Myosin Isozymes The major questions remaining to be answered as to how myosin functions as a molecular motor surround the actin-activated product release steps. We propose that we now have high-resolution structures that show us the starting (transition state) and ending (myosin V closed cleft or rigor-like state) structures of myosin as it goes through its force-producing cycle. What we are lacking is any insight as to how the interaction with actin leads to the sequential release of phosphate and MgADP that is coupled to movement. There are two major goals of this study. Our goal is to characterize key structural elements of myosin that are involved in the product release mechanisms. We will make use of the fact that myosin V, non-muscle myosin MB, smooth muscle myosin II and Dictyostelium myosin II have kinetics that are fundamentally different from each other. These kinetic differences may allow these motors to work alone, in small numbers or in large ensembles. Functional evaluation of the expressed myosin will include determination of enzyme kinetic parameters, in vitro motility (translocation of actin filaments by myosin), and single molecule optical trap assays. FRET measurements based on cysteine-lite constructs will allow monitoring of subdomain movements that accompanying changes in the biochemical state of the motors. Time-resolved FRET and EPR will be used to determine distances between subdomain landmarks in defined kinetic states. High resolution (2-3A) X-ray structures will be generated in collaboration with Anne Houdusse. The goals of this project will be realized by addressing the following specific aims: (1) validate the myosin V rigor state and test its generality;(2) analyze the mechanism of MgADP release;(3) assess the mechanism of phosphate release and the weak to strong transition on actin;and (4) generate high resolution (X-ray) structures of myosin isoforms.