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 degrees 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 similar to 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, These results are consistent with the following scheme for the binding of Ca2+ to the regulatory site: [GRAPHICS] where the asterisks denote states with enhanced fluorescence, The apparent second-order rate constant for calcium binding is K(o)k(1) = 1.4 x 10(8) M(-1) s(-1). The two first-order transitions occur with observed rates of k(1) + k(-1) approximate to 590 s(-1) and k(2) + k(-2) approximate to 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.