Calcium binding by troponin-C. A proton magnetic resonance study

The conformational changes induced by the binding of Ca(II) to rabbit skeletal muscle troponin-C (TNC) have been followed by proton magnetic resonance spectroscopy. Ca(II)-free TNC (apo-TNC) contains definite ordered regions. Ca(II) titration of the high affinity sites (cf. Potter , Gergely, 1975) causes a large folding of the backbone, some of which involves refolding of an ordered region(s) and changes in several side-chains e.g. Glu, Asp and Phe. Titration of the low affinity sites does not alter the backbone but leads to changes among hydrophobic side-chains (one or more Val, Leu, Ile; two or more Phe, Glu and Asp) that define an ordered region(s) of apo-TNC. The rate constants for the conformation changes of the low and high affinity sites are approximately 10 s −1 and < 20 s −1, respectively. Final stages of the titration include a downfield shifted methyl group (likely Ile) and a Phe residue. The thermal stabilities of apo-TNC, TNC · Ca 2(II) and native TNC were compared. It was concluded that Ca(II) binding by the two high affinity sites both directs and stabilizes much of the structure. The role of the changes of the low affinity sites, which are thought to activate contraction, are briefly discussed.