Figure 8. Summary of results from hydrogen-deuterium exchange mass spectrometry (HDX-MS) experiments.

Average peptide ¹H/²H exchange levels at the 10 second time point in TKM buffer (pH 8.0) are plotted onto ribbon diagrams of the corresponding WT enzymes (PDB id 1M74 for BsSecA and PDB id 2FSG for EcSecA). The catalytic glutamates are shown in green sphere representation. The panels on the left (A,C,E,G) show the absolute percent exchange of observable protons in peptides in the WT enzyme, while the panels on the right (B,D,F,H) show the difference in this value in the E-to-Q mutant minus that in the corresponding WT enzyme at the same time point; these latter images highlight the regions in which backbone dynamics are altered by the mutation. The exchange levels are color-coded as indicated in the bar at the bottom of each column (10–20% exchange in blue, 21–40% in cyan, 41–60% in magenta, 61–80% in yellow, and 81–100% in red for the absolute exchange levels shown in the left column, and ≥ 21% reduction in exchange in blue, 20–11% reduction in cyan, 10% reduction to 10% increase in dark gray, 11–20% increase in yellow, and ≥ 21% increase in red for the differences in exchange levels shown in the right column). Experiments were conducted on BsSecA at 25 °C (panels A and B) where the DSC data (Fig. 7A,C) indicate that the WT enzyme is in the conformational ground state but the E208Q mutant is undergoing the ECT. Experiments were conducted on EcSecA at 25 °C (panels C and D), 31 °C (panels E and F), and 37 °C (panels G and H). The DSC data on WT-EcSecA (Fig. 7E) indicate that this enzyme is in the conformational ground-state at all three temperatures, while the fine-structure in the broad DSC peak for the E210Q mutant (Fig. 7G) suggests that it sequentially occupies different conformational sub-states as it undergoes the ECT from 30–40 °C.