Transition Metals Induce Quenching of Monomeric Near-Infrared Fluorescent Proteins

Transition metals such as zinc and copper are essential in numerous life processes, and both deficiency and toxic overload of these metals are associated with various diseases. Fluorescent metal sensors are powerful tools for studying the roles of metal ions in the physiology and pathology of biological systems. Green fluorescent protein (GFP) and its derivatives are highly utilized for protein-based sensor design, but application to anaerobic systems is limited because these proteins require oxygen to become fluorescent. Bacteriophytochrome-based monomeric near-infrared fluorescent proteins (miRFPs) covalently bind a bilin cofactor, which can be added exogenously for anaerobic cells. miRFPs can also have emission wavelengths extending to >700 nm, which is valuable for imaging applications. Here, we evaluated the suitability of miRFP670 and miRFP709 as platforms for single fluorescent protein metal ion sensors. We found that divalent metal ions like Zn²⁺, Co²⁺, Ni²⁺, and Cu²⁺ can quench from ∼6-20% (Zn²⁺, Co²⁺, and Ni²⁺) and up to nearly 90% (Cu²⁺) of the fluorescence intensity of pure miRFPs and have similar impacts in live Escherichia coli cells expressing miRFPs. The presence of a 6× histidine tag for purification influences metal quenching, but significant Cu²⁺-induced quenching and a picomolar binding affinity are retained in the absence of the His₆ tag in both cuvettes and live bacterial cells. By comparing the Cu²⁺ and Cu⁺-induced quenching results for miRFP670 and miRFP709 and through examining absorption spectra and previously reported crystal structures, we propose a surface metal binding site near the biliverdin IXα chromophore.