Calcium regulates cardiac and skeletal muscle contraction by binding to the thin filament, which contains many copies of actin, tropomyosin, and the three subunits of troponin: TnT, TnI, and Ca2+-binding TnC. Multiple, complex, allosteric interactions among these proteins are responsible for regulating muscle contraction. This project involves measurement of protein-protein interactions within the thin filament, perturbation of these interactions by production of mutant recombinant proteins, and study of their effects on regulation. The experiments will provide insights concerning the conformational states of the thin filament, their relationship to regulation, and their thermodynamic linkage to ligand binding and to thin filament assembly. The long range goal is to understand regulation in terms of transitions in thin filament quaternary structure.
The Aims are (1) Determine the importance of specific regions of tropomyosin for regulation, for thin filament assembly (protein-protein binding) under several distinct conditions, for myosin-actin binding, and for Ca2+ binding. (2) Perform mutagenesis of the inhibitory region of TnI to determine this regions role in the intact thin filament regarding troponin-thin filament binding in the presence or absence of Ca2+ and myosin S1, and for regulation of both myosin ATPase activity and in vitro sliding and force. (3) Obtain structural information regarding troponin within the thin filament, by electron microscopy of thin filaments engineered to have more troponin than normally, and by study of selected actin mutations. (4) Investigate the mechanism of muscle activation s cooperativity, by studying in vitro actin-myosin motility and force using thin filaments with engineered TnC that permits precise control of fractional Ca2+ binding, and also by studying the effects of NH2-terminal truncation of TnT. These four Aims address the process that most directly regulates contraction, and will aid understanding of normal, adaptive, and pathological cardiac and skeletal muscle function.
