1. Fig. 3 Differential binding of Cu1+ and Cu2+to PcuC studied by NMR.
Differential binding of Cu1+ and Cu2+to PcuC studied by NMR. (A to F) Overlays of 2D-[13C,1H]-HMQC NMR spectra of Met-(methyl-13C)–labeled apo-PcuC and PcuC loaded with Cu1+ and/or Cu2+. Cartoons of PcuC are shown at the bottom of each panel, depicting the core domain (filled oval) and the flexible C-terminal extension (curved line). Cu1+ and Cu2+ are represented by yellow and orange balls, respectively. The colored circles around the cartoons indicate the color of the spectrum of each species. (A) C-terminally truncated PcuC (apo-PcuC ΔC) was used for identifying methionine signals from the flexibly disordered C-terminal segment (“C-term”) and the structured core domain. (B) Upon addition of 1 meq of Cu1+ to PcuC, only signals from the core domain shift, indicating preferential Cu1+ binding to the core domain. (C) Cu2+ binds to the C-terminal extension of PcuC, evidenced by the quenching of the signals from this region due to the paramagnetic properties of Cu2+. (D) In the presence of excess Cu1+, shifts of the methionine peaks in both the core and C-terminal extension indicate binding of Cu1+ ions to both sites. The sample contained 50 mM sodium dithionite to suppress Cu1+ oxidation. (E) In presence of 1.5 meq of both Cu1+ and Cu2+, Cu1+ preferentially binds to the conserved, primary Cu1+-binding site in the PcuC core. (F) Same sample of PcuC·Cu1+·Cu1+ as in (D) but in the absence of sodium dithionite. The Cu1+ bound at the C terminus became oxidized to Cu2+, leading to quenching of the C-terminal methionine peaks. In contrast, the Cu1+ bound to the PcuC core stayed resistant against oxidation.
Fabia Canonica et al. Sci Adv 2019;5:eaaw8478
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