Membrane fluidity profiles as deduced by saturation-recovery EPR measurements of spin-lattice relaxation times of spin labels

Abstract

There are no easily obtainable EPR spectral parameters for lipid spin labels that describe profiles of membrane fluidity. The order parameter, which is most often used as a measure of membrane fluidity, describes the amplitude of wobbling motion of alkyl chains relative to the membrane normal and does not contain explicitly time or velocity. Thus, this parameter can be considered as nondynamic. The spin-lattice relaxation rate (T(1)(-1)) obtained from saturation-recovery EPR measurements of lipid spin labels in deoxygenated samples depends primarily on the rotational correlation time of the nitroxide moiety within the lipid bilayer. Thus, T(1)(-1) can be used as a convenient quantitative measure of membrane fluidity that reflects local membrane dynamics. T(1)(-1) profiles obtained for 1-palmitoyl-2-(n-doxylstearoyl)phosphatidylcholine (n-PC) spin labels in dimyristoylphosphatidylcholine (DMPC) membranes with and without 50 mol% cholesterol are presented in parallel with profiles of the rotational diffusion coefficient, R(⊥), obtained from simulation of EPR spectra using Freed's model. These profiles are compared with profiles of the order parameter obtained directly from EPR spectra and with profiles of the order parameter obtained from simulation of EPR spectra. It is shown that T(1)(-1) and R(⊥) profiles reveal changes in membrane fluidity that depend on the motional properties of the lipid alkyl chain. We find that cholesterol has a rigidifying effect only to the depth occupied by the rigid steroid ring structure and a fluidizing effect at deeper locations. These effects cannot be differentiated by profiles of the order parameter. All profiles in this study were obtained at X-band (9.5 GHz).

Fig. 1

Chemical structures of n-PC spin labels, DMPC, and cholesterol. Structures are aligned to illustrate approximate locations of these molecules and nitroxide moieties of spin-labels across the membrane. However, since alkyl chains tend to have many gauche conformations, the chain-length projection to the membrane normal would be shorter than depicted here and the rigid structure of cholesterol would sink somewhat differently in the fluid phase membranes.

Fig. 2

Experimental (solid line) and simulated (broken line) X-band EPR spectra of n-PC in DMPC and DMPC/50 mol% cholesterol membranes at 165 °C (rigid-limit conditions).

Fig. 3

Experimental (solid line) and simulated (broken line) X-band EPR spectra of n-PC in DMPC and DMPC/50 mol% cholesterol membranes at 27 °C (fluid phase membranes).

Fig. 4

Representative SR signals with fitted curves and the residuals (the experimental signal minus the fitted curve) for 5-PC (A) and 16-PC (B) in DMPC membranes without and with 50 mol% cholesterol at 27 °C. The fits to the single-exponential curves for 5-PC give recovery times of 4.61 μs and 5.73 μs for membranes without and with cholesterol, respectively (A); and recovery times for 16-PC of 2.11 μs and 1.61 μs for membranes without and with cholesterol, respectively (B). All samples were equilibrated with nitrogen. Upper residuals are for membranes without cholesterol and lower residuals are for membranes with cholesterol. These results indicate the opposite effects of cholesterol on spin label dynamics at membrane surface and in the membrane center.

Fig. 5

Profiles of T⁻¹₁ (the spin-lattice relaxation rate) obtained for n-PC spin labels in DMPC membranes without and containing 50 mol% cholesterol as a function of nitroxide moiety position “n” on the alkyl chain.

Fig. 6

Plots of the best fit values of the rotational diffusion coefficient (R_⊥) for n-PC in DMPC membranes without and containing 50 mol% cholesterol as a function of nitroxide moiety position “n” on the alkyl chain.

Fig. 7

Profiles of the order parameter as a function of the nitroxide moiety position “n” on the alkyl chain obtained for n-PC spin labels in DMPC membranes without and containing 50 mol% cholesterol. The order parameter S was calculated from experimental EPR spectra using Eq. (2).

Fig. 8

Plots of the best fit values of the order parameter (S_o) for n-PC in DMPC membranes without and containing 50 mol% cholesterol as a function of nitroxide moiety position “n” on the alkyl chain.