The molecular motor myosin plays crucial-roles in contraction and various forms of cell movement and changes in cell shape. Cytokinesis, directed cell migration by chemotaxis, capping of ligand-bound cell surface receptors, morphogenetic changes in cell shape, and muscle contraction are examples of events that clearly involve this motor. In spite of decades of investigations on myosin, the molecular basis of the conversion of the chemical energy of hydrolysis of ATP into mechanical movement is not understood. A new approach was out development of in vitro assays for quantitating myosin movement along actin filaments. In the last grant period, we succeeded in establishing a feedback-enhanced laser trap assay that allows the direct measurement of force and displacement produced by a single myosin molecular pulling on a single actin filament.
The specific aims of this proposal are to continue to develop the laser trap single molecule assay, and to use it and other in vitro assays to examine the mechanical and biochemical parameters of native and mutated forms of the myosin molecule. Our demonstrations that we can use Dictyostelium to express large amounts of functional myosin and soluble fragments that contain the motor domain allow us to combine molecular genetic manipulation of the motor with measurements of its biochemical and mechanical properties. The experimental plan is divided into two parts; 1. Wild type and mutagenized forms of myosin will be expressed in Dictyostelium, purified to homogeneity, and analyzed in vitro using a variety of quantitative biochemical assays, including our in vitro motility assays. 2. A third generation feedback-enhanced laser trap system will be developed to measure, with higher precision than available previously, displacement and force produced by a single myosin molecule, and the duration of the strongly-bound state of the actin-myosin complex. Specific questions for the next grant period are; 1. Does the light chain binding domain of myosin act as a lever arm to produce about 10nm step in motion? 2. Does the gear box zone undergo large changes in structural rearrangements during the ATPase cycle? 3. How do the various domains of the myosin motor 'talk' to one another to effect the chemomechanical energy transduction? 4. How can we improve the feedback-enhanced laser trap system? 5. How will we use the improved feedback-enhanced laser trap system to study myosin function?
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