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Probing Membrane Lipids:

a Perspective from Solid-State NMR Study

Michio Murata 村田道雄

大阪大学大学院理学研究科

JST ERATO脂質活性構造プロジェクト

1

2015.4.28

ASIAA/CCMS/IAMS/LeCosPA/NTU-Phys Joint Colloquia

(2)

Biomembrane comprise diverse lipids and proteins and form complex structures

without proteins

2

(3)

3

Contents

I Model Membrane of Lipid Rafts

II Domain Formation in Membrane

III Raman Imaging of Raft Model Membrane

Sphingomyelin and Cholesterol Binary System

Sphingomyelin and Phosphatidylcholine System

Development of new Raman Tagged Sphingomyelin

I Model Membrane of Lipid Rafts

II Domain Formation in Membrane

III Raman Imaging of Raft Model Membrane

Sphingomyelin and Cholesterol Binary System

Sphingomyelin and Phosphatidylcholine System

Development of new Raman Tagged Sphingomyelin

(4)

Approaches towards membrane lipids with variable time and spacious scales

Artificial lipid membranes

biomembrane

Ternary system

Unary and binary systems

Solid state NMR (

2

H NMR, REDOR)

Molecular level 10 μs – 10 ms time scale

3

Our objective: Elucidation of the molecular basis of lipid raft formation

Raman Imaging

Macroscopic membrane

Fluorescent lifetime

Molecular level several ns time scale

SM

Lipid behavior in various membranes (mobility and intermolecular interaction)

Cho

(5)

Lipid rafts

Glycosphingolipid GPI-anchored

protein

Transmembrane protein

Sphingomyelin (SM, SSM)

・Resistance to solubilization with Triton X-100 (DRM)

・ Ordered lipids (L

o

phase) undergoes domain formation

・Implication in many cellular processes (signal transduction etc.)

1) Simons,K.; Ikonen,E. Nature, 1997, 387, 569-572. 2) Pike, L. J. J. Lipid Res. 2006, 47, 1597-1598.

5

Molecular basis of lipid raft formation

Cholesterol (Cho)

(6)

Elucidation of 3D structures and interactions of lipids in membrane is essential

Difficulties in structure elucidation

X-ray Crystallography

×

Solution NMR

Lipid bilayer membranes

But…

Solid state NMR

works in such weird systems ?

6

How can we elucidate the conformatoin and interactions of lipids in membrane?

Drug-membrane interaction Lipid-lipid & lipid-protein interaction in membrane

(7)

Micelles Bicelles

Detergent Phospholipid

Detergent Sterol

・Many examples

・High curvature

・Bilayer-like structure

・Strick conditions;

temp. & conc.

Micelles vs Bicelles

for membrane mimic

7

(8)

10’-d

2

-SM/DHPC(4/1) 33.6 kHz

B

0

35.3 kHz

10’-d

2

-SM/DHPC/Chol (4/1/0.4)

10% Cho siginificantly ehnaces the ordering of SM bicelle membranes

+ Chol

8

B

0

Ordering of SM-d 2 bound

in Cho-containing large bicelles

(9)

q = [SSM] / [DHPC]

DHPC (short chained FA) Stearoyl SM (major constituent)

q > 2.0 q < 2.0

O O

O P O

N O

O O

O

Size-dependent orientation of bicelles along magnetic field

・ Planar bilayer structure

・Non-orientation along B

0

Small Bicelles

High mobility of small bicelles enables high resolution NMR spectra even for

1

H nucleus.

9

(10)

5 4 1,3 2

a b g 2’ 6

a

SM liposome

MAS: 5 kHz, Mixing time: 30 ms, Temp.:37 ºC

SM/DHPC (1/2) bicelle

10

1 H NMR NOESY of bicelles under MAS

(11)

11

Conformation of SM head group deduced from NOEs and J coupling in small bicelles

Yamaguchi, T. Suzuki, T., Yasuda, T. Oishi,T., Matsumori, N., Murata, M. Bioorg. Med. Chem. 20, 270-278 (2012).

