The reactivity of sulfhydryl groups of bovine cardiac troponin C.
Bovine cardiac troponin C (cTnC) contains 2 cysteine residues, Cys-35 located in the nonfunctional Ca2+-binding loop I and Cys-84 in the N-terminal segment of the central helix. We have studied the reactivity of Cys residues in cTnC with 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) and 7-diethylamino-3-(4'-maleimidylphenyl)-4-methylcoumarin (CPM). The latter compound fluoresces only when reacted with the protein. The reaction with DTNB followed second order kinetics with respect to DTNB, the rate constants being 3.37 s-1 M-1 and 1.82 s-1 M-1 in the presence and absence of Ca2+, respectively. These rates are much slower than the rate of reaction with Cys-98 of skeletal TnC (sTnC) or with the urea-denatured cTnC, indicating that both Cys residues are partly buried within the structure of the protein. The increase in reactivity was induced by binding of Ca2+ to the single low affinity Ca2+ binding site (site II). The fluorescence increase upon reaction of cTnC with CPM in the absence of Ca2+ could be fitted with a single exponential equation indicating that both cysteine residues are equally available to the reagent. The reaction in the presence of Ca2+ was biphasic. Analysis of CNBr fragments of cTnC labeled with CPM under various conditions indicated that in the presence of Ca2+ the reactivity of Cys-84 is increased while that of Cys-35 is slightly decreased. This finding is consistent with the model of Herzberg et al. (Herzberg, O., Moult, J., and James, M. N. G. (1986) J. Biol. Chem. 261, 2638-2644) and the data of Ingraham and Hodges (Ingraham, R. H., and Hodges, R. S. (1988) Biochemistry 27, 5891-5898), suggesting that the Ca2+-induced conformational change in the N-terminal half of TnC involves separation of the helix C from the central helix, thereby increasing the accessibility of Cys-84. The slow overall kinetics, however, indicates that the structure in the vicinity of Cys residues is relatively compact regardless of Ca2+. We interpret the increase in reactivity towards CPM as consistent with a Ca2+-induced exposure of a hydrophobic pocket in the vicinity of Cys-84.
Fuchs
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Department of Physiology, University of Pittsburgh School of Medicine, Pennsylvania 15261.
Liou
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Grabarek
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D000596:Amino Acids / Q000032:analysis; D000818:Animals; D001665:Binding Sites; D002118:Calcium / Q000494:pharmacology; D002417:Cattle; D003488:Cyanogen Bromide; D004228:Dithionitrobenzoic Acid / Q000378:metabolism*; D007700:Kinetics; D009206:Myocardium / Q000378:metabolism*; D009579:Nitrobenzoates / Q000378:metabolism*; D010446:Peptide Fragments / Q000302:isolation & purification; D011485:Protein Binding; D013050:Spectrometry, Fluorescence; D013438:Sulfhydryl Compounds / Q000378:metabolism; D014336:Troponin / Q000378:metabolism*; D019209:Troponin C
D000596:Amino Acids; D009579:Nitrobenzoates; D010446:Peptide Fragments; D013438:Sulfhydryl Compounds; D014336:Troponin; D019209:Troponin C; D004228:Dithionitrobenzoic Acid; D003488:Cyanogen Bromide; D002118:Calcium