The contractile interaction in the filament array of the muscle f fiber is complex, and the identification of unique model of this interaction will require a large amount of dat. We will measure actomyosin mechanics in permeable fibers and in single motor assays under a variety of conditions that are designed to provide data on the kinetics and energetics of this interaction. In one set of experiments we will measure the mechanics of fibers which have increased populations of cross-bridges in pre-power- stroke states. These states will be populated using a remarkable series of s substrate analogs that have been identified, which have graded efficacies for activating muscle fibers, and by analogs of phosphate. We will measure how rapidly cross-bridges in these states interact with actin, and we will investigate why some of these states present a drag on filament motion while others don't. We will use these analogs to determine which steps in the cycle limit the rate of force development. These steps will also be disected using photoactivatable phosphate to measure the rates associated with phosphate release in fibers, and by measuring the rates of the hydrolysis step and acto-S1 binding in solution. The studies of fiber mechanics will be complemented by measurements of the forces and displacements generated by single motor proteins activated in this series of analogs. These data will determine how a perturbation in the nucleotide-protein interaction affects the power-stroke. They will provide more definitive information on the states populated by the analogs and may define the relation between force and position within the powerstroke. In another set of experiments, photoactivatible nucleotide analogs that either don't generate force or generate little force will provide a measure of a single nucleotide turnover in an active fiber. The data will also help determine the fraction of myosin heads that are generating force, a parameter that remains controversial and which is important for the interpretation of both mechanical and structural studies. Recently we have developed techniques that allow us to obtain reproducible data from fibers that are activated at higher temperatures 15-35 degrees C, and have shown that changes in pH have a different effect at higher temperatures. We will complete our characterization of fiber mechanics, investigating the effects of increased phosphate, increased [ADP] and decreased [ATP]. These studies will also provide additional data defining cross-bridge kinetics, contributing to the long term goal of this grant, which is to explain the complex physiological behaviour of active muscle fibers in terms of the kinetics and energetics of the actomyosin interaction.