NOEs are similar between the Cho-containing and Cho-free bicelles

Conformation of SM is

similar between pure SM

and SM-Cho

(12)

How REDOR works I

15

N

Accuracy is <0.1 Angstrom !

Synchronous irradiation to magic angle spinning

12

T. Gullion, J. Schaefer, J. Magn. Reson., 1989, 13, 57

*REDOR data; A. Naito, et al., J. Phys. Chem. 1996, 100, 14995

S

0

S DS

15

N-non- irradiated

15

N-irradiated REDOR Decay

- =

d 13C

r

15

N

13

C

N

C

T

r

(ms)

⊿S/S

0

0 4 8 12 16 20

0

1.0

(13)

Jn

: Vessel Function, n t

r

: REDOR dephasing time g

I

: Gyromagnetic Ratio of I nucleus. g

S

: That of S nucleus

h: Plank const.,

m

0

: Permeability of Vacuum

How REDOR works II

MAS (Magic Angle Spinning): S

0

REDOR: S

N S N S

13C r

B0

t

13Cが31Pから 受ける磁場

2

3 4

Tr

Magic

Angle N

S1 N S2

3

4

1 1

180°

パルス

Integration = 0

Integration > 0

180O

Pulse

Magnetic field Strength

Gullion, T. et al. Adv. Magn. Reson. 13, 57 (1989); Gullion, T. et al. J. Magn. Reson. 89, 479 (1990).

 D 

1

2 2 r

2 r 0

0

)]

n 2 ( 1 [ 16

2 1 )]

n 2 ( [ 1

k

k

D

k J D

S J

S t 

t

 D 

1

2 2 r

2 r 0

0

)]

n 2 ( 1 [ 16

2 1 )]

n 2 ( [ 1

k

k

D

k J D

S J

S t t

Dipole Coupling

D

=

g

I

g

S h

m

0

16 

3

r

3

Magnetic field Strength

15N

13

NCTr (ms)

⊿S/S0

0 4 8 12 16 20

0 1.0

(14)

13 C{ 15 N}REDOR used for evaluation of mobility and orientation

14

Dipole coupling of rapidly moving mol.

depends on

Mol. axis angle q

Wobbling S

mol

θ

2

1 cos

3

2

0

  q    

mol N

C

D S

D

Wobbling S

mol

(15)

θ

Mol. axis Wobbling S

13C

15N

C1’

Non-irr. S

0 Irradiated S

SM/Cho: D

C-N

=265 Hz SM only: D

C-N

=158 Hz

1’-13C,2-15N-SM (D0=1302 Hz)

REDOR data for 13 C- 15 N-labeled SM in SM/Chol and SM only membranes

15

1’-13C,2-15N-SM/Chol (1/1) 50 % wt D2O

MAS 5 kHz, Temp. 45˚C, Scan 2896,

t

4.0 ms

1) Gullion, T et al. Adv. Magn. Reson. 1989, 13, 57-83.

DC-N=265 Hz

DC-N=150 Hz

(16)

16

Major conformer in SM-Chol: S

mol

: 0.94, (a, b) = 166

o

, 32

o

Major conformer in SM only : S

mol

: 0.70, (a, b) = 158

o

, 35

o

θ

265

C1’ /

15

N C2’ /

15

N

12

1302 232

D

0

(Hz)

D

C-N

SM/Chol 63

233

82 33

229

C1 /

15

N C2 /

15

N C3 /

15

N

158 2

D

C-N

SM only 69 55 48

1073

REDOR reveals S mol and orientation for amide bond

2

1 cos

3

2

0

  q    

mol N

C

D S

D

Not only conformation but orientation

is not affected by Cho. Difference is in mobility

(17)

17

Cho ordering effect and orientation lead to intermolecular H-bonds

Formation

Disruption

Lo domain

(raft-like)

SM/Chol

H-bonding

(18)

