ANALYSIS OF MOTOR PROTEIN FUNCTION

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Description: (Verbatim from the applicant's abstract) The fundamental goal of this proposal is to obtain biochemical, biophysical, and structural data from both kinesin-family and myosin-family motor proteins to better define the changes in protein conformation that produce force and work. The crystal structures of motor domains from both families have suggested plausible hypotheses for force production involving the co-ordination of conformational changes at the nucleotide site, the converter region, and neck of the motor. Testing these hypotheses, however, will require both correlating the presently known structures with specific intermediates in the biochemical/mechanical cycle, and determining whether other, currently unresolved structures exist. Electron paramagnetic resonance (EPR) spectroscopy will be used to monitor changes in conformation and orientation at the nucleotide site and converter region during the transition from weak to strong states, information crucial for understanding how force is produced. The corresponding regions in kinesin-family motors will also be probed with spin labels. Orientation information from EPR probes will be obtained using protocols I have developed to flow-align microtubules. The study of ncd builds on previous data that identified an endogenous specific site for probes and a state with cross-linked cysteines, possibly corresponding to the well-studied cross-linked state in myosin. I will also explore a previously unanticipated domain movement in S1-the opening of the Pi-tube. A structural comparison suggests that the complementary domain movement occurs in the kinesin-family of motors-a closing of the Pi-tube. A related set of experiments will examine motions of the Pi tube of the kinesin-family motor. Opening and closing of the Pi-tube will be monitored by measuring distances across it and by gluing it shut using cross-linking reagents. The movement of this region may play an important role in determining rates of nucleotide binding and release in both myosin and kinesin family motors. Together, the data obtained above will link structural information with kinetic and energetic data, providing a more complete picture of how motor proteins function to produce force and displacement. A fundamental motivation for this proposal is that recognition of crystal structure differences and similarities between the X-ray structures of myosin and MT motors can significantly enhance experimental design. Although this approach will be shown to be a two-way street, the most significant advantage clearly accrues to the much less studied microtubule motor family.