Free energy for the generation of force and motion in kinesin- and myosin-family motors comes from the interaction of the motor protein, the nucleotide triphosphate substrate, ATP, and the polymer along which directed motion occurs. Actin is the polymer for myosin, and microtubules for kinesin. Conformational changes at the nucleotide site associated with hydrolysis are postulated to be propagated to peripheral elements of the proteins, which function as the actual motor elements. Identification of these conformational changes and their relationship to motility, thus defining an atomic level description of motor function, remains a fundamental goal of the field. X-ray crystallographic structures have provided fascinating insights into motor function. However, each x-ray structure is only a single fixed picture in time of a dynamic process, and the crystallographic database has yet to define a coherent picture of free energy transduction. Significantly, the x-ray structures are also all obtained in the absence of polymer. Electron paramagnetic resonance (EPR) spectroscopy using nucleotide-analog spin probes bound at the active site will demonstrate a previously unanticipated conformational change in kinesin-family motors – the nucleotide binding site closes when the motor binds to microtubules. Using existing x-ray structures as a starting point, I will use structural homologies between kinesin- and myosin-family motors to suggest the conformational changes involved. Molecular dynamics simulations will show the new conformation of the motor to be compatible with experimental observations of EPR probe mobility, and demonstrate that the closing of the nucleotide pocket is crucial for hydrolysis and force transduction. Related observations on myosin will also be discussed.
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