脂質膜上でのナノドメインの静的および動的構造
Y. Sakuma and M. ImaiOchanomizu UniversityCell Membranes

Components of cell membrane(red blood cell)
Protein 49.2%
Lipid 43.6%
Phospholipid 32.5%
Cholesterol 11.1%
Carbohydrate 7.2%
Glycoprotein 6.7%
Glycolipid 0.5%

Lateral diffusion and electron transfer in the mitochondrial inner membrane

Cytochrome c oxidaze
M =140,000
D = 8.3 x 10-14 m2/s

Cytochrome c
M = 12,000
D =1.0 x 10-12 m2/s
C. Hackenbrock, TIBS (1981)

Saffman-Delbrück Modelηw
ηmηw

r <<rhηm h= r*ηwkBT ⎡ ⎛ ηm h ⎞ ⎤D(r ) =⎢ln⎜⎟ − γ⎥
4 πηmh ⎣ ⎝ ηw r ⎠ ⎦Lateral Phase Separation in Ternary Lipid Membrane
Unsaturated and Saturated Phospholipids
and Cholesterol
DOPCDPPC

Cholesterol
One phase region
20μm

Two phase region

S. Veatch and S. Keller,
Phys. Rev. Lett. 89, 268101 (2002).

Preparation of Nanometer-Sized Domains
Preparation ofSmall Unilamellar Vesicle[sample]Gentle Hydration MethodGVs

SUVs

60 °C

hydration

sonication

・ DPPC(saturated lipid)

・ DOPC(unsaturated lipid)

・ cholesterolEstimation of SUV size
[SANS under film contrast]

Nanometer Sized Vesicle
I (q) : Intensity
P(q) :form factor
2P(q) = 16π 2Δρ 2 R 3 f 0 (qR) − (R − δ ) 3 f 0 (q(R − δ )) q : scattering vector (= 4π sinθ /λ)Δρ : scattering length density
3f 0 (x) = (sin x − x cos x) / xf (r) : Schulz distribution of vesicle radiusp : size polydispersity ( = <R2>/<R>2 - 1 )∞I (q) = N ∫0 f (r ) P (q, r ) dr{}Intensity / cm-110010Rδ1

R = 11.2 nm

0.1
8 90.0123456 7 8 9q / Å-1

Instrument : SANS-U (ISSP, JPN)

0.1

Fitting results

δ = 4.0 nm
p = 0.3

Structure of nanometer sized domains
Matching contrast of neutron scattering[sample]One phase region(T > Tmix)

・ DPPC-d62(deuterated saturated lipid)

・ DOPC(unsaturated lipid)

・ cholesterol+_Structure of nanometer sized domains
Matching contrast of neutron scattering[sample]Two phase region(T < Tmix)

・ DPPC-d62(deuterated saturated lipid)

・ DOPC(unsaturated lipid)

・ cholesterolΔρdΔρmSANS Profiles of the SUVs at the Contrast Matching Point

0.1

44 °C

Intensity /cm-1

Tmix

0.08

26 °C
1 hour2 hour3 hour

0.06

0.04

0.02

00

0.02

0.04

0.06

q /Å-1

0.08

0.1

0.12

Nanometer Sized Domains
Temperature dependenceof SANS profiles

Molecular Dynamics Simulation
Red particle(domain) : 750
Green particle(matrix) : 750[potential]
・Lennard-Jones potential

32.5

60 °C
30 °C
20 °C
10 °C
ε22 = 3.4
ε22 = 2.6
ε22 = 3.0

Intensity / cm-12

1.5U LJ1

ε 22 =

0.5

00

0.02

0.04

0.06

0.08

0.1

q / Å-1

Instrument : SANS-U (ISSP, JPN)⎪⎧⎛ σ ⎞12 ⎛ σ ⎞ 6 ⎫⎪= 4 ε ij ⎨⎜ ⎟ − ⎜ ⎟ ⎬⎪⎩⎝ r ⎠⎝ r ⎠ ⎭⎪ε11 = ε0
ε22 = 2.6ε0 ~ 3.4ε0

ε 12 =ε1 ⋅ ε 2k BTT *ε22 = 2.6

3.0

[Model scattering function]Nρ (r) = ∑ δ (r - ri )i=1

ρ q = ∫ ρ (r)e− iq⋅r dr
S(q) =1
ρ q ⋅ ρ− qN

3.4

Monte Carlo Simulation

θcθcOOMono domainTwo domains

P(q) = ∑α,β ρα ρβ Sαβ (q)Nα N βsin(qr)Nαr
2Sαβ (q) =δαβ +4πρ∫rg(r)
dr0αβNN2qrmax

α : domain particle
β : matrix particle
Nα : number of domain particle
Nβ : number of matrix particle
N : total number of particle
gαβ(r) : partial radial distribution function

2.5

Chol-20

Intensity / cm-1

21.5
285 K
289 K
293 K
298 K
303 K
333 K

10.5

000.020.04

0.06

q / Å-1

0.080.1

dddφ d-DPPCΔρ d-DPPC + φDOPCΔρDOPC + φChol
ΔρCholΔρ d =ddφ dd-DPPC + φDOPC+ φCholΔρd-DPPC=3.36 x 1014 m-2
ΔρDOPC= -2.22 x 1014 m-2
ΔρChol= -2.31 x 1014 m-2

Dynamic Structure of Phase Separated Vesicles
S(q,t)/S(q,0) = Aexp(-D0q2t) + (1-A)exp(-(D0+Dd)q2t)
Diffusion of Vesicles
S(q,t)/S(q,0) = Aexp(-D0q2t)

20x10-3

1.0q = 0.0610 Å-1

0.90.0858 Å-10.7

Γ / ns-1

0.0727 Å-1

0.8
S(q,t)/S(q,0)Γ vs q2151050.0926 Å-1
0.6

0-10.1162 Å

0.5024681012 14x10-3

q2 / Å-20.4
01020t / ns

3040

Instrument : NG5-NSE (NIST, USA)50

D0 = (1.31 ± 0.02)x10-11 m2/s

conclusion
Using a contrast matching technique, a neutron spin echo spectroscopy and aphase separation on nanometer-sized vesicle we addressed static and dynamic
structures of nanometer-sized domains on membrane.
Nanometer-sized domains are agitated by the thermal fluctuations and thecomposition depends on the temperature.
The diffusion coefficient of nanometer-sized domains in fluid membranes is almost
constant versus the domain size, which well described by the hydrodynamic model.