Abstract
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 language | English (US) |
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Pages (from-to) | 688-694 |
Number of pages | 7 |
Journal | Journal of Biological Chemistry |
Volume | 271 |
Issue number | 2 |
DOIs | |
State | Published - Jan 12 1996 |
ASJC Scopus subject areas
- Biochemistry
- Molecular Biology
- Cell Biology