion of molecules.
3) Solution-state NMR for structure and interaction of
biomolecules.
4) Ultra-high field NMR.
Solid-state NMR is a suitable technique to investigate
biomolecules in solid-like environment, such as membraneproteins. Comparing with the crystallographic study, theirnative environment can be kept in the NMR study.
Heterogeneous system in terms of its environment or
structure can be the target of solid-state NMR. Comparing
with solution NMR, the difficulty coming from molecularsize is less severe. Biomolecules in the large complex willbe the anticipated target. Recently, by the development of
instruments and methods, we can obtain fairly good
spectra of complicated membrane proteins.
Aquaporin Z from E. coli is a water channel protein,consisting of 231 amino acids. This size is challenging forsolid-state NMR. The molecule is fully labelled with 13C
and 15N, and we can get assignments by connecting
neighboring 13C and 15N atoms sequentially. The lack of
1H signals (comparing with solution NMR) brings about
the problem poor separation. To get rid of overlaps of
NMR signals, we record 3-dimensional spectra to separate
signal in the 2 dimensions and connect to the next atom inthe last dimension. We combine the following connections:
N-CA-CO connection by NCACX, CA-CO-N connectionby NCOCX, CO-N-CA connection by CANCO. Then, we
can connect to the backbone signals 1 residue ahead.

Fig. 2. Detecting binding of drug molecule to Aquaporin Z in
3D spectrum. Measured on 700MHz NMR with 3.2mm HCN
probe.

Though the crystal structure of Aquaporin Z is known, A few
of the chemical shifts observed in the current study showed
unexpected values. This might be an indication of protein
structure of native environment. We are closely analyzingthem.
The current standard techniques presented here required
high cost of sample and measurement time, because thesensitivity and resolution are not sufficient. New techniquesof ultra-high field NMR(ref. 1) and 1H detection NMR will
renew the efficiency of solid-state NMR. DNP is also most
expected technique. In future, we will be able to apply
solid-state NMR to variety of biomolecules with much low
cost and time.
࠙Referencesࠚ
1. Achievement of 1020MHz NMR. Hashi K et al. (2015) J.
Magn. Res. in press.







Fig. 1.

Three 3-dimensional experiments for assignment

Actual analysis was hard. There are still many overlaps in 2
dimensional space. We also recorded spectra to connect 4
atoms, by which we skip the overlapped signal and connect
to the next atom. Transmembrane helices are uniform instructure and amino acid composition, leading to failure of
assignments. The assignments of other parts are promising.
Laboratory of Visiting Scientist

39

Division of International Collaborative ResearchTsukihara Laboratory
Visiting Professor

Tomitake TSUKIHARA

CorrespondenceTel: +81-6-6879-4331; Fax: +81-6-6879-4331
E-mail: tsuki@protein.osaka-u.ac.jp

Three-dimensional protein structure brings us the beautiful
structural biology. X-ray crystallography that is the best
method to determine atomic coordinates of protein
molecules is a familiar tool of protein science. However,
X-ray
structure
determinationsofbiologicalmacromolecular assemblies and membrane proteins thathave key roles in biological cells still contain difficulties tobe conquered. The main aim of us is the X-ray structure
determinations of respiratory complexes residing in the
mitochondrial innermembrane in order to elucidate themolecular mechanism of the highly organized biologicalprocesses at atomic level.

that a peroxide anion with an O-O distance of 1.55 Å exhibits
multiple conformations in the binuclear center, in which the main
component, with 95% occupancy, has O-CuB and O-Fea3
distances of 2.14 Å and 2.35 Å, respectively, and the minor
component has analogous distances of 1.95 Å and 3.76 Å.
Consequently, the compound in the dioxygen reduction center ofthe fully oxidized state is a peroxide anion.
࠙Referencesࠚ
1. Structures of metal sites of oxidized bovine heart cytochromec oxidase at 2.8 Å.Tsukihara et al. (1995) Science 269,
1069-1074.
2. The whole structure of the 13-subunit oxidized cytochrome coxidase at 2.8 Å. Tsukihara et al. (1996) Science 272,1136-1144.
3. Redox-coupled crystal structural changes in bovine heart
cytochrome c oxidase. Yoshikawa et al. (1998) Science 280,
1723-1729.
4. The low-spin heme of cytochrome c oxidase as the driving
element of the proton-pumping process.Tsukihara et al.
(2003) PNAS. 100, 15304-15309
5. The proton pumping pathway of bovine heart cytochrome coxidase. Shimokata et al. (2007) PNAS. 104, 4200-4205.
6. A peroxide bridge between Fe and Cu ions in the O2
reduction site of fully oxidized cytochrome c oxidase
could suppress the proton pump. Aoyama et al. (2009)
PNAS. 106, 2165-2169.
7. The structure of rat liver vault at 3.5 angstrom resolution.
Tanaka et al. (2009) Science 323, 384-388.
8. Structure of the connexin 26 gap junction channel at 3.5 Å
resolution. Maeda et al. (2009) Nature 458, 597-602.
9. A high-resolution structure of the pre-microRNA nuclear
export machinery. Okada et al. (2009) Science 326,
1275-1279.
10. Bovine cytochrome c oxidase structures enable O2 reduction
with minimization of reactive oxygens and provide a
proton-pumping gate. Muramoto et al. (2010) PNAS. 107,
7740-7745.
11. Distinguishing between Cl- and O2(2-) as the bridging
element between Fe3+ and Cu2+ in resting-oxidized
cytochrome c oxidase. Suga et al. (2011) Acta Cryst., D67,
742-744.
12. Structural studies of large nucleoprotein particles, vaults.
Tanaka and Tsukihara, (2012) Proc Jpn Acad Ser B Phys Biol
Sci., 88, 416-33.
13. New features of vault architecture and dynamics revealed bynovel refinement using the deformable elastic network
approach. Casañas et al. (2013) Acta Cryst., D69, 1054-61.
14. Determination of damage-free crystal structure of an
X-ray-sensitive protein using an XFEL. Hirata et al. (2014)
Nat Methods. 11, 734-736.
15. Higd1a is a positive regulator of cytochrome c oxidase.
Hayashi et al. (2015) Proc. Natl. Acad. Sci. U. S. A. 112,1553-1558.

