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 opp osite sides of the cleft that separates the proposed actin-binding interfac e 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 tho se of wild-type MDE. The steady-state fluorescence properties of W546-MDE [ emission peak (lambda(max)) = 344, quantum yield = 0.20, and acrylamide bim olecular quenching constant (k(q)) = 6.4 M-1 . ns(-1)] and V413W-MDE [lambd a(max) = 338, quantum yield = 0.27, and k(q) = 3.6 M-1 . ns(-1)] demonstrat e that Trp-546 and Trp-413 are nearly fully exposed to solvent, in agreemen t with the crystallographic data on these residues. In the presence of acti n, Trp-546 shifts to a more buried environment in both the ADP-bound and nu cleotide-free (rigor) actomyosin complexes, as indicated by an average lamb da(max) of 337 or 336 nm, respectively, and protection from dimethyl(2-hydr oxy-5-nitrobenzyl) sulfonium bromide (DHNBS) oxidation. In contrast, Trp-41 3 has a single conformation with an average lambda(max) of 338 nm in the AD P-bound complex, but in the rigor complex it is 50% more accessible to DHNB S oxidation and can adopt a range of possible conformations (lambda(max) = 341-347 nm). Our results suggest a structural model in which the A-site rem ains tightly bound to actin and the R-site adopts a more flexible and solve nt-exposed conformation upon ADP release.