ESR Spectroscopy Determines the Affinity of Cholesterol for Lipids with Varying Degrees of Unsaturation

Abstract

Membrane organization and the formation of lipid rafts have been studied extensively to provide a better understanding of cellular signaling and function. Previous studies have shown that cell and model membranes segregate into raft-like domains, regions rich in cholesterol and saturated lipids that play a role in membrane signaling, and cholesterol-poor regions, rich in unsaturated lipids. Cholesterol, an abundant lipid in mammalian cell membranes, orders lipid motion and has an aversion for the disordered acyl chains of polyunsaturated phospholipids, thus, cholesterol-lipid interactions are thought to play a key role in promoting lateral segregation in membranes. Here we present a new method for measuring the relative affinity of cholesterol between different lipids using electron spin resonance (ESR) spectroscopy and a phospholipid spin-label, 1-palmitoyl-2-stearoyl-(5-doxyl)-sn-glycero-3-phosphocholine (P(5DSA)PC). Using this technique, we measure the partitioning of cholesterol between large unilamellar vesicles (LUVs) of 1-palmitoyl-2-oleyol-phosphatidylcholine (POPC), our reference, and LUVs composed of lipids with varying degrees of unsaturation. These partitioning experiment results will be reported, as well as preliminary experiments showing the first, to our knowledge, partitioning experiments for vitamin E.