Calcium-induced conformational change in cardiac troponin C studied by fluorescence probes attached to Cys-84.

Residue Cys-84 of bovine cardiac troponin C (cTnC) located at the C-terminal end of helix D was selectively labeled in the presence of Ca2+ with two fluorescent probes: IAANS (2-(4-(iodoacetamido)anilino)naphthalene-6-sulfonic acid) and acrylodan (6-acrylol-2-(dimethylamino)naphthalene). The fluorescence of the attached probes was studied by the steady-state and time-resolved methods to gain an insight about the nature of Ca(2+)-induced conformational changes in the N-domain regulatory region of cTnC. Changes in the experimental emission spectra, quantum yields, and excited-state lifetimes suggested that bound Ca2+ at the single regulatory site induced a less polar microenvironment for both probes attached to Cys-84. However, a twofold increase in the bimolecular collisional quenching constant was observed for both probes in the presence of activator Ca2+, indicating an increased exposure of the probes to solvent. These data were interpreted with reference to the origins of the observed Stokes' shifts. In the apo and 2Mg states of cTnC, the attached probes were partially shielded by helices B and C, and their excited-states were highly quenched in the tertiary structure through strong interactions of a dipolar nature with neighboring amino-acid side chains. In the 3Ca state, these interactions were disrupted so that nonradiative decay processes were suppressed and radiative processes were enhanced, leading to the observed increases in quantum yields and lifetimes and blue-shifts of the emission spectra. As the disruption of internal quenching resulted from separation of helices B and C from helix D, the attached probes became more accessible to solvent and experienced increases in the rate of collisions with external molecules in the solvent. Although this increased exposure to solvent would lead to suppression of radiative decay processes, this effect apparently was overcompensated by the effect of elimination of internal quenching. The present results are consistent with a Ca(2+)-induced open conformation of the N-domain in cTnC.