Saturation Transfer Electron Paramagnetic Resonance Research Articles

A Highly Ordered Nitroxide Side Chain for Distance Mapping and Monitoring Slow Structural Fluctuations in Proteins.

The online version contains supplementary material available at 10.1007/s00723-023-01618-8.

Structural Dynamics of Protein Interactions Using Site-Directed Spin Labeling of Cysteines to Measure Distances and Rotational Dynamics with EPR Spectroscopy.

Here we review applications of site-directed spin labeling (SDSL) with engineered cysteines in proteins, to study the structural dynamics of muscle and non-muscle proteins, using and developing the electron paramagnetic resonance (EPR) spectroscopic techniques of dipolar EPR, double electron electron resonance (DEER), saturation transfer EPR (STEPR), and orientation measured by EPR. The SDSL technology pioneered by Wayne Hubbell and collaborators has greatly expanded the use of EPR, including the measurement of distances between spin labels covalently attached to proteins and peptides. The Thomas lab and collaborators have applied these techniques to elucidate dynamic interactions in the myosin-actin complex, myosin-binding protein C, calmodulin, ryanodine receptor, phospholamban, utrophin, dystrophin, β-III-spectrin, and Aurora kinase. The ability to design and engineer cysteines in proteins for site-directed covalent labeling has enabled the use of these powerful EPR techniques to measure distances, while showing that they are complementary with optical spectroscopy measurements.

ESR, STESR, DFT, and MD Study of the Dynamical Structure of Cucurbituril[7]-Spin Probe Guest-Host Complexes.

We study the molecular dynamics and structures of the guest–host complexes of cucurbituril, CB[7], with spin probes through the conventional electron spin resonance (ESR), saturation transfer ESR (STESR), density functional theory (DFT), and molecular dynamics (MD) computations. Protonated TEMPOamine (I), a derivative of TEMPO having a positive charge and an octyl group on the quaternary nitrogen atom (II), and the neutral spin-labeled indole (III) are used as guests. To eliminate the overall complex rotation, the solutions of complexes in a solid CB[7] matrix were prepared. Resultantly, for all of the spin probes, the combined study of the conventional ESR and STESR spectra indicates the librational character of the rotational motion within the CB[7] cavity as opposed to the diffusional rotation over the whole solid angle. The kinetic accessibilities of the reporter NO groups to the paramagnetic complexes in aqueous solutions, determined by Heisenberg exchange broadening of the ESR spectra, together with the environment polarities from the hyperfine interaction values, as well as DFT computation results and MD simulations, were used to estimate the spin probe location relative to CB[7]. Utilizing the concept of the aqueous clusters surrounding the spin probes and CB[7] molecules and MD simulations has allowed the application of DFT to estimate the aqueous environment effects on the complexation energy and spatial structure of the guest–host complexes.

Slow Dynamics around a Protein and Its Coupling to Solvent.

Solvent is essential for protein dynamics and function, but its role in regulating the dynamics remains debated. Here, we employ saturation transfer electron spin resonance (ST-ESR) to explore the issue and characterize the dynamics on a longer (from μs to s) time scale than has been extensively studied. We first demonstrate the reliability of ST-ESR by showing that the dynamical changeovers revealed in the spectra agree to liquid–liquid transition (LLT) in the state diagram of the glycerol/water system. Then, we utilize ST-ESR with four different probes to systematically map out the variation in local (site-specific) dynamics around a protein surface at subfreezing temperatures (180–240 K) in 10 mol % glycerol/water mixtures. At highly exposed sites, protein and solvent dynamics are coupled, whereas they deviate from each other when temperature is greater than LLT temperature (∼190 K) of the solvent. At less exposed sites, protein however exhibits a dynamic, which is distinct from the bulk solvent, throughout the temperature range studied. Dominant dynamic components are thus revealed, showing that (from low to high temperatures) the overall structural fluctuation, rotamer dynamics, and internal side-chain dynamics, in turn, dominate the temperature dependence of spin-label motions. The structural fluctuation component is relatively slow, collective, and independent of protein structural segments, which is thus inferred to a fundamental dynamic component intrinsic to protein. This study corroborates that bulk solvent plasticizes protein and facilitates rather than slaves protein dynamics.

Interaction of Huntingtin Exon-1 Peptides with Lipid-Based Micellar Nanoparticles Probed by Solution NMR and Q-Band Pulsed EPR.

