Fig. 4. ¹³C-Lys ClC-ec1.

a¹H-¹³C HSQC spectra of methylated ClC-ec1 at pH 7.5 (left), pH 4.5 (middle) and reversed to pH 7.5 (right). The spectral window is zoomed in on the ¹³C methyl signals arising from the Lys side chains (with the ¹³C signal from the methylated N-terminus not shown). Changes in [H⁺] induce spectral shifts, the most notable being the peak splitting and shift of the peak at δ_H = 2.7 ppm (highlighted by the red boxes) at low pH.

b pH titration of methylated ClC-ec1. Left: Overlaid ¹H-¹³C HSQC spectra at pH 7.5, 6.5, 6.0, 5.5, 5.0, 4.5, and 4.0, as indicated in the figure. Right: ¹H chemical shifts for the peak of interest as a function of pH. The solid curve is a fit to a single-site H⁺-binding isotherm, with an apparent pKa of 5.6.

c¹H-¹³C HSQC spectra of methylated channel-like mutant at pH 7.5 (left), pH 4.5 (middle) and reversed to pH 7.5 (right). The spectrum of the channel-like mutant exhibits little [H⁺] sensitivity, and the peak at δ_H = 2.70 ppm and δ_C = 44.9 ppm is notably missing at both high and low pH.