Multifrequency ESR Studies of the Complex Dynamics of Membranes

ESR has been extensively used to study membrane structure and dynamics with the aid of spin-labeled lipid additives. By means of careful line shape analysis, one can obtain detailed information on the ordering and motion of the lipids in the membrane. Also, recent studies on membranes have shown that high-frequency ESR provides improved orientational resolution. It is certainly true that the dynamical structure of lipid membranes is very complex. The lipid molecules are locally ordered and engaged in overall reorientation. In addition, the internal motions of the chain segments around the many C-C bonds leads to complex dynamics. It could be expected that a combined study at a low frequency (9 GHz) and a high frequency (250 GHz) would enable one to distinguish between the overall motion of the lipids and the internal modes of motion affecting the local site to which the spin label is attached, (cf. Figure). A combined 250 and 9 GHz ESR study was performed on membrane vesicles composed of pure lipid (DPPC) and of DPPC: cholesterol in a 1:1 molar ratio using the end chain labeled lipid, 16-PC (cf. Figure). The former are in the liquid crystalline phase, and the latter are in the liquid ordered phase. This liquid-ordered phase is believed to play an important role in facilitating phase segregation in cell membranes, wherein important biological processing may occur. It is found that the 250 GHz spectra represent a “fast time-scale” such that the overall restricted motion of the lipid in the membrane is frozen out, but it is sensitive to the internal dynamics of the end chain. The 9 GHz spectra are, however, sensitive to the overall motion as well. This combined study, utilizing an analysis based on these coupled modes, permitted the separation of both types of motion. It was shown that the addition of cholesterol has a large effect on the end chain dynamics by restricting its motion while increasing motional rates, whereas its effects on the overall lipid motion are modest. This leads to a clearer characterization of the dynamic structure of the cholesterol-rich liquid-ordered phase as compared to the liquid crystalline phase. This study thus shows the significant potential of a multifrequency ESR approach to the complex dynamics of membranes.