別紙41 (課程博士(英文))Date of submission (month day, year)

Janual'y 7小,2022

Depa11menlof

Applied chemistl}、 andSluden11D NⅡnlber

D153444

Life science

Ryugo Tel'0
SupeTⅥSors
RikaNumano

PP11CaΠ1、s nanlc

Melvin Goh wei shel'nAbstmct (Doctor)
Title ofThesis

Non・1'aR submicl'on domain fonnation in lipid bilay引's induced by polyⅡnsatul'ated lipids
and its application t01'econstitution ofmelnbl'ane 訓、oteinsApprox.80o wm'ds
CeⅡ membl、anes al'e complex matrices composed of a 1ω'ge va"ety of ⅡPids and 111embl'ane pl'oteins,Sa'ving aS 介lnctional bal'riers bet、Neen ce11S and theil' envil'onment. Apa11 介om providing some stnlctul'al
SUPP011 for ce11S, t11ey constitute reaction fields for t11e u'ansp011ation of mate"als, signals and enel'gy intoand out of ce11S {11rough the m引n喩'anes. Tha'ef01'e, ceⅡ melnbl'anes al'e l、esponsible for seV引'al ce11Ulal'Processes, such as imlnuniw, metabolism and infection. val'ious types oflipids exist in ce11 membranes.1heirOrganization a仟ects the dynamics and physical pl'ope11ies ofule lipid bilayel'1nembl'anes. These manbl'ane
Characterislics in t11rn al'e responsible for the flmctions of the ceⅡ membl'anes. The fonnation of two・dimensiona11ipid dolnains due to prefel'ential association ofspecific lipid molecules and membrane pl'oteinshas been 、Nidely studied to {mderstand dle mechanisln of ceⅡ membl'ane reaction and l'elated path010gies.
The repl'esentative lipid donlain is dle "1'a負",、Nhich has been extensively investigated using a11ificia11ipid
bilayer systems.

