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. 2016 Jan 22;5:e11189. doi: 10.7554/eLife.11189

Figure 3. 19F NMR detects H+-dependent solvent accessibility at Y419.

(A) Y419only ClC-ec1. In this variant, all native Tyr residues except for Y419 have been mutated to Phe, so that only Y419 will carry a 19F label. Y419 is highlighted in the ClC-ec1 structure shown from the point of view of the membrane (left) and from the extracellular side (right). The lower panels illustrate that Y419 lies in a buried position (left: thin slice through the protein at Y419; right, surface representation viewed from the extracellular side. (B) 19F NMR spectra of Y419only. The prominent peak centered at -60 ppm shifts upfield (-61 and -63 ppm) when the pH is shifted from 7.5 to 4.5 to enrich the OF state. (C) Y419 becomes substantially more exposed to solvent at increased [H+], as indicated by susceptibility to line-broadening by the water-soluble TEMPOL at pH 4.5 (bottom spectra, green vs black trace) compared to pH 7.5 (top spectra, orange vs cyan trace). (D) The change in the Y419 exposure to solvent is reversible, as revealed by return of the signal (to the expected chemical shift) when the pH is raised to 7.5 (bottom trace, orange). (E) Y419 in the channel-like ClC-ec1 background is accessible to TEMPOL at both pH 7.5 and 4.5.

DOI: http://dx.doi.org/10.7554/eLife.11189.006

Figure 3.

Figure 3—figure supplement 1. Functional characterization of ClC-ec1 variants.

Figure 3—figure supplement 1.

Top: Cl- turnover rates for ClC-ec1 variants examined in this study, with the exception of D417C mutants which are summarized in Table 1. Bottom: Stoichiometry of transport for WT ClC-ec1, Y419only (used in NMR studies), D417C (used in cross-linking studies), and Helix-N mutants F357A and L361A (hypothesized to transmit conformational change from D417C to the Cl--transport pathway). Data represent average ± SEM (n=3–7).
DOI: http://dx.doi.org/10.7554/eLife.11189.007
Figure 3—figure supplement 2. Reproducibility of TEMPOL-NMR experiments.

Figure 3—figure supplement 2.

(A) Repeat of the experiment demonstrating (1) TEMPOL causes line-broadening at pH 4.5 and (2) that this line-broadening is reversible by a change to pH 7.5 (cf Figure 3D). The signal is enhanced and returns to the expected chemical shift when the pH is raised to 7.5 (bottom trace, orange). (B) Repeat of the experiment demonstrating that Y419 in the channel-like ClC-ec1 background is accessible to TEMPOL at both pH 7.5 and 4.5 (cf Figure 3E).
DOI: http://dx.doi.org/10.7554/eLife.11189.008
Figure 3—figure supplement 3. Overlay of WT and channel-like ClC-ec1 structures.

Figure 3—figure supplement 3.

Overlay of WT ClC-ec1 (grey, 1OTS) and channel-like variant E148A/Y445A (purple, 3DET), RMSD 0.52 Å. The left panel shows a view from within the membrane; the middle and right panels shows views from the extracellular side, with the Y419 side chain depicted in pink (channel-like) and grey (WT). The side chain is in an identical (overlapping) position in the two structures.
DOI: http://dx.doi.org/10.7554/eLife.11189.009