Measurement of rotational dynamics by the simultaneous nonlinear analysis of optical and EPR data.

In the preceding companion article in this issue, an optical dye and a nitroxide radical were combined in a new dual function probe, 5-SLE. In this report, it is demonstrated that time-resolved optical anisotropy and electron paramagnetic resonance (EPR) data can be combined in a single analysis to measure rotational dynamics. Rigid-limit and rotational diffusion models for simulating nitroxide EPR data have been incorporated into a general non-linear least-squares procedure based on the Marquardt-Levenberg algorithm. Simultaneous fits to simulated time-resolved fluorescence anisotropy and linear EPR data, together with simultaneous fits to experimental time-resolved phosphorescence anisotropy decays and saturation transfer EPR (ST-EPR) spectra of 5-SLE noncovalently bound to bovine serum albumin (BSA) have been performed. These results demonstrate that data from optical and EPR experiments can be combined and globally fit to a single dynamic model.     

Dynamics and ordering in a spin-labeled oligonucleotide observed by 220 GHz electron paramagnetic resonance

The dynamics of a newly synthesized cytosine spin-label and the spin-labeled pentamer TTC*TT have been observed by high-frequency (220 GHz) electron paramagnetic resonance (EPR) in aqueous solution at ambient temperature using only nanomolar amounts of spin-label. Temperature studies were carried out for both labeled species in buffer containing glycerol. The motion of the spin-labeled monomer could be fitted using a model of fully anisotropic rotation (FAR) over the entire temperature range studied. In the single-stranded pentamer, the high-field spectra are best interpreted using a model of microscopic ordering with macroscopic disorder (MOMD) with the probe in a highly nonpolar environment. The observed local order parameters of 0.60-0.70 suggest a micelle-like structure in which the label is tightly packed with the hydrophobic bases. These preliminary studies illustrate how the excellent orientation selectivity of high-field EPR provides new dynamic information about local base motions in DNA, and also how high-field EPR of spin-labels allows one to discriminate accurately between the effects of local versus global motions in spin-labeled macromolecules.     

An EPR study of the rotational dynamics of actins from striated and smooth muscle and their complexes with heavy meromyosin

The rotational motions of the actin from rabbit skeletal muscle and from chicken gizzard smooth muscle were measured by conventional and saturation transfer electron paramagnetic resonance (EPR) spectroscopy using maleimide spin-label rigidly bound at Cys-374. The conventional EPR spectra indicate a slight difference in the polarity of the environment of the label and in the rotational mobility of the monomeric gizzard actin compared to its skeletal muscle counterpart. These differences disappear upon polymerization. The EPR spectra of the two actins in their F form and in their complexes with heavy meromyosin (HMM) did not reveal any difference in the rotational dynamic properties that might be correlated with the known differences in the activation of myosin ATPase activity by smooth and skeletal muscle actin. Our results agree with earlier EPR studies on skeletal muscle actin in showing that polymerization stops the nanosecond rotational motion of actin monomers and that F-actin undergoes rotational motion having an effective correlation time of the order of 0.1 ms. However, our measurements show that complete elimination of the nanosecond motions requires prolonged incubation of F-actin, suggesting that the slow formation of interfilamental cross-links in concentrated F-actin solutions contributes to this process. We have also used the EPR spectroscopy to study the interaction between HMM and actin in the F and G form. Our results show that in the absence of salt one HMM molecule can cooperatively interact with eight monomers to produce a polymer which closely resembles F-actin in its rotational mobility but differs from the complex of F-actin with HMM. The results indicate that salt is necessary for further slowing down, in a cooperative manner, the sub-millisecond internal motion in actin polymer and for a non-cooperative change in the intramonomer conformation around Cys-374 on the binding of HMM.     

Large-scale rotational motions of proteins detected by electron paramagnetic resonance and fluorescence.

