The mechanism by which muscle or any other molecular motor converts chemical energy (ATP) into mechanical work, force, and shortening (chemomechanical transduction) will be studied. Current theories of this process are based on a four step reaction mechanism in which ATP binds to and dissociates actomyosin, followed by strong binding of the myosin products complex to actin and the associated sequential release of Pi and ADP. The release of products powers the rotation of the myosin head, the power stroke, and produces the motive force and sliding of the thick and thin filaments. The rate of ATP hydrolysis, the force produced, and the velocity of shortening are all dependent on the rate constants controlling the transition between different AM states and the rate constants themselves are a function of the strain on the crossbridge. Using transient kinetics in solutions of isolated proteins to study the rates of steps in dissociating or dissociated proteins, and glycerinated fibers to examine the rate constant for actomyosin transitions and ATP hydrolysis, and the recently developed in vitro motility assays, we will test a number of these ideas. A major challenge to conventional wisdom are experiments indicating that the power stroke is much larger than possible by the rotating crossbridge concept. The power stroke size will be estimated by measuring the time course of ATP hydrolysis, shortening, and stiffness in single shortening glycerinated muscle fibers. In comparative studies (using motility assays) the power stroke size produced by myosins from skeletal and smooth muscles and platelets will be estimated. Second, using ATP analogues to produce contractions in which force, rate of force development, Pi transients, and velocity of shortening are different, we will determine to what extend these differences are explicable by measured changes in the rate constants of the actomyosin reaction mechanism. These same studies, when applied to motility assays, will allow determination of the extent to which the motility assay is a paradigm of muscle contraction. Attempts will be made to develop a technique by which the number of strongly attached crossbridges to a length of moving actin filament can be estimated. If successful this would allow evaluation as to what extent stiffness is an adequate measure of the number of attached crossbridges. Finally an attempt will be made to load crossbridges and examine their velocity-pCa relationship.
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