ATP analogs of various types will be prepared to help characterize the nature of nucleotide-protein interactions in contractile proteins with the long-term goal of explaining the molecular basis of muscle contraction. A new class of second generation ATP photoaffinity analogs will be synthesized which carry with them attached reporter groups. These groups will include spin labels, fluorescent and luminescent probes. ATP analogs will be photo-incorporated after first trapping them at the active site by use of the phosphate analogs, vanadate, AlF4, or BeFx. The attached analogs can be displaced from the active site by actin treatment to yield fully active myosin either in solution or in glycerinated muscle fibers. The probes, attached to either side of the active site, will be used to measure distances to other defined parts of myosin (or to actin) by use of the new technique of luminescence resonance energy transfer in collaboration with P. Selvin and R. Cooke. Specifically, changes in the relative orientation of the motor and light chain domains of subfragment 1 as influenced by nucleotides or actin binding will be assessed. Related experiments will utilize a new class of non-nucleoside triphosphate analogs to introduce specific spin probes at myosin's active site in muscle fibers to probe for global orientation changes in heads of myosin during the contraction cycle utilizing EPR spectroscopy. The properties of new thiol mutants of Dictyostelium myosin will be determined with the goal of preparing crystals of thiol-crosslinked subfragment for x-ray structure determination.
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