Molecular mechanisms of myosin II-actin interactions

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Background. Muscle contraction is driven by cyclical interactions of the molecular motor myosin II with actin. After myosin binds to actin, ATP is hydrolyzed; phosphate (Pi) and subsequently ADP are released from myosin. These biochemical reactions fuel the mechanical work produced by myosin, occurring simultaneously to changes in the structure of the molecule. A small conformational change in the myosin produces the so-called "power stroke" and sliding the actin filament towards the center of sarcomeres. The action of many myosin motors arranged  in filaments ultimately causes muscle shortening. There are critical issues that remain unknown and hamper our understanding of the myosin-actin interaction. The long-term goal of my research program is to understand the molecular mechanisms of muscle contraction. The specific goals of this proposal are: (i)To determine the relation between the force production by myosin during the power stroke, ATP hydrolysis, and Pi release (ii)To determine the relation between myosin structural changes and the power stroke (iii)To determine if myosin molecules work cooperatively in filaments (iv)To determine if there is cooperative among sarcomeres in a myofibril Methods. My laboratory will use a variety of methods to measure the mechanics and kinetics of myosin molecules and filaments. Goal (ii) will be achieved by testing myosin molecules with different structures that change Pi release form the active site, using a newly acquired laser-trap system with the capability of measuring molecular force in the pN scale with a time resolution of nanoseconds. Goal (ii) will be achieved by testing and visualizing myosin molecules with different structural alterations interacting with actin using a High-Speed Atomic Force Microscope, that allows the visualization of the myosin molecule dynamics in real time Goal (iii) will be achieved by doublets of myosins to check if they influence each other during interactions with action, and filaments of myosin molecules while interacting with actin Gola (iv) will be evaluated by testing myofibrils, comprised of several sarcomeres in series, to check if activation or deactivation of one sarcomere changes the behavior of adjacent sarcomeres. Significance. The relationship between the myosin power stroke and ATP kinetics is the most fundamental step in muscle contraction. Conformational changes in myosin molecules ultimately lead to contraction at the macro scale, and define the power output of muscles. Although this proposal is directed towards the mechanisms of muscle contraction, myosin is just one among many molecular motors with similar structures, which travel along biological paths while converting chemical energy to produce mechanical work. Molecular motors are the engines of life, responsible for cell division, cell migration, tissue growth, intracellular trafficking, neuronal development, to cite a few.