In dlis P11.D. t11esis,1 ailned to cla"1 the f0Πηation meC11anism ofa ne、¥1y discovel'ed non・ra負 domain in
Iipid bilaya'S, aiming to apply t11e donlain as a platf0Π11 f01' the reconstitution of ion channels into a11ificial
Iipid bilayel'S. The non・1'aft domain formation in tl]is study is a ne、 , concept, postulating a possibility ofthe
existence of such domains in ceⅡ lnelnbl'anes. The f0Π11ation mechanism ofthis non・1'a介 domain is 、N011helucidating h'om the vie、Npoints of physical chemistry of lipid self・assembly and engineel'ing ofbionleln价'anes.1 also ainled to im御'ove the mem血'ane fusion e価Ciency ofpl'oteoliposome (PL)into a11ificial
Iipid bilayel'、vhich is vital f01'ion channel studies.1Π my ph.D. study, tl〕e ion channel that l foC11Sed on washuman elheルa・go・g0 1"elated gene (hERG) channel, a voltage・dependent potassium channel. Many dnlgs、vith dlenlicaⅡy diverse str11Ctures exhibit advel'se e仟ect on t11e hERG channel,1'esU11ing in potential cardiac
alThyt11mias. Hence, obsa'ving its active structul'e at physi010gical condition is desil'able f01' m01'e accurate
dl'ug design to avoid t11e advel'se e什ect, and thus is valuable from the vie、 'point of medical andPhaΠ11aceutical f01' men1励'ane protein studies.
First,1 Cla"fied the colnposition and mechanism of t11e non・1'a負 domain f0Π11ation in Hpid bilayer
Comprising t、和o types of lipids 、Nith lo、N phase transition temperature (LO、¥・Z11) and cholesta'01 (choD, bynUⅨ'escence lnicl'oscopy and atomic force micl'oscopy (AFM). LO、¥・710 lipid is in the liquid cl}/staⅡine phaseat the ambienttemP引'atul'e of expel'iments ( 25 ゜C).1、No di仟a'ent L0圦ノ・rm lipids homogeneously mixed at
any ratio. ch01・induced dolnain fonl〕ation in these completely miscible lipids is a unique phenomenon to bediscovel'ed.1 PI'eP印'ed t引'nary supp011ed lipid bilayers (SLBS) using egg・del'ived phosphatidylcholine(eggpc), ch01, and one of t111'ee di11'el'ent types of phosphatidylethanolamine (PE): egg・derived pE,1・Palmitoyl‑2‑oleoyl・PE (16:0‑18:1PE), and l,2・didocosa11exaenoyl・PE (22:6‑22:6PE). The non・ra介 domains、 a'e obsel'ved as de桝'essions in t11e AFM topogl'aphies.1heil' al'ea fl'action (θ) incl'eased 、 i{h concentl'ation
Of pE, and 22:6‑22:6PE yield the 1ω'gest e among the U11'ee pES,乳唱gesting the non・1'a介 domains in thePE+eggpC十Ch01・SLBS、¥引'e enriched with polyunsatul'ated pE.
Next,1 dl01'OUσhly elucidated the f0Π11ation mechanism of the non・ra介 domain by dal'ifying the l'ole of
Polyunsaturated acyl chains.1 explored the e仟ects of dle degree of unsaturation and the double bond
distribution atthe pE Sπ・position on the f0Π11ation ofthe non・1'a介 domains.1he tenlal>, SLBs containing pE、¥ith various degl'ees of unsatⅢ'ation 、Nel'e investigated by the nu01'escence micl'oscopy, AFM, and fm'ce‑
distance CⅢ've measul'emalt. The al'ea fl'action ofthe non・ra介 domain increased 、Nith dle concentration andde即'ee of unsatul、ation ofpE, suggesting t11e ilnp011ance oft11e double bonds at the acyl chains fm'the non・ra負 domain fonnation. The force・distance cul've measurement sho、、/s that the non・1'aR dolnains 、¥ere in theIiquid・dis01'dered・1ike state,、¥ha'eas theil' sun'ounding l'egions 、¥a'e in dle liquid・01'd引'ed・1ike state. The
1'esults suggest that the se即'egation of pE fl'om ch01・rich l'egion,、¥hich mainly conlpl'ises monounsatul'ated
Pc and ch01, induces a non・1'aft domain f0Πηation.1 also found that polyunsaturated pc is also capable toinduce t11e non・1'a介 domain fonnation, depending on its degl'ee ofunsatul'ation and concentration.1 PI'oposed
the mechanism for the f0Π11ation ofthese non・ra介 domains based on moleculal'interactions involYing thehydrophobic and hydrophilic pa11S ofthe bilay引' membl'ane
Ihen,1 investigated d〕e e行'ects of22:6‑22:6PE,、¥hich induced ule larσest e, on t11e fusion e仟iciency ofPLs into pE+PC十Ch01・SLB. The findings clearly sho、¥ that the large al'ea of non・1'a負 domahls in sLB
induced by 22:6‑22:6PE dl'astica11y impl'oved t11e pL fusion e伍Ciency, colnPω'ed to the e行'ects of eggpE on
the pL fusion e価Ciency. This enables a pl'omising method for the l'econstitution of m引11愉'ane 即'otein into
a11i6CiaΠゆid bilayer.
Lasuy,10bserved hERG channel molecules by AFM at physi010gical condition. pure hERG channel with
f11Ⅱ・1ength amino acid residues 、Nas synthesized using ceⅡ・fl'ee u'anslation systenl (by coU11esy of pl'ofYUZUI'U Toza、¥a).1 reconstituted dle 11ERG channels in sLB and observed t11eir association states at

Physi010gical condition.1he molecular ilnages of hERG channels in val'ious oligomel'ic states 、¥引'e
SuccessfU11y obsel'ved, and dleir association states were examined quantitatively. This study is expected to
be va111able and useful for dnlo desion to avoid dl'ug・induced side e仟ects on hERG channel.
In summaly, ulis ph.D. thesis descl'ibes the composition and mechanism ofnon・ra介 domain fonnati0Π,its
application to iln御'ove pL fusion e価Ciency into a11ificia11ipid bilayer, and the molecU1磁'images of hERG
Cha11nel at physi010gical condition. These l'eseal'ch 、Norks are expected to stinlulate furthel' studies on lipid
dolnains in bi010gical membrane, and to contl'ibute to t11e fields of hlt引face chaηistl'y, and phan11aceuticala11d medicalindustries