Direct spectroscopic measurements of rotational motions of proteins and large protein segments are crucial to understanding the molecular dynamics of protein function. Fluorescent probes and spin labels attached to proteins have proved to be powerful tools in the study of large-scale protein motions. Fluorescence depolarization and conventional electron paramagnetic resonance (EPR) are applicable to the study of rotational motions in the nanosecond-to-microsecond time range, and have been used to demonstrate segmental flexibility in an antibody and in myosin. Very slow rotational motions, occurring in the microsecond-to-millisecond time range, are particularly important in supramolecular assemblies, where protein motions are restricted by association with other molecules. Saturation transfer spectroscopy (ST-EPR), a recently developed electron paramagnetic resonance (EPR) technique that permits the detection of rotational correlation times as long as 1 ms, has been used to detect large-scale rotational motions of spin-labeled proteins in muscle filaments and in membranes, providing valuable insights into energy transduction mechanisms in these assemblies.     

Spin-label studies of the lipid and protein components of erythrocyte membranes. A comparison of electron paramagnetic resonance and saturation transfer electron paramagnetic resonance methods.

We have used both a protein spin label and a lipid spin probe to study some of the slow motions of proteins and of lipids, respectively, in intact erythrocyte membranes. Three electron paramagnetic resonance (EPR) methods, conventional (V1) EPR, second harmonic out-of-phase absorption saturation transfer (ST) EPR (V'2), and first harmonic out-of-phase dispersion ST EPR (U'1) were used to compare the experimental methods and spectral sensitivities with different kinds of molecular motions in human erythrocyte membranes under different experimental conditions. The results show that the V'2 display is relatively more sensitive to the protein motion, while the U'1 display appears more sensitive to the lipid motions, and the V'2 display is substantially more convenient to obtain than the U'1 display.     

Time-dependent phosphorescence anisotropy measurements of the slow rotational motions of proteins in viscous solution

A method of monitoring slow rotational motions of proteins from the decay of the intrinsic phosphorescence is described. The phosphorescence is excited with a 10-μsec pulse of vertically polarized light from an air gap lamp, and the anisotropy was computed as a function of time from the simultaneously detected vertically and horizontally polarized components of the emission. The approach is illustrated with time-dependent measurements of the anisotropy of the tryptophan phosphorescence of Staphylococcus aureus nuclease, bovine carbonic anhydrase, and liver alcohol dehydrogenase in glycerol-phosphate buffer between ?90 and ?70°C. The temperature- and molecular-weight dependence of the exponential decays in the anisotropy indicate that overall rotation of the proteins is at the origin of the depolarization. The potential of the approach as a probe of the slow rotational motions of proteins in membranes and other macromolecular complexes is stressed.     

Rotational dynamics of the Fc epsilon receptor on mast cells monitored by specific monoclonal antibodies and IgE.

The rotational motions of the type I receptor for the Fc epsilon domains (Fc epsilon RI) present on mast cells were investigated by measuring the phosphorescence emission and anisotropy decay kinetics of erythrosin (Er) covalently bound to several Fc epsilon RI-specific macromolecular ligands. The latter consisted of three murine monoclonal antibodies (IgG class) raised against the Fc epsilon RI of rat mast cells (RBL-2H3 line), their Fab fragments, and a murine monoclonal IgE. Different anisotropy decay patterns were observed for the three monovalent Er-Fab fragments bound to the Fc epsilon RI, reflecting the rotational motion of the Fe epsilon RI reported by each specific macromolecular probe bound to its particular epitope. Internal motions of the tethered Er-labeled ligands may also contribute to the observed anisotropy decay, particularly in the case of cell-bound IgE. The results corroborate an earlier study with rat Er-IgE in which the Fc epsilon RI-IgE complex was shown to be mobile throughout the temperature range examined (5-37 degrees C). The anisotropy decays of the three Er-labeled, Fc epsilon RI-specific intact mAbs bound to cells also differed markedly. Whereas the decay curves of one mAb (H10) were characterized by temperature-dependent positive amplitudes and rather short rotational correlation times, the decay of a second mAb (J17) showed complex qualitative variations with temperature, and in the case of the third antibody (F4), there was no apparent decay of anisotropy over the time and temperature ranges examined.(ABSTRACT TRUNCATED AT 250 WORDS)     

Rotational dynamics of spin-labeled F-actin in the sub-millisecond time range.

