Cholesterol-Lipid Affinity Determined By Epr

Cholesterol-lipid interactions are thought to play an intrinsic role in determining lateral organization within cellular membranes. Rafts enriched in cholesterol are “glued” together by the high affinity that the sterol has for sphingolipids, whereas poor affinity for cholesterol is hypothesized to drive the formation of polyunsaturated fatty acid (PUFA)-containing phospholipid domains depleted in the sterol. Here we describe a novel method using electron paramagnetic resonance (EPR) to measure the affinity of cholesterol for model membranes. Our method determines the relative amount of cholesterol that partitions between large unilamellar vesicles (LUV) and cyclodextrin by analyzing EPR spectra recorded with the spin label, cholestane, in terms of a superposition of spectral components from the two environments. We compare our results on 1-palmitoyl-2-oleoylphosphatidylcholine (POPC) to other methods and measure the affinity of cholesterol for phospholipids with increasing levels of unsaturation.