࠙Current Research Programsࠚ
1) Structural life science of mitochondrial inner
membrane2) Development of methods of high resolution andtime-resolved structural analyses by XFEL for
biological macromolecular assemblies

Fig. 1. A respiratory supper complex consisting of Complexes I,
III and IV pumps proton to create efficiently an electrochemical
proton gradient across the mitochondrial inner membrane andmay reduce harmful superoxide production. Complex V
generated ATP by using the electrochemical proton gradient. We
are currently working on crystallization of the respiratory suppercomplexes from various organisms.
1.70A

CuB
2.14A

1.55A
2.35A

3.67A

Fea3

Fig. 2. The X-ray structural analysis of bovine cytochrome coxidase using radiation damage free data at the SACLA indicates
Tsukihara Laboratory40

Research Center forState-of-the-Art Functional Protein Analysis

Research Center for State-of-the-art Functional Protein Analysis

Research Center for State-of-the-art Functional Protein AnalysisThe research Center for State-of-the-art Functional Protein Analysis (RCSFP) was established in 2012, inheriting the
success of its predecessor The Research Center for Functional and Structural Proteomics. In recognition of the importanceof expanding the scope of research outside the field of proteomics, the Center now aims to answer various scientificquestions by incorporating a full range of cutting-edge technologies of protein analysis and world-class analytical
instruments. The Center consists of six divisions and one open space laboratory and covers following research areas:
protein profiling and functional proteomics, protein synthesis and expression, supramolecular crystallography, protein
informatics, advance protein characterization, and database development.

Laboratory of Supramolecular
Crystallography

Laboratory of Protein Informatics

Laboratory ofLaboratory of
Advanced Protein
Protein
Characterization
Databases

Open Space Lab
for Advanced
Protein Science

Laboratory of Protein Profilingand Functional Proteomics

Laboratory of Protein
Synthesis and Expression

Research Center for State-of-the-art Functional Protein Analysis

41

Research Center for State-of-the-art Functional Protein Analysis Laboratory of Protein Profiling and Functional Proteomics

Professor     Toshifumi TAKAO    Assistant Professor   Caroline Donzeli PereiraCorrespondence

Tel: +81-6-6879-4312; Fax: +81-6-6879-4332
E-mail:tak@protein.osaka-u.ac.jp

Mass spectrometry (MS) is well accepted technique for the
analyses of chemical structures of biological compounds.
For the last three decades, we have been working todevelop methods for determining primary structures andpost-translational modifications of proteins by using MS.
In conjunction with accumulating protein and genesequence databases, we are using state-of-the-art MS forlarge-scale protein identification which is indispensable forproteomics research. We apply the following developed

methods to the structural analysis of micro quantities of
peptides, proteins, and their related substances. We havefound several novel post-translational modifications suchas farnesyl moiety at the C-terminal Cys, heterogeneous
fatty acids at the N-terminal Gly, H-(J-glutamyl)lysine incore histones, phosphatidylethanolamine linked to the
C-terminal Gly, palmitoleoyl moiety at Ser, etc.

࠙Current Research Programsࠚ
1) Development of chemical/analytical methods and
software for analysis of protein primary structure by mass
spectrometry2) Mass spectrometric analysis of post-translational
modifications3) Development of chemical and analytical methods forproteomics
4) Study on fragmentation in mass spectrometry of peptides
and carbohydrates5) Hardware development for high-sensitivity andhigh-accuracy mass spectrometry

Asn42

Posttranslational Modifications of Wnt Protein-Wnt Lipid Modifications: Not as Saturated as We Thought(Takada R et al, 2006; Yamamoto H et al, 2013, 2015)347 days (t1/2)
O

