Troponin regulatory function and dynamics revealed by H/D exchange-mass spectrometry.
Muscle contraction is tightly regulated by Ca(2+) 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 NH(2) domain of subunit TnC. Troponin was found to be highly dynamic, with 60% of amides exchanging H for D within seconds of exposure to D(2)O. In contrast, portions of the TnT-TnI coiled-coil exhibited high protection from exchange, despite 6 h in D(2)O. The data indicate that the most stable portion of the trimeric troponin complex is the coiled-coil. Regulatory site Ca(2+) 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 NH(2) domain. More notably, Ca(2+) 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 Ca(2+) 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.
Kowlessur
Devanand
D
Department of Medicine, University of Illinois at Chicago, Chicago, Illinois 60612, USA.
Tobacman
Larry S
LS
D008841:Actin Cytoskeleton / Q000737:chemistry / Q000378:metabolism; D000595:Amino Acid Sequence; D001665:Binding Sites; D002118:Calcium / Q000378:metabolism; D041961:Deuterium Exchange Measurement; D006801:Humans; D006859:Hydrogen / Q000378:metabolism; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D054792:Muscle, Striated / Q000737:chemistry / Q000378:metabolism; D009206:Myocardium / Q000737:chemistry* / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D014336:Troponin / Q000737:chemistry* / Q000378:metabolism*; D019209:Troponin C / Q000737:chemistry / Q000378:metabolism; D019210:Troponin I / Q000737:chemistry / Q000378:metabolism; D020107:Troponin T / Q000737:chemistry / Q000378:metabolism
D014336:Troponin; D019209:Troponin C; D019210:Troponin I; D020107:Troponin T; D006859:Hydrogen; D002118:Calcium