Lipid-based micellar nanoparticles promote aggregation of huntingtin exon-1 peptides. Here we characterize the interaction of two such peptides, httNTQ 7 and httNTQ 10 comprising the N-terminal amphiphilic domain of huntingtin followed by 7 and 10 glutamine repeats, respectively, with 8 nm lipid micelles using NMR chemical exchange saturation transfer (CEST), circular dichroism and pulsed Q-band EPR. Exchange between free and micelle-bound httNTQ n peptides occurs on the millisecond time scale with a KD ∼ 0.5-1 mM. Upon binding micelles, residues 1-15 adopt a helical conformation. Oxidation of Met7 to a sulfoxide reduces the binding affinity for micelles ∼3-4-fold and increases the length of the helix by a further two residues. A structure of the bound monomer unit is calculated from the backbone chemical shifts of the micelle-bound state obtained from CEST. Pulsed Q-band EPR shows that a monomer-dimer equilibrium exists on the surface of the micelles and that the two helices of the dimer adopt a parallel orientation, thereby bringing two disordered polyQ tails into close proximity which may promote aggregation upon dissociation from the micelle surface.

Chain Dynamics, Relaxation Times, and Conductivities of Bithiophene--Acene Copolymers Measured Using High Frequency Saturation Transfer EPR.

We present a study to probe the formation of localized aromatic sextets and their effects on the charge transport properties in polymers with acene cores. Bithiophene-acene copolymers containing benzene, naphthalene, or anthracene as acene cores were synthesized using Yamamoto polymerization. Drop-casted polymer films were chemically doped and analyzed using high frequency saturation transfer EPR (HF ST-EPR), a method which has proven useful in the study of conducting polymers. The spin-spin and spin-lattice relaxation times were determined for these polymers at low temperatures (4 to 20 K) and used to obtain inter- and intrachain spin diffusion rates and conductivities. Similar interchain spin diffusion rates were seen across all polymer systems; however, anthracene containing polymer poly(hexylTTATT) was found to have the largest intrachain spin diffusion rate. The poly(hexylTTATT) intrachain spin diffusion rate may be artificially high if the anthracene ring restricts the diffusion of spin to the hexylated quaterthiophene segment in poly(hexylTTATT) whereas the spins diffuse through the acene cores in the benzene and naphthalene derivatives. Alternatively, as both the spin diffusion rates and conductivities vary unpredictably with temperature, it is possible that the π-electron localization previously seen in the anthracene core could be relieved at lower temperatures.

FASCIN and alpha-actinin can regulate the conformation of actin filaments

BackgroundActin filament bundling proteins mediate numerous processes in cells such as the formation of cell membrane protrusions or cell adhesions and stress fiber based locomotion. Among them alpha-actinin and fascin are the most abundant ones. This work characterizes differences in molecular motions in actin filaments due to the binding of these two actin bundling proteins. MethodsWe investigated how alpha-actinin and fascin binding modify the conformation of actin filaments by using conventional and saturation transfer EPR methods. ResultsThe result characteristic for motions on the microsecond time scale showed that both actin bundling proteins made the bending and torsional twisting of the actin filaments slower. When nanosecond time scale molecular motions were described the two proteins were found to induce opposite changes in the actin filaments. The binding of one molecule of alpha-actinin or fascin modified the conformation of numerous actin protomers. ConclusionAs fascin and alpha-actinin participates in different cellular processes their binding can serve the proper tuning of the structure of actin by establishing the right conformation for the interactions with other actin binding proteins. Our observations are in correlation with the model where actin filaments fulfill their biological functions under the regulation by actin-binding proteins. General significanceSupporting the general model for the cellular regulation of the actin cytoskeleton we showed that two abundant actin bundling proteins, fascin and alpha-actinin, alter the conformation of actin filaments through long range allosteric interactions in two different ways providing the structural framework for the adaptation to specific biological functions.

Bifunctional Spin Labeling of Muscle Proteins: Accurate Rotational Dynamics, Orientation, and Distance by EPR.

While EPR allows for the characterization of protein structure and function due to its exquisite sensitivity to spin label dynamics, orientation, and distance, these measurements are often limited in sensitivity due to the use of labels that are attached via flexible monofunctional bonds, incurring additional disorder and nanosecond dynamics. In this chapter, we present methods for using a bifunctional spin label (BSL) to measure muscle protein structure and dynamics. We demonstrate that bifunctional attachment eliminates nanosecond internal rotation of the spin label, thereby allowing the accurate measurement of protein backbone rotational dynamics, including microsecond-to-millisecond motions by saturation transfer EPR. BSL also allows for accurate determination of helix orientation and disorder in mechanically and magnetically aligned systems, due to the label's stereospecific attachment. Similarly, labeling with a pair of BSL greatly enhances the resolution and accuracy of distance measurements measured by double electron-electron resonance (DEER). Finally, when BSL is applied to a protein with high helical content in an assembly with high orientational order (e.g., muscle fiber or membrane), two-probe DEER experiments can be combined with single-probe EPR experiments on an oriented sample in a process we call BEER, which has the potential for ab initio high-resolution structure determination.