The rotational motions of F-actin filaments and myosin heads attached to them have been measured by saturation transfer electron paramagnetic resonance spectroscopy using spin-labels rigidly bound to actin, or to the myosin head region in intact myosin molecules, heavy meromyosin, and subfragment-1. The spin-label attached to F-actin undergoes rotational motion having an effective correlation time of the order of 10^?4 seconds. This cannot be interpreted as rotation of the entire F-actin filament or local rotation of the spin-label, but must represent an internal rotational mode of F-actin, possibly a bending or flexing motion, or a rotation of an actin monomer or a segment of it. The rate of this rotational motion is reduced approximately fourfold by myosin, HMM or S-1; HMM and S-1 are equally effective, on a molar basis, in slowing this rotation and both produce their maximal effect at a ratio of about one molecule of HMM or S-1 per ten actin monomers. With chymotryptic S-1, the effect is partially reversed at higher concentrations. With S-1 prepared with papain in the presence of Mg²⁺, the reversal is smaller, while with HMM or myosin there is no reversal at higher concentrations. Tropomyosin slightly decreases the actin rotational mobility, and the addition of HMM to the actin-tropomyosin complex produces a further slowing. The rotational correlation time for acto-HMM is the same whether the spin-label is on actin or HMM, indicating that the rotation of the head region of HMM when bound to F-actin is controlled by a mode of rotation within the F-actin filaments.     

Human serum albumin. Spectroscopic studies of binding and proximity relationships for fatty acids and bilirubin.

Binding and proximity relationships of hydrophobic ligands on human serum albumin have been studied using absorption, fluorescence, circular dichroism, and electron paramagnetic resonance spectroscopy. The ligands studied were bilirubin, two conjugated linear polyene fatty acids, cis-parinaric acid and cis-eleostearic acid, and three nitroxide derivatives of stearic acid with doxyl groups at positions 5, 10, and 12, respectively. Binding of polyene fatty acids was monitored by absorption peak shifts, induced circular dichroism, enhancement of fluorescence, and energy transfer between albumin's single tryptophanyl residue and the polyene chromophore. Induced circular dichroism studies indicate excitonic ligand-ligand interaction between bound fatty acids. Fluorescence enhancement of cis-parinaric acid was analyzed using a stepwise multiple equilibrium model, and six binding constants in the range 10(8) to 10(6) M-1 were obtained, in agreement with previous measurements for other fatty acids. The temperature dependence of the equilibrium constants indicates that the binding enthalpy is nearly zero. Fluorescence energy transfer was similarly used to quantitate bilirubin binding to albumin. Energy transfer, nitroxide quenching of fluorescence, and electron paramagnetic resonance spectroscopy were used to elucidate binding geometries which support and extend proposed structural models for albumin. It is suggested that the first two fatty acids bind side-by-side in an antiparallel fashion in domain III of human serum albumin.     

Microsecond rotational motions of eosin-labeled myosin measured by time-resolved anisotropy of absorption and phosphorescence

