High-field (i.e., 94 GHz) electron paramagnetic resonance is used to characterize the nonaxial ordering of spin-labeled lipid chains in membranes containing cholesterol. Employing high magnetic fields (and microwave frequencies) allows investigation of both the lateral and transverse ordering of the phospholipid chains by cholesterol, from the x-y and z-elements, respectively, of the spin-label g-tensor. Transverse ordering is described by the conventional order parameter, (P2(cosβ)), where β is the instantaneous inclination of the chain axis to the membrane normal; and lateral ordering is described by the order parameter (cos2(φ-φ)), where β is the azimuthal angle about the chain axis and φ is the mean azimuthal orientation about which angular fluctuations take place. To obtain high positional resolution, phosphatidylcholines spin labeled at all odd and even positions from n = 4 to n = 14 in the sn-2 chain (1-acyl-2-[n-(4,4′ -dimethyloxazolidine-N-oxyl)]stearoyl-sn-glycero-3-phosphocholine) are used at probe amounts in membranes of dimyristoyl phosphatidylcholine containing either high (40 mol%) or low (5 mol%) concentrations of cholesterol. At high-cholesterol content, lateral ordering of the spin-labeled lipid chains is detected over a wide range of temperature throughout the liquid-ordered phase. The transverse profile of lateral φ-ordering with position, n, of chain labeling follows the profile of the rigid steroid nucleus of cholesterol. It becomes progressively averaged toward the terminal methyl group of the sn-2 chain, in the region of the flexible hydrocarbon chain of cholesterol. At low-cholesterol content, lateral chain ordering is prominent at low temperature, but diminishes at progressively higher chain positions with increasing temperature. The nonaxial lipid ordering may be related to the formation of in-plane lipid domains in membranes containing cholesterol and saturated lipids.
Kurad, D., Jeschke, G., & Marsh, D. (2004). Lateral Ordering of Lipid Chains in Cholesterol-Containing Membranes: High-Field Spin-Label EPR. Biophysical Journal, 86(1 I), 264–271. https://doi.org/10.1016/S0006-3495(04)74102-8