The Orientation of the H1 and H2 Helices of Troponin I in Ventricular Trabeculae

Abstract

Contraction of cardiac muscle is triggered and modulated by structural changes in the heterotrimeric troponin complex (TnC, TnI and TnT) on the actin filaments. The changes in conformation of the N-terminal lobe of TnC induced by Ca2+ binding are well understood, but the structural basis of the downstream signaling pathway via TnI and TnT to tropomyosin and actin is much less clear, as is that of the modulation of contractility by TnI phosphorylation. To address these questions we are investigating the orientation of the TnI H1 helix (residues 43-79, human cardiac TnI; hcTnI) and H2 helix (residues 93-135) of the troponin core domain (Takeda et al., Nature 424: 35, 2003) in a cardiac muscle cell using bifunctional fluorescent probes (Corrie et al., Nature 400:425, 1999). The hcTnI cysteines 80 and 97 were replaced by serines, and pairs of cysteines were introduced for covalent attachment of bifunctional sulphorhodamine (BSR) at surface-accessible positions 60 and 67 or 119 and 126. Full-length troponin complex was formed from these modified hcTnIs in combination with cysteine-null hcTnC and hcTnT. The cysteine pair on hcTnI in each complex was labeled with BSR, and the hcTnI-BSRs were characterized by HPLC and mass spectroscopy. The labeled troponin complex will be exchanged into skinned trabeculae from rat ventricle for determination of the orientation of the H1 and H2 helices with respect to the thin filament axis by polarized fluorescence (Dale et al., Biophys. J. 76:1606, 1999; Julien et al., Biophys. J. 93:1008, 2007).Supported by the British Heart Foundation.