We have studied submicrosecond and microsecond rotational motions within the contractile protein myosin by observing the time-resolved anisotropy of both absorption and emission from the long-lived triplet state of eosin-5-iodoacetamide covalently bound to a specific site on the myosin head. These results, reporting anisotropy data up to 50 microseconds after excitation, extend by two orders of magnitude the time range of data on time-resolved site-specific probe motion in myosin. Optical and enzymatic analyses of the labeled myosin and its chymotryptic digests show that more than 95% of the probe is specifically attached to sulfhydryl-1 (SH1) on the myosin head. In a solution of labeled subfragment-1 (S-1) at 4 degrees C, absorption anisotropy at 0.1 microseconds after a laser pulse is about 0.27. This anisotropy decays exponentially with a rotational correlation time of 210 ns, in good agreement with the theoretical prediction for end-over-end tumbling of S-1, and with times determined previously by fluorescence and electron paramagnetic resonance. In aqueous glycerol solutions, this correlation time is proportional to viscosity/temperature in the microsecond time range. Furthermore, binding to actin greatly restricts probe motion. Thus the bound eosin is a reliable probe of myosin-head rotational motion in the submicrosecond and microsecond time ranges. Our submicrosecond data for myosin monomers (correlation time 400 ns) also agree with previous results using other techniques, but we also detect a previously unresolvable slower decay component (correlation time 2.6 microseconds), indicating that the faster motions are restricted in amplitude. This restriction is not consistent with the commonly accepted free-swivel model of S-1 attachment in myosin. In synthetic thick filaments of myosin, both fast (700 ns) and slow (5 microseconds) components of anisotropy decay are observed. In contrast to the data for monomers, the anisotropy of filaments has a substantial residual component (26% of the initial anisotropy) that does not decay to zero even at times as long as 50 microseconds, implying significant restriction in overall rotational amplitude. This result is consistent with motion restricted to a cone half-angle of about 50 degrees. The combined results are consistent with a model in which myosin has two principal sites of segmental flexibility, one giving rise to submicrosecond motions (possibly corresponding to the junction between S-1 and S-2) and the other giving rise to microsecond motions (possibly corresponding to the junction between S-2 and light meromyosin).(ABSTRACT TRUNCATED AT 400 WORDS)     

Simulation of nitroxide electron paramagnetic resonance spectra from brownian trajectories and molecular dynamics simulations

A simulated continuous wave electron paramagnetic resonance spectrum of a nitroxide spin label can be obtained from the Fourier transform of a free induction decay. It has been previously shown that the free induction decay can be calculated by solving the time-dependent stochastic Liouville equation for a set of Brownian trajectories defining the rotational dynamics of the label. In this work, a quaternion-based Monte Carlo algorithm has been developed to generate Brownian trajectories describing the global rotational diffusion of a spin-labeled protein. Also, molecular dynamics simulations of two spin-labeled mutants of T4 lysozyme, T4L F153R1, and T4L K65R1 have been used to generate trajectories describing the internal dynamics of the protein and the local dynamics of the spin-label side chain. Trajectories from the molecular dynamics simulations combined with trajectories describing the global rotational diffusion of the protein are used to account for all of the dynamics of a spin-labeled protein. Spectra calculated from these combined trajectories correspond well to the experimental spectra for the buried site T4L F153R1 and the helix surface site T4L K65R1. This work provides a framework to further explore the modeling of the dynamics of the spin-label side chain in the wide variety of labeling environments encountered in site-directed spin labeling studies.     

Slow-motion ESR study of order and dynamics in oriented lipid multibilayers: effects of unsaturation and hydration

Electron spin resonance (ESR) experiments were carried out on 3-doxyl-5 alpha-cholestane spin-label (CSL) molecules embedded in macroscopically oriented multibilayers of dimyristoylphosphatidylcholine (DMPC), palmitoyloleoylphosphatidylcholine (POPC), dioleoylphosphatidylcholine (DOPC) and dilinoleoylphosphatidylcholine (DLPC). For these lipids we studied the effects of temperature, hydration and unsaturation on the orientational order parameters and rotational motions of the probe molecules in the liquid crystalline phase. The experimental ESR spectra were simulated by a numerical solution of the stochastic Liouville equation (SLE) for the density matrix of a spin-label molecule. This allows extraction of detailed information about both molecular order and rotational dynamics. The data show that, in our temperature range, the lipid systems are in the slow-motion regime, thereby precluding a motional narrowing interpretation. This is illustrated by a simple model calculation which shows that a fast-motion interpretation seriously overestimates the order parameters. We have compared our results with data obtained independently from angle-resolved fluorescence depolarization (AFD) experiments on oriented bilayers in which 1-[4-(trimethylammonio)phenyl]-6-phenyl-1,3,5-hexatriene (TMA-DPH) molecules were used as fluorescent probes (Deinum et al., (1988) Biochemistry 27, 852-860). It is found that the orientational order and the rotational dynamics obtained with both techniques agree well. This shows that the probe molecules do not perturb the local bilayer structure to any large extent and that they indeed reflect the intrinsic behaviour of the lipid molecules. Upon increase in temperature or hydration, we observe faster reorientational motion and lower molecular ordering. In contrast, we do not find any systematic effect of unsaturation on molecular reorientational motion. Our results indicate that changes in membrane molecular order and reorientational dynamics have to be considered separately and are not necessarily correlated as implied by the common concept of membrane fluidity.     