Conformationally Trapping the Actin-binding Cleft of Myosin with a Bifunctional Spin Label

We have trapped the catalytic domain of Dictyostelium (Dicty) myosin II in a weak actin-binding conformation by chemically crosslinking two engineered cysteines across the actin-binding cleft, using a bifunctional spin label (BSL). By connecting the lower and upper 50 kDa domains of myosin, the crosslink restricts the conformation of the actin-binding cleft. Crosslinking has no effect on the basal ATPase activity of isolated myosin, but it impairs rigor actin binding and actin-activation of myosin ATPase. EPR spectra of BSL provide insight into actomyosin structural dynamics. BSL is highly immobilized within the actin-binding cleft and is thus exquisitely sensitive to the global orientation and rotational motions of the myosin head. Conventional EPR shows that myosin heads bound to oriented actin filaments are highly disordered with respect to the actin filament axis, in contrast to the nearly crystalline order of myosin heads in rigor. This disorder is similar to that of weakly bound heads induced by ATP, but saturation transfer EPR shows that the disorder of crosslinked myosin is at least 100 times slower. Thus this cleft-crosslinked myosin is remarkably similar, in both actin affinity and rotational dynamics, to SH1-SH2 crosslinked BSL-myosin S1. We conclude that, whether myosin is trapped at the actin-myosin interface or in the force-generating region between the active site and lever arm, the structural state of myosin is intermediate between the weak-binding state preceding phosphate release and the strong-binding state that succeeds it. We propose that it represents the threshold of force generation.

Antifungal activity of the primycin complex and its main components A1, A2 and C1 on a Candida albicans clinical isolate, and their effects on the dynamic plasma membrane changes

The in vitro antifungal activities of the macrolide lactone antibiotic complex primycin (PC) and its main components, A1 (50%), A2 (7.3%) and C1 (13%), against the opportunistic pathogenic fungus Candida albicans 33erg(+)were determined by microdilution testing. The MIC(100) (the minimal concentration required for 100% growth inhibition) values found, A2 (2 μg ml(-1)), PC (32 μg ml(-1)), A1 (32 μg ml(-1)) and C1 (64 μg ml(-1)), suggested that the biological activity of PC is highly dependent on the proportions of its constituents. In vivo measurements of the biophysical properties of plasma membranes were carried out by electron paramagnetic resonance (EPR) spectroscopic methods, using the spin probe 5-(4,4-dimethyloxazolidine-N-oxyl)stearic acid. Conventional EPR measurements demonstrated altered phase transition temperatures (Tm) of the plasma membrane of strain 33erg(+) as a consequence of treatment with PC or its constituents: for cells treated with 128 μg ml(-1) PC, A1, A2 or C1 for 15 min, Tm was 17, 21, 14.5 and 15 °C, respectively; that is significantly higher than the Tm of untreated cells, 12 °C. The molecular motions of the near-surface hydrophobic region of the plasma membrane, estimated by saturation transfer EPR spectroscopy, reflected changes in the membrane phases after the treatment. Two physiological membrane phases were detected in control samples: liquid-ordered and liquid-disordered, characterized by molecular movements ∼10(-6)-10(-8) s and ≥10(-9) s. The cells treated with the investigated compounds showed the strong presence of a non-physiological gel phase additional to the above phases, characterized by movements ≤10(-5) s.

A study of the photoinduced conformational mobility of spin-labeled regenerated rhodopsin by ESR spectroscopy

Saturation transfer EPR spectroscopy is used to study the photoinduced conformational changes of spin-labeled (Cys140 and Cys316) rhodopsin in a photoreceptor membrane. Illumination of rhodopsin with “yellow” light (λ > 450 nm), converting it from the dark form into metarhodopsin II, a physiologically active form, is accompanied by an increase in the mobility of the cytoplasmic loop (Cys140) and eighth alpha-helix (Cys316), whereas the subsequent illumination with “blue” light (λ < 445 nm), which converts metarhodopsin II into a mixture of inactive photoregenerated products, results in a decrease in the mobility. The restoration of the specific features of the photoinduced changes in the conformational mobility of the cytoplasmic loops of rhodopsin regenerated in the dark is demonstrated.

