Zoe CourniaZoe Cournia

25 October 200425 October 2004

Cholesterol vs.Ergosterol / Lanosterol

in Membrane S(t)imulations

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

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 membranesof fungi and protozoans

Lanosterol evolutionary precursor of cholesterol / ergosterolfound in prokaryotes

18 enzymatic steps

9 enzymatic steps

Cholesterol vs. Ergosterol & LanosterolCholesterol

1. Saturated side chain2. One hydrogen at C243. One hydrogen at C144. C5- C6 double bond5. C7 – C8 single bond6. C8 – C9 single bond

Ergosterol1. Unsaturated side chain2. One methyl at C243. One hydrogen at C144. C5- C6 double bond5. C7 – C8 single bond6. C8 – C9 double bond

Lanosterol1. Unsaturated side chain2. On hydrogen at C243. One methyl at C144. C5 – C6 single bond5. C7 – C8 double bond6. C8 – C9 double bond

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

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

Systems + Simulation Details

• Cholesterol/DPPC - 28ns 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

• Ergosterol/DPPC - 10ns production run

• Lanosterol/DPPC - 1ns production run

• Pure DPPC - 10ns production run

Snapshots of the Lipid and Sterol in the Membrane

Chol - DPPC Erg - DPPC Lan - DPPC

Deuterium Order Parameters

)1cos3(2

1 2 iCD

iCDS CDq S

h

qQe2

2

3

In Simulation: In NMR:

C - HC - H

C - H

C - H

z-axis

Pure DPPC Chol/DPPC, Erg/DPPC, Lan/DPPC

Electron Density Profiles

X-Ray Franks, Chol-Dppc 40%mol, T = 24C

hpure-DPPC = 46.0 ÅhCHOL-DPPC = 50.0 ÅhERG-DPPC = 50.0 ÅhLAN-DPPC = 49.0 Å

h

Chol/DPPC, Erg/DPPC, Lan/DPPCPure DPPC

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° 5.1Avg. erg. tilt angle: 8.6° 4.3Avg. lan. Tilt angle: 15.5° 12.2

Radial Distribution Function

drr

rn

N

VrgRDF

24

)()(

Cholesterol OH – water O: 3

Ergosterol OH – water O: 2.5Lanosterol OH – water O: 2

Trans/Gauche Populations of DPPC Chains

PTNkG AB ln

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

Gliss, Bayerl et al., Biophys.J., 1999 / Endress et al, Biochemistry 2002

Lateral/transversal diffusion rate: cholesterol > lanosterol > ergosterol

possible geometrical models

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

movie

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

Quasielastic Neutron Scattering

),(]exp[2

1),( tQFtidtQS inc

N

iinc RQitRQi

NF

1

)0(exp[)](exp[1

QENS of Oriented Lipid Bilayers

inout kkq

)sin,0,cos(

)sin,0,cos(

),,(

kkink

kk

kkkoutk zyx

)sin2,0,0(2

1

Q

q

ink

outk

θ45º

θ

θ

2sin|||

2|

ik

q

|||| outi kk Scattering is Considered elastic:

Outlook: Calculation of spectra Calculation of EISF

From QENS Experiment:

)()(

)()(0 LorentzianIpeakI

peakIQA

EISF gives us information

on the geometry of the motions

S(q,

w=

0)

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)

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 hourTotal time spent reading/writing: 176 hours

Work hours: 4 hours/dayTotal working days of reading(once!)/writing: 44 days !!!

Some Statistics for the end…

Solution? : Write DVDs

CMB Teufelskreis

Problem !!

Thanks !!!

… and Special Thanks to:

Jeremy

Matthias

Emil Endress

Torsten

AlexBogdan

VandanaLars