Molecular Mechanism of Troponin Function in Health and Disease

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DESCRIPTION (provided by applicant): Our long-term objective is to elucidate the molecular mechanisms whereby intracellular Ca2+ and cross- bridge interactions with the thin filaments of striated muscle regulate myofilament activity. Elucidation of the molecular mechanisms of the muscle regulation is critical to understand the genetic disease, such as familial hypertrophic cardiomyopathy, and to develop drugs, such as Ca2+-sensitizers. Ca2+-dependent regulation of striated muscle contraction requires the protein complex, tropomyosin (Tm), and troponin (Tn), which consists of Tnl, TnC, and TnT. Ca2+-dependent interactions among thin filament proteins are the key events in striated muscle regulation. At low Ca2+, Tnl interacts with actin through at least two regions and inhibits actin-activated myosin ATPase activity. The molecular mechanism of this inhibition, however, remains to be solved. The important unanswered question is: How do actin and Tnl interact with each other to prevent the activation of the thin filament? At high [Ca2+], the regulatory site of Tnl interacts with the newly exposed hydrophobic surface of the N-lobe of TnC. This interaction causes the actin-interacting sites of Tnl to move away from actin and allows Tm to move to the inner domain of the actin filament. There is evidence that indicates that the interaction between the regulatory site of Tnl and the N- lobe of TnC should cause more than a simple release of the inhibitory region/second actin-Tm site from actin. There is evidence that the inhibitory region of Tnl may interact with the central linker region of TnC at high [Ca2+]. Recent structural and biochemical data suggest that the interaction between three Tn components at the linker region of Tn complex consists of the central linker of TnC, the inhibitory region of Tnl and TnT, may play a role in Ca2+-activation. My hypothesis, based on these data, is that the energetically balanced Ca2+-dependent interactions between the thin filament proteins are critical in the Ca2+-dependent regulation of myofilament activity. We will investigate using site-directed mutations, synthetic peptide-array assay, a solid phase-based protein binding assay, a photocross-linking, Bioinformatics analysis, Ca2+-binding measurement, acto-S1 ATPase assay, SPR, and FRET measurements.