Time-resolved fluorescence microscopy.

In fluorescence microscopy, the fluorescence emission can be characterised not only by intensity and position, but also by lifetime, polarization and wavelength. Fluorescence lifetime imaging (FLIM) can report on photophysical events that are difficult or impossible to observe by fluorescence intensity imaging, and time-resolved fluorescence anisotropy imaging (TR-FAIM) can measure the rotational mobility of a fluorophore in its environment. We compare different FLIM methods: a chief advantage of wide-field time-gating and phase modulation methods is the speed of acquisition whereas for time-correlated single photon counting (TCSPC) based confocal scanning it is accuracy in the fluorescence decay. FLIM has been used to image interactions between proteins such as receptor oligomerisation and to reveal protein phosphorylation by detecting fluorescence resonance energy transfer (FRET). In addition, FLIM can also probe the local environment of fluorophores, reporting, for example, on the local pH, refractive index, ion or oxygen concentration without the need for ratiometric measurements.     

Protein structural dynamics revealed by site-directed spin labeling and multifrequency EPR

Multifrequency electron paramagnetic resonance (EPR), combined with site-directed spin labeling, is a powerful spectroscopic tool to characterize protein dynamics. The lineshape of an EPR spectrum reflects combined rotational dynamics of the spin probe's local motion within a protein, reorientations of protein domains, and overall protein tumbling. All these motions can be restricted and anisotropic, and separation of these motions is important for thorough characterization of protein dynamics. Multifrequency EPR distinguishes between different motions of a spin-labeled protein, due to the frequency dependence of EPR resolution to fast and slow motion of a spin probe. This gives multifrequency EPR its unique capability to characterize protein dynamics in great detail. In this review, we analyze what makes multifrequency EPR sensitive to different rates of spin probe motion and discuss several examples of its usage to separate spin probe dynamics and overall protein dynamics, to characterize protein backbone dynamics, and to resolve protein conformational states.     

Differential dynamic behavior of actin filaments containing tightly-bound Ca2+ or Mg2+ in the presence of myosin heads actively hydrolyzing ATP.

We have investigated how Ca2+ or Mg2+ bound at the high-affinity cation binding site in F-actin modulates the dynamic response of these filaments to ATP hydrolysis by attached myosin head fragments (S1). Rotational motions of the filaments were monitored using steady-state phosphorescence emission anisotropy of the triplet probe erythrosin-5-iodoacetamide covalently attached to cysteine 374 of actin. The anisotropy of filaments containing only Ca2+ increased from 0.080 to 0.137 upon binding S1 in a rigor complex and decreased to 0.065 in the presence of ATP, indicating that S1 induced additional rotational motions in the filament during ATP hydrolysis. The comparable anisotropy values for Mg(2+)-containing filaments were 0.067, 0.137, and 0.065, indicating that S1 hydrolysis did not induce measurable rotational motions in these filaments. Phalloidin, a fungal toxin which stabilizes F-actin and increases its rigidity, increased the anisotropy of F-actin containing either Ca2+ or Mg2+ but not the anisotropy of the 1:1 S1-actin complexes of these filaments. Mg(2+)-containing filaments with phalloidin bound also displayed increased rotational motions during S1 ATP hydrolysis. A strong positive correlation between the phosphorescence anisotropy of F-actin under specific conditions and the extent of the rotational motions induced by S1 during ATP hydrolysis suggested that the long axis torsional rigidity of F-actin plays a crucial role in modulating the dynamic response of the filaments to ATP hydrolysis by S1. Cooperative responses of F-actin to dynamic perturbations induced by S1 during ATP hydrolysis may thus be physically mediated by the torsional rigidity of the filament.     

