Biomedical EPR Part-B Methodology Instrumentation and Dynamics - Sandra R. Eaton

The second set of calibrations is given for the out-of-phase/in-phase ratios of the first-harmonic integrated spectral intensities (Tables A3-A5) and low-field spectral amplitudes (Tables A3, A6, A7). For all calibrations, the ratio depends on according to the following semi-empirical expression (Livshits et al., 1998a; Livshits and Marsh, 2000):

where the parameters and depend quite strongly on and the Zeeman modulation frequency, but less strongly on and the spin label rotational rate, The exponent m in Eq. A.4 depends on the modulation frequency and also on whether signal amplitudes or integrated intensities are measured. Table A3 gives calibrations established for the “no-motion” situation, which applies to both quasi-rigid limit and extreme motional narrowing spectra (Livshits et al., 1998a). Tables A4-A7 give corresponding calibrations that specifically take account of molecular motion (Livshits and Marsh, 2000). For the first-harmonic out-of-phase method, calibrations are given not only for the standard modulation frequency but also for a modulation frequency of 25 kHz. Calibrations for show enhanced sensitivity to longer relaxation times.

364 DEREK MARSH ET AL.

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370 ALBERT H. BETH AND ERIC J. HUSTEDT

membranes. EPR, in particular, has provided unique characterization of the structure and dynamics of the lipids that solvate the periphery of intrinsic membrane proteins, the so-called boundary lipids (e.g. Griffith and Jost, 1976; Ge and Freed, 1993; Borbat et al., 2001). Most recently, these basic techniques and in particular EPR have been utilized to investigate the lateral organization of lipids in cell membranes including the sizes and stabilities of microdomains (e.g. Kawasaki et al., 2001). These investigations have taken on added importance with the discovery of specialized structures including lipid “rafts” that are hypothesized to play important roles in modulating protein-membrane interactions and in cellular signaling pathways (e.g. Zacharias et al., 2002; Anderson and Jacobson, 2002).

Less is known about the organization and dynamics of intrinsic membrane proteins despite the central role that they play in regulating cellular functions. Studies of membrane protein global dynamics are particularly challenging due in large part to the anisotropic, viscous nature of the bilayer, the number of different proteins that are present, and the very high local concentrations of total protein. The latter property merits serious attention. While there is variability in the percentage by weight of lipid and protein in different specialized membranes (e.g. myelin versus inner mitochondrial), the plasma membranes of cells and many subcellular organelle membranes are often composed of roughly equal percentages of lipid and protein by weight. Using average values for molecular weights and sizes of lipids and intrinsic membrane proteins, on the order of 30% of the outer membrane surface area can be occupied by protein and the effective concentration of protein within the volume of the bilayer can exceed 100 mg/mL! Given this extremely crowded two-dimensional environment, there are obvious questions regarding the extent of weak interactions between membrane proteins that would not be observed following extraction with non-denaturing detergents and how such interactions might modulate the functions of membrane proteins. Currently, there is unprecedented interest in determining the static structures of membrane proteins and in understanding their functional dynamics. As the structural database of individual membrane proteins continues to expand, studies designed to determine the structural consequences of interactions between proteins in the crowded environment of the cell membrane will undoubtedly take on increased importance.

A number of different experimental approaches have been utilized to examine the oligomeric state and higher order lateral organization of membrane proteins in situ including chemical cross-linking, visualization by freeze-fracture electron microscopy, visualization by fluorescence microscopy, and target size analysis by neutron inactivation to cite a few. Each of these approaches has provided valuable information but each is limited in its ability to provide information on dynamic aspects of protein-

protein interactions under native conditions. Fluorescence resonance energy transfer (FRET; Stryer, 1978) between labeled subunits has been utilized extensively to provide direct information on the stable assembly and dynamic proximity of membrane proteins (e.g. Blackman et al., 1998) including changes in assembly associated with cellular signaling events in living cells (e.g. Martin-Fernandez et al., 2002).

