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 Cu²⁺ ions. Here, we have investigated Cu²⁺ binding to SUP using a combination of electron paramagnetic resonance, fluorescence and circular dichroism spectroscopies. Our results provide evidence for two Cu²⁺ 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.