Dynamics and interactions of the anion channel in intact human erythrocytes: an electron paramagnetic resonance spectroscopic study employing a new membrane-impermeant bifunctional spin-label

We have developed a new membrane-impermeant, bifunctional spin-labeling reagent, bis-(sulfo-N-succinimidyl) doxyl-2-spiro-4'-pimelate (BSSDP), and employed it in an electron paramagnetic resonance (EPR) study of the rotational diffusion of the anion-exchange channel (band 3) in intact human erythrocytes. BSSDP reacts in a covalent manner and with high specificity with the extracytoplasmic domain of band 3, forming a complex in which the spin-label is immobilized on the protein. The linear EPR spectrum of BSSDP-labeled intact erythrocytes is characteristic of a highly immobilized, spatially isolated nitroxide probe. The saturation-transfer EPR spectrum of the same sample indicates that the anion channel in intact erythrocytes exhibits rotational dynamics in the 0.1-1 ms correlation time range at 20 degrees C. Rotational dynamics in this motional domain are consistent with a strong interaction of the anion-exchange channel with the erythrocyte cytoskeleton. The saturation-transfer EPR spectrum of ghosts prepared from BSSDP-labeled erythrocytes indicates a significant increase in rotational mobility of the anion channel, suggesting a significant disruption on lysis of interactions between the anion channel and the cytoskeleton.     

Time-resolved rotational dynamics of phosphorescent-labeled myosin heads in contracting muscle fibers.

We have measured the microsecond rotational motions of myosin heads in contracting rabbit psoas muscle fibers by detecting the transient phosphorescence anisotropy of eosin-5-maleimide attached specifically to the myosin head. Experiments were performed on small bundles (10-20 fibers) of glycerinated rabbit psoas muscle fibers at 4 degrees C. The isometric tension and physiological ATPase activity of activated fibers were unaffected by labeling 60-80% of the heads. Following excitation of the probes by a 10-ns laser pulse polarized parallel to the fiber axis, the time-resolved emission anisotropy of muscle fibers in rigor (no ATP) showed no decay from 1 microsecond to 1 ms (r infinity = 0.095), indicating that all heads are rigidly attached to actin on this time scale. In relaxation (5 mM MgATP but no Ca2+), the anisotropy decayed substantially over the microsecond time range, from an initial anisotropy (r0) of 0.066 to a final anisotropy (r infinity) of 0.034, indicating large-amplitude rotational motions with correlation times of about 10 and 150 microseconds and an overall angular range of 40-50 degrees. In isometric contraction (MgATP plus saturating Ca2+), the amplitude of the anisotropy decay (and thus the amplitude of the microsecond motion) is slightly less than in relaxation, and the rotational correlation times are about twice as long, indicating slower motions than those observed in relaxation. While the residual anisotropy (at 1 ms) in contraction is much closer to that in relaxation than in rigor, the initial anisotropy (at 1 microsecond) is approximately equidistant between those of rigor and relaxation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Orientation dependence and motional properties of spin labels in cardiac and skeletal muscle fibres

