Low temperature dynamic mapping reveals unexpected order and disorder in troponin.

Troponin is a pivotal regulatory protein that binds Ca(2+) reversibly to act as the muscle contraction on-off switch. To understand troponin function, the dynamic behavior of the Ca(2+)-saturated cardiac troponin core domain was mapped in detail at 10 °C, using H/D exchange-mass spectrometry. The low temperature conditions of the present study greatly enhanced the dynamic map compared with previous work. Approximately 70% of assessable peptide bond hydrogens were protected from exchange sufficiently for dynamic measurement. This allowed the first characterization by this method of many regions of regulatory importance. Most of the TnI COOH terminus was protected from H/D exchange, implying an intrinsically folded structure. This region is critical to the troponin inhibitory function and has been implicated in thin filament activation. Other new findings include unprotected behavior, suggesting high mobility, for the residues linking the two domains of TnC, as well as for the inhibitory peptide residues preceding the TnI switch helix. These data indicate that, in solution, the regulatory subdomain of cardiac troponin is mobile relative to the remainder of troponin. Relatively dynamic properties were observed for the interacting TnI switch helix and TnC NH(2)-domain, contrasting with stable, highly protected properties for the interacting TnI helix 1 and TnC COOH-domain. Overall, exchange protection via protein folding was relatively weak or for a majority of peptide bond hydrogens. Several regions of TnT and TnI were unfolded even at low temperature, suggesting intrinsic disorder. Finally, change in temperature prominently altered local folding stability, suggesting that troponin is an unusually mobile protein under physiological conditions.