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