1. Zoe Cournia 25 October 2004 Cholesterol vs. Ergosterol / Lanosterol in Membrane S(t)imulations

2. I.Motivation and Basics about Sterols in Membranes II.Structural Analysis of the Membrane III.Towards Understanding the Dynamics of the System … IV.Conclusions - Outlook Overview

3. The role of Cholesterol in the membrane Regulates: membrane fluidity membrane permeability lateral mobility of proteins Cholesterol ~40% in mammalian plasma membrane Ergosterol found in membranes of fungi and protozoans Lanosterol evolutionary precursor of cholesterol / ergosterol found in prokaryotes 18 enzymatic steps 9 enzymatic steps

4. Cholesterol vs. Ergosterol & Lanosterol Cholesterol 1.Saturated side chain 2.One hydrogen at C24 3.One hydrogen at C14 4.C5- C6 double bond 5.C7 – C8 single bond 6.C8 – C9 single bond Ergosterol 1.Unsaturated side chain 2.One methyl at C24 3.One hydrogen at C14 4.C5- C6 double bond 5.C7 – C8 single bond 6.C8 – C9 double bond Lanosterol 1.Unsaturated side chain 2.On hydrogen at C24 3.One methyl at C14 4.C5 – C6 single bond 5.C7 – C8 double bond 6.C8 – C9 double bond

5. Determining why nature needs sterols  Why did nature select cholesterol for eukaryotic cells?  What is the role of cholesterol in plasma membranes?  Quasielastic neutron scattering (QENS) + MD simulations  dynamics of similar sterols in membranes (cholesterol, lanosterol, ergosterol)   structure - function relationships of cholesterol + lipids

6. Nielsen, Europhys.Lett., (2000) 52:368-374 Sterol-Lipid Phase Diagram (a)lipid-cholesterol: stable region of coexistence between ld-lo phases at high concentrations  liquid-ordered phase (lo) (a)lipid-lanosterol: no lo formation  Cholesterol stabilizes the liquid-ordered phase

7. Systems + Simulation Details Cholesterol/DPPC - 28ns production run General Characteristics - PME, NPT - 200 DPPC, 1600 H 2 O - T = 309 K, P = 1atm - 2ns equilibration (A) DPPC (B) Cholesterol (C) Ergosterol (D) Lanosterol Ergosterol/DPPC - 10ns production run Lanosterol/DPPC - 1ns production run Pure DPPC - 10ns production run

8. Snapshots of the Lipid and Sterol in the Membrane Chol - DPPC Erg - DPPCLan - DPPC

9. Deuterium Order Parameters In Simulation: In NMR: C - H z-axis  Pure DPPC Chol/DPPC, Erg/DPPC, Lan/DPPC

10. Electron Density Profiles X-Ray Franks, Chol-Dppc 40%mol, T = 24C h pure-DPPC = 46.0 Å h CHOL-DPPC = 50.0 Å h ERG-DPPC = 50.0 Å h LAN-DPPC = 49.0 Å h Chol/DPPC, Erg/DPPC, Lan/DPPCPure DPPC

11. Smondyrev et al. (simulation): 10.6º (Chol-DMPC, 50%mol., T=50C) Smondyrev et al. (simulation): 22.2º (Chol-DMPC, 11%mol., T=50C) Cholesterol / Ergosterol / Lanosterol Tilt Angle Avg. chol. tilt angle: 9.8°  5.1 Avg. erg. tilt angle: 8.6°  4.3 Avg. lan. Tilt angle: 15.5°  12.2

12. Radial Distribution Function Cholesterol OH – water O: 3 Ergosterol OH – water O: 2.5 Lanosterol OH – water O: 2

13. Trans/Gauche Populations of DPPC Chains

14. QENS study of the motion of cholesterol / ergosterol /lanosterol in DPPC bilayers Characteristics 2 orientations: (a) membrane normal (z), (b) in the plane (x-y) 3 energy resolutions (1, 8 and14eV)  three time scales T = 20, 36, 50 o C 40% sterol concentration Results  motional anisotropy of cholesterol: long-range motions in the membrane normal: (a) out-of-plane diffusion parallel to membrane normal (for T  36 o C cholesterol can move in opposite leaflets) (b) locally confined motion within the bilayer plane Gliss, Bayerl et al., Biophys.J., 1999 / Endress et al, Biochemistry 2002 Lateral/transversal diffusion rate: cholesterol > lanosterol > ergosterol possible geometrical models

15. Towards understanding the dynamics of the system … Center of mass motion in the z-direction Cholesterol: 2 types of motion: high amplitude/low amplitude Ergosterol: more confined motions 4ns, saved every 0.2ps Restricted diffusion Long-range diffusion

16. movie

17. Center of mass motion in the x-y plane (view from top) 4ns, saved every 1ps 2-6ns6-10ns z-plane motion: long range ~10Ǻ (x-y)-plane motion: restricted ~4Ǻ

18. Quasielastic Neutron Scattering

19. QENS of Oriented Lipid Bilayers θ 45º θ θ Scattering is Considered elastic: 

20. Outlook: Calculation of spectra  Calculation of EISF From QENS Experiment: EISF gives us information on the geometry of the motions S(q,w=0)

21. Conclusions Overall good agreement with experiment: Reproduced structural and dynamical properties of the lipid/sterol systems Cholesterol/Ergosterol induce order in the lipid bilayer / Inhibit rotation of the middle carbons of the lipid acyl chain Lanosterol has smaller ordering effect on the membrane / Is located closed to the bilayer center Two different types of diffusion for cholesterol/ergosterol: long-range motions in the z-axis / restricted in x-y plane Calculate lateral/transverse diffusion coeff. from MSD/QENS  Geometrical models should be fitted to the EISF to describe cholesterol motion (rotational, translational)

22. A 100ps-trajectory = 630 MB Need 30ns  189 GB For my 4 systems, need: 189GB x 4 = 756 GB 1 DVD = 4.3 GB I need to write 176 DVDs !!!! To write/read DVD ~1 hour Total time spent reading/writing: 176 hours Work hours: 4 hours/day Total working days of reading(once!)/writing: 44 days !!! Some Statistics for the end … Solution? : Write DVDs

23. CMB Teufelskreis Problem !!

24. Thanks !!! … and Special Thanks to: Jeremy Matthias Emil Endress Torsten AlexBogdan Vandana Lars