D

ν Δνmax 63.8 kHz Quadrupolar

Compling Small

Mobility Large

Molecular motion capture

Rotation axis

Library of site-specifically

2

H-labeled SM

6

Motion capture of alkyl chains of membrane lipid by 2 H NMR

Palmitoyl-sphingomyelin (PSM)

Perdeutero-acyl chain

(19)

Synthesis of 2 H-labeled fatty acids

19

(20)

Synthesis of 2 H-labeled SM

20

(21)

Depth-dependent order of SM by 2 H NMR

0 10 20 30 40 50 60

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 0

10 20 30 40 50 60

1 2 3 4 5 6 7 8 9 101112131415161718 2’ 3’ 4’ 6’ 8’ 10’ 12’ 14’ 16’ 18

Q sp litt in g D n (kH z)

Raft

(SM/Chol =1/1)

Non-Raft (100% SM)

Non-Raft (100% SM)

Both alkyl chains interact with Cho

similarly.

Cho cyclic core

45℃ 45℃

Matsumori, N.; Yasuda, T. et al. Biochemistry 2012, 51, 8363-8370.

3 5 6 8 10 12 14 16 18

<Sphingosin chain> carbon number <Acy chain>

carbon number

Raft (SM/Chol =1/1)

21

Mobilit y

(22)

22

Contents

I Model Membrane of Lipid Rafts

II Domain Formation in Membrane

III Raman Imaging of Raft Model Membrane

Sphingomyelin and Cholesterol Binary System

Sphingomyelin and Phosphatidylcholine System

Development of new Raman Tagged Sphingomyelin

I Model Membrane of Lipid Rafts

II Domain Formation in Membrane

III Raman Imaging of Raft Model Membrane

Sphingomyelin and Cholesterol Binary System

Sphingomyelin and Phosphatidylcholine System

Development of new Raman Tagged Sphingomyelin

(23)

The structure properties make SM preferentially form lipid rafts Both SM and saturaed PC are known to form L o domains

Comparison between SM and PC

Systematic comparison between SM and saturated PC

Space : Atomic ~ Molecular ~Entire membrane Time : nanosecond ~ millisecond

Lipid constituents : Unary system ~ Ternary system

23

PSPC

SM

What is the difference between SM and

PC in formation of L o domains.

(24)

0 10 20 30 40 50 60

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18

0 10 20 30 40 50 60

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18

50 ℃

<SM acyl chain> <PSPC acyl chain>

50 ℃

2’ 3’ 4’ 6’ 8’ 10’ 12’ 14’ 16’ 18

carbon number carbon number

Comparison of chain mobility between SM and PSPC

2) Yasuda, T. et al. Biophys. J. 2014, 106, 631-638.

<PSPC-Chol>

< SM-Chol>

2’ 3’ 4’ 6’ 8’ 10’ 12’ 14’ 16’ 18

Raft

(SM/Chol =1/1)

Non-Raft (100% SM)

Raft (PC/Chol =1/1)

Non-Raft (100% PC)

1) Matsumori, N.; Yasuda, T. et al. Biochemistry 2012, 51, 8363-8370.

Probably due to hydrogen bond network by amide groups of SMs

The rigid tetracycle of Cho is located more deeply in SM membrane

24

Q sp litt in g D n (kH z)

Mobilit y

(25)

35 40 45 50 55 60

15 20 25 30 35 40 45 50 55

35 40 45 50 55 60

15 20 25 30 35 40 45 50 55

Temperature (℃)

33 mol% Cho 20 mol% Cho

Temperature (℃)

SM-Cho membrane is more tolerant to temperature change than PC-Cho membrane.

(Lesser temperature dependence)

Yasuda, T. et al. Biophys. J. 2014, 106, 631-638.

