Fig. 2: HDX-MS analysis of CLC-ec1 reveals low pH-driven dynamics.

a CLC-ec1 structure (PDB: 1OTS) rainbow-colored according to back-exchange corrected deuteration levels at the beginning (20 s) and end (63,000 s) of the labeling experiment under different pH conditions. These views are presented without pH correction. Visually, these simple representations illustrate how lowering the pH increases deuteration, a trend opposite to the expected effect on chemical exchange, indicating that the observed changes arise from changes in protein dynamics. b pH-corrected heatmap to more sensitively visualize the change in protein dynamics occurring between pH 6.5 and 4.5, because the intrinsic deuterium-exchange rate at pH 4.5 is 100-fold slower than at pH 6.5, comparison of time points 100-fold different (e.g., 20 s at pH 6.5 vs. 2000 s at pH 4.5) reveals differences in exchange that are due to differences in protein dynamics. c Selected uptake plots of representative peptides were mapped by color onto the CLC-ec1 structure (views from the membrane plane and from the intracellular side). Standard deviations are shown for replicated time points (20 s (n = 6), 633 s (n = 3), 20,000 s, n = 3). Plots at the top represent regions undergoing little or no change, whereas those at the sides depict peptides capturing major pH-driven transitions. The uptake data were not corrected for pH-dependent differences in intrinsic exchange rates, and therefore, the curves are not directly comparable. Nevertheless, the common trend of increased dynamics (manifested as higher deuteration/faster kinetics) at lower pH illustrates the structural changes in CLC-ec1. The complete set of uptake plots is available in Supplementary Fig. 3, which also contains a set of plots where the differences in intrinsic exchange rates were compensated for by applying theoretically calculated correction factors (parts B and C). d Membrane and intracellular views of CLC-ec1 with helices labeled.