The long-term goals of the proposed research are to determine how the structural dynamics of the cytoskeletal protein myosin are coupled to the interactions at the acto-myosin interface during force generation and unloaded shortening, and to combine mechanical, biochemical and biophysical data into a cohesive model for force generation in skeletal muscle. We will specifically modify myosin in situ using site-directed probes (spin labels, fluorescent probes and/or cross-linkers). Labeling will be followed by ATPase, muscle mechanics (tension, stiffness, velocity of contraction) and/or spectroscopic (electron paramagnetic resonance [EPR], confocal microscopy) measurements to extract information at both the cellular and molecular level. Sequential and/or concomitant mechanical and spectroscopic measurements will provide a unique system for comparisons of cellular level responses with conformational dynamics of contractile protein. Experiments on control and probe-labeled chemically skinned muscle fibers will directly test the role of conformational changes of myosin during energy transduction. Site-directed modifications of myosin and /or nucleotide analogs will be employed to trap a variety of myosin conformations so that a biochemical and mechanical pathway from ATP binding through the crossbridge power- stroke can be constructed. EPR spectroscopy of spinlabeled myosin will be used to reveal the conformational dynamics of myosin, and the dependence of stress on the duration of stretch will be used to quantify changes in the affinity of myosin for actin. The three aims of this project are to: (1) Determine whether the two myosin heads of striated muscle interact cooperatively; (2) develop methodology for coordinated EPR spectroscopy and muscle mechanics; and (3) map the conformational transitions of myosin related to energy acquisition from ATP and energy release during force production. This combination of muscle mechanics and spectroscopy will provide new insights into basic aspects of muscle function. Our approach is an interdisciplinary one, where the data from biochemical, biophysical and physiological techniques will be used to address fundamental questions in muscle biology. These include: How is chemical energy converted to mechanical work by myosin? How do motor proteins respond to muscle stress (e.g., a rapid change in load)? What is the physiological advantage of the redundancy of having two crossbridge domains in the structure of skeletal muscle myosin? Despite gains in our knowledge, in many ways the function of muscle at the molecular level is still treated as a black box where a stimulus from the nerve results in force output by the muscle. The construction of a comprehensive model of how skeletal muscle functions, addressing the questions outlined above, will provide not only a better understanding of healthy muscle function, but also a more complete understanding of muscle illness and failure.
