Cholesterol dynamics in membranes

Time-resolved fluorescence anisotropy of the sterol analogue, cholestatrienol, and 13C nuclear magnetic resonance (NMR) spin lattice relaxation time (T,,) measurements of [13C41 labeled cholesterol were exploited to determine the correlation times characterizing the major modes of motion of cholesterol in unsonicated phospholipid multilamellar liposomes. Two modes of motion were found to be important: (a) rotational diffusion and (b) time dependence of the orientation of the director for axial diffusion, or "wobble." From the time-resolved fluorescence anisotropy decays of cholestatrienol in egg phosphatidylcholine (PC) bilayers, a value for -r, the correlation time for wobble, of 0.9 x 10-9 s and a value for S_, the order parameter characterizing the same motion, of 0.45 s were calculated. Both r, and S, were relatively insensitive to temperature and cholesterol content of the membranes. The T1C measurements of 113C41 labeled cholesterol did not provide a quantitative determination of -r1, the correlation time for axial diffusion. T1C from the lipid hydrocarbon chains suggested a value for rsimilar to that for cholesterol. Steady-state anisotropy measurements and time-resolved anisotropy measurements of cholestatrienol were used to probe sterol behavior in a variety of pure and mixed lipid multilamellar liposomes. Both the lipid headgroups and the lipid hydrocarbons chains contributed to the determination of the sterol environment in the membrane, as revealed by these fluorescence measurements. In particular, effects of the phosphatidylethanolamine (PE) headgroup and of multiple unsaturation in the lipid hydrocarbon chains were observed. However, while the steady-state anisotropy was sensitive to these factors, the timeresolved fluorescence analysis indicated that Ti was not strongly affected by the lipid composition of the membrane. S, may be increased by the presence of PE. Both steady-state anisotropy measurements and timeresolved anisotropy measurements of cholestatrienol were used to probe sterol behavior in three biological membranes: bovine rod outer segment (ROS) disk membranes, human erythrocyte plasma membranes, and light rabbit muscle sarcoplasmic reticulum membranes. In the ROS disk membranes the value for S, was marginally higher than in the PC membranes, perhaps reflecting the influence of PE. The dramatic difference noted was in the value for Ti. In both the ROS disk membranes and the erythrocyte membranes, Ti was one-third to one-fifth of Ti in the phospholipid bilayers. This result may reveal an influence of membrane proteins on sterol behavior.