In lipid bilayers, the spin-labeled alkyl chain of 1-palmitoyl-2-(n
-PC) spin label or n
-doxylstearic acid spin label (n
-SASL) (with the nitroxide moiety attached at the Cn
position) undergoes rapid anisotropic motion about the long axis of the spin label and also a wobbling motion of the long axis within the confines of a cone imposed by the membrane environment. The anisotropic rotational motion of the spin label gives rise to unique features of the EPR spectra that can be used to calculate the order parameter for the alkyl chain [1
]. The order parameter is a measure of the amplitude of the wobbling motion of the alkyl chain, with an increase in order parameter indicating a decrease in the angle of the cone. Moreover, as the spin label is moved from the bilayer surface to the membrane interior, deviations in the alkyl-chain-segment direction from the bilayer normal accumulate. Thus, ordering of the alkyl chain close to the membrane surface (induced, for example, by contact with the plate-like portion of cholesterol), also causes an apparent ordering of the distal fragment of the alkyl chain [3
]. Although the order parameter indicates an amplitude of wobbling motion of alkyl chains in the lipid bilayer, change in the order parameter is most often described as a change in spin-label mobility, and thus as a change in membrane fluidity. Profiles of the order parameter have been routinely used as a measure of the fluidity of membrane samples [4
]. With some restrictions, profiles of the dynamic parameter R
, which is the rotational diffusion coefficient of the nitroxide moiety around an axis perpendicular to the mean symmetry axis for the rotation, can be also obtained from computer simulation of EPR spectra [6
We propose here an alternative approach in displaying membrane fluidity, namely, profiles of the spin-lattice relaxation rate (T−11). This parameter can be obtained from saturation-recovery (SR) EPR measurements. In deoxygenated samples, this parameter depends primarily on the motion of the nitroxide moiety within the lipid bilayer and thus characterizes the dynamics of the membrane environment at the depth at which the nitroxide moiety is located.
Robinson et al. [9
] discuss various mechanisms for spin-lattice relaxation of nitroxide spin labels. For an isotropic rotational correlation time constant, they found good agreement between experiment and a theoretical model that was dominated by the so-called electron-nuclear dipolar (END) mechanism for rotational correlation times from about 10−11
s. In this range, Tend1e
, which provides the fundamental basis for the use of the electron spin-lattice relaxation time as a fluidity parameter. In subsequent work, Mailer et al. [10
] extended the theoretical framework of Ref. 
to include anisotropic motion and explicitly considered stearic acid spin labels in lipid bilayers. For data at X-band, fits were not improved. However, for multifrequency T1
data, consideration of anisotropic motion was necessary. Thus, it seems possible that an improved fluidity parameter that reflects anisotropic motion could be developed based on multifrequency saturation recovery measurements.
values of lipid spin labels (1–10 μs) are much longer than the correlation time for reorientation of the nitroxide group of lipid spin labels as measured using conventional EPR (0.1–10 ns). Because of the short correlation time of different modes of reorientation, motional effects are superimposed and spectral parameters obtained from conventional EPR are affected by these motions in complicated ways. However, because of the long T1
the complicated motional effects should be averaged in SR measurements. Thus, T−11
can be used as a convenient quantitative measure of membrane fluidity that indicates this averaged motion of phospholipid alkyl chains (or nitroxide free radical moieties attached to those chains). If T−11
is measured for n
-SASL or n
-PC spin labels, a fluidity profile across the lipid bilayer can be obtained that reflects membrane dynamics [11
]. In addition, because T−11
can be measured in coexisting membrane domains and membrane phases [12
], these fluidity profiles can be obtained in coexisting domains and phases without the need for their physical separation.
We previously used SR in dual-probe pulse EPR experiments in which small paramagnetic molecules (e.g., molecular oxygen or paramagnetic metal complexes) were introduced into the membrane and bimolecular collision rates with lipid-analog spin labels determined [14
]. The rate of bimolecular collision between the nitroxide moiety of a lipid-type spin label placed at a specific location in the membrane and a small paramagnetic probe molecule (like molecular oxygen) is a useful monitor of membrane fluidity that reports on translational diffusion of the small molecule probe, but not on motion of lipid alkyl chains [14
]. Those initial experiments were performed at X-band. However, we recently showed that spin-lattice relaxation times of lipid-analog spin labels initially increase when the microwave frequency is increased above X-band, reaching maximum values at Q-band (35 GHz) [17
], and then decrease again as the frequency is further increased to W-band (94 GHz) [18
]. Nonetheless, the observed trend of a decreasing spin-lattice relaxation time for n
-SASL or n
-PC spin labels with increasing membrane depth was independent of microwave frequency [17
]. We conclude that the longest values of T1
will generally be found at Q-band, noting that long values are advantageous for measurement of T1
-dependent membrane processes. These new capabilities have the potential to be a useful tool for studying membrane dynamics.
In this paper, we present profiles of the spin-lattice relaxation time of phospholipid spin labels for fluid-phase dimyristoylphosphatidylcholine (DMPC) membranes without and with 50 mol% cholesterol obtained at X-band. In parallel, for the same systems, we present profiles of another dynamic parameter—namely, the rotational diffusion coefficient obtained from computer simulation of conventional EPR spectra. We chose the DMPC/cholesterol membranes to demonstrate this new approach for characterizing membrane fluidity because other membrane properties for this system are readily available in the literature, thus allowing our new results to be broadly compared with commonly used parameters.