ASIAA/CCMS/IAMS/LeCosPA/NTU-Phys Joint Colloquia
2015.4.28
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
Biomembrane comprise diverse lipids and
proteins and form complex structures
without proteins
2
Contents
I Model Membrane of Lipid Rafts
Sphingomyelin and Cholesterol Binary System
II Domain Formation in Membrane
Sphingomyelin and Phosphatidylcholine System
III Raman Imaging of Raft Model Membrane
Development of new Raman Tagged Sphingomyelin
3
Approaches towards membrane lipids with
variable time and spacious scales
Our objective: Elucidation of the molecular
basis of lipid raft formation
biomembrane
Solid state NMR
(2H NMR, REDOR)
Molecular level
10 μs – 10 ms time scale
Raman Imaging
Ternary
system
Unary and binary
systems
Macroscopic membrane
Fluorescent lifetime
Molecular level
several ns time scale
Artificial lipid membranes
Cho
SM
Lipid behavior in various membranes
(mobility and intermolecular interaction)
3
Lipid rafts
GPI-anchored
protein
Glycosphingolipid
Sphingomyelin (SM, SSM)
Cholesterol (Cho)
Transmembrane
protein
・Resistance to solubilization with Triton X-100 (DRM)
・Ordered lipids (Lo phase) undergoes domain formation
・Implication in many cellular processes
(signal transduction etc.)
Molecular basis of
lipid raft formation
1) Simons,K.; Ikonen,E. Nature, 1997, 387, 569-572. 2) Pike, L. J. J. Lipid Res. 2006, 47, 1597-1598.
？
5
How can we elucidate the conformatoin
and interactions of lipids in membrane?
Lipid-lipid & lipid-protein
interaction in membrane
Drug-membrane interaction
Elucidation of 3D structures
and interactions of lipids
in membrane is essential
But…
Lipid bilayer membranes
Difficulties in structure elucidation
× X-ray Crystallography
△ Solution NMR
Solid state NMR
works in such weird systems ?
6
Micelles vs Bicelles
for membrane mimic
Micelles
Detergent
Sterol
・Many examples
・High curvature
Bicelles
Phospholipid
Detergent
・Bilayer-like structure
・Strick conditions;
temp. & conc.
7
Ordering of SM-d2 bound
in Cho-containing large bicelles
B0
B0
33.6 kHz
35.3 kHz
+ Chol
10’-d2-SM/DHPC(4/1)
10’-d2-SM/DHPC/Chol (4/1/0.4)
10% Cho siginificantly ehnaces the ordering of SM bicelle
membranes
8
Size-dependent orientation of
bicelles along magnetic field
O
O
N
O
O
P
O
O
O
O
DHPC (short chained FA)
Stearoyl SM (major constituent)
Small Bicelles
・ Planar bilayer structure
・Non-orientation along B0
q > 2.0
q < 2.0
q = [SSM] / [DHPC]
High mobility of small
bicelles enables high
resolution NMR spectra
even for 1H nucleus.
9
1H
5 4
NMR NOESY of bicelles under MAS
a 1,3 b
2
g
2’ 6
SM liposome
SM/DHPC (1/2) bicelle
a
MAS: 5 kHz, Mixing time: 30 ms, Temp.:37 ºC
10
Conformation of SM head group deduced
from NOEs and J coupling in small bicelles
NOEs are similar between the
Cho-containing and Cho-free
bicelles
Conformation of SM is
similar between pure SM
and SM-Cho
11
Yamaguchi, T. Suzuki, T., Yasuda, T. Oishi,T., Matsumori, N., Murata, M. Bioorg. Med. Chem. 20, 270-278 (2012).
How REDOR works I
⊿S/S 0
1.0
15N
0
0
4
8
12
16
NCTr (ms)
20
Synchronous irradiation
to magic angle spinning
S0
15N
r
13C
d
－
S
DS
＝
13C
15N-non-
15N-irradiated
REDOR Decay
irradiated
Accuracy is <0.1 Angstrom !
