Cholesterol at different bilayer concentrations can promote or antagonize lateral segregation of phospholipids of differing acyl chain length

Fourier-transform infrared-spectroscopic and fluorescence measurements have been combined to examine the effect of cholesterol on the intermixing of short-chain dilauroyl phosphatidylcholine (DLPC) and its bromo-substituted derivative (12BrPC) with longer-chain (C16- or C18-) phosphatidylcholines (PCs) in hydrated lipid bilayers. Infrared spectroscopy of mixtures combining protonated DLPC or 12BrPC with chain-perdeuterated dipalmitoyl PC reveals that cholesterol at lower concentrations in the bilayer modifies the resolved thermal melting profiles for both phospholipid components and, at high bilayer concentrations, produces a convergence of the thermal transitions for the two PC species. Fluorescence-quenching measurements using a short-chain fluorescent PC (1-dodecanoyl-2-[8-[N-indolyl]octanoyl] PC) in ternary mixtures combining 12BrPC, dipalmitoyl or distearoyl PC, and cholesterol confirm that very high cholesterol levels (50 mol %) abolish the lateral segregation of the PC components at 25 degrees C, a temperature where the phospholipids extensively phase-separate in the absence of sterol. By contrast, under these same conditions cholesterol at lower concentrations in the bilayer is found to enhance the tendency of the PC components to exhibit lateral segregation. We show that these seemingly contradictory effects of cholesterol can be readily explained in the light of a ternary phase diagram that is fully consistent with out current understanding of the nature of cholesterol-phospholipid interactions in binary mixtures.