The myosin superfamily of molecular motors plays crucial roles in contraction, diverse forms of cell movement, and changes in cell shape. Myosin-II is a non-processive motor that drives cytokinesis and muscle contraction, and myosin-V is a processive motor that drives vesicular movements in neurons and other cells. The molecular basis of myosin function has reached a detailed level of analysis, due to the advent of in vitro motility assays and the establishment of a dual-beam laser trap assay that allows the direct measurement of force and displacement produced by a single myosin molecule 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 both myosin-II and myosin-V. We will also initiate studies on members of other classes of the myosin superfamily, to compare their mechanochemical properties with those of myosin-II and myosin-V. Our demonstrations that Dictyostelium can be used to express large amounts of functional myosin and its motor domain (Subfragment 1) allow us to combine molecular genetic manipulation of the myosin-II motor with measurements of its biochemical and mechanical properties. Specific questions for the next grant period are: 1) What conformational changes occur within the S1 motor domain coincident with displacements and force transients measured at the single molecule level? 2) Can we visualize directly processive movement of myosin-V molecules along actin filaments? 3) What relationships are there between orientations of the S2 head on a surface and its measured mechanical properties using single molecule analysis? 4) What are the effects of load on the functioning of myosin-V and on its thermodynamic efficiency? 5) How does the step size (d) of myosin and its strongly bound state time (ts) to actin vary with Dictyostelium myosin mutants altered in lever arm length? 6) How does a single point mutation near the nucleotide binding site of the motor domain of myosin result in uncoupling of the actin-activated ATPase activity from mechanical movement?
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