Calcium structural transition of human cardiac troponin C in reconstituted muscle fibres as studied by site-directed spin labelling

The in situ structure of human cardiac troponin C (hcTnC) has been studied with site-directed, spin labelling, electron paramagnetic resonance (SDSL-EPR). Analysis of the in situ structures of hcTnC is essential for elucidating the molecular mechanism behind its Ca2+-sensitive regulation. We prepared two hcTnC mutants (C35S and C84S) containing one native cysteine residue (84 and 35, respectively) for spin labelling. The mutants were labelled with a methane thiosulfonate spin label (MTSSL) and the TnC was reconstituted into permeabilized muscle fibres. The mobility of Cys84-MTSSL changed markedly after addition of Ca2+, while that of the Cys35 residue did not change in the monomer state or in fibres. The rotational correlation time of Cys84-MTSSL decreased from 32 ns to 13 ns upon Ca2+-binding in the monomer state, whereas in fibres the spectrum of Cys84-MTSSL was resolved into mobile (16 ns) and immobile (35 ns) components and the addition of Ca2+ increased the immobile component. Moreover, the accessibility of Cys84-MTSSL to molecular oxygen increased slightly in the presence of Ca2+. These data suggest that Cys35 remains in the same location regardless of the addition of Ca2+, whereas Cys84 is located at the position that interacts with B and C helices of hcTnC and interacts with troponin I (TnI) at high concentrations of Ca2+. we determined the distances between Cys35 and Cys84 by measuring pulsed electron-electron double resonance spectra. The distances were 26.0 angstrom and 27.2 angstrom in the monomer state and in fibres, respectively, and the addition of C 2 Ca2+ decreased the distance to 23.2 angstrom in fibres but only slightly in the monomer state, showing that Ca2+ binding to the N-domain of hcTnC induced a larger structural change in muscle fibres than in the monomer state. (c) 2005 Elsevier Ltd. All rights reserved.