Rotational Diffusion of Membrane Proteins: Characterization of Protein-Protein Interactions in Membranes

It has long been appreciated that the rotational diffusion coefficient (Dr) and hence the rotational correlation time (τr) of a transmembrane protein about its membrane normal axis is predicted to be highly sensitive to its cylindrical radius (e.g., Saffman and Delbruck (1)). Due to the highly viscous nature of a cellular membrane or a membrane bilayer, the correlation times for most transmembrane proteins are predicted to be in the microsecond or longer correlation time range. This time range is not readily accessible to classical spectroscopic techniques like time-resolved or frequency domain fluorescence anisotropy when using conventional fluorescence probes or by continuous-wave electron paramagnetic resonance (EPR) when using nitroxide spin labels. However, as shown by the early work of Hyde and Dalton (2) and in the seminal study by Thomas et al. (3), the range of motional sensitivity of EPR could be extended into the microsecond-to-millisecond time range by saturation transfer EPR (ST-EPR) spectroscopy using conventional nitroxide spin labels. Since its introduction, ST-EPR has been utilized to study the very slow rotational motions of a wide range of membrane proteins and other large protein assemblies.

Protein-Protein Interactions in Calcium Transport Regulation Probed by Saturation Transfer Electron Paramagnetic Resonance

We have used electron paramagnetic resonance (EPR) to probe the homo- and heterooligomeric interactions of reconstituted sarcoplasmic reticulum Ca-ATPase (SERCA) and its regulator phospholamban (PLB). SERCA is responsible for restoring calcium to the sarcoplasmic reticulum to allow muscle relaxation, whereas PLB inhibits cardiac SERCA unless phosphorylated at Ser16. To determine whether changes in protein association play essential roles in regulation, we detected the microsecond rotational diffusion of both proteins using saturation transfer EPR. Peptide synthesis was used to create a fully functional and monomeric PLB mutant with a spin label rigidly coupled to the backbone of the transmembrane helix, while SERCA was reacted with a Cys-specific spin label. Saturation transfer EPR revealed that sufficiently high lipid/protein ratios minimized self-association for both proteins. Under these dilute conditions, labeled PLB was substantially immobilized after co-reconstitution with unlabeled SERCA, reflecting their association to form the regulatory complex. Ser16 phosphorylation slightly increased this immobilization. Complementary measurements with labeled SERCA showed no change in mobility after co-reconstitution with unlabeled PLB, regardless of its phosphorylation state. We conclude that phosphorylating monomeric PLB can relieve SERCA inhibition without changes in the oligomeric states of these proteins, indicating a structural rearrangement within the heterodimeric regulatory complex.

Three Distinct Actin-Attached Structural States of Myosin in Muscle Fibers

We have used thiol cross-linking and electron paramagnetic resonance (EPR) to resolve structural transitions of myosin's light chain domain (LCD) and catalytic domain (CD) that are associated with force generation. Spin labels were incorporated into the LCD of muscle fibers by exchanging spin-labeled regulatory light chain for endogenous regulatory light chain, with full retention of function. To trap myosin in a structural state analogous to the elusive posthydrolysis ternary complex A.M′.D.P, we used pPDM to cross-link SH1 (Cys707) to SH2 (Cys697) on the CD. LCD orientation and dynamics were measured in three biochemical states: relaxation (A.M.T), SH1-SH2 cross-linked (A.M′.D.P analog), and rigor (A.M.D). EPR showed that the LCD of cross-linked fibers has an orientational distribution intermediate between relaxation and rigor, and saturation transfer EPR revealed slow rotational dynamics indistinguishable from that of rigor. Similar results were obtained for the CD using a bifunctional spin label to cross-link SH1-SH2, but the CD was more disordered than the LCD. We conclude that SH1-SH2 cross-linking traps a state in which both the CD and LCD are intermediate between relaxation (highly disordered and microsecond dynamics) and rigor (highly ordered and rigid), supporting the hypothesis that the cross-linked state is an A.M′D.P analog on the force generation pathway.

