Molecular biophysics of motility in cytoskeletal motor proteins

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This project will investigate the structure and function of two proteins, kinesin and myosin, which belong to category of proteins that are designated as 'molecular motors'. These enzymes use the energy derived from hydrolysis of one of the most common energy storage molecules in cells, adenosine triphosphate, to drive muscle contraction, intracellular transport, cell motility, and cell division. The goal of the project is to understand how these proteins generate motion to perform cellular work. This project will utilize a 'metal-rescue' technology for controlling and modulating the activity of the motor protein by varying the ratio of metal ions that are necessary components for the proteins. This innovative technology will be useful for researchers to reversibly control the activity of multiple different enzymes. The educational aims of this project will fill a gap in the local education system to promote robust science teaching and learning for the next generation of scientists. Underrepresented minorities at the high school level will be educated and trained in STEM research.The research objective of this project is to define the nucleotide hydrolysis mechanisms of unconventional kinesin and myosin motor proteins using biochemical, structural, biophysical, and advanced mathematical analysis. Myosin and kinesin superfamily motors are cytoskeletal filament-stimulated ATPases that share structural motifs in their active sites which directly interact with the nucleotide and divalent metal cofactor, typically Mg(II). A 'metal-rescue' strategy will be used to control the enzymatic activity and motility of kinesins and myosins and by taking advantage of the differential affinities of Mg(II) and Mn(II) for serine or cysteine residues. Specifically, manipulation of the protein-metal interaction will provide a direct and experimentally reversible strategy to modulate switch-1 closure and, thus, motor motility upon its filament. The ATPase mechanism of wild type and the metal-rescue mutant kinesin-5 motors from Saccharomyces cerevisiae will be defined and the allosteric mechanochemistry of the wild type and metal-rescue mutant of myosin-II motors from Dictyostelium discoideum will be determined. This project will develop a biophysical tool to reversibly control the activity of molecular motors or other P-loop NTPases. This project is supported by the Molecular Biophysics Cluster of the Molecular and Cellular Biosciences Division in the Directorate for Biological Sciences.