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For many proteins, especially for molecular motors and other enzymes, the functional mechanisms remain unsolved due to a gap between static structural data and kinetics. We have filled this gap by detecting structure and kinetics simultaneously. This structural kinetics experiment is made possible by a new technique, (TR) 2 FRET (transient time-resolved FRET), which resolves protein structural states on the submillisecond timescale during the transient phase of a biochemical reaction. (TR) 2 FRET is accomplished with a fluorescence instrument that uses a pulsed laser and direct waveform recording to acquire an accurate subnanosecond time-resolved fluorescence decay every 0.1 ms after stopped flow. To apply this method to myosin, we labeled the force-generating region site specifically with two probes, mixed rapidly with ATP to initiate the recovery stroke, and measured the interprobe distance by (TR) 2 FRET with high resolution in both space and time. We found that the relay helix bends during the recovery stroke, most of which occurs before ATP is hydrolyzed, and two structural states (relay helix straight and bent) are resolved in each nucleotide-bound biochemical state. Thus the structural transition of the force-generating region of myosin is only loosely coupled to the ATPase reaction, with conformational selection driving the motor mechanism.

We have used two complementary time-resolved spectroscopic techniques, dipolar electron–electron resonance and fluorescence resonance energy transfer to determine conformational changes in a single structural element of the myosin motor domain, the relay helix, before and after the recovery stroke. Two double-Cys mutants were labeled with optical probes or spin labels, and interprobe distances were determined. Both methods resolved two distinct structural states of myosin, corresponding to straight and bent conformations of the relay helix. The bent state was occupied only upon nucleotide addition, indicating that relay helix, like the entire myosin head, bends in the recovery stroke. However, saturation of myosin with nucleotide, producing a single biochemical state, did not produce a single structural state. Both straight and bent structural states of the relay helix were occupied when either ATP (ADP.BeF x ) or ADP.P i (ADP.AlF 4 ) analogs were bound at the active site. A greater population was found in the bent structural state when the posthydrolysis analog ADP.AlF 4 was bound. We conclude that the bending of the relay helix in the recovery stroke does not require ATP hydrolysis but is favored by it. A narrower interprobe distance distribution shows ordering of the relay helix, despite its bending, during the recovery stroke, providing further insight into the dynamics of this energy-transducing structural transition.

We have used transient time-resolved FRET (TR2-FRET) to detect structural changes within the relay helix in real time during the myosin recovery stroke. The most intriguing problem in myosin's function is the mechanism of signal transduction between the nucleotide binding site and the force-generating domain of myosin during the recovery stroke. Atomic structures of myosin provide clues, but structural measurements during transient kinetics are needed to reveal the details of this mechanism. Kinetics of nucleotide binding is reliably determined from the fluorescence of mant nucleotides or single Trp myosin mutants (W129, W131). Single Trp mutant W501 has provided insight into structural kinetics in the force-generating domain, but the complex fluorescence signals observed during the recovery stroke do not yield clear structural interpretation. To overcome this problem, we have performed FRET measurements that monitor well-defined structural changes in the relay helix, which is proposed to be a key structural element in myosin's force-generating domain. We engineered two double-Cys myosin mutants in the Cys-lite D. discoideum myosin construct (gift from J. Spudich), with one Cys placed at the C-terminus of the relay helix (K498C) and the other at one of two stable positions in the lower 50K domain (D515C or A639C). We labeled these mutants with a donor-acceptor FRET pair (IAEDANS-DABSYL), then used time-resolved FRET (TR-FRET, detects donor lifetime at nanosecond timescale) to detect interprobe distances in equilibrium states trapped by nucleotide analogs, reflecting pre- and post-powerstroke structures of myosin. We then performed these nanosecond time-resolved measurements repetitively every 0.1 ms, in the transient phase after ATP addition using stopped flow. These transient time-resolved FRET (TR2-FRET) signals showed directly that the relay helix changes from straight to bent conformation in the recovery stroke, from the post-powerstroke to the pre-powerstroke state.

