Intrinsic Tryptophan Fluorescence Identifies Specific Conformational Changes at the Actomyosin Interface upon Actin Binding and ADP Release

The helix−loop−helix (A-site) and myopathy loop (R-site) are located on opposite sides of the cleft that separates the proposed actin-binding interface of myosin. To investigate the structural features of the A- and R-sites, we engineered two mutants of the smooth muscle myosin motor domain with the essential light chain (MDE), containing a single tryptophan located either in the A-site (W546-MDE) or in the R-site (V413W MDE). W546- and V413W-MDE display actin-activated ATPase and actin-binding properties similar to those of wild-type MDE. The steady-state fluorescence properties of W546-MDE [emission peak (λmax) = 344, quantum yield = 0.20, and acrylamide bimolecular quenching constant (k q) = 6.4 M-1·ns-1] and V413W-MDE [λmax = 338, quantum yield = 0.27, and k q = 3.6 M-1·ns-1] demonstrate that Trp-546 and Trp-413 are nearly fully exposed to solvent, in agreement with the crystallographic data on these residues. In the presence of actin, Trp-546 shifts to a more buried environment in both the ADP-bound and nucleotide-free (rigor) actomyosin complexes, as indicated by an average λmax of 337 or 336 nm, respectively, and protection from dimethyl(2-hydroxy-5-nitrobenzyl)sulfonium bromide (DHNBS) oxidation. In contrast, Trp-413 has a single conformation with an average λmax of 338 nm in the ADP-bound complex, but in the rigor complex it is 50% more accessible to DHNBS oxidation and can adopt a range of possible conformations (λmax = 341−347 nm). Our results suggest a structural model in which the A-site remains tightly bound to actin and the R-site adopts a more flexible and solvent-exposed conformation upon ADP release.