T. Gullion, J. Schaefer, J. Magn. Reson., 1989, 13, 57
*REDOR data; A. Naito, et al., J. Phys. Chem. 1996, 100, 14995
12
How REDOR works II
MAS (Magic Angle Spinning): S0
Magnetic field
Strength
Dipole Coupling
gI gS h m0
D=
16 3 r3
Integration ＝ ０
15N
DS
1
2
1 [ J 0 ( 2nt r D )] 2
[ J k ( 2nt r D )]2
2
S0
k 1 16k 1
REDOR: S
13Magnetic
31
field
⊿S/S 0
Cが Pから
Strength
受ける磁場
N
2
1.0
S
4
2
S
3
B0
Magic
Angle
N
N
1
O
S
S
13
C
r
180
180°
パルス
Pulse
4
N
1
1
3
Tr
Integration > ０
t
0
0
4
8
12
16
NCTr (ms)
20
Jn: Vessel Function, ntr: REDOR dephasing time gI: Gyromagnetic Ratio of I nucleus. gS: That of S nucleus
h: Plank const., m0: Permeability of Vacuum
Gullion, T. et al. Adv. Magn. Reson. 13, 57 (1989); Gullion, T. et al. J. Magn. Reson. 89, 479 (1990).
13
13C{15N}REDOR
DC N
used for evaluation of mobility
and orientation
3 cos 2 q 1
D0 Smol 2
Wobbling Smol
Dipole coupling of
rapidly moving mol.
depends on
Mol. axis angle q
Wobbling Smol
θ
14
REDOR data for 13C-15N-labeled SM
in SM/Chol and SM only membranes
Mol. axis
Wobbling S
θ
DC-N=265 Hz
13C
15N
DC-N=150 Hz
C1’
1’-13C,2-15N-SM
(D0=1302 Hz)
SM/Cho: DC-N=265 Hz
SM only: DC-N=158 Hz
Non-irr. S0
Irradiated S
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.
15
REDOR reveals Smol and orientation
for amide bond
Not only conformation but orientation
is not affected by Cho. Difference is in mobility
C1’ / 15N
C2’ / 15N
C1 / 15N
C2 / 15N
C3 / 15N
1302
232
233
1073
229
DC-N SM/Chol
265
12
63
82
33
DC-N SM only
158
2
69
55
48
D0 (Hz)
DC N
θ
3 cos 2 q 1
D0 Smol 2
Major conformer in SM-Chol: Smol: 0.94, (a, b) = 166o, 32o
Major conformer in SM only : Smol: 0.70, (a, b) = 158o, 35o
16
Cho ordering effect and orientation lead to
intermolecular H-bonds
●
H-bonding
Formation
Lo domain
(raft-like)
SM/Chol
Disruption
17
Motion capture of alkyl chains of membrane
lipid by 2H NMR
Quadrupolar
Compling
Small
Δνmax
63.8 kHz
Rotation axis
Dν
Mobility
Large
Molecular motion capture
Perdeutero-acyl chain
Palmitoyl-sphingomyelin (PSM)
Library of site-specifically 2H-labeled SM
6
Synthesis of 2H-labeled fatty acids
19
Synthesis of 2H-labeled SM
20
Depth-dependent order of SM by 2H NMR
60
大
Q splitting Dn (kHz)
Mobility
小
Raft (SM/Chol =1/1)
45℃
60
50
50
40
40
30
30
20
Non-Raft
(100% SM)
20
10
10
0
0
1 2 3 4 5 6 7 8 9 101112131415161718
10 12 14 16 18
carbon number
<Sphingosin chain>
Raft
(SM/Chol =1/1)
45℃
Non-Raft
(100% SM)
1 2’
2 33’ 44’ 5 6’
6 7 88’ 9 10
14 15 16
10’11 12
12’13 14’
16’17 18’
carbon number
<Acy chain>
Cho cyclic core
Both alkyl chains
interact with Cho
similarly.
Matsumori, N.; Yasuda, T. et al. Biochemistry 2012, 51, 8363-8370.
21
Contents
I Model Membrane of Lipid Rafts
Sphingomyelin and Cholesterol Binary System
II Domain Formation in Membrane
Sphingomyelin and Phosphatidylcholine System
III Raman Imaging of Raft Model Membrane
Development of new Raman Tagged Sphingomyelin
22
Comparison between SM and PC
Both SM and saturaed PC are known to form Lo domains
SM
PSPC
What is the difference between SM and
PC in formation of Lo domains.
