X-ray and Neutron diffraction studies of lipid bilayers

1. X-ray and Neutron diffraction studies of lipid bilayers V A Raghunathan Raman Research Institute, Bangalore

2. Phospholipids Phosphatidylcholine (PC) Major component of cell membranes Amphiphilic molecules Self-assemble to form bilayers Critical micellar concentration (CMC) ~ 1 n M

3. Morphologies of lipid bilayers Unilamellar vesicles (ULV) Multilamellar vesicles (MLV) liposomes Multilamellar stacks (on a substrate)

4. Phase diagram of DPPC-water Janiak et al., Biochemistry 15 4575 (1976) Chain melting transition

5. Diffraction geometries 1. Unaligned samples (MLV) 2. Multilayers on a substrate Geometric corrections

6. The fluid phase Occurs above the chain melting transition One dimensional periodicity Liquid-like in-plane order d =d/f , d - bilayer thickness f - lipid volume fraction

7. The gel phase phase – no chain tilt phase – tilted chains No trans-bilayer correlation of tilt direction

8. Phase diagram of hydrated DMPC NNN Arb. NN Smith et al., Phys. Rev. Lett. 60 813 (1988)

9. The sub-gel phase Occurs below the gel phase on long incubation Slow transition kinetics Appearance of a few additional peaks in the diffraction pattern Molecular superlattice Advantage of oriented samples VAR & J Katsaras Phys Rev Lett (1995)

10. Intensity of the scattered beam Structure factor Form factor density-density correlation function

11. 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

12. Paracrystalline model (A. Guinier) Stack of parallel layers with mean separation D mean square fluctuation – Uncorrelated fluctuations Tails Decreasing peak height with increasing order

13. Thermal fluctuations in the lamellar phase (de Gennes & Prost; Chaikin & Lubensky) Density Fluctuations in the phase Normal modes - equipartition of energy

14. Landau – Peierls instability No long-range order Power-law decay of correlations – quasi-long-range order

15. The structure factor Caille, C.R. Hebdo. Acad. Sci. Paris (1972) Approximate relation valid far from the peaks = 0, 0.1, 0.2 Nallet et al., J. Phys. II (1993) Broadening – resolution function - finite size

16. Unoriented (powder) samples Rounding due to finite size Power-law decay Safinya et al., Phys. Rev. Lett. (1986)

17. A better approximation for S(q) Zhang et al., Phys. Rev. E (1994)

18. 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)

19. 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)

20. Modeling I(q) Calculate S(q) and f(q) from models Model parameters from the best fit Pabst et al., Phys. Rev. E (2000)

21. Determination of K and B Oriented samples Parameters In-plane correlation length ~ K/B Lyatskaya et al., Phys. Rev. E (2000)

22. The ripple phase

23. Electron density map of the ripple phase Vary the model parameters to get the best fit with observed data Center of symmetry – phases 0 or p Calculated phases, observed magnitudes Packing of chains in the bilayer? Sun et al., PNAS (1996); Sengupta et al. Phys. Rev. Lett. (01)

24. 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

25. The “bicelle” mixture Mixtures of long-chain and short-chain lipids: DMPC-DHPC Different morphologies preferred by the two DMPC – bilayersDHPC – micelles Leads to novel behavior of the mixtures DMPC DMPC DHPC Used for orienting macromolecules in High-resolution NMR studies Sanders and Prosser, Structure 6, 1227 (1998) DHPC Bicelle – disc-like micelle

26. The Magnetically Alignable Phase Raffard et al, Langmuir 16, 7655 (2000) DMPC-DHPC Phase diagram from NMR Ф = 20 wt % I - isotropic B - ? Aligns in a field L – fluid lamellar

27. Bicelles Dilute solutions Below chain melting transition Nieh et al., Biohys J. (2001)

28. Monodisperse unilamellar vesicles Very dilute solutions Above chain melting transition Nieh et al., Langmuir (2001)

29. ULV 55 45 T(oC) Bilayers 35 25 10 Bicelles 0.15 0.05 0.25 0.0025 0.01 0.1 Lipid Con. (g/mL) Phase behaviour – dilute regime M.-P. Nieh, et al. Biophys. J., 82, 2487 (2002) Charged ‘bicelle’ mixture - DMPC+ DHPC+ DMPG

30. Concentrated solutions [DMPC]/[DHPC] = 3.2 I (q) ~ |f (q)|² S(q) Porod’s law Linear aggregate: |f (q)|² ~ q -1 High viscosity - ribbons (worm-like micelles) Bicelles (disc-like micelles) Nieh et al., Biophys. J. 82, 2487 (2002)

31. The phase diagram From microscopy and SANS No bicelles at higher T Nematic phase of ribbons - high viscosity - magnetic field induced alignment [DMPC]/[DHPC] = 3.2 M.-P. Nieh et al., Langmuir (2004)

32. Antimicrobial peptides in bilayers Alamethicin – 20 amino acid peptide - produced by a fungus Amphipathic – hydrophilic on one side and hydrophobic on the other Brogden, Nature (2005)

33. SANS studies of pores in bilayers In-plane scattering Solvent – heavy water He et al., Biophys. J. (1996)

34. The form factor He et al., Biophys. J. (1996)

35. The structure factor Lipid /peptide ~ 10 Determined from simulations

36. Effect of contrast variation He et al., Biophys. J. (1996)

37. The structure of the pore