title: The effects of deletion of the amino-terminal helix on troponin C function and stability.
abstract: Troponin C has a 14-residue alpha-helix at the extreme amino terminus (the N-helix) which is absent in calmodulin. To learn the significance of this region in troponin C, residues 1-14 were deleted using site-directed mutagenesis. Analysis of the mutant troponin C (delta 14-TnC) showed that deletion of the N-helix did not alter the secondary structure of troponin C. Like wild type troponin C, it exhibited Ca(2+)-dependent conformational changes based on electrophoretic mobility and increases in alpha-helix content. The thermal stability of delta 14-TnC, however, was 20 degrees C lower than wild type troponin C in the presence or absence of divalent cations because of destabilization of the amino-terminal domain. To determine the functional consequences of the deletion, its ability to relieve troponin I and IT inhibition of the actomyosin ATPase was assayed. The results show that the mutant could relieve troponin I inhibition in the presence and absence of Ca2+ but could relieve troponin IT inhibition only to 45-50% of the wild type level, even at high concentrations. Also, the calcium affinity of the low affinity sites is reduced as evidence by the 2.4-2.8-fold increase in Ca2+ concentration required to achieve half-maximal activation of the MgATPase and calcium titration of the metal-induced conformation monitored by far UV circular dichroism measurements. In addition, the N-helix is required for the full conformational change to take place upon the binding of Ca2+, but not Mg2+, to the high affinity sites. The results indicate that the N-helix of troponin C is important for the stability of troponin C and may play a vital role in the Ca(2+)-switching mechanism.
authors.affiliation: Department of Neuroscience and Cell Biology, Robert Wood Johnson Medical School, Piscataway, New Jersey 08854.
authors.forename: L
authors.initials: L
authors.lastname: Smith
authors.forename: N J
authors.initials: NJ
authors.lastname: Greenfield
authors.forename: S E
authors.initials: SE
authors.lastname: Hitchcock-DeGregori
mesh_terms: D000251:Adenosine Triphosphatases / Q000737:chemistry / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002118:Calcium / Q000494:pharmacology; D002645:Chickens; D002942:Circular Dichroism; D003285:Contractile Proteins / Q000737:chemistry; D007700:Kinetics; D008969:Molecular Sequence Data; D009132:Muscles / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D009838:Oligodeoxyribonucleotides; D011487:Protein Conformation / Q000187:drug effects; D011489:Protein Denaturation; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism; D017384:Sequence Deletion; D013816:Thermodynamics; D014336:Troponin / Q000096:biosynthesis / Q000737:chemistry* / Q000378:metabolism*; D019209:Troponin C
chemical_list: D003285:Contractile Proteins; D009838:Oligodeoxyribonucleotides; D011994:Recombinant Proteins; D014336:Troponin; D019209:Troponin C; D000251:Adenosine Triphosphatases; D002118:Calcium