Ca2+-dependent regulation of contraction in striated muscle is mediated through a heterotrimer called the troponin complex. Troponin C is the Ca2+-binding subunit of the troponin complex and is responsible for initial reception of the Ca2+ signal and transmission of this information through a cascade of myofibril regulatory and contractile proteins. The critical role for troponin C in regulation of muscle contraction emphasizes a need to fully understand structure/function relationships that enable it to bind Ca2+ and interact with other troponin subunits in a tissue-specific manner. Two isoforms of troponin C exist, one is present in fast skeletal muscle while the other is found in slow skeletal and cardiac muscle. Major regions of sequence divergence between the isoforms occur in the N-terminal helix and the first Ca2+- binding loop which is inactive in the cardiac protein. In this proposal, molecular biology and recombinant DNA techniques will be used to generate genes that encode cardiac troponin C with specific mutations. The mutant proteins will be produced in bacteria and characterized in vitro with the goal of identifying regions that contribute overall function and tissue-specific characteristics. Three regions in cardiac troponin C will be selected for mutagenesis. First, N-terminal helix, which is divergent between troponin C isoforms, will be deleted to determine its participation in the Ca2+-dependent triggering of the troponin complex. Second, the inactive first Ca2+-binding site in cardiac troponin C will be activated and the other three sites systematically inactivated to determine their relative contribution to the overall function of the protein. Finally, specific acidic amino acids in helices C, D and F will be selected for mutagenesis to determine if they represent critical sites of interaction with other troponin subunits. All mutant proteins will be characterized for Ca2+-binding properties, alterations inprtein dynamics and ability to interact with other troponin subunits and regulate acto-heavy meromyosin ATPase. Long-term goals will include characterization of selected mutants by NMR and X-ray crystallography and transfection of muscle cells in culture with mutant genes to evaluate structure/function relationships in vivo. The sum of this information may facilitate the development of therapeutic agents that modify muscle contraction and possibly aid in the management of disease states.
| Roberts, Thomas J; Marsh, Richard L (2003) Probing the limits to muscle-powered accelerations: lessons from jumping bullfrogs. J Exp Biol 206:2567-80 |
| Brito, R M; Krudy, G A; Negele, J C et al. (1993) Calcium plays distinctive structural roles in the N- and C-terminal domains of cardiac troponin C. J Biol Chem 268:20966-73 |
| Krudy, G A; Brito, R M; Putkey, J A et al. (1992) Conformational changes in the metal-binding sites of cardiac troponin C induced by calcium binding. Biochemistry 31:1595-602 |
| Brito, R M; Putkey, J A; Strynadka, N C et al. (1991) Comparative NMR studies on cardiac troponin C and a mutant incapable of binding calcium at site II. Biochemistry 30:10236-45 |
| George, S E; VanBerkum, M F; Ono, T et al. (1990) Chimeric calmodulin-cardiac troponin C proteins differentially activate calmodulin target enzymes. J Biol Chem 265:9228-35 |