We have used site-directed spin labeling and EPR spectroscopy to detect structural changes within the regulatory light chain (RLC) of smooth muscle myosin upon phosphorylation. Smooth muscle contraction is activated by phosphorylation of S19 on RLC, but the structural basis of this process is unknown. There is no crystal structure containing a phosphorylated RLC, and there is no crystal structure for the N-terminal region of any RLC. Therefore, we have prepared single-Cys mutations throughout RLC, exchanged each mutant onto smooth muscle heavy meromyosin, verified normal regulatory function, and used EPR to determine dynamics and solvent accessibility at each site. A survey of spin-label sites throughout the RLC revealed that only the N-terminal region (first 24 aa) shows a significant change in dynamics upon phosphorylation, with most of the first 17 residues showing an increase in rotational amplitude. Therefore, we focused on this N-terminal region. Additional structural information was obtained from the pattern of oxygen accessibility along the sequence. In the absence of phosphorylation, little or no periodicity was observed, suggesting a lack of secondary structural order in this region. However, phosphorylation induced a strong helical pattern (3.6-residue periodicity) in the first 17 residues, while increasing accessibility throughout the first 24 residues. We have identified a domain within RLC, the N-terminal phosphorylation domain, in which phosphorylation increases helical order, internal dynamics, and accessibility. These results support a model in which this disorder-to-order transition within the phosphorylation domain results in decreased head–head interactions, activating myosin in smooth muscle.

We have used pulsed EPR spectroscopy (DEER) to determine the structural effects of point mutations in the force generation region of myosin (I499A, F506A, F692A, D.discoideum sequence). As it was previously reported, these myosin mutants maintain basal ATPase activity, but completely lose their motor function. It was proposed that F506A, located in the relay loop, disrupts communication between the nucleotide binding site and the force generation region in myosin, and F692A and I499A, located on the interface with the converter domain, affect coordination of the converter domain relative to the myosin head. In this study we assayed the structure of the relay helix in these mutants and in wild-type myosin. The mutations were introduced into the A639C:K498C myosin construct, both Cys were labeled with maleimide spin probes, and interprobe distance was measured. Previous studies showed that probes at these sites detect the nucleotide-induced bending of the relay helix. Observed spin echo decays were interpreted in terms of one or two Gaussian distance distributions, corresponding to one or two myosin structural states. Nucleotide analogs were used to trap myosin in several biochemical states. In the wild type, two structural states, corresponding to pre- and post-recovery stroke, are present in a single biochemical state. The mutations affect the equilibrium between these two structural states and increase the conformational disorder, indicating that these mutants destabilize the structure of the force-generation region, particularly in the post-recovery state. These results provide a structural explanation for the functional perturbation, introduced to myosin.

We have used transient time-resolved FRET (TR2FRET) to monitor the conformation of the relay helix in a myosin II functional mutant during the recovery stroke in real time. Myosin was perturbed with the F506A mutation (Dictyostelium discoideum sequence), located within the relay loop in the force-generating region. F506 is a highly conserved residue in myosin II and is a hypertrophic cardiomyopathy mutation site. Previous studies [Tsiavaliaris, EMBO Rep, 2002, 3(11), 1099] showed a significant effect of the F506A mutation on myosin function. Actin affinity in the presence of ATP was increased, and the mutant did not move actin filaments in in vitro motility assays. A small decrease in intrinsic fluorescence was observed upon addition of excess ATP, but ATP binding and hydrolysis were not affected by the mutation. It was proposed that the F506A disrupts the communication between the active site and the lever arm. We engineered a double-Cys myosin mutant (A639C:K498C) in the Cys-less background with the F506A functional mutation, and labeled the mutant with optical probes. We used TR-FRET to determine the interprobe distance, and TR2FRET measurements after rapid mixing with ATP revealed changes in the relay helix conformation during the recovery stroke in real time. The mutation induced significant disorder of the relay helix in the force-generating region, but myosin still produces a recovery stroke, changing the relay helix conformation from straight to bent. We conclude that (a) the relay helix is disordered in myosin functional mutant F506A, which demonstrates the importance of the relay loop - relay helix interaction in the relay helix stabilization, and (b) the relay helix is the major structural element in the force-generating region of myosin, responsible for communication from the active site to the converter domain and the lever arm.

We have used dipolar electron-electron resonance (DEER) and time-resolved fluorescence resonance energy transfer (TR-FRET) to investigate the role of the myosin relay helix in coupling between the active site and the force-generating region of myosin II. Two double-Cys Dictyostelium myosin constructs have been engineered, and the structure of the relay helix was monitored by measuring interprobe distances in MSL/MSL or IAEDANS/Dabcyl-labeled myosin. Experiments were performed on WT myosin and on F506A, a functional mutant that has close to normal enzymatic activity but completely lacks motor functions (e.g. unable to move actin filaments or support cell development). We found that the WT myosin relay helix adopts two distinct states (straight and bent), with the bent conformation populated when ATP and ADP.Pi analogs are bound at the active site. In contrast, binding of nucleotide analogs had very little effect on relay helix conformation in the F506A mutant. In addition, the width of the distance distribution was significantly larger in the F506A compared to WT myosin, indicating loss of structural organization. Our results demonstrate that the relay helix plays a key role in coupling of myosin ATPase and motor activities. Loss of functionality observed in F506A myosin can be explained by the disruption of the relay helix-relay loop interactions that normally stabilize well-defined conformations of the myosin force-generating region allowing it to switch between distinct structural states.

