Direct Observation of Cholesterol Dimers and Tetramers in Lipid Bilayers

Cholesterol is a ubiquitous component of mammalian cell membranes and affects membrane protein function. Although cholesterol-mediated formation of ordered membrane domains has been extensively studied, molecular-level structural information about cholesterol self-association has been absent. Here, we combine solid-state nuclear magnetic resonance (NMR) spectroscopy with all-atom molecular dynamics simulations to determine the oligomeric structure of cholesterol in phospholipid bilayers. Two-dimensional C-13-C-13 correlation spectra of differentially labeled cholesterol indicate that cholesterol self-associates in a face-to-face fashion at membrane concentrations from 17 to 44 mol %. 2D C-13 and F-19 spin-counting experiments allowed us to measure the average oligomeric number of these cholesterol clusters. At low cholesterol concentrations of similar to 20%, the average cluster size is centered on dimers. At a high cholesterol concentration of 44%, which is representative of virus lipid envelopes and liquid-ordered domains of cell membranes, both dimers and tetramers are observed. The cholesterol dimers are found in both phase-separated membranes that contain sphingomyelin and in disordered and miscible membranes that are free of sphingomyelin. Molecular dynamics simulations support these experimental observations and moreover provide the lifetimes, stabilities, distributions, and structures of these nanoscopic cholesterol clusters. Taken together, these NMR and MD data strongly suggest that dimers are the basic structural unit of cholesterol in phospholipid bilayers. The direct observation of cholesterol dimers and tetramers provides a revised framework for studying cholesterol interactions with membrane proteins to regulate protein functions and for understanding the pathogenic role of cholesterol in diseases.

Automated summary

Using solid-state nuclear magnetic resonance (NMR) spectroscopy and molecular dynamics simulations, the study identified the oligomeric structure of cholesterol in phospholipid bilayers, confirming dimers as the basic structural unit. This has implications for understanding cholesterol interactions with membrane proteins and its role in diseases.