Three-dimensional Dynamic Structure Of Phospholipid Bilayers Saturated With Cholesterol

Abstract

Membranes made of synthetic phospholipids, as well as total phospholipids extracted from the eye lenses of young and old animals and containing saturating amounts of cholesterol (close to or exceeding the cholesterol solubility threshold), were investigated using conventional and saturation-recovery EPR spin-labeling methods. Profiles of the order parameter and hydrophobicity were obtained from conventional EPR spectra. Profiles of the oxygen transport parameter (oxygen diffusion-concentration product) were obtained from saturation-recovery curves. All of these profiles provide unique information about the depth-dependent physical properties and the three-dimensional dynamic organization of the membrane. Additionally, saturation-recovery measurements allow discrimination of membrane domains because the collision rate of molecular oxygen with the nitroxide spin label may differ in these domains. All membranes saturated (but not oversaturated) with cholesterol are homogenous on the EPR timescale. When properties of the phospholipid-cholesterol membrane are monitored with phospholipid analogue spin labels (by measuring the alkyl chain order parameter), the membrane shows high rigidity that decreases gradually toward the membrane center. However, when membrane properties are measured by monitoring movement and/or concentration of small molecules like molecular oxygen or water, the monitored properties change abruptly between the C9 and C10 positions (depth to which the rigid cholesterol ring-structure is immersed), showing low membrane fluidity and hydrophobicity to the depth of the ninth carbon and high membrane fluidity and hydrophobicity in the membrane center. Based on these observations, it can be concluded that the bulk physical properties of membranes saturated with cholesterol (with a cholesterol-to-phospholipid mole ratio close to one) are mainly determined by the presence of the saturating amount of cholesterol and are practically independent on membrane phospholipid composition.