Allostery in myosins studied at the molecular level

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DESCRIPTION (provided by applicant): In the crowded environment of a typical eukaryotic cell, any object larger than 50 nm is effectively immobile and cannot rely on diffusion to arrive at its destination. As a result, myosin motors evolved to generate force for the transport of cargoes, cell motility, and cell division, processes that are critical for life itself. These motors convert chemical free energy by changing shape in a controlled manner while bound to actin filaments. A fundamental feature of all motor proteins is that their movements are carefully coordinated, so that the motor travels through a specific sequence of coupled biochemical and mechanical states. The nature of this coordination remains obscure, but it is clearly essential for proper motor function. The recent recognition that each myosin class has evolved specific structural and kinetic features allows for the comparison of different coordination mechanisms across the myosin super family. These individual adaptations may now be characterized by a host of advanced motility assays. The work proposed here will determine the allosteric coordination mechanisms using new single-molecule manipulations to alter the intramolecular strain felt by the motor. The role of the motor track in transmitting strain will be established, by mechanically altering the actin filament geometry. In addition, communication mechanisms will be identified for myosin V and myosin VI through load dependent stepping measurements under conditions where the two heads of the motor are uncoupled. These mechanisms will be contrasted with those for myosin X, a motor with unusual in vivo motility that suggests a novel form of strain sensing. The long term goal of this research is to reveal how motors, and systems of motors, have been optimized for their cellular trafficking tasks. Myosin motility is required for various normal and pathological cell functions, including development of polarized cellular structures, formation of cellular adhesions and tissue development, wound healing, intracellular trafficking, and cell division. This research will address critical questions about the underlying biology behind motor-related disease, including, among others, several forms of inherited deafness related to the proper development and maintenance of stereo cilia in sensory hair cells.