We will address several fundamental questions concerning the mechanics and energetics of the actomyosin interaction in active muscle fibers: 1. What are the fractions of myosin heads attached strongly to actin, and weakly to actin?. 2) What are the properties of the weakly bound states at the beginning of the power stroke? 3. How is the force produced in the power stroke related to the energetics of the formation of the actomyosin interface? We have recently developed two spectroscopic techniques, which will monitor the interaction of myosin with actin in the fibers. The fluorescent probe pyrene bound to the Cys-374 of actin senses the strong binding of myosin to actin, but not the weak binding. We have shown that we can label actin in a fiber and measure the fraction of myosin heads bound strongly to actin in an active fiber. We propose to measure the fraction attached strongly under a variety of conditions, e.g. during activation, or isotonic releases or changes in solvent conditions, etc. Knowledge of the fraction of myosin heads attached strongly to actin is essential for relating fiber mechanics to dat obtained from single molecules experimentally and for interpreting a number of structural results. The total fraction of myosin heads bound to actin will be determined using luminescence resonance energy transfer (LRET), by measuring a distance between a probe on myosin and a probe on the thin filament. LRET is capable of measuring the large distances involved, and we can isolate a signal that arises solely from transfer between myosin heads and actin. This signal will also provide information on the structure of the complex, and in particular it should be sensitive to changes that occur in the actomyosin interface. We have recently found that a polymer, polyethylene glycol (PEG), perturbs the actomyosin interaction. In particular, it can selectively populate the weakly bound, putative pre-power stroke states thought to exit at the beginning of the power stroke. Using PEG and analogs of phosphate, we will characterize the mechanics of these stakes, and using LRET we will investigate their structure. These spectroscopic techniques will be combined with mechanical measurements to define the energetics of the actomyosin interaction, and how the force produced correlates with the energetics of the strongly bound states. The data will connect mechanical data and structural dat to provide a more complete picture of how force is produced by actin and myosin in both skeletal and cardiac muscles.
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