Fig. 3.

NMR spectroscopy verifying that the His-tail region contributes to cadmium binding in solution. (A) NMR spectra of the ¹⁹F-labeled His-tail variant (CadR^G142F) titrated by Cd(II) at 298 K. The peaks contributed by 4-¹⁹F-Phe49 and 4-¹⁹F-Phe142 are assigned as black and red arrows, respectively. The His-tail region is represented by red dashed lines (“off” state) or arrows (“on” state). (B) NMR spectra of full ¹¹³Cd-bound CadR^WT (Left) and CadR^WT-DNA (Right) performed on a 500-MHz spectrometer at 308.5 K in phosphate buffer (relaxation delay, 10 s; number of scans, 8,000). The ¹¹³Cd-loaded protein sample is divided into 2 equal parts. One part has the added 25-bp promoter DNA (¹¹³Cd-CadR^WT-DNA), while the other does not (¹¹³Cd-CadR^WT). The chemical shift of cadmium-binding sites and the ratio of relative integrated intensity of peaks are indicated by the numbers in black.