Studies of the translational or rotational dynamics of membrane proteins have been carried out under a wide range of experimental conditions including in living cells and these studies have provided important insights into static and dynamic aspects of their assembly and interactions. Most studies of translational diffusion have utilized fluorescence recovery after photobleaching (FRAP; Frye and Edidin, 1970; Webb, 1981) due in large part to the excellent signal-to-noise ratio and dynamic range that this methodology provides. More recently, single particle tracking experiments have provided new insights into the constraints on the lateral motion of membrane proteins (e.g. Cherry et al., 1998). While these optical techniques provide outstanding capabilities for investigating the molecular basis for constraints to the long range lateral movement of membrane proteins on the cell surface (e.g. Koppel et al., 1981), the translational diffusion coefficient is weakly dependent on the effective size of the diffusing species (Saffman and Delbrück, 1975) and hence, to oligomeric state or to other localized, transient interactions. On the other hand, the rotational diffusion coefficient, of an integral membrane protein undergoing global uniaxial rotational diffusion (URD) is inversely proportional to the square of the cross sectional radius of the protein complex (Saffman and Delbrück, 1975; Jähnig, 1986). Thus, measurements of rotational diffusion should be very sensitive to the presence of homo and hetero oligomeric protein complexes in the membrane.

Given the predicted high sensitivity of the correlation time for URD, which can be defined as on the effective sizes of membrane proteins and their complexes in membranes, it is not surprising that considerable effort during the past three decades has been devoted to developing spectroscopic methods that can measure their rotational dynamics in systems ranging from reconstituted proteoliposomes to intact, living cells (Edidin, 1974). These efforts have led to the development of two complementary approaches that have been successfully applied to measuring the very slow rotational dynamics, on the timescale of to msec, that are characteristic of many integral membrane proteins in their natural membranes or in proteoliposomes.

The first approach is a collection of related optical methods that measure the transient optical anisotropy (TOA) or dichroism, following a short excitation pulse of light, of long lived triplet probes coupled to a target

372 ALBERT H. BETH AND ERIC J. HUSTEDT

protein (Cone, 1972; Naqvi et al., 1973; Cherry et al., 1976; Cherry and Schneider, 1976). TOA methods and their applications to measure the rotational diffusion of membrane proteins have been reviewed previously (e.g. Cherry, 1978; 1979; 1981; Thomas et al., 1985). Analytical expressions have been derived for the amplitudes and rates of anisotropy decays for a variety of diffusion models including URD (Cherry, 1979), constrained URD (Wahl, 1975, Szabo, 1984), wobble in a cone (Szabo, 1984), and generalized anisotropic diffusion (Weber, 1971; Chuang and Eisenthal, 1972; Belford et al., 1972; Ehrenberg and Rigler, 1972). These various models provide a sound theoretical base upon which experimental data can be analyzed in a model dependent fashion to determine the values of of membrane proteins as well as the number of different rotational species that are present. Unique interpretation of TOA data from anisotropic systems requires determination of the orientation of the molecular probe reference frame relative to the molecular reference frame and the uniqueness of this orientation. Determination of the orientations of the absorption and emission dipoles of a chromophore relative to the molecular reference frame can be quite challenging (e.g. Blackman et al., 1996) in comparison with the relative ease with which the orientation of a spin label can be determined by EPR (e.g. Hustedt and Beth, 1996).

Typically, TOA data are analyzed by fitting to a multi-exponential decay curve. The biggest challenge in interpreting the anisotropy decays from membrane proteins is the assignment of the multiple decay components to distinct molecular species and to specific dynamic processes. Even for simple unconstrained URD, two decay components are predicted for each distinct oligomeric species that is present (Nigg and Cherry, 1980). Experimentally, one often observes many decay components and it is possible to fit the complex decays to a variety of different multi-component models with similar statistical agreement between experiment and theory. Matayoshi and Jovin (1991) and Blackman et al., (1996; 2001) have addressed some of the complexities that are involved in uniquely interpreting TOA data from membrane proteins in cell membranes.

The second approach that has been utilized to characterize the rotational diffusion of intrinsic membrane proteins is an EPR method, developed by Hyde and Dalton (1972) which measures the effects of rotational diffusion on spectral lineshapes from nitroxide spin labeled proteins under conditions of continuous, partially saturating microwave excitation. The EPR method, which derives its sensitivity to motions from the “transfer” of microwave “saturation” between different orientational resonance positions of the spectrum due to rotational diffusion, has been universally referred to as saturation transfer EPR (ST-EPR; Thomas et al., 1976). A number of reviews of ST-EPR and its applications to studies of very slow rotational