Energy-transfer measurements of the Cys 35-Cys 84 distance in bovine cardiac troponin C

Abstract

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 Ca 2+ binding to site II (Liou, Y.-M. and Fuchs, F (1992) Biophys. J. 61, 892–901). This result would suggest that Ca 2+ 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 Cys 35-Cys 84 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-dimethylamino)-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 Å 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 Å. The distances at pCa 8 and pCa 4.0 were not significantly altered when labeled cTnC was complexed with cardiac troponin I (cTnI). Thus, Ca 2+ has little, if any, effect on the Cys 35-Cys 84 distance. These results are consistent with a model in which Ca 2+ 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.