Current theories of the force generating step in the contractile cycle involve a reorientation of the myosin head relative to the actin filament during sliding. The inability to detect any large conformational changes in the globular, catalytic domain of myosin has focused interest on the neck region, which consists of a long alpha-helical heavy chain segment that is stabilized by the regulatory (RCL) and essential ELC light chains. It has been proposed that small structural changes in the motor domain serve to rotate this light chain binding domain. This hypothesis will be tested by: (1) Using an in vitro motility assay to measure the working stroke and unitary force of single myosin molecules from which the RCL or ELC has been removed by biochemical methods, or from which the LC-binding site has been deleted by recombinant techniques. The mechanical properties of single-headed myosin will be compared to the two-headed species. (2) Light chain interactions within a head and between heads will be determined by labelling mutant RLCs and ELCs with fluorescent probes. Changes in distance between fluorescent LCs bound to myosin will be measured by resonance energy transfer, and changes in orientation of the LC-binding region in fibers will be measured by transient fluorescence polarization. (3) The effect of light and heavy chain isoforms on force and movement will be determined by (a) analyzing the interaction of a labelled N-terminal region of ELC with actin by fluorescence spectroscopy and cro-electron microscopy, and (b) mutating variable regions in myosin isoforms to identify sequences in the head that are responsible for coupling ATP hydrolysis to movement.
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