Disparate fluorescence properties of 2-[4'-(iodoacetamido)anilino]-naphthalene-6-sulfonic acid attached to Cys-84 and Cys-35 of troponin C in cardiac muscle troponin.
Two monocysteine mutants of cardiac muscle troponin C, cTnC(C35S) and cTnC(C84S), were genetically generated and labeled with the fluorescent probe 2-[4'-(iodoacetamido)anilino]naphthalene-6-sulfonic acid (IAANS) at Cys-84 and Cys-35, respectively. Cys-84 is located on helix D in the regulatory N-domain, and Cys-35 is at the -y position of the inactive 12-residue loop of site I. These labeled mutants were studied by a variety of steady-state and time-resolved fluorescence methods. In the absence of divalent cation, the fluorescence of the attached IAANS indicated an exposed environment at Cys-35 and a relatively less-exposed environment at Cys-84. The binding of Ca2+ to the single regulatory site elicited a large enhancement of the emission of IAANS attached to Cys-84, but only marginal fluorescence changes of the probe at Cys-35. Upon reconstitution of the labeled cTnC mutants with troponin I and troponin T to form the three-subunit troponin, the fluorescence of IAANS-Cys-84 in apo-troponin was spectrally similar to that observed with the Ca(2+)-loaded uncomplexed cTnC mutant. Only very moderate changes in the fluorescence of IAANS-Cys-84 were observed when the regulatory site in reconstituted troponin was saturated. The exposed Cys-35 environment of the uncomplexed cTnC mutant became considerably less exposed and less polar when the mutant was incorporated into apo-troponin. In contrast to the Cys-84 site, saturation of the regulatory site II by Ca2+ in reconstituted troponin resulted in a reversal of the environment of the Cys-35 site toward a more exposed and more polar environment. These results indicated involvement of the inactive loop I in the Ca2+ trigger mechanism in cardiac muscle. The fluorescence of IAANS at both Cys-84 and Cys-35 was sensitive to phosphorylation of cTnl in reconstituted troponin, and the sensitivity was observed with both apo-troponin and Ca(2+)-loaded troponin.
Dong
W J
WJ
Department of Biochemistry and Molecular Genetics, University of Alabama at Birmingham 35294-2041, USA. wdong@bmg.bhs.uab.edu
Wang
C K
CK
Gordon
A M
AM
Cheung
H C
HC
D000818:Animals; D002118:Calcium / Q000378:metabolism; D002417:Cattle; D003545:Cysteine / Q000378:metabolism*; D005456:Fluorescent Dyes / Q000138:chemical synthesis / Q000378:metabolism; D007700:Kinetics; D009154:Mutation; D009206:Myocardium / Q000737:chemistry; D009282:Naphthalenesulfonates / Q000378:metabolism*; D010766:Phosphorylation; D011485:Protein Binding; D011487:Protein Conformation; D019209:Troponin C / Q000737:chemistry / Q000378:metabolism*; D019210:Troponin I / Q000737:chemistry / Q000378:metabolism*
D005456:Fluorescent Dyes; D009282:Naphthalenesulfonates; D019209:Troponin C; D019210:Troponin I; C011488:IAANS; D003545:Cysteine; D002118:Calcium