Description (taken from the application): One major goal will be to investigate the dynamic flexibility of the motor proteins in solution. We will determine whether skeletal muscle myosin level arm motion is coupled to ATP hydrolysis, and if an energized level arm conformation is sufficient for force generation. In collaboration with Don Eden and Roger Cooke, we will correlate the production of force by muscle fibers with the substrate hydrolysis-driven conversion of the isolated motor domain a more compact structure for the same substrate. If the hypothesis is correct, only substrates that support contract should rotate the lever arm to convert S1 to a more compact structure, and substrates that form compact intermediates should support contraction. Kinesin and NCD motor domain segmental flexibility in solution will also be characterized. The dynamic flexibility in solution of their motor domain attachments to the stalk region, and the effects of nucleotides on that flexibility will be determined using monomeric and dimeric constructs of NCD and kinesin. A second major goal will be characterize properties of the complexes of the motor proteins with their polymers. We are currently studying the ternary complex between a tublin dimer and a motor monomer (NCD) with the goal of crystallizing it. The equilibrium association constants, Ka, for these complexes in the presence of different nucleotides, and the kinetic constants, complexes in the presence of different nucleotides, and the kinetic constants, Km and Vmax, for tubulin-activated MgATPase activity, will be measured. The crystallization trials will be done in collaboration with Elena Sablin and Robert Fletterick. Electrostatic and energetic interactions for both systems will be characterized. For NCD binding to tubulin, the ionic strength dependence will be used to quantify nucleotide- induced charge changes t the binding interface. For NCD binding to tubulin, the ionic strength dependence will be used top quantify nucleotide-induced charge changes at the binding interface. For myosin binding to actin, using loop 2 mutants in collaboration with Jim Spudich and Joel Cohen, nucleotide-induced electric charge changes at the actin binding site will be measured and compared to net electric charge changes of the motor domain.
NIH ExPorter
NIH ExPorter