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 myofibrils regulatory and contractile proteins. The critical role for troponin C in regulation of muscle contraction emphasizes a need to fully understand structure/function relationship 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, the N-terminal helix, which is divergent between troponin C isoforms, will be deleted to determine its participation in the C12+-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 or Ca2+-binding properties, alterations in protein 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 relationship 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.
