(taken from the application): We will address two major areas relating to the function of myosin. First we will take dynamic measurements of actin and myosin to reveal changes in conformation of myosin that are of the size required to explain the observed displacements. Conformational changes in myosin during the ATPase cycle will be quantitated using fluorescence energy formation of a complex between a single myosin head and actin oligomers, leading to an X-ray crystal structure of the actin-bound form of S1, a critical state in the kinetic cycle. The preliminary data using fluorescence resonance energy transfer (FRET) suggests that the myosin lever arm may indeed function as a mechanical amplifier for motility by swinging through an arc greater than 50 degrees. We propose to further refine this data using FET approaches that allow one to ascertain different populations of myosin head conformations and thereby determine the maximum swing angle of the leer arm and the resultant maximum step size of one power stroke. The number of conformation states in the presence of ATP and various ATP analogs will be examined. The Vale laboratory has developed a custom build laser microscope that can measure FRET at the single molecule level, and we plan to collaborate with him to make such measurements for the myosin motor. Together with Roger Cooke's group, we will measure other aspects of conformational changes in the myosin head by placing various probes on chosen sites in the molecule. In all cases, we will use our cysteine-light myosin construct, which is a functional motor containing essentially no cysteine residues. Chosen sites will be changed to cysteine residues for direct labeling with probes. The above techniques will also be applied to myosin heads arrested in various states of the cycle via mutagenesis of the protein. For example, mutational changes that result in failure to hydrolyze bound ATP can be studied in this way. An F-actin trimer will be created for crystallization and characterization with and without bound myosin motor domain. Atomic structures of F- actin and F-actin with myosin bound are essential for understanding myosin-based motility. Actin monomers do not activate myosin ATPase and the filamentous form of actin has not been crystallized. A major hurdle is creating only the core part of the actin filament, which is an actin trimer, and isolating that in pure form. A mutational approach will be used to attempt to isolate such a species. Its ability to activate myosin ATPase activity and to crystallize with and without the myosin head bound will be pursued. We acknowledge that this is an extremely high risk project. We are optimistic, however, that with some luck we can achieve this goal, and the payoff will be high.
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