O
OH O
OH

N
H

E2-Microglobulin

Asn17
7

18
18

N
H
O

R
H
N

18

N
H

R

L-As
L-Asp
As
sp

OH O
OH

O

L-is
L-isoAsp
soA
oAsp
sp

33 days (t1/2)
Quantitative Analysis of Deamidation and Isomerization in β2-Microglobulin by 18O Labeling. (Fukuda M & Takao T, 2012)࠙Referencesࠚ1. Basolateral secretion of Wnt5a in polarized epithelial cells is required for apical lumen formation. Yamamoto H, et al (2015)
J Cell Sci. 128, 1051.
2. The apical and basolateral secretion of Wnt11 and Wnt3a in polarized epithelial cells is regulated by different mechanisms.
Yamamoto H, et al (2013) J Cell Sci. 126, 2931.
3. Identification of cargo proteins specific for importin-β with importin-α applying a stable isotope labeling by amino acids incell culture-based in vitro transport system. Kimura M, et al (2013) J Biol Chem 288, 24540.
4. Identification of cargo proteins specific for the nucleocytoplasmic transport carrier transportin by combination of an in vitrotransport system and stable isotope labeling by amino acids in cell culture-based quantitative proteomics. Kimura M, et al(2013) Mol Cell Proteomics 12,145.
5. Quantitative Analysis of Deamidation and Isomerization in E2-Microglobulin by 18O Labeling. Fukuda M & Takao T (2012)
Anal. Chem 84, 10388.
6. Mono-unsaturated Fatty Acid Modification of Wnt Protein: Its Role in Wnt Secretion. Takada R, et al. (2006) Dev. Cell 11,791.

Laboratory of Protein Profiling and Functional Proteomics

42Research Center for State-of-the-art Functional Protein Analysis

Laboratory of Protein Synthesis and Expression
Professor
Junichi TAKAGIAssociateProfessor
Kenji IWASAKI
Assistant Professor
Yu KITAGO
Spec. App. Assist. Prof. Yukiko MATSUNAGASpec. App. Assist. Prof. Masataka UMITSU
Technical AssistantKeiko KAWAKAMICorrespondenceTel: +81-6-6879-8607; Fax: +81-6-6879-8609; E-mail: takagi@protein.osaka-u.ac.jp
࠙Current Research Programsࠚ
1) Structure and function of extracellular ligands and their
receptors implicated in cell adhesion and neural
guidance/morphogenesis.
2) Structure-guided molecular design of novel proteins.
3) "Correlative" structural analysis by multidisciplinary
approach.
4) Development of high-quality recombinant protein
production system.
5) 3-D visualization of conformationally heterogeneous
macromolecules using electron microscopy.

"How things work?" - This is the question most, if not all,scientists are eager to answer. Our passion is to unravel themechanism of function of proteins in a living organism
where they work as small "molecular machines" with aremarkable precision. This lab can be described as a"structural biology lab", but our goal is not thedetermination of three-dimensional structure of proteins.
Rather, we FIRST solve the structure, THEN performbiochemical, biophysical, and cell biological experiments
to draw novel pictures about molecular mechanism ofproteins, taking advantage of the structural informationthat is not available to anybody else. We are mostly
focused on the molecular interactions between cell surface
receptors and their extracellular ligands implicated in thesignal transduction in a wide variety of biological contexts,
ranging from development, neurobiology, and immunity.
Cellular response to the extracellular environment depends
on the “sensing” the extracellular cues by use of thereceptor-ligand system. Binding of ligands to theextracellular domain of the receptors transduces signals
into cells that initiates various cellular events, ultimatelychanging the cell fate. In spite of a wealth of cellbiological “signal transduction researches” conducted atevery corner of today's biomedical arena, mechanism forthe “signal transmission across the membrane”, the very
first step in the signaling pathway is poorly understood.
Our study focuses on questions such as how receptors
recognize their specific ligands, how this recognition leads
to structural change in the receptor complex, and how the
information cross the plasma membrane withouttransporting chemical entity.
Our approach is multi-faceted. As the methodology forstructural analysis, we utilize X-ray crystallography, which
determines 3D structure of proteins at atomic resolution,and electron microscopy, which can derive structure ofprotein complexes too large for XRD or visualize theshape of proteins in their true biological environment (e.g.,
within cells). The latter expertise includes cutting-edge
technologies such as cryoelectron microscopy and electron
tomography. In order to back these structural efforts, we
also develop an array of in-house technologies critical forthe production of high-quality recombinant proteins using
mammalian cell expression system.

Fig. 1. Crystal structure of complex between reelin (pink) and its
receptor (rainbow)

Fig. 2. Epitope mapping of antibody (green, cyan) against Plexin(gray) using negative stain EM imaging
࠙Referencesࠚ
1. Structural basis for amyloidogenic peptide recognition by
sorLA. Kitago Y, Nagae M, Nakata Z, Yagi-Utsumi M,
Takagi-Niidome S, Mihara E, Nogi T, Kato K, Takagi J. (2015)
Nature Struct. Mol. Biol. 22, 199-206.
2. Giant cadherins Fat and Dachsous self-bend to organize
properly spaced intercellular junctions. Tsukasaki Y, Miyazaki
N, Matsumoto A, Nagae S, Yonemura S, Tanoue T, Iwasaki K,
and Takeichi M. (2014) PNAS,111(45), 16011-16016.
3. New constructs and expression of proteins: Making things
better. Takagi, J. and Tate, CG. (2014) Curr Opin Struct Biol.
26:iv-vi.

Laboratory of Protein Synthesis and Expression

43Research Center for State-of-the-art Functional Protein Analysis

Laboratory of Supramolecular Crystallography
Professor
AssociateProfessor
Assistant Professor
Assistant Professor
Spec. App. Assist. Prof.
Spec. App. Assist. Prof.

