Molecular mechanisms of muscle contraction

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Background. Muscle contraction is driven by the molecular motor myosin II while attached to actin filaments. Myosin molecules transform chemical energy extracted from adenosine tri-phosphate (ATP) into mechanical work. After an initial attachment between myosin and actin, ATP is hydrolyzed, and phosphate (Pi) and subsequently ADP are released from myosin. The molecule undergoes a conformational change - the power stroke - producing force and sliding the actin filament. The molecular mechanisms behind the conversion of chemical energy released from ATP into the myosin power stroke, and its amplification into muscle contraction is not well understood. ***Long-term objective. The long-term objective of this research program is to understand the molecular and cellular mechanisms of muscle contraction and force generation. The specific goals of this proposal are:***1 - To determine the relation between myosin structures and actin sliding ***2 - To determine the sub-structures of myosin responsible for converting chemical energy into mechanical work ***3 - To determine the emerging properties of myosin molecules when working cooperatively in filaments.***Methods. This research program will use a variety of experimental systems and techniques developed in my laboratory. There are domains and sub-domains of the myosin molecule that have been hypothesized to define the power stroke. In specific aim (1) we will experiment with native myosin molecules and also myosin mutants in which some of these sub-domains have been depleted. Molecules will be tested with a motility essay, that allows for measurements of velocity of actin sliding. In specific aim (2) the molecules (native and mutants) will be tested under a High-Speed Atomic Force Microscope (HS-AFM), that allows measurements of force while visualizing structural changes in the myosin-actin system. In specific aim (3) molecules (native and mutants) will be polymerized into filaments in different concentrations, to evaluate the emerging properties of myosins working cooperatively. The filaments will be tested for force measurements using a system of nanofabricated cantilevers. ***Significance. Understanding the myosin power stroke - the most fundamental step of muscle contraction - is essential for understanding muscle contraction, a basic function of life. Beyond the mechanisms of contraction, myosin II is one among many molecular motors (e.g. kinesin, dynein, myosin V), with a variety of physiological functions. Molecular motors share several similarities, including a power-stroke induced motility and force production. Therefore, understanding myosin II mechanisms will provide insight into the functioning of molecular motors in general, with far-reaching implications for biology.**