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. Inclusion of spectra at 34GHz greatly improves precision in determining the D(R//) element of the slow diffusion tensor in these systems.