It has been well established that muscle contraction is driven by structural rearrangements within myosin during its ATPase cycle and interaction with actin. The crystal structures of myosin (Fig. 1) solved in different nucleotide states have provided a framework for examining the structural basis of the ATPase cycle of myosin (1-6). In addition, solution techniques that include monitoring the intrinsic fluorescence of myosin have proven extremely valuable for examining the enzymatic and kinetic properties of the molecule (7-13). The following kinetic scheme of the myosin MgATPase cycle was developed based on observed changes in intrinsic fluorescence corresponding to structural changes within myosin, where M represents myosin and an asterisk represents enhanced protein fluorescence (14).In this reaction myosin first forms a collision complex with ATP followed by an isomerization to the M*⅐ATP complex, which results in the first level of fluorescence enhancement (*). During the rapid reversible process of ATP hydrolysis, a structural change induces an additional fluorescence enhancement (**). Then, following hydrolysis of ATP the fluorescence decreases back to the first level of enhancement (*), which is thought to correspond to the rate-limiting structural change resulting in phosphate release. The phosphate release step then shifts myosin from a weak to strong binding conformation and is the step believed to be associated with force generation during muscle contraction. The release of ADP is a much faster two-step process in which the second step results in a decrease in fluorescence to basal levels. Thus, examining the conformational changes that result in alterations in intrinsic tryptophan fluorescence may lead to important insights about the structural properties of the myosin MgATPase cycle.Dynamic structural information about domain motions within myosin during its MgATPase cycle has been pursued extensively (reviewed in Ref. 15). Indeed, several studies have demonstrated key rearrangements in the light chain-binding region (residues 781-820 highlighted in pink in Fig. 1), also referred to as the lever arm, providing a structural mechanism for force generation (reviewed in Ref. 16). Electron paramagnetic resonance studies, utilizing a probe on the regulatory light chain have shown a 30°rotation of this region (17), suggesting that the lever arm region rotates relative to the catalytic domain during muscle contraction. In addition, fluorescence studies utilizing a fluorescent probe at the regulatory light chain demonstrated rotation of the lever arm in contracting muscle fibers (15). Cryo-electron microscopy studies have revealed that smooth muscle myosin decorated on actin filaments undergoes a large structural change (30 -35 Å) of the lever arm upon ADP release (18). Furthermore, nucleotide analogs, which trap myosin in specific nucleotide states, have been useful in elucidating the structural properties of the normally short-lived stages of the MgATPase cycle. Structural studies suggest that MgADP-BeF ...