(taken from the application): We are interested in understand the precise mechanism of microtubule-based motility by kinesin and dynein. The status of research and knowledge on these two motors is quite different. Kinesin has been intensively studied and much information is known concerning its kinetics, biophysical parameters of movement, and atomic structure. However, the structural changes that lead to motility have not been defined. We intend to pursue this question with Robert Fletterick and Roger Cooke in the next phase of the grant. For dynein, very little is known about the structure of the motor, largely owing to its size. We intend to crystallize the motor domain of dynein with Robert Fletterick and with assistance from Jim Spudich as a second major aim. Regarding kinesin's mechanism, a variety of preliminary data suggests that a region termed the """"""""neck"""""""" functions as a mechanical amplifier for motility. We propose to measure potential conformational changes in the neck region by spectroscopic techniques. Together with Roger Cooke's groups, we will measure distance changes in the 10-25 angstrom range by EPR using appropriately placed spin label probes. We also will look for conformational changes in this region using FRET (fluorescence resonance energy transfer). By comparing data from EPR, FRET x-ray crystallographic 9with R. Fletterick), and cryo-electron microscopy studies (collaboration with Ron Milligan), we hope to formulate a structural model for kinesin-based force-generation. Together with Jim Spudich, we also propose to extend this work to measure conformational changes using a custom-built laser microscope that can measure FRET at the single molecule level. For advancing an understanding of dynein-based motility, we believe that an atomic structure is essential. We propose to crystallize and solve the- structure of dynein's motor domain using a Dictyostelium expression system. Our demonstrated teamwork with Fletterick will be instrumental in solving this challenging protein structure. Our purified dynein motor domain also will be supplied to the Highsmith and Cooke laboratories for biochemical studies of microtuble and nucleotide binding properties, which have been poorly characterized for dynein in comparison with myosin and kinesin.
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