Systematic comparison between SM and saturated PC
Space ： Atomic ～ Molecular ～Entire membrane
Time ： nanosecond ～ millisecond
Lipid constituents : Unary system ～ Ternary system
The structure properties make SM preferentially form lipid rafts
23
Comparison of chain mobility between SM and PSPC
60
大
Q splitting Dn (kHz)
Mobility
小
60
Raft
(SM/Chol =1/1)
50 ℃
50
50
40
40
30
30
Non-Raft
(100% SM)
20
20
10
10
0
0
1 2’
2 3’
3 44’ 5 6’
6 7 8’
8 9 10’
10 11 12
16 17 18’
12’13 14
14’15 16’
carbon number
＜ SM-Chol＞
<SM acyl chain>
Raft
(PC/Chol =1/1)
50 ℃
Non-Raft
(100% PC)
1
2
3
4
2’ 3’ 4’
5
6
6’
7
8
8’
carbon number
9 10 11 12 13 14 15 16 17 18
10’
12’
14’
16’ 18’
<PSPC acyl chain>
＜PSPC-Chol＞
The rigid tetracycle of Cho is located more deeply in SM membrane
Probably due to hydrogen bond network by amide groups of SMs
24
1) Matsumori, N.; Yasuda, T. et al. Biochemistry 2012, 51, 8363-8370. 2) Yasuda, T. et al. Biophys. J. 2014, 106, 631-638.
Temperature dependent ordering of SM and PC
at low Cho concentration
60
60
55
50
45
● 10’,10’-d2-SM - Cho
● 10’,10’-d2-PSPC - Cho
40
35
15
20
25
30
35
40
45
Temperature (℃)
33 mol% Cho
Q splitting Dn (kHz)
Q splitting Dn (kHz)
20 mol% Cho
50
55
55
50
45
● 10’,10’-d2-SM - Cho
● 10’,10’-d2-PSPC - Cho
40
35
15
20
25
30
35
40
45
Temperature (℃)
50
55
SM-Cho membrane is more tolerant to temperature change than PC-Cho membrane.
(Lesser temperature dependence)
Higher thermal stability
SM intermolecular H-bond (membrane surface)
+
Cho ordering effect (membrane interior)
Yasuda, T. et al. Biophys. J. 2014, 106, 631-638.
25
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
Gel phase
Lo phase
Ld phase
Intensity [Counts]
104
Example ：SSM+33 mol% Cho
30 ℃
103
Measuring
data
102
101
100
0
40
80
120
160
200
240
280
320
360 400
time [ns]
Fluidity Low
Lifetime Long
High
Short
※ Deconvolution by two lifetime
components
𝑛
α𝑖 exp −𝑡/τ𝑖
I (t) =
𝑖=1
= α1exp (-t/τ1) + α2exp (-t/τ2)
trans parinaric acid (tPA)
λex = 295 nm, λem = 405 nm
2 mol% of
total lipids
τ1 , τ2 … lifetime of each component
α1 , α2 … fractional amplitudes of
each component
26
Membrane fluidity on nanosecond time scale
SM/33 mol% Cho
lifetime τ (ns)
70
The fluidity of phase state : Gel ＜ Lo ＜ Ld
Lo + Ld
Cho-poor gel-like + Lo
60
No gel phase exists above Tm.
50
40
30
Over Tm
(> 45 ℃)
Lo domain
Ld domain
Under Tm
(< 45 ℃)
Cho-poor
gel-like
domain
Lo domain
20
10
0
15
20
25
30
35
40
45
50
55
Temperature (℃)
τ１ ● ● Longer lifetime
→ Lower fluid domain
τ2 ● ● Shorter lifetime
→ Higher fluid domain
Gel phase
Membrane heterogeneity
NMR cannot detect the coexistence of domains.
Lipid cluster with short lifetime
Low concentration of Cho →
Similar behavior with gel phase
27
Hypothetical model for interconversion of nano-domains
Below Tm
(< 20 ℃)
Gel-like domain
Lo domain Gel-like domain
Lo domain
Gel-like domain
Lo domain
～100 ns 1)
Above Tm
(> 49 ℃)
Ld domain
Lo domain
1) Chachaty, C. et al. Biophys. J. 2005, 88, 4032-4044.
Ld domain
Lo domain
Ld domain
Lo domain
30
Difference in dynamic behavior between SM and
PC in Cho-containing binary systems
PSPC
SM
2H
NMR
Lipid mobility
at atomic level
Fluorescent
lifetime
Membrane fluidity in
nanosecond time domain
Present
Hydrogen bond
network
Absent
Deeper
Location of Cho
Shallower
Temperature dependence of
lipid ordering
Larger
Smaller
Lo domain-forming
ability Lower
Higher
These data
suggest
that SM-SM
H-bonding plays major roles rather than
Coexistence of cho-poor clusters with short lifetime
SM-Cho interaction.
