Determining Structure and Composition of a Model Phospholipid Bilayer ORANGE TEAM Monica Branco Suh...
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Transcript of Determining Structure and Composition of a Model Phospholipid Bilayer ORANGE TEAM Monica Branco Suh...
Determining Structure and Composition of a Model Phospholipid Bilayer
ORANGE TEAMMonica BrancoSuh Joon Han
Matthew HelgesonKathryn KryckaJong Keun ParkChu Xiangiang
Phospholipid Bilayers
• Phospholipid bilayers serve as biomimetic membranes
• Lipids self-assemble into ordered, multi-layer structure
• Our system: DMPC– Phospho-choline head group
– C14 hydrocarbon chains
Advanced Neutron Diffractometer/Reflectometer
• Reflection of incident beam based on Bragg diffraction:
Slit #2Slit #1
Slit #3
Slit #4
monitor
monocromator
sample
detector
sin2dn
θ2θ
ki kr
kt
Qz
Experiment Objectives
• Determine quantitative structural information
– Bilayer thickness
– Bilayer composition profiles
– Solvent penetration
– Extent of thermal fluctuation
Sample Preparation• Deposition of lipid solution on glass slide• Solvent evaporation• Hydration of bilayer film in sample chamber (75% RH)• Allow for sample equilibration at 37ºC
I(Q)
Q (Å-1)Glass slide
Determination of Bilayer Spacing
• Phase problem:
2/12sin nn IF )2
cos()(2
0)( n d
nznF
dz
100% H2O50/50 H2O/D2O100% D2O
I(Q)
Q (Å-1)
n=1
n=2
n=3
n=4n=5
Phospholipid bilayer spacing:d = 49.3 Å
nQ
nddn
2sin2
Solving the Phase Problem
• Variation of solvent conditions: H2O D2O
100% H2O50/50 H2O/D2O100% D2O
I(Q)
Q (Å-1)
Scattering Length Density Profiles
• SLD profiles for lipid C-H chains:
-2
-1.5
-1
-0.5
0
0.5
1
1.5
2
2.5
0 10 20 30 40 50
Z (A)
SL
D
100:0 H2O:D2O
)2
cos()(2
0)( n d
nznF
dz
Bilayer Composition Determination
• Selective deuteration of solvent and lipid:
Hydrogenated Hydrocarbon DMPC lipid bilayer:SLD Profile
-4
-3
-2
-1
0
1
2
3
4
0 10 20 30 40 50
Z (A)
SL
D
100:0 H2O:D2O50:50 H2O:D2O0:100 H2O:D2O
Hydrogenated vs Deuterated DMPC Lipid Chains
-2
-1
0
1
2
3
4
5
6
0 10 20 30 40 50
Z (A)
SL
D
Hydrogenated Hydrocarbon Lipid
20% Deuterated Hydrocarbon Lipid
Water and Hydrocarbon Profiles
• Subtraction of profiles allows for identification of species profiles:
DMPC Lipids: Species SLD Profiles
-1
0
1
2
3
4
5
6
0 10 20 30 40 50
Z (A)
SL
D
Hydrocarbon
Water
i
li V
b
VCH2=27 Å3
VCH3=54 Å3
Conclusions
-1
0
1
2
3
4
5
6
0 10 20 30 40 50
Z (A)
SL
D Hydrocarbon
Water
• AND/R allows for detailed, quantitative structural and compositional characterization of biomimetic bilayers
– Solving the phase problem
– Selective contrast variation
• Further experiments
– Site-specific D-labeling
– Protein incorporation