Investigation of Lipid Organization in Biological Membranes by Two-Dimensional Nuclear Overhauser Enhancement Spectroscopy

Abstract

The cross-peaks between lipid resonances in two-dimensional nuclear Overhauser enhancement spectroscopy 1H NMR spectra, recorded with magic-angle spinning, contain valuable information about the structure and dynamics of the lipid bilayer. We have attempted a quantitative analysis of magnetization exchange between lipid resonances in the biologically relevant liquid crystalline lamellar phase. Spectra of 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) water dispersions were recorded at mixing times from 0.005 to 1 s, and all diagonal and cross-peak volumes were determined by integration. The full relaxation-rate matrix was computed for the 10 lipid resonances by a matrix equation algorithm. Results of this mathematically rigorous approach were compared with simplified approaches to calculate cross-relaxation rates. In a second series of experiments, the DMPC was mixed with increasing amounts of perdeuterated DMPC-d67 to determine the percentage of intra- vs intermolecular magnetization transfer. With the exception of transfer between nearest neighbor protons by chemical bond, cross-relaxation in DMPC bilayers is exclusively intermolecular. There is no evidence for the transfer of magnetization over several bonds within lipid molecules by spin diffusion. Lipids exist in multiple conformations with rapid transitions between them, and, therefore, cross-relaxation rates do not represent stable molecular arrangements with fixed distances. Instead, the cross-relaxation rates reflect the probability of close approach between protons of neighboring lipid molecules. We established experimentally that cross-relaxation rates are proportional to the statistical average of lateral lipid−lipid contacts in binary mixtures. The location of lipid segments along the bilayer normal is best described by a distribution function. Segments which are located at the same depth in the bilayer approach each other with higher probability and have higher rates of magnetization transfer, compared with groups which are, on average, more distant and approach each other less frequently. The per-proton intermolecular cross-relaxation rates vary over the bilayer by only 1 order of magnitude, indicating surprisingly high probabilities of close approach even between the most distant groups, like the choline methyl groups and the terminal methyl groups of hydrocarbon chains. The results reflect the high degree of lipid motional disorder and the substantial variations in location of neighboring lipid molecules.