29
Can SM form macroscopic domains without Cho？
SM: Sphingomyelin (C18)
DHSM: Dihydrosphingomyelin (C18)
a)
eSM
a) SM
c)
DHSM
b) DHSM
vs
b)・Major
tSMSM in human
・Raft model lipid
d) tripleSM
・Relatively abundant SM homologues
・Form more stable Lo domains than SM
DOPC： Unsaturated PC, a typical Ld lipid in the presence of SM and Cho
Kinoshita, M., Goretta, S., Tsuchikawa, H., Matsumori, N., Murata, M., Biophysics 9, 37-49 (2013).
30
DHSM forms macroscopic domains without Cho
DHSM
SM
Temp (℃）
Temp (℃）
Uniform
Uniform
Phase separated
Phase separated
Mol. Ratio of DOPC
Mol. Ratio of DOPC
DHSM-rich
DHSM
DOPC-rich
H-bond
Pure
+DOPC
Mixted
Separated
Mixed again
Kinoshita, M., Matsumori, N., Murata, M. Biochim. Biophys. Acta 1838, 1372-1381 (2014).
31
Approaches towards Membrane Lipids with
Variable Time and Spacious Scales
Elucidation of the molecular basis of
lipid rafts formation
biomembrane
2H
solid state NMR
(2H NMR)
Molecular level
10-1000 μs time scale
Raman Imaging
Entire membrane level
Ternary
system
Fluorescent lifetime
Several ns time scale
Unary and binary
systems
Artificial lipid membranes
SM
DOPC
Cho
34
Domain separation of ternary SM/Cho/DOPC
as observed by microscope and 2H NMR
Solid state 2H NMR
Fluorescence microscope
GUV Sample : SM/Cho/DOPC (1/1/1)
+ 0.2 mol% Bodipy- PC (λex = 488 nm)
T : 30 ℃
10’,10’-d2-SM
51.5 kHz
30 ℃
36.0 kHz
Ld-specific fluorescent dye
SM rich ラフト相
(Lo 相)
DOPC rich 液晶相
(Ld 相)
GUV of SM/Cho/DOPC (1/1/1)
10’,10‘-d2-SM/Cho/DOPC (1/1/1) の2H
NMRスペクトル
Two pairs of doublets
33
Fractional abundance of Ternary SM/Cho/DOPC
system as revealed by 2H NMR
Lo Domain
Ld非ラフト相
Domain
34
Depth-dependent order of Lo and Ld domains
in SM-Cho-DOPC system
Lo domains of ternaryCarbon
and number
binary systems showed similar ordering
↓
Occurrence
SM-only
domains
evenN. in
ternary
systems
Yasuda,of
T., Kinoshita,
M., Murata,
M., Matsumori,
Biophys.
J. 106, 631-638
(2014). 35
Contents
I Model Membrane of Lipid Rafts
Sphingomyelin and Cholesterol Binary System
II Domain Formation in Membrane
Sphingomyelin and Phosphatidylcholine
PhosphatidylChoine System
III Raman Imaging of Raft Model Membrane
Development of new Raman Tagged Sphingomyelin
36
Labelled lipids for fluorescence spectroscopy
do not reproduce original lipids due to balky
substituents
Fluorescence Imaging
Raman Imaging
Small Raman tag
37
Small Raman tags of SM for imaging
O
R
O
OH
O
P
O
HN
O
R:
N
D3C
HO
N
N
SM
alkyne-SM (1)
diyne-SM (2)
D3C
N
CD3
SM-d9 (3)
Goretta, S. A., Kinoshita, M., Mori, S., Tsuchikawa, H., Matsumori,N., Murata, M.
Bioorg. Med. Chem. 2012, 20, 4012-4019.
38
Diyne moiety shows strong intensity in
background-free area
Triple bond
Diyne
Deuterated
Cui, J., Lethu, S., Yasuda, T., Matsuoka, S., Matsumori, N., Sato, F., Murata, M. Bioorg. Med. Chem. Lett.
25, 203-206 (2015).