Atsushi NAKAGAWA
Mamoru SUZUKI
Eiki YAMASHITA
Mayumi AMANO
Kohei TAKESHITA
Akifumi HIGASHIURACorrespondenceTel: +81-6-6879-4313; Fax: +81-6-6879-4313; E-mail: atsushi@protein.osaka-u.ac.jp
There exist various biological macromolecular assemblies
S1
S2
S2
S1
consisting of proteins, nucleic acids, sugars, lipids, andS3other substances in living cells. These macromolecularassemblies play key roles in all living system. Our
S4
90
S3
S4
laboratory works on structure determination of biologicalmacromolecular assemblies, as well as proteins, whichplay important roles in biological system, using X-ray
S0
S0
crystallography. Development of tools for X-ray crystal
structure determination of biological macromolecular
coiled-coilcoiled-coilassemblies, including synchrotron radiation beamtime at
SPring-8, is also one of our main works.
o࠙Current Research Programsࠚ
1) X-ray structure determination of macromolecularassemblies and proteins
2) Development methodologies for X-ray structure
determination of biological macromolecular assemblies
using synchrotron radiation and X-ray free electron
laser3) Development of data processing algorithm of diffraction
data from micro-crystalsFig. 2. Structure of Voltage-gated Proton Channel(VSOP/Hv1). We have succeeded to solve the atomic
structure of the voltage-gated ion channel (VSOP/Hv1)
by X-ray crystallography at 3.45 Å resolution. This is
the first structure of the resting-state of thevoltage-sensor protein family and it gave valuable
information on the mechanism of voltage-sensor
domain. In addition, the structure showed theinhibition mechanism by binding of zinc ion.

Fig. 1. Synchrotron radiation beamline for Biological
Macromolecualr Assemblies (SPring-8 BL44XU). This
beamline utilizes high-brilliant undulator radiation ofSPring-8 to collect high quality diffraction data from
biological macromolecular assembly crystals. Abouthalf of the user time is opened for the research groups
outside of the IPR.

࠙Referencesࠚ
1. X-ray crystal structure of voltage-gated proton channel.
Takeshita K, Sakata S, Yamashita E, Fujiwara Y, Kawanabe A,
Kurokawa T, Okochi Y, Matsuda M, Narita H, Okamura Y,
Nakagawa A (2014) Nat. Struct. Mol. Biol. 21, 352-357.
2. High-resolution X-ray crystal structure of bovine H-proteinusing the high-pressure cryocooling method. Higashiura A,
Ohta K, Masaki M, Sato M, Inaka K, Tanaka H, Nakagawa A(2013) J. Synchrotron Rad. 20, 989-993.
3. A new protein complex promoting the assembly of Rad51
filaments. Sasanuma H, Tawaramoto MS, Lao JP, Hosaka H,
Sanda E, Suzuki M, Yamashita E, Hunter N, Shinohara M,
Nakagawa A, Shinohara A (2013) Nat. Commun. 4, 1676.
4. Crystal structure of the C-terminal domain of Mu phage central
spike and functions of bound calcium ion. Harada K,
Yamashita E, Nakagawa A, Miyafusa T, Tsumoto K, Ueno T,
Toyama Y, Takeda S (2013) Biochim. Biophys. Acta: Protein
Proteomics 1834, 284-291.

Laboratory of Supramolecular Crystallography

44Research Center for State-of-the-art Functional Protein Analysis

Laboratory of Protein Informatics
Professor
AssociateProfessor
Assistant Professor
Technical Assistant

Haruki NAKAMURA
Akira R. KINJO
Yuko TSUCHIYA
Takashi KOSADACorrespondenceTel: 06-6879-4310; Fax: 06-6879-4310; E-mail: harukin@protein.osaka-u.ac.jp

edge indicates a proximal residue pair with a highly positive
Laboratory of Protein Informatics is engaged in the
dynamic correlation observed during molecular dynamics
following protein informatics researches: (1) Structural
simulation. B. A 3D representation of the core network. Eachbioinformatics studies covering molecular modeling andsphere shows the atom having large Betweenness value, which isdesign, (2) Development of new databases and Weba measure of the importance of each residue for the connection ofservices, and (3) Large scale molecular simulations by
the entire network. (Ref. 5)parallel computers with GPGPUs to examine free energy
landscapes of biomolecular systems to study theprocedures of protein folding and of forming protein
complexes.
࠙Current Research Programsࠚ
1) Bioinformatics studies focused on protein structuresand protein-protein interactions (Refs. 1-3)
2) Development of new algorithms for molecularsimulations to examine the free energy landscapes onprotein folding and formation of protein complexes(Refs. 3-6)


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

Fig. 3. Free energy landscape and corresponding structures at
300K for homo-dimer formation of two Alzheimer's peptides
by V-AUS (Virtual-system coupled Adaptive Umbrella
Sampling) method. The horizontal axis is the distance between
the centers of the two peptides, and the longitudinal axis is theinner product of the unit direction of each peptide. Graph
colors correspond to the free energy, and the red circle at theupper left corresponds to the crystal structure. (Ref.6)

Fig. 1. Composite Structural Motifs of Binding Sites forDelineating Biological Functions of Proteins (C). They aredefined by integrating the elementary motifs (A) associated withindividual subunits having binding sites for ligands including
small molecules, proteins and nucleic acids. These 3 composite
motifs share the same elementary motif for FAD binding (labeled
N2 in B). (Ref. 1)

