Kinetic studies of calcium binding to the regulatory site of troponin C from cardiac muscle

Wen Ji Dong, Steven S. Rosenfeld, Chien Kao Wang, Albert M. Gordon, Herbert C. Cheung

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53 Scopus citations


We have studied the kinetics of the structural transitions induced by calcium binding to the single, regulatory site of cardiac troponin C by measuring the rates of calcium.mediated fluorescence changes with a monocysteine mutant of the protein (C35S) specifically labeled at Cys-84 with the fluorescent probe 2-[4'-(iodoacetamido)anilino]naphthalene-6-sulfonic acid. At 4 °C, the binding kinetics determined in the presence of Mg2+ was resolved into two phases with positive amplitude, which were completed in less than 100 ms. The rate of the fast phase increased linearly with [Ca2+] reaching a maximum of 590 s-1, and that of the slow phase was approximately 100 s-1 and did not depend on Ca2+ concentration. Dissociation of bound Ca2+ from the regulatory site occurred with a rate of 102 s-1, whereas the dissociation from the two high affinity sites was about two orders of magnitude slower. The apparent second-order rate constant for calcium binding is K0k1 = 1.4 x 108 M-1 s-1. The two first-order transitions occur with observed rates of k1 + k-1 ≃ 590 s-1 and k2 +k-2 ≃ 100 s-1, and the binding of Ca2+ to the regulatory site is not a simple diffusion- controlled reaction. These transitions provide the first information on the rates of Ca2+-induced conformational changes involving helix movements in the regulatory domain.

Original languageEnglish (US)
Pages (from-to)688-694
Number of pages7
JournalJournal of Biological Chemistry
Issue number2
StatePublished - Jan 12 1996

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology


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