An engineered azurin with a lanthanide binding site capable of copper sensing

Proteins with hetero-bimetallic metal centers can catalyze important reactions and are challenging to design. Azurin is a mononuclear copper center that has been extensively studied for electron transfer. Here we inserted the lanthanide binding tag (LBT), which binds lanthanide with sub μM affinity, into the copper binding loop of azurin, while keeping the type 1 copper center unperturbed. The resulting protein, Az-LBT, which has two metal bonding centers, shows strong luminescence upon coordination with Tb³⁺ and luminescence quenching upon Cu²⁺ binding. The in vitro luminescence quenching has high metal specificity and a limit-of-detection of 0.65 μM for Cu²⁺. With the low background from lanthanide's long luminescence lifetime, bacterial cells expressing Az-LBT in the periplasm also shows sensitivity for metal sensing.