The effects of deletion of the amino-terminal helix on troponin C function and stability

Lula Smith, Norma J. Greenfield, Sarah E. Hitchcock-DeGregori

Research output: Contribution to journalArticlepeer-review

35 Scopus citations

Abstract

Troponin C has a 14-residue α-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 (Δ14-TnC) showed that deletion of the N-helix did not alter the secondary structure of troponin C. Like wild type troponin C, it exhibited Ca2+-dependent conformational changes based on electrophoretic mobility and increases in α-helix content. The thermal stability of Δ14-TnC, however, was 20 °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 evidenced 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 Ca2+-switching mechanism.

Original languageEnglish (US)
Pages (from-to)9857-9863
Number of pages7
JournalJournal of Biological Chemistry
Volume269
Issue number13
StatePublished - Apr 1 1994

All Science Journal Classification (ASJC) codes

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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