Lewis, John H

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  • Publication
    Examining the Coupling of the Mechanical and Chemical Functions of Myosin Family Members Using Single Molecule and Bulk Solution Techniques
    (2011-05-16) Lewis, John H
    Myosins are actin-activated ATPases that convert the chemical energy stored in ATP into the mechanical swing of its lever-arm. The members of the myosin family exhibit a wide range of cellular functions. Myosin Ib (myo1b) is single-headed and may link the cell membrane to the actin network acting as a tension sensor; while myosin V (myoV) is double-headed and can act as a cargo transporter in cells. The very different functions of myo1b and myoV arise from differences in their chemical and mechanical activities. We examined the chemomechanical properties of myo1b using stopped flow and optical trap experiments, from which were determined mechanical step sizes and kinetic rates associated with the chemomechanical steps of the myo1b crossbridge cycle. Most importantly, we found that the rates are slow and the rate associated with ADP release during actin attachment is greatly decreased by force, which could allow it to act as a tension sensor. These kinetic rates and force sensitivity of myo1b are strongly regulated by the signaling molecule, calcium. MyoV steps along actin in a complex and dense cellular environment; how this is done can be understood from its intrinsic stepping behavior as measured from the changes in lever-arm conformation as it steps along actin using single molecule techniques and a novel analytic tool I developed. From this we find that myoV mainly walks straight along actin, but can take steps around the long axis of actin. The frequency of these azimuthal steps depends on the length of the myoV lever-arm.