X-ray and Neutron diffraction studies of lipid bilayers
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Transcript of X-ray and Neutron diffraction studies of lipid bilayers
X-ray and Neutron diffraction studies of lipid bilayers
V A Raghunathan
Raman Research Institute, Bangalore
Phospholipids
Major component of cell membranes
Amphiphilic molecules Self-assemble to form bilayers
Critical micellar concentration (CMC) ~ 1 n M
Phosphatidylcholine (PC)
Morphologies of lipid bilayers
Unilamellar vesicles (ULV)
Multilamellar vesicles (MLV) liposomes
Multilamellar stacks (on a substrate)
Phase diagram of DPPC-water
Janiak et al., Biochemistry 15 4575 (1976)
Chain melting transition
Diffraction geometries
1. Unaligned samples (MLV)
2. Multilayers on a substrate
Geometric corrections
The fluid phase
Occurs above the chain melting transitionOne dimensional periodicityLiquid-like in-plane order
d - bilayer thickness - lipid volume fraction
The gel phase
phase – no chain tilt
phase – tilted chains
No trans-bilayer correlation of tilt direction
Phase diagram of hydrated DMPC
Smith et al., Phys. Rev. Lett. 60 813 (1988)
NN NNN Arb.
The sub-gel phaseOccurs below the gel phase on long incubationSlow transition kineticsAppearance of a few additional peaks in the diffraction pattern
Molecular superlatticeAdvantage of oriented samples
VAR & J Katsaras Phys Rev Lett (1995)
Intensity of the scattered beam
Structure factor
Form factor
density-density correlation function
Models for the lamellar structure factor
1D crystal
f(q) sampled at the reciprocal lattice points
bilayer - center of symmetry – f(q) real
determination of |f(q)| from swelling expts
equal weight for all reflections
Paracrystalline model
Stack of parallel layers with mean separation D
mean square fluctuation –
Uncorrelated fluctuations
Decreasing peak height with increasing order
Tails
(A. Guinier)
Thermal fluctuations in the lamellar phase (de Gennes & Prost; Chaikin & Lubensky)
Density
Fluctuations in the phase
Normal modes - equipartition of energy
Landau – Peierls instability
No long-range order
Power-law decay of correlations – quasi-long-range order
The structure factor
= 0, 0.1, 0.2
Nallet et al., J. Phys. II (1993)Broadening – resolution function - finite size
Caille, C.R. Hebdo. Acad. Sci. Paris (1972)
Approximate relation valid far from the peaks
Unoriented (powder) samples
Safinya et al., Phys. Rev. Lett. (1986)
Rounding due to finite size
Power-law decay
A better approximation for S(q)
Zhang et al., Phys. Rev. E (1994)
Electron density profiles
|F(h)| obtained from integrating the data over a q-range about the peak
Correct it by integrating S(q) over the same range
Phases from trial and error or modeling
Corrections not too important
Nagle et al., Biophys. J. (1996)
Modeling the electron density
Models with a few adjustable parameters
Their values from the best fit between calculated and observed |F(h)|
Also gives the phases
Data from different samples with differing water contents can be used
No truncation errors (Fourier wiggles)
Nagle et al., Biophys. J. (1996)
Modeling I(q)
Calculate S(q) and f(q) from models Model parameters from the best fit
Pabst et al., Phys. Rev. E (2000)
Determination of K and B
Oriented samples
Parameters
In-plane correlation length ~ K/B
Lyatskaya et al., Phys. Rev. E (2000)
The ripple phase
Electron density map of the ripple phase
Sun et al., PNAS (1996); Sengupta et al. Phys. Rev. Lett. (01)
Vary the model parameters to get the best fit with observed data
Center of symmetry – phases 0 or
Calculated phases, observed magnitudes
Packing of chains in the bilayer?
Small angle neutron scattering
I (q) ~ |f (q)|² S(q)
Systems with short-range order
High dilution S(q) ~ 1
Neutrons – scattering cross section different for isotopes contrast variation deuterated chains and solvent
The “bicelle” mixture
Mixtures of long-chain and short-chain lipids: DMPC-DHPC
DMPC
DHPC
DHPC
DMPCUsed for orienting macromolecules inHigh-resolution NMR studies
Sanders and Prosser, Structure 6, 1227 (1998)
Bicelle – disc-like micelle
Different morphologies preferred by the two DMPC – bilayers DHPC – micelles
Leads to novel behavior of the mixtures
The Magnetically Alignable Phase
Ф = 20 wt %
I - isotropic
B - ? Aligns in a field
L – fluid lamellar
Raffard et al, Langmuir 16, 7655 (2000)
DMPC-DHPC Phase diagram from NMR
Bicelles
Dilute solutions Below chain melting transition
Nieh et al., Biohys J. (2001)
Monodisperse unilamellar vesicles
Very dilute solutions
Above chain melting transition
Nieh et al., Langmuir (2001)
Phase behaviour – dilute regime
Lipid Con. (g/mL)
0.0025 0.01 0.05 0.1 0.15 0.25
ULV
Bilayers
Bicelles
T(oC)
55
45
35
25
10
Charged ‘bicelle’ mixture
- DMPC+ DHPC + DMPG
M.-P. Nieh, et al. Biophys. J., 82, 2487 (2002)
Concentrated solutions
[DMPC]/[DHPC] = 3.2
I (q) ~ |f (q)|² S(q)
Linear aggregate: |f (q)|² ~ q-1
Bicelles (disc-like micelles)
Nieh et al., Biophys. J. 82, 2487 (2002)
High viscosity - ribbons(worm-like micelles)
Porod’s law
The phase diagram
[DMPC]/[DHPC] = 3.2
From microscopy and SANSNo bicelles at higher T
Nematic phase of ribbons - high viscosity - magnetic field induced alignment
M.-P. Nieh et al., Langmuir (2004)
Antimicrobial peptides in bilayers
Brogden, Nature (2005)
Alamethicin – 20 amino acid peptide
- produced by a fungus
Amphipathic – hydrophilic on one side and hydrophobic on the other
SANS studies of pores in bilayers
In-plane scattering
Solvent – heavy water
He et al., Biophys. J. (1996)
The form factor
He et al., Biophys. J. (1996)
The structure factor
Lipid /peptide ~ 10
Determined from simulations
Effect of contrast variation
He et al., Biophys. J. (1996)
The structure of the pore