Orientation dependence and rotational motion of maleimide spin labels attached to the fast reacting thiol sites of myosin were studied in glycerinated cardiac and skeletal muscle fibres in rigor and in relaxing medium. The probe order in skeletal muscle was shown to be about one order of magnitude higher than that in cardiac muscle. In skeletal muscle in rigor the orientational order is static on the time scale of the saturation transfer electron paramagnetic resonance measurement (ST EPR, rotational correlation time of the label is greater than 1 ms), but in cardiac muscle fibres, a disorder was observed which was at least partly dynamical, the rotational correlation time being about 100 microseconds. In relaxing solution the degree of order of probe molecules in both types of muscle was strongly reduced at and above the resting length. The disorder was at least partly dynamical on the ST EPR time scale, the apparent rotational correlation times being 200 microseconds for skeletal muscle and 60 microseconds for cardiac muscle, respectively. According to the results of ST EPR the rotational behavior of cross-bridges was identical in cardiac and skeletal muscle in relaxing medium.     

Interactions of 14N:15N stearic acid spin-label pairs: effects of host lipid alkyl chain length and unsaturation

Electron-electron double resonance (ELDOR) and saturation recovery electron paramagnetic resonance (EPR) spectroscopy have been employed to examine the interactions of 14N:15N stearic acid spin-label pairs in fluid-phase model membrane bilayers composed of a variety of phospholipids. The [14N]-16-doxylstearate:[15N]-16-doxylstearate (16:16) pair was utilized to measure lateral diffusion of the spin-labels, while the [14N]-16-doxylstearate:[15N]-5-doxylstearate (16:5) pair provided information on vertical fluctuations of the 16-doxylstearate nitroxide moiety toward the membrane surface. Three saturated host lipids of varying alkyl chain length [dimyristoylphosphatidylcholine (DMPC), dipalmitoylphosphatidylcholine (DPPC), and distearoylphosphatidylcholine (DSPC)], an alpha-saturated, beta-unsaturated lipid [1-palmitoyl-2-oleoylphosphatidylcholine (POPC)], and phosphatidylcholine from a natural source [egg yolk phosphatidylcholine (egg PC)] were utilized as host lipids. Lateral diffusion of the stearic acid spin-labels was only slightly affected by alkyl chain length at a given reduced temperature (Tr) in the saturated host lipids but was significantly decreased in POPC at the same Tr. Lateral diffusion in DMPC, POPC, and egg PC was quite similar at 37 degrees C. A strong correlation was noted between lateral diffusion constants and rotational mobility of [14N]-16-doxylstearate. Vertical fluctuations were likewise only slightly influenced by alkyl chain length but were strongly diminished in POPC and egg PC relative to the saturated systems. This diminution of the 16:5 interaction was observed even under conditions where no differences were discernible by conventional EPR. These studies indicate that vertical fluctuation of 16-doxylstearate is quite sensitive to host lipid unsaturation and that ELDOR studies of interactions between 14N:15N spin-label pairs can provide information on spin-label motion beyond that given by conventional EPR.     

Microsecond rotational dynamics of phosphorescent-labeled muscle cross-bridges

We have measured the microsecond rotational motions of myosin heads in muscle cross-bridges under physiological ionic conditions at 4 degrees C, by detecting the time-resolved phosphorescence of eosin-maleimide covalently attached to heads in skeletal muscle myofibrils. The anisotropy decay of heads in rigor (no ATP) is constant over the time range from 0.5 to 200 microsecond, indicating that they do not undergo rotational motion in this time range. In the presence of 5 mM MgATP, however, heads undergo complex rotational motion with correlation times of about 5 and 40 microsecond. The motion of heads in relaxed myofibrils is restricted out to 1 ms, as indicated by a nonzero value of the residual anisotropy. The anisotropy decay of eosin-labeled myosin, extracted from labeled myofibrils, also exhibits complex decay on the 200-microsecond time scale when assembled into synthetic thick filaments. The correlation times and amplitudes of heads in filaments (under the same ionic conditions as the myofibril experiments) are unaffected by MgATP and very similar to the values for heads in relaxed myofibrils. The larger residual anisotropy and longer correlation times seen in myofibrils are consistent with a restriction of rotational motion in the confines of the myofibril protein lattice. These are the first time-resolved measurements under physiological conditions of the rotational motions of cross-bridges in the microsecond time range.