Determining Ethanol Distribution in Phospholipid Multilayers with MAS−NOESY Spectra

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Abstract
The location of an ethanol molecule within a membrane, an issue of considerable controversy, was investigated directly by NMR with two-dimensional NOESY. Lipid and ethanol 1H NMR resonances of multilamellar liposomes were resolved by magic-angle spinning (MAS). We observed strong proton lipid−ethanol crosspeaks in dispersions of saturated dimyristoylphosphatidylcholine and monounsaturated stearoyloleoylphosphatidylcholine and in polyunsaturated stearoyldocosahexaenoylphosphatidylcholine. Crosspeak intensity has been interpreted in terms of an ethanol distribution function over the lipid bilayer. Ethanol resides with the highest probability at the lipid water interface near the lipid glycerol backbone and upper methylene segments of lipid hydrocarbon chains. Chain unsaturation has only a minor influence on the ethanol distribution function. In all cases, the ethanol concentration in the bilayer core is significantly lower. At ambient temperature all lipid−ethanol crosspeaks are positive. Crosspeak intensity decreases with increasing water content and increasing temperature most likely because of shorter correlation times of lipid and ethanol reorientation. This suggests a lifetime for specific lipid−ethanol contacts of about 1 ns. Lipid−ethanol and lipid−lipid crosspeaks reflect the high degree of motional disorder of lipids and incorporated ethanol in membranes and the rather arbitrary nature of the location of the lipid−water interface.