Low temperature dynamic mapping reveals unexpected order and disorder in troponin

Abstract

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.

FIGURE 1.

Kinetics of H/D exchange in troponin. TnC peptides are shown. TnC peptide mass increases were determined after exposure of troponin to D₂O at 10 °C for times between 5 s and 48 h. Data points are shown, as well as best fit kinetic curves. Dashed lines indicate 100% exchange. Thin lines show representative, previously published patterns for several peptides at 25 °C. In the thumbnail images of troponin, the locations of graphed peptides are indicated as black space-filling sections.

FIGURE 2.

Patterns of H/D exchange completion after long time intervals. Maximum H/D exchange values are shown for those TnC, TnI, or TnT peptides that reached a mass increase plateau by the time of the last measured time point. Open squares, 25 °C data. Filled circles, 10 °C data. The lower temperature data tended to fall short of 100% exchange. Most of the 25 °C results reached 100% exchange. Deviations of the open squares from the line of identity indicate the effect of the imprecision in back exchange corrections: ± 0.95 Da. See text.

FIGURE 3.

Kinetics of H/D exchange in troponin. TnI peptides are shown. TnI peptide mass increases were determined after exposure of troponin to D₂O at 10 °C for times between 5 s and 48 h. In the thumbnail images, the locations of graphed peptides are indicated as black space-filling sections, and/or by gray circles for regions not identified by x-ray crystallography.

FIGURE 4.

Kinetics of H/D exchange in troponin. TnT peptides are shown. TnT peptide mass increases were determined after exposure of troponin to D₂O at 10 °C for times between 5 s and 48 h. Note that the rightmost panel shows fractional exchange, rather than mass increase. In the thumbnail images, the locations of graphed peptides are indicated as black space-filling sections, and/or by gray circles for regions not identified by x-ray crystallography.

FIGURE 5.

Primary sequence map of troponin dynamics. Color-coded summary of all of the results in the present study, using a logarithmic scale to convey the wide range of dynamic behavior is shown. Unprotected regions are shown in red. Regions that took longer than 2 days to undergo H/D exchange are shown in violet. Green line segments indicate the assignable regions for the various transitions. See text.

FIGURE 6.

Troponin dynamic behavior mapped onto the troponin structure. The linear map from Fig. 5 was applied to the high resolution structure of troponin (23), using the molecular graphic program Pymol. Panels A and B show either cartoon-ribbon or dots representations of troponin, respectively. Their orientation is the same as shown in Fig. 1. Panel C shows a different orientation, and is zoomed-in relative to the other panels. TnC is shown in dots format, and the other subunits are shown in cartoon-ribbon format.