Instability of cholesterol clusters in lipid bilayers and the cholesterol's Umbrella effect

Abstract

The instability of cholesterol clusters and the Umbrella effect of cholesterol in dipalmitoylphosphatidylcholine (DPPC) and dioleoylphosphatidylcholine (DOPC) lipid bilayers were investigated via atomistic molecular dynamics (MD) simulation. Cholesterol clusters in phosphatidylcholine (PC) bilayers are found to be very unstable and to readily disperse into cholesterol monomers. This instability results from the difficulty of the bilayer system in preventing water exposure to cholesterol's bulky hydrophobic bodies in a cluster. The system responds to artificially arranged cholesterol clusters in several interesting manners: (i) cholesterol clusters quickly form a "frustum" shape to reduce water penetration between cholesterol headgroups; (ii) many clusters bury themselves deeper into the bilayer interior, causing bilayer deformation; and (iii) cholesterol fluctuates rapidly, both laterally and vertically, to escape clusters. These fluctuations result in the disintegration of clusters and, in one incidence, a highly unusual flip-flop event of cholesterol across the DOPC bilayer. Our results show that cholesterols have a strong tendency to avoid forming clusters in lipid bilayers and that the fundamental cholesterol-cholesterol interaction is unfavorable. Furthermore, the radial distribution functions of cholesterol hydroxyl oxygen to various headgroup atoms of PC reveal that the PC headgroups surrounding cholesterol have a clear tendency to reorient and to extend toward cholesterol. This reorientation has a layered structure that extends 2-3 nm from the cholesterol molecule. This study demonstrates that the Umbrella hypothesis is valid in both saturated and unsaturated lipid bilayers.

Figure 1

The Umbrella model: (A) At low cholesterol concentration, headgroups of neighboring PCs easily cover the hydrophobic bodies of cholesterol. Motion of acyl chains next to cholesterol is restricted by the rigid body of cholesterol; (B) At high cholesterol concentration, headgroups of PCs work together to cover more cholesterol. The acyl chains of PCs become highly ordered. (C) The frustum shaped cluster: A cholesterol cluster adopts the “frustum shape” to reduce water penetration between cholesterol headgroups. The cluster also moves deeper into bilayer interior allowing a better coverage of its outskirts by surrounding PC headgroups.

Figure 2

The initial and final snapshots of the cholesterol distribution in the cluster and the random systems. Cholesterol (in yellow) is represented by the space-filling model. Only the lipids on the top leaflet are shown.

Figure 3

The average cluster size vs. time for the cluster and the random systems. The inserts: The total number of cholesterol clusters vs. time in the two systems.

Figure 4

Left: An artificially arranged cholesterol cluster in DOPC cluster system at the start of the simulation. Right: At 1ns, the cluster has already adopted the frustum shape.

Figure 5

The radial distribution functions (RDF) in the cluster systems over the first 20 ns. Solid line: The RDF between cholesterol oxygen O6. Dashed line: The RDF between the center of mass (COM) of cholesterol.

Figure 6

The electron density profiles of PC, cholesterol and solvent across the bilayer in the cluster and the random systems.

Figure 7

The snapshots of an unusual flip-flop of cholesterol across the DOPC bilayer in the cluster system. Inserts: the snapshots of the membrane surface viewed from the top aqueous phase. 0 ns: An artificially arranged cholesterol cluster at the start of the simulation. 4 ns: The cluster adopted the frustum shape and also moved deeper into the bilayer. One cholesterol molecule was half way into the bottom leaflet. 9 ns: The cholesterol was completely inside the bottom leaflet, but was also up side down. 12 to 26 ns: The cholesterol completed the flip entirely within the bottom leaflet. At the same time, the cluster dispersed into smaller pieces.

Figure 8

RDFs over the last 50 ns in the random system. Solid line: The RDF between cholesterol oxygen O6 and the nitrogen atom of PC choline group. Dashed line: The RDF between cholesterol oxygen O6 and PC phosphate residue P8.