10’,10’-d

2

-SM - Cho

● 10’,10’-d2

-PSPC - Cho

10’,10’-d

2

-SM - Cho

● 10’,10’-d2

-PSPC - Cho

Temperature dependent ordering of SM and PC at low Cho concentration

Higher thermal stability

SM intermolecular H-bond (membrane surface) +

Cho ordering effect (membrane interior)

25

Q splittingD

n

(kHz) Q splittingD

n

(kHz)

(26)

Intensity [Counts]

Example :SSM+33 mol% Cho 30 ℃

100 104

103

102

101

Measuring data

0 40 80 120 160 200 240 280 320 360 400

time [ns]

trans parinaric acid (tPA) λex = 295 nm, λem = 405 nm

α𝑖exp −𝑡/τ𝑖

𝑛

𝑖=1

I (t) =

= α

1

exp (-t/τ

1

) + α

2

exp (-t/τ

2

)

α

1

, α

2

… fractional amplitudes of each component

※ Deconvolution by two lifetime components

τ

1

, τ

2

… lifetime of each component

Ld phase

Gel phase Lo phase

Fluidity Low High

Lifetime Long Short

Evaluation of membrane fluidity in nanosecond time domain -Fluorescent lifetime experiment

Fluorescent lifetime τ

The average time that fluorophore remains in the excited state (ns)

Dependence on lipid phase state

2 mol% of total lipids

26

(27)

0 10 20 30 40 50 60 70

15 20 25 30 35 40 45 50 55

lif etime τ (ns )

τ ●●

Longer lifetime

→ Lower fluid domain

τ2

● Shorter lifetime

→ Higher fluid domain Temperature (℃)

NMR cannot detect the coexistence of domains.

SM/33 mol% Cho

Membrane heterogeneity

Lipid cluster with short lifetime

Under Tm (< 45 ℃)

Over Tm

(> 45 ℃)

L

o

domain L

o

+ L

d

Cho-poor gel-like + L

o

Gel phase

Low concentration of Cho → Similar behavior with gel phase

L

d

domain The fluidity of phase state : Gel < L

o

< L

d

No gel phase exists above T

m

.

Cho-poor gel-like

domain

L

o domain

Membrane fluidity on nanosecond time scale

27

(28)

Hypothetical model for interconversion of nano-domains

Below Tm (< 20 ℃)

Above Tm (> 49 ℃)

Gel-like domain Lo domain Gel-like domain Lo domain Gel-like domain Lo domain

Ld domain

Ld domain Lo domain Lo domain Ld domain Lo domain

~100 ns

1)

1) Chachaty, C. et al. Biophys. J. 2005, 88, 4032-4044.

30

(29)

Difference in dynamic behavior between SM and PC in Cho-containing binary systems

SM PSPC

2

H NMR

Temperature dependence of lipid ordering

Location of Cho

Coexistence of cho-poor clusters with short lifetime

Shallower Deeper

Smaller Larger

Lipid mobility at atomic level

Fluorescent lifetime

Membrane fluidity in

nanosecond time domain

Higher

L

o

domain-forming ability

Lower

Hydrogen bond

network

Absent

Present

29

These data suggest that SM-SM H-bonding plays major roles rather than

SM-Cho interaction.

(30)

DHSM: Dihydrosphingomyelin (C

18

) SM: Sphingomyelin (C

18

)

・Major SM in human

・Raft model lipid ・Relatively abundant SM homologues

・Form more stable L

o

domains than SM

DOPC: Unsaturated PC, a typical L

d

lipid in the presence of SM and Cho

vs

Can SM form macroscopic domains without Cho?

Kinoshita, M., Goretta, S., Tsuchikawa, H., Matsumori, N., Murata, M., Biophysics 9, 37-49 (2013).

a) eSM

b) tSM

c) DHSM

d) tripleSM

a) SM b) DHSM

30

(31)

DHSM forms macroscopic domains without Cho

Kinoshita, M., Matsumori, N., Murata, M. Biochim. Biophys. Acta 1838, 1372-1381 (2014).