39
Diyne SM shows similar behavior to
original lipid on 2H NMR
d-Cho
d2-SM vs Diyne-d2-SM
2H
NMR Spectra
DOPC
SM
SM
Diyne-SM
(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
(a)d2-SM/DOPC/Cho (1/1/1 mol), and
(b) d2-diyne SM/DOPC/Cho (1/1/1 mol) at 25 oC.
Cui, J., Lethu, S., Yasuda, T., Matsuoka, S., Matsumori, N., Sato,
F., Murata, M. Bioorg. Med. Chem. Lett. 25, 203-206 (2015).
40
Diyne probe mimics SM in Lo domains
on supported monolayer
１０mm
=
Bodipy-PC
ジイン-SM
Quartz-supported monolayer Diyne-SM/Cho/DOPC (1/1/1 mol)
41
Concentration graduation of SM revealed
by Raman imaging
Raman Image of diyne-SM
0
Monolayer of diyne-SM/DOPC/Cho (1:1:1)
2
4 mm
42
Summary
Site-selective 2H labeling precisely discloses depthdependent mobility of alkyl chains of SM and PC in Lo and Ld
membranes
Intermolecular hydrogen-bonds play a key role in SM-SM
interaction, which may lead to formation of raft-like Lo
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
A
B
C1
C2
44
大阪大学理学研究科
化学専攻
JST ERATO, 理研
（九州大学教授）
Dr. Sodeoka
Å bo Akademi Univ. (Finland)
Prof. Matsumori
Prof. Slotte
Dr. Yasuda
Dr. Jin Cui
Dr. Yamaguchi
45
Thank you for your attention
46
Osaka is the 2nd
largest city in Japan.
Our Campus
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
SM-Cho interaction results in stable SM-SM
hydrogen bond formation while SM-Cho
affinity is not high
Hydrogen Bond
Lo phase
association
(raft model)
SM/Cho
dissociation
Ld phase
(non-raft)
SM 100%
50
Lo domain-forming ability in SM and PSPC memb.
70
SM
60
lifetime τ (ns)
lifetime τ (ns)
70
50
40
30
20
PSPC
60
50
40
30
Lo domain
20
Lo domain
10
10
15
20
25
30
Temperature (℃)
35
40
15
▲ SSM (Gel phase) ● SSM-33 mol%Cho
Cho-poor gel-like domain
25
30
35
Temperature (℃)
Lo domain
45
・
・
＞
・
40
▲ PSPC (Gel phase) ● PSPC-33 mol%Cho
Cho-poor gel-like domain
The affinity
with Cho
・
Cho-poor gel-like domain
20
Gel phase
Cho-poor gel-like domain
SM has a higher Lo domain-forming ability.
51
Temperature dependence of mean lifetime
in SM and PSPC membrane containing Chol
Mean lifetime : the weighted average of fluidity in the bilayer on nanosecond time domain
60
60
50
50
Mean lifetime (ns)
Mean lifetime (ns)
20 mol% Chol
40
30
20
● SSM-Chol
● PSPC-Chol
10
0
15
20
25
33 mol% Chol
40
30
20
● SSM-Chol
● PSPC-Chol
10
0
30
35
40
45
Temperature (℃)
50
55
15
20
25
30
35
40
45
50
55
Temperature (℃)
Similar behavior to the data from 2H NMR
The decreasing degree of lifetime in SM membrane is smaller with increasing temperature.
The local mobility of acyl chain in phospholipids is closely correlated
to the entire membrane order.
52
ERATO脂質活性構造プロジェクト
本研究の３つの目標
膜タンパク質周辺脂質
(around protein)
３つグループで協力
固体NMR, 結晶X線回折,
合成化学, XAFS
周辺脂質の立体構造と機能
脂質活性構造
異分野融合の必要性
タンパク質内部脂質
杉山G(in
と protein)
松岡Gが担当
マイクロドメイン
主に村田Gが担当
(as a field)
結晶X線回折, 固体NMR,
カロリメータ, 計算機科学
脂質リガンドとの相互作用
Spring-8, SACLA,
固体NMR, 化学合成,
表面プラズモン共鳴
物理化学計測,
脂質集合体の分子基盤
共焦点顕微鏡,
ラマンイメージング
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
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
54
偏光減衰全反射赤外分光法（pATR-FTIR)
Z
YTX含有または非含有
GpA-TM再構成重水和DMPC膜
θ1
θ2
αα1
α
α
2
エバネッセント波
0o
x
45°
90o
赤外入射
ATRプリズム（Ge）
検出器
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
偏光フィルター
例：
二色比： R ＝ΔA 90° /ΔA 0°
Abs./a.u.
