Cloning, expression, and site-directed mutagenesis of chicken skeletal muscle troponin C.
Skeletal muscle troponin C (TNC) is structured into two separate domains linked by a nine-turn alpha-helix (D/E helix). It has been demonstrated that calcium binding to the regulatory sites within the N-terminal domain induces conformational changes in the C-terminal domain of isolated TNC. Since the only contact between the two domains is the long D/E helix, the transfer of information must involve conformational changes within this helix. The center of the helix is occupied by a glycine (Gly-92). A postulated mechanism for allowing interdomain interaction involves a conformational change of the D/E helix around Gly-92 (Herzberg, O., and James, M. N. G. (1985) Nature 312, 653-659). We tested this hypothesis using site-directed mutants of troponin C. Two separate mutants containing an alanine and a proline replacing Gly-92 were constructed and compared with wild type TNC. Calcium binding studies showed no significant differences among the TNC species. The different TNC were assembled into thin filaments and used to assay the calcium regulation of actin-activated ATPase of myosin. All TNC species were able to mediate the calcium regulation of ATPase. Under the conditions used for the assays, no differences were detected among the TNC species. These results show that Gly-92 is not essential for the proper interaction of the calcium regulatory sites with the other components of the thin filament, and therefore exclude a large rotation around Gly-92 as the mechanism of information transfer between the two domains of troponin C.
D000251:Adenosine Triphosphatases / Q000378:metabolism; D000818:Animals; D001483:Base Sequence; D002118:Calcium / Q000378:metabolism; D002645:Chickens; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D005786:Gene Expression Regulation; D008969:Molecular Sequence Data; D009154:Mutation; D014336:Troponin / Q000235:genetics*; D019209:Troponin C
D014336:Troponin; D019209:Troponin C; D000251:Adenosine Triphosphatases; D002118:Calcium