Fig. 2. A dynamic cross correlation
network in (Ets1)2-DNA complex.
A. The two circles correspond to the two Ets1 proteins and thepink nodes are the DNA. Each node indicates a residue and each࠙Referencesࠚ
1. Composite structural motifs of binding sites for delineating
biological functions of proteins. Kinjo AR, Nakamura H (2012)
PLoS One 7, e31437.
2. 3D flexible alignment using 2D maximum common
substructure: dependence of prediction accuracy ontarget-reference chemical similarity. Kawabata T, Nakamura H,(2014) J. Chem. Info. Model. 54, 1850-1863.
3. High-resolution modeling of antibody structures by a
combination of bioinformatics, expert knowledge, andmolecular simulations. Shirai H, Ikeda K, Yamashita K,
Tsuchiya Y, Sarmiento J, Liang S, Morokata T, Mizuguchi K,
Higo J, Standley DM, Nakamura H, (2014) Proteins 82
1624-1635.
4. The Zero-multipole summation method for estimating
electrostatic interactions in molecular dynamics: analysis of theaccuracy and application to liquid systems. Fukuda I, Kamiya
N, Nakamura H (2014) J. Chem. Phys. 140, 194307.
5. A Novel Approach of Dynamic Cross Correlation Analysis on
Molecular Dynamics Simulations and its Application to Ets1
dimer–DNA Complex. Kasahara K, Fukuda I, Nakamura H,(2014) PLoS One 9, e112419.
6. Virtual-system coupled adaptive umbrella sampling to computefree-energy landscape for flexible molecular docking. Higo J,
Dasgupta B, Mashimo T, Kasahara K, Fukunishi Y, Nakamura
H, (2015) J. Comput. Chem., in press.
Laboratory of Protein Informatics

45

Research Center for State-of-the-art Functional Protein Analysis

Laboratory of Advanced Protein Characterization
Professor
Atsushi NAKAGAWA
Professor
Junichi TAKAGIProfessor
Toshifumi TAKAO
Associate Professor
Kenji IWASAKI
Associate Professor (Lecturer) Takeshi SATO
Assistant Professor
Eiki YAMASHITA
Assistant Professor
Naotoshi MIMURA
Assistant Professor
Toshihiko SUGIKI
Assistant Professor
Risa MUTOH
Spec. App. Assist. Prof.
Akifumi HIGASHIURA
Technical staff
Naoko NORIOKA
Technical staff
Keiko KAWAKAMI
Technical staff
Naoyuki ABE

CorrespondenceE-mail: atsushi@protein.osaka-u.ac.jp
This laboratory is working on development and application of several important methodologies for advanced
characterization of protein molecules and plays an important role in technical innovations for protein analyses andmeasurements. Faculty members in this laboratory are organized into three research groups, a group using NMR with superhigh magnet field, a group of X-ray crystallography using synchrotron radiation at SPring-8 and an electron microscopic
imaging group, and one technical support group for chemical analyses of protein molecules.


Synchrotron Radiation Research Group
Atsushi NAKAGAWA, Eiki YAMASHITA and
Akifumi HIGASHIURA
Correspondence
Tel: +81-6-6879-8635; Fax: +81-6-6879-4313
E-mail: bladmin@protein.osaka-u.ac.jp
We are working on development of new methodologies for
X-ray protein crystallography using synchrotron radiation
beamline for biological macromolecular assemblies atSPring-8 (BL44XU). This beamline utilizes high-brilliant
undulator radiation of SPring-8 to collect high quality
diffraction data from biological macromolecular assembly
crystals. About half of the user time is opened for the
research groups outside of the IPR.
Synchrotron
Radiation
Beamlinefor
Biological
Macromolecular Assemblies at SPring-8 (BL44XU). This
beamline utilizes high-brilliant undulator radiation ofSPring-8 to collect high quality diffraction data from
biological macromolecular assembly crystals. About half of
the user time is opened for the research groups outside of
the IPR.

䛆Current Reearch Programs䛇
1) Management of the Synchrotron Radiation Beamline
for Biological Macromolecular Assemblies atSPring-8 (BL44XU)
2) Development of methodologies for X-ray crystal
structure determination of biological macromolecularassemblies using synchrotron radiation
3) Structural studies on membrane proteins andmacromolecular complexes

Ogura T, Sugimoto H, Shen J-R, Yoshikawa S, Ago H(2014) Nat. Methods 11, 734-736.
2. High-resolution X-ray crystal structure of bovine
H-protein using the high-pressure cryocooling method.
Higashiura A, Ohta K, Masaki M, Sato M, Inaka K,
Tanaka H, Nakagawa A (2013) J. Synchrotron Rad. 20,
989-993.

䛆References䛇
1. Determination of damage-free crystal structure of an
X-ray-sensitive protein using an XFEL. Hirata K,
Shinzawa-Itoh K, Yano N, Takemura S, Kato K,
Hatanaka M, Muramoto K, Kawahara T, Tsukihara T,
Yamashita E, Tono K, Ueno G, Hikima T, Murakami H,
Inubushi Y, Yabashi M, Ishikawa T, Yamamoto M,
Laboratory of Advanced Protein Characterization46

Research Center for State-of-the-art Functional Protein Analysis
NMR Research Group
Toshihiko SUGIKI, Risa MUTOH and
MIMURA
Correcpondence
Tel: +81-6-6879-4417; Fax: +81-6-6879-8599
E-mail: sugiki@protein.osaka-u.ac.jp

Naotoshi

We mainly determine the three-dimensional structures ofproteins and peptides using nuclear magnetic resonancespectroscopy (NMR). In addition, we are studying thefunctions based on the interaction of a protein with anotherprotein or its substrate. NMR is also suitable for analysesof flexibility, providing information as to which parts ofproteins fluctuate. Furthermore, the developments of NMR
methodologies to facilitate the above studies are also
important tasks.

