Troponin regulatory function and dynamics revealed by H/D exchange-mass spectrometry

Abstract

Muscle contraction is tightly regulated by Ca2+ binding to the thin filament protein troponin. The mechanism of this regulation was investigated by detailed mapping of the dynamic properties of cardiac troponin using amide hydrogen exchange-mass spectrometry. Results were obtained in the presence of either saturation or non-saturation of the regulatory Ca 2+ binding site in the NH2 domain of subunit TnC. Troponin was found to be highly dynamic, with 60% of amides exchanging H for D within seconds of exposure to D2O. In contrast, portions of the TnT-TnI coiled-coil exhibited high protection from exchange, despite 6 h in D 2O. The data indicate that the most stable portion of the trimeric troponin complex is the coiled-coil. Regulatory site Ca2+ binding altered dynamic properties (i.e. H/D exchange protection) locally, near the binding site and in the TnI switch helix that attaches to the Ca 2+-saturated TnC NH2 domain. More notably, Ca2+ also altered the dynamic properties of other parts of troponin: the TnI inhibitory peptide region that binds to actin, the TnT-TnI coiled-coil, and the TnC COOH domain that contains the regulatory Ca2+ sites in many invertebrate as opposed to vertebrate troponins. Mapping of these affected regions onto the troponin highly extended structure suggests that cardiac troponin switches between alternative sets of intramolecular interactions, similar to previous intermediate resolution x-ray data of skeletal muscle troponin. © 2010 by The American Society for Biochemistry and Molecular Biology, Inc.