Zoe Cournia 25 October 2004

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

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

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

4. Cholesterol vs. Ergosterol & Lanosterol • Cholesterol • Saturated side chain • One hydrogen at C24 • One hydrogen at C14 • C5- C6 double bond • C7 – C8 single bond • C8 – C9 single bond • Ergosterol • Unsaturated side chain • One methyl at C24 • One hydrogen at C14 • C5- C6 double bond • C7 – C8 single bond • C8 – C9 double bond • Lanosterol • Unsaturated side chain • On hydrogen at C24 • One methyl at C14 • C5 – C6 single bond • C7 – C8 double bond • 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. Sterol-Lipid Phase Diagram • lipid-cholesterol: stable region of coexistence between ld-lo phases • at high concentrations  liquid-ordered phase (lo) • lipid-lanosterol: no lo formation  Cholesterol stabilizes the liquid-ordered phase Nielsen, Europhys.Lett., (2000) 52:368-374

7. Systems + Simulation Details • Cholesterol/DPPC • - 28ns production run • Ergosterol/DPPC • - 10ns production run • Lanosterol/DPPC • - 1ns production run • Pure DPPC • - 10ns production run • General Characteristics • - PME, NPT • - 200 DPPC, 1600 H2O • - T = 309 K, P = 1atm • - 2ns equilibration (A) DPPC (B) Cholesterol (C) Ergosterol (D) Lanosterol

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

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

10. Electron Density Profiles Pure DPPC Chol/DPPC, Erg/DPPC, Lan/DPPC h hpure-DPPC = 46.0 Å hCHOL-DPPC = 50.0 Å hERG-DPPC = 50.0 Å hLAN-DPPC = 49.0 Å X-Ray Franks, Chol-Dppc 40%mol, T = 24C

11. 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 Smondyrev et al. (simulation): 10.6º (Chol-DMPC, 50%mol., T=50C) Smondyrev et al. (simulation): 22.2º (Chol-DMPC, 11%mol., T=50C)

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, 50oC • 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  36oC cholesterol can move in opposite leaflets) (b) locally confined motion within the bilayer plane possible geometrical models Lateral/transversal diffusion rate: cholesterol > lanosterol > ergosterol Gliss, Bayerl et al., Biophys.J., 1999 / Endress et al, Biochemistry 2002

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

16. movie

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

18. Quasielastic Neutron Scattering

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

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

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. Some Statistics for the end… A 100ps-trajectory = 630 MB Need 30ns  189 GB For my 4 systems, need: 189GB x 4 = 756 GB Solution?: Write DVDs 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 !!!

23. CMB Teufelskreis Problem !!

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