SM

Temp(℃)

Mol. Ratio of DOPC

Mol. Ratio of DOPC

DHSM

Temp(℃)

Uniform

Phase separated

Uniform

Phase separated

31

DHSM

H-bond

Separated Mixted

+DOPC

Pure Mixed again

DOPC-rich DHSM-rich

(32)

Approaches towards Membrane Lipids with Variable Time and Spacious Scales

biomembrane

Ternary system

2

H solid state NMR (

2

H NMR)

Molecular level 10-1000 μs time scale

34

Elucidation of the molecular basis of lipid rafts formation

Raman Imaging

Entire membrane level

Artificial lipid membranes

Unary and binary systems

Fluorescent lifetime

Several ns time scale

DOPC SM

Cho

(33)

Two pairs of doublets

10’,10‘-d2-SM/Cho/DOPC (1/1/1) の2H NMRスペクトル

Solid state

2

H NMR

10’,10’-d

2

-SM

30 ℃

SM rich ラフト相 (Lo 相)

DOPC rich 液晶相 (L

d

相)

GUV of SM/Cho/DOPC (1/1/1)

Domain separation of ternary SM/Cho/DOPC as observed by microscope and 2 H NMR

GUV Sample : SM/Cho/DOPC (1/1/1)

+ 0.2 mol% Bodipy- PC (λ

ex

= 488 nm) T : 30 ℃

Fluorescence microscope

L

d

-specific fluorescent dye

51.5 kHz

36.0 kHz

33

(34)

Fractional abundance of Ternary SM/Cho/DOPC system as revealed by 2 H NMR

34

非ラフト相

L o Domain L d Domain

(35)

Depth-dependent order of L o and L d domains in SM-Cho-DOPC system

Yasuda, T., Kinoshita, M., Murata, M., Matsumori, N. Biophys. J. 106, 631-638 (2014).

35

Carbon number

L o domains of ternary and binary systems showed similar ordering

Occurrence of SM-only domains even in ternary systems ↓

(36)

Contents

I Model Membrane of Lipid Rafts

II Domain Formation in Membrane

III Raman Imaging of Raft Model Membrane

Sphingomyelin and Cholesterol Binary System

Sphingomyelin and PhosphatidylChoine System

Development of new Raman Tagged Sphingomyelin

36 I Model Membrane of Lipid Rafts

II Domain Formation in Membrane

III Raman Imaging of Raft Model Membrane

Sphingomyelin and Cholesterol Binary System

Sphingomyelin and Phosphatidylcholine System

Development of new Raman Tagged Sphingomyelin

(37)

Labelled lipids for fluorescence spectroscopy do not reproduce original lipids due to balky

substituents

37

Small Raman tag Raman Imaging

Fluorescence Imaging

(38)

O

HN

OH O P

O O

O R

N N

N HO

D

3

C N

D

3

C CD

3

R:

SM alkyne-SM (1) diyne-SM (2) SM-d

9

(3)

Small Raman tags of SM for imaging

Goretta, S. A., Kinoshita, M., Mori, S., Tsuchikawa, H., Matsumori,N., Murata, M. 38

Bioorg. Med. Chem. 2012, 20, 4012-4019.

(39)

Cui, J., Lethu, S., Yasuda, T., Matsuoka, S., Matsumori, N., Sato, F., Murata, M. Bioorg. Med. Chem. Lett.

39

25,

203-206

(2015).

Diyne moiety shows strong intensity in background-free area

Triple bond

Diyne

Deuterated

(40)

(a)d-Cho/DOPC (1/1 mol),

(b)SM/d-Cho/DOPC (1/1/1 mol), and

(c)diyne SM/d-Cho/DOPC (1/1/1 mol) at 25 oC.

Diyne SM shows similar behavior to original lipid on 2 H NMR

40

DOPC SM

Diyne

-SM

(a)d2-SM/DOPC/Cho (1/1/1 mol), and

(b) d2-diyne SM/DOPC/Cho (1/1/1 mol) at 25 oC.

