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
Biomembrane comprise diverse lipids and proteins and form complex structures
without proteins
2
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
Approaches towards membrane lipids with variable time and spacious scales
Artificial lipid membranes
biomembrane
Ternary system
Unary and binary systems
Solid state NMR (
2H 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
Lipid rafts
Glycosphingolipid GPI-anchored
protein
Transmembrane protein
Sphingomyelin (SM, SSM)
?
・Resistance to solubilization with Triton X-100 (DRM)
・ Ordered lipids (L
ophase) 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)
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
Micelles Bicelles
Detergent Phospholipid
Detergent Sterol
・Many examples
・High curvature
・Bilayer-like structure
・Strick conditions;
temp. & conc.
Micelles vs Bicelles
for membrane mimic
7
10’-d
2-SM/DHPC(4/1) 33.6 kHz
B
035.3 kHz
10’-d
2-SM/DHPC/Chol (4/1/0.4)
10% Cho siginificantly ehnaces the ordering of SM bicelle membranes
+ Chol
8
B
0Ordering of SM-d 2 bound
in Cho-containing large bicelles
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
0Small Bicelles
High mobility of small bicelles enables high resolution NMR spectra even for
1H nucleus.
9
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
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
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
0S DS
15
N-non- irradiated
15
N-irradiated REDOR Decay
- =
d 13C
r
15
N
13C
N
CT
r(ms)
⊿S/S
00 4 8 12 16 20
0
1.0
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
0REDOR: 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
180OPulse
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
Ig
S hm
016
3r
3Magnetic field Strength
15N
13
NCTr (ms)
⊿S/S0
0 4 8 12 16 20
0 1.0
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
molθ
2
1 cos
3
20
q
mol NC
D S
D
Wobbling S
molθ
Mol. axis Wobbling S
13C
15N
C1’
Non-irr. S
0 Irradiated SSM/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 ms1) Gullion, T et al. Adv. Magn. Reson. 1989, 13, 57-83.
DC-N=265 Hz
DC-N=150 Hz
16
Major conformer in SM-Chol: S
mol: 0.94, (a, b) = 166
o, 32
oMajor conformer in SM only : S
mol: 0.70, (a, b) = 158
o, 35
oθ
265
C1’ /
15N C2’ /
15N
12
1302 232
D
0(Hz)
D
C-NSM/Chol 63
233
82 33
229
C1 /
15N C2 /
15N C3 /
15N
158 2
D
C-NSM only 69 55 48
1073
REDOR reveals S mol and orientation for amide bond
2
1 cos
3
20
q
mol NC
D S
D
Not only conformation but orientation
is not affected by Cho. Difference is in mobility
17
Cho ordering effect and orientation lead to intermolecular H-bonds
●
Formation
Disruption
Lo domain
(raft-like)
SM/Chol
H-bonding
D
ν Δνmax 63.8 kHz QuadrupolarCompling Small
Mobility Large
Molecular motion capture
Rotation axis
Library of site-specifically
2H-labeled SM
6
Motion capture of alkyl chains of membrane lipid by 2 H NMR
Palmitoyl-sphingomyelin (PSM)
Perdeutero-acyl chain
Synthesis of 2 H-labeled fatty acids
19
Synthesis of 2 H-labeled SM
20
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
大 小
Mobilit y
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
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.
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)
大 小
Mobilit y
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 splittingDn
(kHz)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
1exp (-t/τ
1) + α
2exp (-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 phaseGel 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
0 10 20 30 40 50 60 70
15 20 25 30 35 40 45 50 55
lif etime τ (ns )
τ1 ●●
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
odomain L
o+ L
dCho-poor gel-like + L
oGel phase
Low concentration of Cho → Similar behavior with gel phase
L
ddomain The fluidity of phase state : Gel < L
o< L
dNo gel phase exists above T
m.
Cho-poor gel-like
domain
L
o domainMembrane fluidity on nanosecond time scale
27
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
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
HigherL
odomain-forming ability
LowerHydrogen bond
network
AbsentPresent
29
These data suggest that SM-SM H-bonding plays major roles rather than
SM-Cho interaction.
DHSM: Dihydrosphingomyelin (C
18) SM: Sphingomyelin (C
18)
・Major SM in human
・Raft model lipid ・Relatively abundant SM homologues
・Form more stable L
odomains than SM
DOPC: Unsaturated PC, a typical L
dlipid 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
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
Approaches towards Membrane Lipids with Variable Time and Spacious Scales
biomembrane
Ternary system
2
H solid state NMR (
2H 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
Two pairs of doublets
10’,10‘-d2-SM/Cho/DOPC (1/1/1) の2H NMRスペクトル
Solid state
2H 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
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
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 ↓
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
Labelled lipids for fluorescence spectroscopy do not reproduce original lipids due to balky
substituents
37
Small Raman tag Raman Imaging
Fluorescence Imaging
O
HN
OH O P
O O
O R
N N
N HO
D
3C N
D
3C CD
3R:
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.
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
(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).
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
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
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
Hypothetical nano-sized cluster of SM
44 A B
C1 C2
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
Thank you for your attention!
Our Campus
Osaka is the 2
ndlargest city in Japan.
47
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
OF OSAKA UNIV.
49
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
Temperature (℃)
1020 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
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
2H 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
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
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
90
o0
oAmide I
Wavenumber/cm
-11800 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 0°
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
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
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
DOPC のモル比(x
DOPC)
T empera ture ( ℃ )
x
px
q1. 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
α2domain (x
DOPC=x
q)
L
α2domain
(x
DOPChomogeneous L =x
qDOPC vesicle )
α2domain
58
DHMS相挙動のDOPC依存性
Molar fraction of DOPC ( x
DOPC)
Temperature (℃)
DHSM/DOPC
(Mason, 1988)
Temperature (℃)
DSPC/C
18C
10PC
(Wu & McConnel, 1975)
Temperature (℃)
DEPC/DPPE
59
DHSM/DOPC 系の相分離は珍しい例
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
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
0.0 0.2 0.4 0.6 0.8 1.0 -20
0 20 40 60
-20 0 20 40 60
62
x
cholParti 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.
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
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)