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, ΔAzzqx and Δgzzqx, in effective Azz-hyperfine splittings and gzz-values between 34- (or 94-GHz) and 9-GHz spectra; (iv) using such calibrations for ΔAzzqx and Δgzzqx 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, DR⊥ and DR//, and the long-axis order parameter, Szz, that characterize the slow axial motion of the lipid chains, from spectra at both 34 and 9 GHz. Inclusion of spectra at 34 GHz greatly improves precision in determining the DR// element of the slow diffusion tensor in these systems.