Diyne

-SM SM

d-Cho d

2

-SM vs Diyne-d

2

-SM

2

H NMR Spectra

Cui, J., Lethu, S., Yasuda, T., Matsuoka, S., Matsumori, N., Sato, F., Murata, M. Bioorg. Med. Chem. Lett. 25, 203-206 (2015).

(41)

Diyne probe mimics SM in L o domains on supported monolayer

=

Bodipy-PC ジイン-SM

Quartz-supported monolayer Diyne-SM/Cho/DOPC (1/1/1 mol)

41

10mm

(42)

Monolayer of diyne-SM/DOPC/Cho (1:1:1)

42

Concentration graduation of SM revealed by Raman imaging

Raman Image of diyne-SM

0 2 4 mm

(43)

Summary

Site-selective 2 H labeling precisely discloses depth-

dependent mobility of alkyl chains of SM and PC in L o and L d membranes

Intermolecular hydrogen-bonds play a key role in SM-SM interaction, which may lead to formation of raft-like L o

domains.

Nano-domains largely consisting of SM can be formed in the presence or absence of Cho.

Formation mechanism of SM/Cho-rich rafts in

biological membranes 43

(44)

Hypothetical nano-sized cluster of SM

44 A B

C1 C2

(45)

大阪大学理学研究科 化学専攻

(九州大学教授)

JST ERATO, 理研

Prof. Matsumori

Dr. Sodeoka

Dr. Yamaguchi Dr. Jin Cui

Dr. Yasuda 45

Å bo Akademi Univ. (Finland)

Prof. Slotte

(46)

46

Thank you for your attention!

(47)

Our Campus

Osaka is the 2

nd

largest city in Japan.

47

(48)

Stat. of Osaka University

Graduate schools: 20 Faculty members : 2600

Undergraduate students: 12000 Graduate students: 7800

(including 1000 foreign students)

The largest national university in Japan in terms of the number of undergraduate students.

48

(49)

OF OSAKA UNIV.

49

(50)

Ld phase

(non-raft)

SM 100%

SM-Cho interaction results in stable SM-SM hydrogen bond formation while SM-Cho

affinity is not high

association

dissociation

50

Lo phase

(raft model)

SM/Cho

Hydrogen Bond

(51)

Temperature (℃)

10

20 30 40 50 60 70

15 20 25 30 35 40 45

L o domain-forming ability in SM and PSPC memb.

Temperature (℃)

▲ SSM (Gel phase) ● SSM-33 mol%Cho

Cho-poor gel-like domain ▲ PSPC (Gel phase) ● PSPC-33 mol%Cho Cho-poor gel-like domain

SM has a higher L o domain-forming ability.

10 20 30 40 50 60 70

15 20 25 30 35 40

lif etime τ (ns ) lif etime τ (ns )

Lo domain Lo domain

・ ・

Cho-poor gel-like domain

Lo domain Gel phase

The affinity with Cho

Cho-poor gel-like domain

51

SM PSPC

(52)

0 10 20 30 40 50 60

15 20 25 30 35 40 45 50 55

0 10 20 30 40 50 60

15 20 25 30 35 40 45 50 55

Mean lifetime (ns) Mean lifetime (ns)

33 mol% Chol

Temperature (℃)

● SSM-Chol

● PSPC-Chol

20 mol% Chol

Temperature (℃)

● SSM-Chol

● PSPC-Chol

Mean lifetime : the weighted average of fluidity in the bilayer on nanosecond time domain

Similar behavior to the data from

2

H NMR

The local mobility of acyl chain in phospholipids is closely correlated to the entire membrane order.

The decreasing degree of lifetime in SM membrane is smaller with increasing temperature.

