Histidine peaks change with pH in the [¹H,¹⁵N]HSQC spectrum of WT KcsA ΔC. (A) pH-dependent chemical shift changes of the C-terminal H124 peaks (labeled peaks A and B) at pH 4.5 (blue), 5.5 (orange), 6.5 (green), and 7.5 (pink). Peaks A and B are observed through the pH range measured (pH 3–8). (B) Raw intensities plotted for C-terminal H124 peaks A and B as a function of pH. (C) pH-dependent chemical shift changes of the primary H124 peaks (labeled C1 and C2) at pH 3.0 (yellow), 4.5 (blue), 5.5 (orange), 6.5 (green), and 7.5 (pink). C1 and C2 peaks are marked with an arrow for pH 3.0 and 4.5. At pH 5.5, 6.5, and 7.5 only C2 is visible. (D) Raw intensities plotted for primary H124 peaks C1 and C2 as a function of pH. (E) pH-dependent chemical shift changes of the H20 peaks (labeled peaks D–F) at pH 3.7 (red), 4.0 (blue), 5.0 (black), and 5.5 (pink) showing their pH-dependent changes in chemical shift. (F) Raw intensities plotted for H20 peaks D–F.