Fluorescence lifetime and acrylamide quenching studies of the interactions between troponin subunits

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Abstract
Fluorescence lifetime and acrylamide quenching studies were carried out to characterize the interactions between the subunits of [rabbit muscle] troponin under various conditions of metal ion binding. Troponin C was labeled at Cys-98 with N-(iodoacetyl)-N''-(5-sulfo-1-naphthyl)ethylenediamine. In the presence of Ca2+, the fluorescence decay of labeled troponin C (TnC*) was monoexponential, lifetime .tau. = 15.5 ns and quenching rate constant kq = 2.97 .times. 108 M-1 s-1. In the absence of Ca2+, the decay was resolvable into a major component with .tau. = 11.9 ns and a minor component with .tau. = 20.5 ns, with corresponding values of kq = 4.80 .times. 108 and 0.66 .times. 108 M-1 s-1, respectively. Upon the binding of either troponin I (TnI) or troponin T (TnT) in the presence of Ca2+, .tau. increased to .apprx. 18 ns, and kq decreased to .apprx. 0.8 .times. 108 M-1 s-1. For the Ca2+ form of the TnC*-TnI-TnT ternary complex, values of .tau. = 17.6 ns and kq = 1.73 .times. 108 M-1 s-1 were obtained. These values did not vary significantly when Ca2+ was removed, or when Mg2+ replaced Ca2+. These findings were interpreted as follows: the region around Cys-98 of TnC* adopts a looser conformation upon the removal of Ca2+ from the high-affinity sites. Both TnI and TnT bind to TnC* in the region containing Cys-98. The probe is shielded from the solvent to a greater extent in the binary complexes than in the ternary complex. The lack of metal-induced changes in the ternary complex indicates that conformational changes in this region of the molecule, if they do occur, do not result in changes in the environment of the probe.

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