Amide I
90o
0o
1800
1700
1600
Wavenumber/cm-1
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
55
リン脂質膜中でYTXによるGpA-TMの配向変化の解析
配向
脂質分子DMPCのアシル鎖
GpA-TMのαヘリックス軸
CH2対称伸縮振動
アミドⅠ
（αヘリックス）
バンド
試料名
α (°)
重水和
GpATM-DMPC
(1:50)
27.30
重水和
GpATM-DMPCYTX (1:50:1)
YTX
26.84
重水和
GpATM-DMPC
(1:50)
30.61
重水和
GpATM-DMPCYTX (1:50:1)
33.23
￮ 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。
GpA二量体
GpA単量体 GpA-YTX複合体
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
56
細胞膜内ステロールと相互作用する天然物
TMN
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
DHMS相挙動のDOPC依存性
仮定
1.
2.
DHSMは強い分子間水素結合を形成する(H-bonds).
DHSM はDOPCより大きな曲率をもつ膜を形成する
DHSM vesicle
xq
Temperature (℃)
xp
Lα2 domain
attenuation
(xDOPC=xq)
of H-bond
DOPC
add
DOPC のモル比(xDOPC)
Lα2 domain
vesicle
homogeneous
Lα2 domain
(xDOPC
=xqDOPC
)
58
DHSM/DOPC系の相分離は珍しい例
DEPC/DPPE
Temperature (℃)
Temperature (℃)
DSPC/C18C10PC
(Mason, 1988)
(Wu & McConnel, 1975)
Temperature (℃)
DHSM/DOPC
Molar fraction of DOPC (xDOPC)
59
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？
SM: Sphingomyelin (C18)
a) SM
DHSM: Dihydrosphingomyelin (C18)
a) eSM
a) eSM b)
c) DHSM
DHSM
c) DHSM
c)
b) tSM
tSM
d) tripleSM
tripleSM
d)
vs
b) tSM
d) tripleSM
・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
Kinoshita, M., Goretta, S., Tsuchikawa, H., Matsumori, N., Murata, M., Biophysics 9, 37-49 (2013). 60
SM誘導体の物理学的膜物性の測定
v (mL/g)
1.02
1.03
a)
a SM
1.02
v (mL/g)
1.03
1.01
1.00
0.99
1.03
0.1
0.2
xchol
0.3
0.4
0.98
0.0
0.5
1.03
c)b tSM
0.1
0.2
xchol
0.3
0.4
0.5
0.4
0.5
d) tripleSM
d
1.02
1.01
1.00
1.01
1.00
0.99
0.99
0.98
0.0
1.00
0.1
0.2
0.3
0.4
0.98
0.0
0.5
0.1
0.2
x chol
xchol0.3
1270
1250
ｘ： tripleSM
VPMVSM (Å3)
v (mL/g)
1.02
1.01
0.99
v (mL/g)
0.98
0.0
c DHSM
b)
1230
□：
1210
DHSM
SM
△： tSM
〇：
1190
1170
0
0.1
0.2
0.3
xchol
0.4
0.5
0.6
61
60
40
40
20
20
0
0
-20
0.0
-20
1.0
Partial molecular area
of cholesterol (Å2)
60
0.2
0.4
0.6
0.8
xchol
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 CSM-1 (mN/m)
at 5 mN/m.
diyneSM
SSM
xchol=0
(LE phase)
xchol≧0.5
(ordered phase)
33±10
47±10
130±20
120±20
62
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
ラフト相にSMとCholが多く分配
SM分子間水素結合のネットワーク
相分離が高温まで安定に保持
SM
SM
Ld ドメイン (周辺膜) Lo ドメイン (ラフト膜)
膜界面
ナノ秒スケールのラフト相内部
DOPC
短寿命の
脂質クラスター
(SM/Chol)
速い生成と崩壊を繰り返す
Chol
膜の深い位置に
分布するChol
Chol
Chol のステロイド骨格に
よるオーダー効果
アルキル鎖中央部へのオーダー効果最大
16
位置選択的重水素標識PSPCの合成
Stearoyl-sphingomyelin (SSM)
アシル鎖長 : C18
相転移温度(Tm) : 44 ℃
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(Tm) : 48.8 ℃
8
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