[Role of Ca-specific binding sites in the induction of conformational changes in troponin structure]

A new technique for the registration of troponin conformational changes by hydrophobic fluorescent dye, dimethylnaphteirodin, is described. The increase of Ca2+ concentration from pCa 8 to pCa 5 is accompanied by 4--5-fold increase of fluorescence of troponin--dimethylnaphteirodin complex and by the shift of the fluorescence maximum by 10 nm into the short-wave region. Isolated troponin components and troponin I--troponin T and troponin I--troponin C complexes do not bind dimethylnaphteirodin. The troponin T--TROPONIN C complex binds dimethylnaphteirodin, and fluorescence of the bound dye increases under the increase of Ca2+ concentration, as it does in the case of complete froponin complex. The apparent Ca2+-binding constant by the whole troponin complex (K=8.10(6) M(-1)) and by troponin T--troponin C complex (K=8.10(5) M(-1)), and the value of fluorescence of troponin- and troponin components-bound dimethylnaphteirodin do not depend on the magnesium concentration. Highly cooperative dependence of the fluorescence intensity of troponin-dimethylnaphteirodin on Ca2+ concentration (the Hill coefficient is 2.0--2.4) suggests, that the changes in the troponin structure, registered using dimethylnaphteirodin, take place only after Ca2+ binding to two Ca-specific sites.