Fig. 1. The 950 and 800MHz NMR machines containing a
superconducting magnet and cryogenic probe for each. The
sensitivity of the 950MHz NMR reaches about 17-times the valuethat is exhibited by a normal 400MHz machine. Therefore, themeasurement time can be saved by 1/300. The machines are also
used for dilute samples within a shorter period, and forisotopically non-labeled samples. The superconducting ismaintained by cooling the coil for the static magnetic field down
to 2K with liquid helium.

࠙Current Research Programsࠚ
1) Development of new NMR methodology by applying
amino acid-selective isotope labeling technique.
2) NMR analysis of the protein interaction between a small
protein and macromolecular complexes withconcomitant administration of X-ray crystallography.
3) Development of fundamental technology for successful,versatile, and reproducible In-cell NMR measurements.
4) Comprehensive structural analyses of variant Laminproteins which causes severe hereditary disorders
Laminopathy by NMR spectroscopy.

࠙Referencesࠚ

1. Latest approaches for efficient protein production in
drug discovery. Sugiki et al. (2014) Expert Opin. Drug
Discov. 9, 1189-1204.
2. The ATP-mediated regulation of KaiB-KaiC interactionin the cyanobacterial circadian clock. Mutoh et al. (2012)
PLoS ONE 8, e80200.

Electron Microscopy Group
Junichi TAKAGI and Kenji IWASAKI
Correspondence
Tel: +81-6-6879-8607 ; Fax: +81-6-6879-8609
E-mail:takagi@protein.osaka-u.ac.jp
We set out to establish a road map for the development of
EM techniques for structural analysis of purified proteinsat functional state, and for the purpose of understanding
the molecular architecture of subcellular structures, such asorganelles, from the perspective of their atomic structures.
Our primary aims in this directive:
࠙ࠚDevelop a single-particle reconstruction system forstructural analysis of protein at atomic resolution using
most advanced direct detection camera.
࠙ࠚExpand EM technology and its peripheral techniques
for a wide range of applications, such as the three
dimensional structure of pleomorphic objects in cells, andorganic materials.
࠙  ࠚ Develop a hybrid approach that combines EM
imaging with computer simulation, biochemistry, X-ray
crystallography, optical microscopes and a variety of othercutting-edge methodologies to extract even further
information from EM images and so as to enhance ourunderstanding of protein function.

Cryo-electron microscope equipped with a most-advanced
direct detector and energy filter.

䛆References䛇
1. Giant cadherins Fat and Dachsous self-bend to organize
properly spaced intercellular junctions. Tsukasaki Y,
Miyazaki N, Matsumoto A, Nagae S, Yonemura S,
Tanoue,T, Iwasaki K, and Takeichi M. (2014) Proc. Natl.
Acad. Sci.,111(45), 16011-16016.

Laboratory of Advanced Protein Characterization

47

Research Center for State-of-the-art Functional Protein Analysis
Molecular Analysis Group
Toshifumi TAKAO, Atsushi NAKAGAWA, Junichi
TAKAGI, Takeshi SATO, Naoko NORIOKA, Keiko
KAWAKAMI and Naoyuki ABE
Correspondence
Tel: +81-6-6879-4312; Fax: +81-6-6879-4332
E-mail: procise@protein.osaka-u.ac.jp
This group is working on analysis of the primary structureof proteins and peptides by using conventional mass
spectrometers and peptide sequencers. Nowadays we often
use a variety of recombinant proteins for structural andfunctional study of target proteins. To obtain solid results,
information on the chemical properties of recombinant
proteins of experimental materials, such as terminal
sequences and post-translational modifications, is very
useful. This group is accumulating various data for suchquality control of proteins to make them appropriate formolecular analysis.

MALDI-TOF Mass Spectrometer

Peptide Sequencer

Laboratory of Advanced Protein Characterization48

Research Center for State-of-the-art Functional Protein Analysis

Laboratory of Protein DatabasesProfessor
Professor
Professor
Associate Professor
Associate Professor
Technical AssistantHaruki NAKAMURA
Toshimichi FUJIWARAKiyotoshi SEKIGUCHI
Akira R. KINJO
Chojiro KOJIMA
Takashi KOSADA

CorrespondenceTel: 06-6879-4311; Fax: 06-6879-8636; E-mail: harukin@protein.osaka-u.ac.jp

This Laboratory develops and manages several databases, which are freely and publicly available for a wide and global
community of protein science: PDBj (Protein Data Bank Japan), for protein atomic structure database, BMRB
(BioMagResBank) for NMR experimental data of biological molecules, Matrixome for mouse basement membrane
bodymap, and a portal for CSD (Cambridge Structural Database).

