PubMed1993
Bovine cardiac troponin C (cTnC) has cysteine residues located in the non-functional Ca(2+)-binding loop I (Cys-35) and at the N-terminal end of the central helix (Cys-84) near site II, the regulatory Ca(2+)-binding site. Recently, we reported that the excimer fluorescence resulting from the dimerization of adjacent pyrene groups attached to the two Cys residues is reduced by Ca2+ binding to site II (Liou, Y.-M. and Fuchs, F. (1992) Biophys. J. 61, 892-901). This result would suggest that Ca2+ binding causes a separation of the two Cys residues, a conclusion at variance with predictions from molecular modeling studies (Herzberg, O., Moult, J. and James, M.N.G. (1986) J. Biol. Chem. 261, 2638-2644). Alternatively, the reduction in excimer fluorescence could be accounted for by an immobilization of the pyrene attached to Cys-84 by a Ca(2+)-induced hydrophobic pocket. To arrive at a more definitive interpretation of these experiments, we carried out steady-state fluorescence resonance energy-transfer measurements of the Cys35-Cys84 distance. We used three different donor-acceptor pairs: 2-(4'-(iodoacetamido)anilino) naphthalene-6-sulfonic acid (IAANS) and 4-dimethylamino-phenylazophenyl-4-maleimide (DABMI), IAANS and N-(4-(dimethyl-amino)-3,5-dinitrophenyl) maleimide (DDPM), and 5-((((2-iodoacetyl)amino)ethyl)amino)naphthalene-1-sulfonic acid (IAEDANS) and DDPM. At pCa 8.0, the distances were 23.8, 21.0, and 22.0 A with the donor-acceptor pairs, IAANS-DABMI, IAANS-DDPM and IAEDAN-DDPM, respectively. At pCa 4.0, the distances were 25.8, 24.1 and 21.2 A. The distances at pCa 8 and pCa 4.0 were not significantly altered when labeled cTnC was complexed with cardiac troponin I (cTnI). Thus, Ca2+ has little, if any, effect on the Cys35-Cys84 distance. These results are consistent with a model in which Ca2+ binding induces a separation of helices B and C from helix D, without any relative movement of the two N-terminal Ca(2+)-binding domains.
Adenosine Triphosphatases↗