Temperature dependence of mean lifetime in SM and PSPC membrane containing Chol

52

(53)

ERATO脂質活性構造プロジェクト 本研究の3つの目標

タンパク質内部脂質 (in protein)

マイクロドメイン (as a field)

脂質リガンドとの相互作用 脂質集合体の分子基盤

膜タンパク質周辺脂質 (around protein)

周辺脂質の立体構造と機能

関連する戦略目標:生命システムの動作原理の解明と活用のための基盤技術の創出

53

異分野融合の必要性

主に村田Gが担当

固体NMR, 化学合成,

物理化学計測, 共焦点顕微鏡, ラマンイメージング

杉山G と 松岡Gが担当

結晶X線回折, 固体NMR, カロリメータ, 計算機科学

Spring-8, SACLA, 表面プラズモン共鳴

3つグループで協力

固体NMR, 結晶X線回折, 合成化学, XAFS

脂質活性構造

(54)

54

II 脂質ラフト形成の分子機構

III SM膜の生物物理学的解析

IV ラマンイメージを用いた液体秩序相の観察

V 脂質二重膜における天然物との相互作用

1.梯子状ポリエーテル系天然物

2.細胞膜内Cholと相互作用する天然物

生体膜中脂質分子Gの研究成果 ④

II 脂質ラフト形成の分子機構

III SM膜の生物物理学的解析

IV ラマンイメージを用いた液体秩序相の観察

V 脂質二重膜における天然物との相互作用

1.梯子状ポリエーテル系天然物

2.細胞膜内Cholと相互作用する天然物

(55)

55

90

o

0

o

Amide I

Wavenumber/cm

-1

1800 1700 1600

Abs./a.u.

偏光減衰全反射赤外分光法(pATR-FTIR)

90o 0o

赤外入射 偏光フィルター

検出器 45°

YTX含有または非含有 GpA-TM再構成重水和DMPC膜

ATRプリズム(Ge)

エバネッセント波

二色比: R ΔA 90° /ΔA

例:

プリズム平面上におけるペプチド 含有脂質二重膜の簡略図

θ1

θ2 α

α

x Z

θ1

θ2 α

α

x Z

α

1 α2

イェッソトキシン (YTX) 4)

有毒渦鞭毛藻によって生産される海洋生物毒

(56)

56

配向

脂質分子DMPCのアシル鎖 GpA-TMのαヘリックス軸

バンド

試料名

重水和 GpATM-DMPC

(1:50)

重水和 GpATM-DMPC-

YTX (1:50:1)

重水和 GpATM-DMPC

(1:50)

重水和 GpATM-DMPC-

YTX (1:50:1)

α (°) 27.30 26.84 30.61 33.23

リン脂質膜中でYTXによるGpA-TMの配向変化の解析

○ 脂質分子のアシル鎖の配向角度はYTXの有無に関わらず一定、ペプチドのαヘリックス軸の配

向はYTXにより約10%変化

○ GpA-TMとYTXが結合することによってペプチドの会合状態や配向が変化した結果と考えられる。

リン脂質膜中におけるYTXとGpA-TMが相互作用することが示唆された

CH2対称伸縮振動 アミドⅠ

(αヘリックス)

YTX

GpA二量体 GpA単量体 GpA-YTX複合体

(57)

細胞膜内ステロールと相互作用する天然物

ペプチド-膜脂質の持つ強い 分子間相互作用と比較的小 さな分子量に着目

・ 膜脂質検出用の低分子 プローブの開発

・ 固体NMR実験の試行

Espiritu, R. A., Matsumori, N., Murata, M., Nishimura, S., Kakeya, H., Matsunaga, S., Yoshida, M., Biochemistry 2013, 52, 2410. 57 TMN

(58)

DOPC のモル比(x

DOPC

)

T empera ture ( ℃ )

x

p

x

q

1. DHSMは強い分子間水素結合を形成する(H-bonds).

2. DHSM はDOPCより大きな曲率をもつ膜を形成する

仮定

DHSM vesicle

DOPC add

attenuation of H-bond

L

α2

domain (x

DOPC

=x

q

)

L

α2

domain

(x

DOPC

homogeneous L =x

q

DOPC vesicle )

