This proposal concerns the function, structure, and dynamics of troponin and tropomyosin, the two proteins that most directly control the contraction and relaxation of the heart. Bound directly to the thin filaments of the contractile apparatus, troponin and tropomyosin cause the actions of the molecular motor myosin to be Ca2+-dependent. Ca2+ binding to troponin changes its structure, and this is propagated to cause changes in thin filament structure: the thin filament switches among states that permit or prohibit muscle contraction. The mechanism of this regulation will be investigated by assessing these proteins' properties and function in an increasingly complex series of settings: troponin (or tropomyosin) internal dynamics, interactions between troponin and tropomyosin, thin filament function, and muscle fiber function. Dynamic, functionally significant features of tropomyosin and troponin will be investigated by native state amide hydrogen exchange, measured by mass spectrometry. Functional abnormalities caused by human cardiomyopathy-inducing mutations in tropomyosin and in the tail domain of troponin will be determined. Regions of troponin subunits Tnl and TnT thought critical for inhibition of contraction in the absence of Ca2+ will be altered by mutagenesis to determine the mechanism by which contraction is halted or prevented. Alterations in dynamics, in protein-protein binding, in myosin MgATPase regulation, in thin filament conformational switching assessed by 3-D reconstructions of electron micrographs, and in fiber mechanics after whole troponin exchange will be determined, with the mechanistic implications considered in concert. Our long-term goal of understanding troponin-tropomyosin-mediated regulation of contraction has fundamental physiological importance in the heart and in skeletal muscle. Also, this goal is significant because this regulation is altered in inherited cardiomyopathy, and because thin filament proteins are potential therapeutic targets in other heart disorders. ? ?
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