Laboratory of Protein Databases

PDBj Group (Haruki NAKAMURA, Akira R. KINJO and Takashi KOSADA㸧
Correspondence Tel: 06-6879-4311; Fax: 06-6879-8636; E-mail: nahokoh@protein.osaka-u.ac.jp;
URL: http://pdbj.org/

Fig. 1. Top page of the PDBj
(Protein Data Bank Japan: http://pdbj.org/)

We, PDBj, curate, edit and process the 3D atomic structure data of
biological macromolecules such as proteins and nucleic acids, andprovide them freely and publicly from our own Web site,
collaborating with wwPDB (worldwide PDB: http://wwpdb.org/) as
one of the members of wwPDB. We also prepare various services forresearchers and students, who are interested in structural
biology. (Refs. 1, 2): Molecular graphics viewer, jV,
molecular surface database for functional sites, eF-site,with the eF-seek service for the search of similarmolecular surface, GIRAF, query service for the similar
ligand binding sites, the navigation of the sequence andstructure neighbours, Spanner, homology modeling server,and EM Navi, image viewer for the structures by electron
microscopy.

࠙Referencesࠚ
1. Protein Data Bank Japan (PDBj): Maintaining a structural data archive and Resource Description Framework format.
Kinjo AR, Suzuki H, Yamashita R, Ikegawa Y, Kudo T, Igarashi R, Kengaku Y, Cho H, Standley DM, Nakagawa A,
Nakamura H. Nucleic Acids Research 40:D453-D460 (2012)
2. PDBj Mine: Design and implementation of relational database interface for Protein Data Bank Japan. Kinjo AR,
Yamashita R, Nakamura H. Database 2010:baq021 (2010)


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Laboratory of Protein Databases

49Research Center for State-of-the-art Functional Protein Analysis
Laboratory of Protein Databases

PDBj-BMRB (BioMagResBank) Group (Toshimichi FUJIWARA and Chojiro KOJIMA)Correspondence Tel: 06-6879-8598; Fax: 06-6879-8599; E-mail: bmrbhelp@protein.osaka-u.ac.jp;
URL: http://bmrbdep.protein.osaka-u.ac.jp

We, PDBj-BMRB, collect and process the NMR experimental data of biological molecules, and provide them from our
Web site, collaborating with BMRB at University of Wisconsin-Madison in USA.
BMRB collects, annotates, archives, and disseminates (worldwide in the public domain) the important spectral andquantitative data derived from NMR spectroscopic investigations of biological macromolecules and metabolites. The goal
is to empower scientists in their analysis of the structure, dynamics, and chemistry of biological systems and to support
further development of the field of biomolecular NMR spectroscopy.

࠙Referenceࠚ
1. An automated system designed for large scale NMR data
deposition
andannotation: Application to over 600
assigned chemical shift data entries to the BioMagResBank fromthe RIKEN Structural Genomics/Proteomics Initiative internal
database. Kobayashi N, Harano Y, Tochio N, Nakatani E, Kigawa
T, Yokoyama S, Mading S, Ulrich EL, Markley JL, Akutsu H,
Fujiwara T. Journal of Biomolecular NMR 53:311-320 (2012)
Fig. 2. The home page of PDBj-BMRB (Protein Data BankJapan - BioResMagBank: http:/bmrb.protein.osaka-u.ac.jp/).

Laboratory of Protein Databases

Matrixome Group (Kiyotoshi SEKIGUCHI)
CorrespondenceTel: 06-6879-8617; Fax: 06-6879-8619; E-mail: matrixome@protein.osaka-u.ac.jp;
URL: http://www.matrixome.com/bm

We organize and maintain a web database "Mouse Basement Membrane Bodymap" which consists of hundreds of highresolution images representing the localization of more than 40 extracellular matrix proteins in whole mouse embryos
analyzed by immunehistochemical technique. (Ref. 1)

࠙Referenceࠚ
1. Transcriptome-based systematic identification of extracellular matrix
proteins. Manabe et al. Proceedings of National Academy of ScienceU S A. 105:12849-12854 (2008)

Fig. 3. The Mouse Basement Membrane Bodymap database(http://www.matrixome.com/bm) consists of a panel of high resolution imagesof whole-body sections of mouse embryos immunostained for extracellularmatrix proteins. Each image provides “virtual slide” on which users canzoom-in/out and move the view seamlessly online.

Laboratory of Protein Databases
CSD (Cambridge Structural Database) Group (Haruki NAKAMURA)
CorrespondenceTel: 06-6879-4311; Fax: 06-6879-8636; E-mail: ynatsuko@protein.osaka-u.ac.jp;
URL: http://www.protein.osaka-u.ac.jp/csd/csd.html

We support Academia researchers in Japan, who want to use CSD (Cambridge Structural Database system), which is adatabase for small organic chemical compounds constructed by CCDC (Cambridge Crystallographic Data Centre). We also
provide a portal for CSD to researchers inside and even outside of Osaka University.
Laboratory of Protein Databases

50Research Center for State-of-the-art Functional Protein Analysis

Open Space Laboratory for Advanced
Protein Science
VisitingProfessor

Midori Takimoto-Kamimura

CorrespondenceTel: +81-42-586-8283; Fax: +81-42-586-8219
E-mail: m.kamimura@teijin.co.jp

