Molecular Dynamics in Lipid Rafts by High-Sensitivity, High-Resolution NMR

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Cholesterol is involved in the formation of dynamic microdomains referred to as lipid rafts in the membrane. Because the inside of a lipid domain is nonpolar and aprotic, it is of great interest to examine how cholesterol molecules are dissolved in low-polarity aprotic organic solvents that mimic the hydrophobic core of a phospholipid bilayer. Self-diffusion coefficients (D) are measured for normal (nondeuterated) and deuterated cholesterol in 1-octanol, chloroform, and cyclohexane at concentrations of 1-700 mM. The pulsed field gradient spin-echo (PGSE) ^1H and ^2H NMR were used, respectively, at 600 and 92 MHz. The hydrodynamic radius (R) obtained from D is the smallest in 1-octanol and it is comparable with the average length of the molecular axes for the cholesterol molecule. In 1-octanol, R is invariant against the concentration variation, whereas in chloroform, R is larger and increases almost linearly with cholesterol concentration. The R value larger than that in hydrogen-bonding 1-octanol indicates that cholesterol forms an aggregate through hydrogen bonding. The aggregate structure is confirmed by comparing NOESY spectra in chloroform and 1-octanol. The NOESY analysis reveals the presence of one extra cross peak (C4-C19) in chloroform compared to 1-octanol. Because the carbon atoms related to the cross peak are close to the hydroxyl group (C3-OH), cholesterol molecules are considered to be not piled but OH centered in the aggregate. This is supported also by larger rotational hydrodynamic radii measured on cholesterol deuterated at positions C2, C3, C4, and C6. This shows that the aggregate formation is driven by the hydrogen bonding between cholesterol molecules.