Direct observation of the properties of cholesterol in membranes by deuterium NMR.

The properties of cholesterol in bilayers of egg phosphatidylcholine (PC) were investigated directly by means of 2H-NMR of specifically-deuterated species (C3, C7, C26, C27). Quadrupole splittings were a measure of molecular ordering, and relaxation times T1 and T2e were indicators of rates of motion. The importance of the use of echoes for spectral acquisition is emphasised, particularly to obtain accurate values of the quadrupole splitting. In the case of overlapping powder patterns from two labelled positions, the use of the absolute value mode of spectral presentation is shown to yield reasonable estimates of the individual quadrupole splittings. Spectral properties were monitored as a function of cholesterol concentration and temperature. Increasing cholesterol concentration led to a high degree of ordering for the rigid ring system of cholesterol, approaching a molecular order parameter of 0.8 at 50 mol% cholesterol. The isopropyl methyl groups were in all cases less ordered and more mobile than the ring system, but responded in a similar fashion to variable cholesterol concentration and temperature. The observation of a minimum in the temperature dependence of T1 for cholesterol-7,7-d2 led to a direct estimate of its correlation time for molecular motion, 3.5 X 10(-9) S rad-1. This indicates that the overall rate of motion of cholesterol is considerably slower than that of the lipids in which it is located. The short T2e values suggest that the motional spectrum of cholesterol is rich in low frequencies. The parallel temperature and cholesterol dependences of quadrupole splittings for different positions on the rigid ring system of cholesterol indicate that the position of the axis of motional averaging of the molecule is not changing, and is the same as that determined in an earlier study. It is emphasised that the steep temperature dependence and small quadrupole splittings for the chain isopropyl methyl groups of cholesterol do not necessarily indicate a high degree of disorder, but may be due to their axes of motional averaging lying at angles close to 54 degrees with respect to the director of the ordered lipids.