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

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.