SMOOTH-MUSCLE MYOSIN MUTANTS CONTAINING A SINGLE TRYPTOPHAN REVEAL MOLECULAR-INTERACTIONS AT THE ACTIN-BINDING INTERFACE

Elucidation of the molecular details of the cyclic actomyosin interact ion requires the ability to examine structural changes at specific sit es in the actin-binding interface of myosin, To study these changes dy namically, me have expressed two mutants of a truncated fragment of ch icken gizzard smooth muscle myosin, which includes the motor domain an d essential light chain (MDE). These mutants were engineered to contai n a single tryptophan at (Trp-546) or near (Trp-625) the putative acti n-binding interface, Both 546- and 625-MDE exhibited actin-activated A TPase and actin-binding activities similar to wild-type MDE, Fluoresce nce emission spectra and acrylamide quenching of 546- and 625-MDE sugg est that Trp-546 is nearly fully exposed to solvent and Trp-625 is les s than 50% exposed in the presence and absence of ATP, in good agreeme nt with the available crystal structure data. The spectrum of 625-MDE bound to actin was quite similar to the unbound spectrum indicating th at, although Trp-625 is located near the 50/20 kDa loop and the 50-kDa cleft of myosin, its conformation does not change upon actin binding. However, a 10-nm blue shift in the peak emission wavelength of 546-MD E observed in the presence of actin indicates that Trp-546, located in the A-site of the lower 50-kDa subdomain of myosin, exists in a more buried environment and may directly interact with actin in the rigor a cto-S1 complex, This change in the spectrum of Trp-546 constitutes dir ect evidence for a specific molecular interaction between residues in the A-site of myosin and actin.