and specific aims): The goal is to understand how the energy from ATP binding and hydrolysis is converted into myosin's force and motion generating capabilities. Purified mutant smooth muscle myosin subfragments will be obtained from insect cells using the baculovirus expression system. All myosin mutants will be characterized by electron microscopy and hydrodynamic methods, their enzymatic properties determined by steady-state and transient kinetic techniques, and their ability to move actin determined by an in vitro motility assay. Unitary step size and force production of the mutant myosins will be measured. The following questions will be asked: (1) is a rigid lever arm model for the neck region of myosin sufficient to explain the effect that changes in neck length have on myosin's force and motion generation, or do kinetic changes in the motor domain contribute to these differences? Mutant heavy meromyosins with shorter and longer neck regions will be used to probe the relative contributions of structural and kinetic changes to these myosin's altered mechanical properties. (2) Do the two heads of myosin function cooperatively in motion and force production? The unitary force and step-size of single and double-headed myosins will be compared to test for independent or cooperative head action. A chimera with a myosin head attached to a stable leucine zipper coiled-coil region will show if flexibility at the junction where the two heads join is an important feature for optimal movement and force generation. (3) Which regions of myosin account for the higher force produced by smooth muscle myosin relative to striated myosin? Three regions of the smooth muscle myosin heavy chain will be mutated to the sequence found in skeletal muscle myosin, to determine which enzymatic or mechanical parameters correlate with the properties of the myosin from which the region was derived.
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