Open space laboratory for advanced protein science has
a role of bridging between academic and pharmaceutical
drug discovery.
The most important process of thesmall molecule drug discovery is (1) How to choose the hit
compound ? (2) How to choose the drug target? (3)How to detect the interaction between hit compounds anddrug targets? Finally these information are compiled as
the protein-ligand interaction data base. We collaborate
with related laboratories in the institute for Protein
Research (Laboratory of Molecular Physics andLaboratory of Protein databases etc.). Under thesecollaborations, we develop the basic techniques which arenecessary to complete above issues.
To figure out these problems, recently Fragment Based
Drug Discovery (FBDD) method grows from the potential
and low molecular weight compound (MW<250) to theclinical candidate. The key of the success is whether wecan pick up a potential hit but weak compoundat the beginning stage. Regarding the sensitivity, NMR ismost appropriate method. But the problem is its low
throughput. To avoid this problem, we focused on the
fluorine compounds. 19F peak shows very characteristic
peak by each compound and we can mix more than ten
compounds at the same time by selecting appropriatecompounds as a cocktail. Last year we selected 125
fluoride commercial available compounds. After watersolubility check and examine the 19F peak profiling, basedon last year experiments another new 125 compounds wereadded to our library as second fragment library. We
selected two kinases (JAK3ࠊGSK-3) as protein target.
They are also attractive target for drug discovery field.
We did fragment screen by our soluble 250 fragment
against two kinases by STDD method using 19F signals.
The fragment hits against two kinases are different. Even
weak hits, They have selectivity for the each ATP site. We
show hit fragments scaffold. It examined by competitive
inhibition by known ATP site binding compound..This year,
we will add other 250 fragments and increase fragment
to 500 selected by Dr. Teruki Honnma (RIKEN) 1)
strategy which is completely different from previous 250
fragments selection method. This year,  I will try
determing the orientation of hit fragments using QCI-F
by RIKEN NMR facility under the collaboration with Prof.
Kojima , IPR.
In addition to that, the protein targets would like to be also
expanded to the membrane proteins.

ligands and some proteins involving ADME and TOX.
But any systematic data base regarding reliable physicoChemical interactions between ligands and drug targets is
not found yet. If we can have such a DB withphysicochemical interaction properties, they must bepractically useful for both academic and industrial drug
discovery. So we cooperate with related laboratories in
Institute for Protein Research (Laboratory of Molecular
Physics, Laboratory of Protein databases etc.) and would
like to realize new approach with the physicochemicalinteraction DB between ligands and proteins.
࠙Current Research Programsࠚ

Physicochemical interaction DB between ligands anddrug target proteins
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Fig.1. Fragment Hit example by 19F STDD
࠙Referenceࠚ
1)Homma T. et al (to be submitted)

On the other hand, some big public data base like
ChEMBL contain the biological assay data between theOpen Space laboratory for Advanced Protein Science51

Equipments

1. General purpose computer system

9. Ultra microtome system &High pressure freezer

15. Fluorescence and radioactivity
imaging system
2. Synchrotron radiation beam line formacromolecular assemblies


3.Ultra-high brilliant X-ray generator
with a high performance imaging
plate diffractometer & Free mounting
system (FMS)
10. Analytical ultracentrifuge

11. Nano-flow liquid chromatography

12. Matrix-assisted laser desorption
ionization tandem time-of-flight mass
spectrometer

16.DNA chip analysis system
17. Time-lapse video microscope

18. Cell sorter


4.High-resolution NMR (400, 500, 600,
800, 950 MHz) &High-resolution
solid-state NMR (500, 600, 700 MHz)13. microLC system/
MALDI plate spotter
5. 395GHz-600MHz
DNP-NMR system

19.Differential scanning calorimeter &Isothermal titration calorimeter
20. Circular dichroism spectrometer
21. Fourier transform infraredabsorption spectrometer
22. PC cluster system with 28 GPGPUs
23. Cryo-electron micoscope forbiological imaging
24. Tandem Mass Spectrometer

14. Protein sequencer
6. Protein-protein interaction analyzer
7. Transmission electron microscope
8. Environmental control typevitrification device for TEM specimen

Equipments

52

25. Octet(Biolayer Interferometry device)Floor Plan

Floor Plan

53

Access

Transportation to IPR from the nearest stations‫ڦ‬Hankyu Railways, Kita Senri Station: About 15 min walk. Or, by taxi a ride of about 5 min from Kita Senri Station‫ڦ‬Subway, Midosuji-line, Senri Chuo Station: By taxi a ride of about 10 min. Or, take Hankyu-bus for “Onohara Higashi”
and get off at “Handai Guchi”, and then about 5 min walk. Take Hankyu-bus for “Handai Honbu Mae” and get off at“Handai Honbu Mae”, and then about 15 min walk. Take Hankyu-bus for “Handai Igakubu Byoin Mae” and get off at“Handai Shigakubu Byoin Mae”, and then about 15 min walk.
‫ڦ‬West Japan Railway (JR) Tokaido-line, Ibaraki Station: By taxi a ride of about 15 min. Or, take Kintetsu-bus for “Handai
Honbu Mae” and get off at Handai Honbu Mae, and then about 15 min walk.
‫ڦ‬Osaka Monorail, Handai Byoin Mae Station: About 20 min walk.



Access54

Institute for Protein Research, Osaka University
3-2 Yamadaoka, Suita, Osaka 565-0871, Japan
Phone: +81-6-6879-8594(General Affairs Section)
Fax: +81-6-6879-8590
http://www.protein.osaka-u.ac.jp