Calcium and peptide binding to folded and unfolded conformations of cardiac Troponin C. Electrospray ionization and Fourier transform ion cyclotron resonance mass spectrometry

Eur. J. Mass Spectrom. 8, 471–481 (2002)
DOI: 10.1255/ejms.523

Calcium and peptide binding to folded and unfolded conformations of cardiac Troponin C. Electrospray ionization and Fourier transform ion cyclotron resonance mass spectrometry

Marjaana Nousiainen and Pirjo Vainiotalo
Department of Chemistry, University of Joensuu, PO BOX 111, FIN-80101 Joensuu, Finland
Helen J. Cooper, Antuan Hoxha and Peter J. Derrick
Institute of Mass Spectrometry, Department of Chemistry, University of Warwick, Coventry CV4 7AL, UK
Dorina Fati
Institute of Mass Spectrometry, Department of Chemistry, University of Warwick, Coventry CV4 7AL, United Kingdom and Department of Chemistry, University of Liège, 4000 Sart-Tilman, Belgium
Hylary R. Trayer, Douglas G. Ward and Ian P. Trayer
School of Biosciences, University of Birmingham, Edgbaston, Birmingham B15 2TT, UK

ABSTRACT:

The binding of Ca²⁺ and of a peptide (N-TnI) to human cardiac troponin C (TnC) and its isolated N- and C- terminal domains has been characterized by electrospray ionization Fourier transform ion cyclotron resonance (ESI FT-ICR) mass spectrometry. The peptide N-TnI corresponds to residues 1–29 of the cardiac-specific N-terminal extension of human cardiac troponin I (TnI). The binding of Ca²⁺ to intact TnC in the absence of the peptide was found to take a bimodal form with preferred stoichiometries of 1 : 1 TnC : Ca²⁺ and 1 : 3 TnC : Ca²⁺. It is concluded that TnC existed in two conformational isomers that had different binding affinities for Ca²⁺: the binding of 3 Ca²⁺ was characteristic of a folded conformation (TnC_A) and the binding of 1 Ca²⁺ was characteristic of a partially unfolded conformation (TnC_B). Both of these conformations contributed to the 8+ (and other) charge states of TnC, and were distinguished on the basis of their different Ca²⁺-binding affinities and not on the basis of the charge state. In the presence of the peptide, a complex with 1 : 1 : 1 TnC : peptide : Ca²⁺ stoichiometry was formed.

Keywords: troponin C; troponin I; Ca²⁺ binding; electrospray ionization; Fourier transform ion cyclotron resonance

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