Probing Metal Ion Discrimination in a Protein Designed to Bind Uranyl Cation With Femtomolar Affinity.

The design of metal-binding sites in proteins that combine high affinity with high selectivity for the desired metal ion remains a challenging goal. Recently, a protein designed to display femtomolar affinity for , dubbed "Super Uranyl-binding Protein" (SUP), was described, with potential applications for removing in water. Although it discriminated most metal ions present in seawater, the protein showed a surprisingly high affinity for Cu ions. Here, we have investigated Cu binding to SUP using a combination of electron paramagnetic resonance, fluorescence and circular dichroism spectroscopies. Our results provide evidence for two Cu binding sites on SUP that are distinct from the binding site, but one of which interferes with binding. They further suggest that in solution the protein's secondary structure changes significantly in response to binding ; in contrast, the crystal structures of the apo- and holo-protein are almost superimposable. These results provide insights for further improving the selectivity of SUP for , paving the way toward protein-based biomaterials for decontamination and/or recovery of uranium.