Calcium-Induced Conformational Changes in the Cardiac Isoform of the Troponin Complex Monitored By Hydrogen/deuterium Exchange and Fourier Transform Ion Cyclotron Resonance Mass Spectrometry

Calcium-induced conformational changes in the cardiac troponin complex have been determined from solvent accessibility data measured by solution-phase hydrogen/deuterium exchange (HDX). The troponin complex was digested with protease XIII and the peptides identified by FT-ICR mass spectrometry. To uniquely assign proteolytic fragments of the same mass, experiments were performed with troponin subunits isotopically depleted in 13C and 15N. For instance, the peptide with monoisotopic m/z = 592.983+could be either TnT fragment 76-91 or to TnI fragment 155-170, also known as the switch peptide. Isotopic depletion uniquely identified the TnT fragment. Comparison of the HDX rates in the isolated subunits and those observed in the reconstituted complex identified inter-subunit interfaces, in general agreement with the x-ray structure of the core complex (Takeda et al. Nature, 2003 424 (6944)). HDX experiments for the complex in the presence and in the absence of calcium revealed a multitude of conformational changes, some of which were not apparent from comparison of Ca-saturated and Ca-free x-ray structures of skeletal troponin (Vinogradova et al. PNAS, 2005 102 (14)). For example, the C-lobe of TnC, which was not expected to change upon Ca binding, showed different HDX rates in the presence and in the absence of Ca for peptides 105-119 and 150-153. Similar effects were observed for residues 24-27 of TnC, but smaller differences were found for the TnI switch peptide (158-162), the N-terminus of TnI, and the IT coiled-coil. These observed changes will be discussed in the context of the cardiac and skeletal crystal structures. This work was supported by NIH (R01 GM78359), NSF Division of Materials Research through DMR-06-54118, and the State of Florida.