Structural Dynamics of Calcium Transport Proteins Detected by Saturation Transfer Electron Paramagnetic Resonance

We have used conventional and saturation transfer electron paramagnetic resonance (EPR) to probe the structural dynamics of the integral membrane protein phospholamban (PLB), as a function of phosphorylation and the presence of its regulatory target, the sarcoplasmic reticulum calcium ATPase (SERCA). Our goal is to elucidate the mechanism of SERCA inhibition by PLB, which is needed for the rational design of therapies to improve calcium transport in muscle cells. We used the monomeric mutant AFA-PLB with the rigid electron spin label TOAC incorporated at position 36 in the transmembrane domain, and reconstituted the protein in lipid vesicles. EPR experiments were performed to determine the nanosecond (conventional EPR) and microsecond (saturation transfer EPR) rotational motions of PLB, as modulated by phosphorylation of PLB at serine 16 (pPLB), and/or addition of excess SERCA. Neither SERCA binding nor PLB phosphorylation caused significant changes in conventional EPR spectra of PLB, indicating no effect on the nanosecond flexibility of PLB's transmembrane domain. However, saturation transfer EPR found that addition of SERCA caused a large decrease in microsecond rotational motion for both PLB and pPLB. Phosphorylation of PLB caused no effect in the absence of SERCA and only slight mobilization in the presence of SERCA. These results indicate that both PLB and pPLB bind to SERCA, and PLB phosphorylation only slightly increases the flexibility and/or the dissociation constant of the SERCA-PLB complex. Therefore, PLB phosphorylation relieves SERCA inhibition primarily by changing the structural dynamics of the SERCA-PLB complex, not by dissociation of the complex. This work was funded by grants from NIH (R01 GM27906 and T32 AR007612).

Direct in vivo interaction of the antibiotic primycin with the plasma membrane of Candida albicans: An EPR study

The direct interaction of the antibiotic primycin with the plasma membrane was investigated by employing the well-characterized ergosterol-producing, amphotericin B-sensitive parental Candida albicans strain 33erg+ and its ergosterol-less amphotericin B-resistant plasma membrane mutant erg-2. The growth inhibition concentration in shaken liquid medium was 64μgml−1 for 33erg+ and 128μgml−1 for erg-2, suggesting that the plasma membrane composition influences the mode of action of primycin. To determine the primycin-induced changes in the plasma membrane dynamic, electron paramagnetic resonance (EPR) spectroscopy methods were used, the spin-labeled fatty acid 5-(4,4-dimethyloxazolidine-N-oxyl)stearic acid) being applied for the in vivo measurements. The phase transition temperatures of untreated strain 33erg+ and its mutant erg-2 were 12.5°C and 11°C, respectively. After 128μgml−1 primycin treatment, these values increased to 17.5°C and 16°C, revealing a significant reduction in the phospholipid flexibility. Saturation transfer EPR measurements demonstrated that, the rotational correlation times of the spin label molecule for the control samples of 33erg+ and erg-2 were 60ns and 100ns. These correlation times gradually decreased on the addition of increasing primycin concentrations, reaching 8μs and 1μs. The results indicate the plasma membrane “rigidizing” effect of primycin, a feature that may stem from its ability to undergo complex formation with membrane constituent fatty acid molecules, causing alterations in the structures of phospholipids in the hydrophobic surface near the fatty acid chain region.

Dynamics of Tropomyosin in Muscle Fibers as Monitored by Saturation Transfer EPR of Bi-Functional Probe

The dynamics of four regions of tropomyosin was assessed using saturation transfer electron paramagnetic resonance in the muscle fiber. In order to fully immobilize the spin probe on the surface of tropomyosin, a bi-functional spin label was attached to i,i+4 positions via cysteine mutagenesis. The dynamics of bi-functionally labeled tropomyosin mutants decreased by three orders of magnitude when reconstituted into “ghost muscle fibers”. The rates of motion varied along the length of tropomyosin with the C-terminus position 268/272 being one order of magnitude slower then N-terminal domain or the center of the molecule. Introduction of troponin decreases the dynamics of all four sites in the muscle fiber, but there was no significant effect upon addition of calcium or myosin subfragment-1.

