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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 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 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.