Parallel measurement of Ca2+ binding and fluorescence emission upon Ca2+ titration of recombinant skeletal muscle troponin C: Measurement of sequential calcium binding to the regulatory sites

Fernando Fortes De Valencia, Adriana Aparecida Paulucci, Ronaldo Bento Quaggio, Ana Cláudia Rasera Da Silva, Chuck S. Farah, Fernando De Castro Reinach

    Research output: Contribution to journalArticle

    10 Scopus citations

    Abstract

    Calcium binding to chicken recombinant skeletal muscle TnC (TnC) and its mutants containing tryptophan (F29W), 5-hydroxytryptophan (F29HW), or 7-azatryptophan (F29ZW) at position 29 was measured by flow dialysis and by fluorescence. Comparative analysis of the results allowed us to determine the influence of each amino acid on the calcium binding properties of the N-terminal regulatory domain of the protein. Compared with TnC, the Ca2+ affinity of N-terminal sites was: 1) increased 6-fold in F29W, 2) increased 3-fold in F29ZW, and 3) decreased slightly in F29HW. The Ca2+ titration of F29ZW monitored by fluorescence displayed a bimodal curve related to sequential Ca2+ binding to the two N-terminal Ca2+ binding sites. Single and double mutants of TnC, F29W, F29HW, and F29ZW were constructed by replacing aspartate by alanine at position 30 (site I) or 66 (site II) or both. Ca2+ binding data showed that the Asp → Ala mutation at position 30 impairs calcium binding to site I only, whereas the Asp → Ala mutation at position 66 impairs calcium binding to both sites I and II. Furthermore, the Asp → Ala mutation at position 30 eliminates the differences in Ca2+ affinity observed for replacement of Phe at position 29 by Trp, 5-hydroxytryptophan, or 7-azatryptophan. We conclude that position 29 influences the affinity of site I and that Ca2+ binding to site I is dependent on the previous binding of metal to site II.

    Original languageEnglish (US)
    Pages (from-to)11007-11014
    Number of pages8
    JournalJournal of Biological Chemistry
    Volume278
    Issue number13
    DOIs
    StatePublished - Mar 28 2003

    ASJC Scopus subject areas

    • Biochemistry
    • Molecular Biology
    • Cell Biology

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