Structure and dynamics of the aliphatic cholesterol side chain in membranes as studied by (2)H NMR spectroscopy and molecular dynamics simulation.

Cholesterol is an evolutionarily highly optimized molecule particularly known for its ability to condense the phospholipids in cellular membranes. Until recently, the accompanying increase in the chain order of the surrounding phospholipids was attributed to the planar and rigid tetracyclic ring structure of cholesterol. However, detailed investigations of cholesterol's aliphatic side chain demonstrated that this side chain is responsible for approximately half of the condensation effect. Therefore, we investigated the structure and dynamics of the aliphatic side chain of cholesterol using (2)H solid-state nuclear magnetic resonance (NMR) spectroscopy and microsecond timescale all-atom molecular dynamics (MD) simulations in four different model membranes: POPC, DPPC, PSM, and POPC/PSM (1 : 1 mol/mol) and at three different temperatures: 5 °C, 37 °C, and 50 °C. A cholesterol variant, in which 11 hydrogens of the aliphatic side chain were exchanged for deuterium, was used and the respective (2)H NMR spectra confirmed the axially asymmetric rotational diffusion of cholesterol in DPPC and PSM. Furthermore, NMR spectra indicated that some hydrogens showed an unexpected magnetic inequivalency. This finding was confirmed by all-atom molecular dynamics simulations and detailed analysis revealed that the hydrogens of the methylene groups at C22, C23, and C24 are magnetically inequivalent. This inequivalency is caused by steric clashes of the aliphatic side chain with the ring structure of cholesterol as well as the branched C21 methyl group. These excluded volume effects result in reduced conformational flexibility of the aliphatic side chain of cholesterol and explain its high order (order parameter of 0.78 for chain motions) and large contribution to the condensation effect. Additionally, the motional pattern of the side chain becomes highly anisotropic such that it shows larger fluctuations perpendicular to the ring plane of cholesterol with a biaxiality of the distribution of 0.046. Overall, our results shed light on the mechanism how the aliphatic side chain is able to contribute about half of the condensation effect of cholesterol.