title: Fluorescence lifetime and acrylamide quenching studies of the interactions between troponin subunits.
abstract: Fluorescence lifetime and acrylamide quenching studies were carried out to characterize the interactions between the subunits of 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 X 10(8) 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 X 10(8) and 0.66 X 10(8) 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 approximately 18 ns, and kq decreased to approximately 0.8 X 10(8) 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 X 10(8) 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.(ABSTRACT TRUNCATED AT 250 WORDS)
mesh_terms: D000818:Animals; D002118:Calcium / Q000494:pharmacology; D007700:Kinetics; D046911:Macromolecular Substances; D008274:Magnesium / Q000494:pharmacology; D009132:Muscles / Q000378:metabolism; D009282:Naphthalenesulfonates / Q000494:pharmacology; D011487:Protein Conformation; D011817:Rabbits; D013050:Spectrometry, Fluorescence; D014336:Troponin / Q000378:metabolism*; D019209:Troponin C
chemical_list: D046911:Macromolecular Substances; D009282:Naphthalenesulfonates; D014336:Troponin; D019209:Troponin C; C006421:1,5-I-AEDANS; D008274:Magnesium; D002118:Calcium