α2

domain

58

DHMS相挙動のDOPC依存性

(59)

Molar fraction of DOPC ( x

DOPC

)

Temperature (℃)

DHSM/DOPC

(Mason, 1988)

Temperature (℃)

DSPC/C

18

C

10

PC

(Wu & McConnel, 1975)

Temperature (℃)

DEPC/DPPE

59

DHSM/DOPC 系の相分離は珍しい例

(60)

DHSM: Dihydrosphingomyelin (C

18

) SM: Sphingomyelin (C

18

)

・代表的SM

・ラフトモデル膜に用いられる

・少量成分SM

・通常のChol存在下SMより固い膜を形成

DOPC不飽和リン脂質: 相分離して軟らかい相を形成

vs

60

SM類縁体だけの相分離の観測

コレステロールがなくても強い相互作用を示すか?

Kinoshita, M., Goretta, S., Tsuchikawa, H., Matsumori, N., Murata, M., Biophysics 9, 37-49 (2013).

a) eSM

b) tSM

c) DHSM

d) tripleSM

a) eSM

b) tSM

c) DHSM

d) tripleSM

a) SM b) DHSM

c) tSM d) tripleSM

(61)

61

a

b

c

d

0.0 0.1 0.2 0.3 0.4 0.5

0.98 0.99 1.00 1.01 1.02 1.03

0.0 0.1 0.2 0.3 0.4 0.5

0.98 0.99 1.00 1.01 1.02 1.03

0.0 0.1 0.2 0.3 0.4 0.5

0.98 0.99 1.00 1.01 1.02 1.03

0.0 0.1 0.2 0.3 0.4 0.5

0.98 0.99 1.00 1.01 1.02 1.03

xchol

xchol xchol

v(mL/g) v(mL/g)

v(mL/g) v(mL/g)

xchol

1170 1190 1210 1230 1250 1270

0 0.1 0.2 0.3 0.4 0.5 0.6

xchol

V

PMVSM

(Å

3

)

SM誘導体の物理学的膜物性の測定

a) SM b) DHSM

c) tSM d) tripleSM

〇: SM

□: DHSM

△: tSM x: tripleSM

(62)

0.0 0.2 0.4 0.6 0.8 1.0 -20

0 20 40 60

-20 0 20 40 60

62

x

chol

Parti al m ole cul ar are a of chole sterol (Å

2

)

x

chol

=0 (LE phase)

x

chol

≧0.5 (ordered phase)

diyneSM 33±10 130±20

SSM 47±10 120±20

Figure 7. The partial molecuar area of chol in (blue) diyneSM/chol and (red) SSM/chol binary monolayers at 5 mN/m was estimated from Figure 5 c and d.

Table 1. Areal compressional modulus of SM C

SM-1

(mN/m)

at 5 mN/m.

(63)

三重結合1つでは感度不足:共役ジインの強度は10倍

-実際の膜で測定してみると-

63

(64)

脂質ラフト形成モデル

Chol

Chol のステロイド骨格に よるオーダー効果 膜の深い位置に

分布するChol

膜界面 Lo ドメイン (ラフト膜)

Ld ドメイン (周辺膜)

相分離が高温まで安定に保持

SM分子間水素結合のネットワーク

アルキル鎖中央部へのオーダー効果最大

ナノ秒スケールのラフト相内部

短寿命の 脂質クラスター

(SM/Chol)

DOPC

速い生成と崩壊を繰り返す

SM SM

Chol

ラフト相にSMとCholが多く分配

16

(65)

位置選択的重水素標識PSPCの合成

1-palmitoyl-2-stearoyl-sn-glycerol-3-phosphocholine (PSPC)

計10種類の標識体ライブラリー

アシル鎖長 : C18

相転移温度(T

m

) : 48.8 ℃ Stearoyl-sphingomyelin (SSM)

アシル鎖長 : C18

相転移温度(T

m

) : 44 ℃ 8

參考文獻

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