Calcium and Peptide Binding to Folded and Unfolded Conformations of Cardiac Troponin C. Electrospray Ionization and Fourier Transform Ion Cyclotron Resonance Mass Spectrometry

Abstract

The binding of Ca2+and of a peptide (N-TnC) 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 (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.