Fig. 4. Mg^IIGTP-CobW binds Zn^II with sub-picomolar affinity.

a Representation of the equilibrium for exchange of Co^II and Zn^II between ligand (L = NTA) and protein (P = Mg^IIGTP-CobW). b–e Absorbance (relative to metal-free solution) of solutions of CobW (17.9–20.4 µM), Mg^II (2.7 mM), GTP (200 µM) and NTA (0.4–4.0 mM) upon (b–d) first addition of Co^II (black trace) then Zn^II (blue trace) or (e) the reverse, at equilibrium (n = 1 for each panel). The absorbance peak at 339 nm corresponds to Co^II-bound protein. An excess of ligand NTA was used to buffer both metals in each experiment: varying the ratios of ligand-bound metal ions ([Co^IINTA]/[Zn^IINTA] = 28–167) shifted the ratios of Co^II- and Zn^II-bound protein as predicted by the equilibrium exchange constant in a. Consistent K_Zn(II) values for Mg^IIGTP-CobW were generated at all tested conditions (Supplementary Table 4). Dashed red lines show expected A_{339 nm} peak intensities for K_Zn(II) of Mg^IIGTP-CobW tenfold tighter or weaker than calculated values.