.) The long-term objective of the proposed studies is to understand how motor proteins work. These enzymes, which include myosin from muscle, dynein from cilia and flagella, and kinesin from eukaryotic cells in general, convert the chemical energy derived from hydrolysis of the gamma phosphate bond of ATP into mechanical work used to power intracellular transport. The strategy of this proposal, which focuses on the microtubule-based motor kinesin, is to combine high-sensitivity single-molecule techniques with biochemical and protein engineering techniques in order to identify the moving parts of the motor-the springs, levers, and axles-and to understand how their coordinated motion is coupled to the hydrolysis of ATP. Kinesin is a processive motor capable of making many steps along a microtubule without dissociating. We will test whether processivity is due to mechanical coordination between kinesin's two motor domains by measuring how force effects the dissociation of individual heads from the microtubule. Putative elastic elements will be localized, and a crucial prediction of the crossbridge cycle model will be tested by comparing the single-motor force with the product of the elastic element's stiffness and the powerstroke distance. Based on the approximate two-fold symmetry of dimeric kinesin when both its heads are in the same nucleotide state, we hypothesize that the power stroke is associated with a rotation of one head with respect to the other: we will use single-molecule fluorescence microscopy to visualize this rotation. To determine how tight is the coupling between chemical and mechanical steps, we will measure the effect of load on the ATP hydrolysis rate. These results will be incorporated into a kinetic model that will relate the speed and processivity of the motor to the load it carries and the ATP concentration. Because of the structural and biochemical similarities between kinesin, myosin, and dynein, the elucidation of the molecular events underlying energy transduction by kinesin should significantly increase the understanding of cellular motility in general. It is hoped that this understanding may lead to more rational treatments of muscle disorders such as heart disease, or to better methods of selectively interfering with pathological cellular movements such as the invasion and proliferation of tumor cells, and the transport of viruses between the cell membrane and the nucleus.
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