1. Laura Toppozini CAP Congress 2014 June 19, 2014 Cholesterol Structure in Lipid Rafts

2. Cholesterol in Lipid Membranes Singer and Nicholson, 1972 Simons, 1997 Mosaic model: Lipids, cholesterol, and proteins homogeneously mixed Lipid raft model: Cholesterol-rich domains What is cholesterol’s role in rafts?

3. Project Details 20,000- molecule simulations What is cholesterol’s role in lipid membranes? Can we see raft formation in a binary system? What can binary system rafts elucidate about rafts in biological membranes? Neutron experiment at Canadian Neutron Beam Centre Coarse-grained MD simulations from collaborators at University of Mainz Neutron diffraction with stacked, oriented bilayers L Toppozini, S Meinhardt, CL Armstrong, Z Yamani, N Kucerka, F Schmid, MC Rheinstadter. The Structure of Cholesterol in Lipid Rafts. Submitted, under review.

4. ~40Å 20 mol% ~100Å Liquid-ordered phase Simulation: CG Molecular Dynamics Liquid-ordered phase

5. Experiment: Neutron Scattering superresolution { nm Larger λ Smaller λ

6. Cholesterol in Lipid Membranes 32.5% cholesterol Liquid–ordered phase

7. Selective Deuteration Selectively highlight molecules

8. Cholesterol Structure

9. Liquid-Disordered Cholesterol-lipid complexes Umbrella model Huang & Feigenson. Biophys J. 1999 76(4):2142-57

10. Cholesterol Structure Cholesterol Plaques -Cholesterol bilayers/plaques -Triclinic unit cell a=b=12.8 Å α=95˚β=91.9˚γ=98.1˚ Ordered-Complexes -Ordered lipid-cholesterol complexes -Monoclinic unit cell a=b= 11Å α= 131˚

11. Cholesterol Structures in Rafts The liquid-ordered phase of a binary system is heterogeneous Domains in binary systems may be nuclei which lead to rafts in biological membranes. Small, (~10nm) transient (~100ns) structures are observed in lipid-cholesterol system Observation of three coexisting cholesterol structures in the l o phase: Liquid disordered Ordered lipid/cholesterol Cholesterol plaques

12. Acknowledgements Maikel C.Rheinstadter Clare Armstrong Norbert Kucerka Zahra Yamani Sebastian Meinhardt Friederike Schmid

14. Lipid Structure a lipid-raft b lipid-raft

15. Simulations: changing chemical potential

16. Lipid/cholesterol phase diagram

17. Membranes, Rafts, and Scattering ~3000 bilayers/wafer give larger signals Hydrated lipid bilayers have diffusive motion: signals are weak Typical neutron scattering setup gives large spatial averaging Gives bulk structure Are small structures being formed but averaged over?

18. Measurement of Rafts neutron Long coherence length ξ Short coherence length ξ wave package ξξ

19. Condensation and the Umbrella Model 0 Phospholipid head-group acts as umbrella, shields hydrophobic regions of cholesterol 0 Cholesterol proximity suppresses tail fluctuations 0 These complexes may form domains in l o phase at moderate cholesterol concentrations

20. Experiment: Neutron Scattering