Multifrequency simulations of the EPR spectra of lipid spin labels in membranes.

Simulations are performed of 34- and 9-GHz EPR spectra, together with 94-GHz EPR spectra, from phospholipid probes spin-labelled at the C4-C14 positions of the sn-2 chain, in liquid-ordered and gel-phase membranes of dimyristoyl phosphatidylcholine with high and low cholesterol contents. The multifrequency simulation strategy involves: (i) obtaining partially averaged spin-Hamiltonian tensors from fast-motional simulations of the 94-GHz spectra; (ii) performing slow-motional simulations of the 34- and 9-GHz spectra by using these pre-averaged tensors with the stochastic Liouville formalism; (iii) constructing, by simulation, slow-motional calibrations for the differences, DeltaA(zz)(qx) and Deltag(zz)(qx), in effective A(zz)-hyperfine splittings and g(zz)-values between 34- (or 94-GHz) and 9-GHz spectra; (iv) using such calibrations for DeltaA(zz)(qx) and Deltag(zz)(qx) and dynamic parameters from stage (ii) as a guide to adjust the extent of pre-averaging of the spin-Hamiltonian tensors; and (v) repeating the 34- and 9-GHz simulations of stage (ii). By using this scheme it is possible to obtain consistent values of the rotational diffusion coefficients, D(R perpendicular) and D(R//), and the long-axis order parameter, S(zz), that characterize the slow axial motion of the lipid chains, from spectra at both 34 and 9GHz.

High-field spin-label EPR of lipid membranes.

High-field EPR of spin-labelled lipid chains has proved to be an extremely productive means for biophysical investigations of phospholipid bilayer membranes. Results on the following three topics are reviewed: 1. Non-axial ordering of lipid chains in cholesterol-containing membranes; 2.

Lateral ordering of lipid chains in cholesterol-containing membranes: high-field spin-label EPR.

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.

Lipid membrane polarity profiles by high-field EPR.

Profiles of polarity across biological membranes are essential determinants of the cellular permeability barrier and of the stability of transmembrane proteins. High-field electron paramagnetic resonance of systematically spin-labeled lipid chains is used here to determine the polarity profiles of cholesterol-containing phospholipid membranes. The polarity dependence of the g(xx)-tensor element is opposite to the dependence on chain dynamics, and additionally has enhanced sensitivity to hydrogen bonding.

High-field electron spin resonance of spin labels in membranes.

High-field electron spin resonance (ESR) spectroscopy is currently undergoing rapid development. This considerably increases the versatility of spin labelling which, at conventional field strengths, is already well established as a powerful physical technique in membrane biology. Among the unique advantages offered by high-field spectroscopy, particularly for spin-labelled lipids, are sensitivity to non-axial rotation and lateral ordering, a better orientational selection, an extended application to rotational dynamics, and an enhanced sensitivity to environmental polarity.