Effects of mutations in the central helix of troponin C on its biological activity

Z. Sheng, J. M. Francois, S. E. Hitchcock-DeGregori, J. D. Potter

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28 Scopus citations

Abstract

To investigate the role of the central helix of skeletal muscle troponin C (TnC), five deletion mutants (Dobrowolski, Z., Xu, G. Q., and Hitchcock-DeGregori, S. E. (1991) J. Biol. Chem. 266, 5703-5710) of chicken TnC in the D/E linker region (K87EDAKGKSEEE97), dEDA, dKG, dKGK, dSEEE, and dKED-AKGK, were assayed for their ability to regulate muscle contraction by testing their effectiveness in restoring force and Ca2+ regulation to TnC-depleted rabbit skinned skeletal muscle fibers. By comparison with rabbit skeletal TnC, wild-type TnC, and chicken TnC, all mutants except dKG equally restored force development and Ca2+ regulation to TnC-depleted skinned muscle fibers. In contrast, ∼4 times more dKG than rabbit skeletal TnC was required to reach 50% force restoration. Also, the pCa50 for dKG activation of force was significantly decreased. Thus, most of the TnC mutants that we studied did not have significantly altered biological activity in the skinned fiber assay. However, the 2-residue deletion in the central helix (dKG) significantly affected TnC activity. This deletion would be expected to produce a 160° rotation in the α-helix versus 60° for dKGK and dEDA, 40° in dSEEE, and 20° in dKEDAKGK. Therefore, the change in orientation of the two Ca2+-binding domains appears to be a major parameter affecting TnC activity. The shift in the Ca2+ dependence in force activation may result from the inability of the Ca2+-specific domain to properly interact with its binding site on troponin I, an interaction which is known to increase the affinity of TnC for Ca2+ (Potter, J. D., and Gergely, J. (1975) J. Biol. Chem. 250, 4628-4633). In addition, the length of the central helix of TnC, Gly92, and the negatively charged cluster, EEE, appear not to be crucial for TnC activity.

Original languageEnglish (US)
Pages (from-to)5711-5715
Number of pages5
JournalJournal of Biological Chemistry
Volume266
Issue number9
StatePublished - Oct 22 1991

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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    Sheng, Z., Francois, J. M., Hitchcock-DeGregori, S. E., & Potter, J. D. (1991). Effects of mutations in the central helix of troponin C on its biological activity. Journal of Biological Chemistry, 266(9), 5711-5715.