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Eur. J. Mass Spectrom. 8, 471 - 481 (2002) |
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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 |
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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 have been characterized by electrospray ionization Fourier transform ion cyclotron resonance (ESI FTICR) mass spectrometry. The peptide (N-TnI) corresponded to residues 129 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 (TnCA) and the binding of 1 Ca2+ was characteristic of a partially unfolded conformation (TnCB). 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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