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 approximately 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: [formula: see text] where the asterisks denote states with enhanced fluorescence. The apparent second-order rate constant for calcium binding is Kok1 = 1.4 x 10(8) M 1 s-1. The two first-order transitions occur with observed rates of k1 + kappa-1 approximately 590 s-1 and kappa 2 + kappa-2 approximately 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 Ca(2+)-induced conformational changes involving helix movements in the regulatory domain.
Dong
W
W
Department of Biochemistry and Molecular Genetics, University of Alabama at Birmingham 35294, USA.
Rosenfeld
S S
SS
Wang
C K
CK
Gordon
A M
AM
Cheung
H C
HC
D000818:Animals; D002118:Calcium / Q000378:metabolism*; D002135:Calcium-Binding Proteins / Q000378:metabolism; D007700:Kinetics; D009154:Mutation; D009206:Myocardium / Q000378:metabolism*; D017510:Protein Folding; D051381:Rats; D014336:Troponin / Q000235:genetics / Q000378:metabolism*; D019209:Troponin C
D002135:Calcium-Binding Proteins; D014336:Troponin; D019209:Troponin C; D002118:Calcium