The Global Analysis of DEER Data

Previously, global analysis has been successfully used to analyze both continuous wave and saturation transfer EPR data1-3. In this work, algorithms have been developed for the global analysis of DEER data. Applications include the analysis of DEER data collected at multiple frequencies or multiple timescales. Analysis of DEER data from the soluble protein CDB3 (MW ≈ 90 kD) has shown that the background DEER signal is not well-fit by an exponential decay due to the large size of the CDB3 dimer. As a result, background correction with an exponential decay prior to analysis results in a poor fit to the data. An algorithm has been developed which explicitly fits the background signal with the radius of the molecule (assuming it is spherical) and the spin concentration as parameters. Using this approach, excellent fits to DEER data can be obtained without prior background correction. Also, DEER data can be globally analyzed to determine changes in the relative populations of components of the distance distribution as a function of experimental conditions. For example, DEER has been previously used to study the structural effects of a proline to arginine mutation at residue 327 of CDB3. Intradimer distances in spin-labelled wild type CDB3 can be fit using a single component distance distribution4. The same measurements on P327R CDB3 indicate the mutation induces a second more disordered component in the distance distribution5. The global analysis of DEER data collected for multiple spin-labelling sites in both the WT and P327R background is being used to further test this two-component model. Supported by NIH GM 080513. 1. Hustedt and Beth, BJ 69:1409–1423 (1995). 2. Hustedt et al., BJ 64:614–621 (1993). 3. Hustedt et al., BJ 72:1861–1877 (1997). 4. Zhou et al., Biochemistry 44:15115–15128 (2005). 5. Zhou et al., BJ 90:528A (2006).

EPR as a Tool to Study the Dynamic of Tropomyosin in the Muscle Fiber - Use of a Bifunctional Spin Label

Tropomyosin (Tm), an alpha-helical coiled-coil protein, is a key regulatory protein in muscle contraction. Little is known about the role of Tm dynamics in muscle regulation and more specifically dynamics in the three states of the thin filament. In this work, the flexibility of four different regions of Tm was determined with Saturation Transfer Electron Paramagnetic Resonance (ST-EPR). The use of bi-functional labels allowed us to immobilize the probe and prevent the motion of the label with respect to protein surface. The rotational correlation time of the bi-functional spin label on Tm was 40 ns compared to 25 ns for a conventional mono-dentate spin label. The spin label was attached to i, i+4 positions of the coiled-coil, obtained by cysteine mutagenesis. The bi-functionally labeled Tm di-mutants were reconstituted into “ghost muscle fibers” from which the myosin filaments and intrinsic regulatory proteins (tropomyosin, troponin) were removed. The filaments were reconsituted with Tn and decorated with myosin S1. We found that there is a gradient of flexibility of Tm along its length in the muscle fiber with the C-terminus mutant A268C/E272C being less mobile (two-fold), as compared to the rest of the mutants. Introduction of troponin decreases the flexibility of the four mutants, specifically the two mid-region mutants H153C/D157C and G188C/E192C by 25% and 30% respectively. The addition of calcium did show a decrease in the flexibility of the C terminus mutant A268C/E272C by 20%. The effect of calcium addition on the two other mutants and the effect of addition of S1 on the four mutants were not significant. Thus, although there is a gradient of flexibility in Tm, its dynamics does not change within different states of thin filament activation.

Myosin Crosslinking and EPR Capture the Start of Force Generation in Muscle Fibers

Crosslinking the two most reactive Cys (SH1 and SH2) of the myosin catalytic domain (CD) inhibits force production and ATP hydrolysis and locks myosin in a weak actin-binding conformation with the CD immobilized and orientationally disordered. These results suggest that crosslinking traps a state in which the myosin head is on the cusp of force generation. In the present study, we measured the structural dynamics of myosin's light chain domain (LCD) in skeletal muscle fibers during rigor, relaxation, and with SH1 and SH2 crosslinked. To measure LCD structural dynamics, we exchanged spin labeled RLC for native RLC in permeabilized muscle fibers, with retention of function, then used EPR spectroscopy to measure structural dynamics. EPR spectra indicate when SH1 and SH2 are crosslinked, the LCD is in an orientation intermediate between relaxation and rigor, indicative of a state beginning to generate force. The saturation transfer EPR (STEPR) spectrum from these fibers does not change with crosslinking, demonstrating that that the LCD undergoes very slow dynamics, as in rigor, and is less dynamic than relaxation. In order to relate LCD structural dynamics with those of the CD, we measured CD structural dynamics in fibers by directly crosslinking SH1 and SH2 with BSL. EPR spectra from these fibers reveal that the CD is highly disordered, with dynamics ten times slower than in relaxation. Thus when SH1 and SH2 are crosslinked, both domains exhibit structural dynamics intermediate between relaxation (pre-power stroke) and rigor (post-power stroke). This supports the conclusion that SH1-SH2 crosslinking traps a state analogous to an initial force-generating state. We propose that this state is the missing link needed to explain how myosin undergoes a transition from dynamic disorder to order as it converts chemical energy to mechanical work.