We have used three complementary spectroscopic techniques, pulsed EPR (DEER), time-resolved fluorescence resonance energy transfer (TR-FRET) and transient time-resolved FRET [(TR)2FRET], to elucidate the mechanisms of energy transduction in myosin - an important motility protein involved in a variety of processes including muscle contraction. We engineered two double-Cys myosin mutants that were labeled with optical probes or spin labels, and determined structural changes in a single structural element of the myosin motor domain, the relay helix, as affected by nucleotide binding and hydrolysis. In all cases, time-domain detection permitted the reliable determination of the interprobe distance distribution, quantitating both order and disorder, and resolving coexisting structural states. While DEER offered superior distance resolution, (TR)2FRET permitted detection of transient structural changes after rapid mixing (stopped-flow) with ATP. All methods demonstrated two structural states of myosin during the recovery stroke, corresponding to straight and bent conformations of the relay helix, in parallel with straight and bent conformations of the entire myosin head. A narrower interprobe distance distribution in the post-recovery state shows ordering of the relay helix structure during the recovery stroke. These experiments reveal changes in both structure and dynamics of the relay helix and identify it as a key player in the interdomain coupling mechanism. This methodology is applicable to any enzyme in which double Cys mutants can be engineered in a key region of energy transduction, permitting resolution of structural states and dynamics in real time during the transient phase of a biochemical reaction.

We have used site-directed time-resolved FRET to determine light-chain domain transitions in expressed Dictyostelium full-length myosin S1. Myosin S1 is proposed to undergo large rotations of the lever-arm in response to the nucleotide state. Previous fluorescence experiments (Shih et al., 2000) have shown that S1 adopts two populations of pre-stroke states and the equilibrium between these states is driven by nucleotide. To observe these population changes in detail, and to observe the effect of actin, we have used time-resolved fluorescence resonance energy transfer (TR-FRET) to measure the distribution of distances between the labeled catalytic domain (A250C) and a labeled RLC (M129C). In the present work, we show that the lever arm adopts multiple orientations in solution. In the absence of nucleotide, the light-chain domain probe is at a mean distance of 8.4 nm from the catalytic domain probe. This distance extends to >9.0 nm when flS1 is bound to actin. The addition of ATP gives three roughly equal populations of distances at 3.6, 4.3, and >9.0 nm. ADP.Vi stabilizes the shorter of these populations, indicating that ADP.Pi induces a conformational change of the light-chain domain. This work was supported by grants from NIH (AR32961, AR07612). We thank Igor Negrashov for excellent technical assistance.

We have used DEER (double electron-electron resonance) and TR-FRET (time-resolved fluorescence resonance energy transfer) to study conformational changes within the myosin II relay helix. Major structural changes during the myosin II ATPase cycle take place in the force-generating domain. Crystal structures show that the converter domain, the relay helix, and SH1 helix have different conformations in the proposed pre- and post- powerstroke structural states of myosin. This dramatic structural change is detected in solution studies by intrinsic fluorescence [1, 2] and EPR of a spin label attached to SH1 [3]. In the present study, we focus on the relay helix as a crucial structural element involved in coupling between the force-generating domain and the nucleotide-binding pocket. Cysteine mutations were introduced into a Cys-lite construct of Dictyostelium discoideum (Dicty) myosin in the lower 50k domain (either D515C or A639C) and the C-terminal end of the relay helix (K498C). These constructs were selectively modified with either MSL/MSL or IAEDANS/DABCYL pairs, and the distance between probes was measured in different myosin conformations trapped with nucleotides or nucleotide analogs Two conformations of the relay helix (with distinct probe-to-probe distances, presumably corresponding to the “straight” and “bent“ states of the relay helix) were resolved. Observed distances were in good agreement with existing crystal structures, but at least two distinct structural states were present in certain biochemical states (e.g, with bound ADP.BeF3, ADP.Vi, ADP.AlF4). The mole fraction of the “bent” conformation was higher with post-hydrolysis analogs (ADP.Vi, ADP.AlF4) bound at the active site. Our results reveal structural rearrangements within a single subdomain of myosin and provide insights into the coupling between ATP binding and changes in the force-generating region.