Investigating Monomeric Near-infrared Fluorescent Proteins as Platforms for New Metal Ion Sensors



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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 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 several miRFPs as platforms for single and multi-fluorescent protein metal ion sensors. Beginning with miRFP670 and miRFP709, we found that divalent metal ions like Zn2+, Co2+, Ni2+, and Cu2+ can quench from ~6%-20% (Zn2+, Co2+, Ni2+) and up to nearly 90% (Cu2+) of the fluorescence intensity of pure miRFPs and have similar impacts in live E. coli cells expressing miRFPs. The presence of a 6x-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 both in 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. In addition, the most red-shifted monomeric NIR fluorescent protein, miRFP720, was expressed, purified, and characterized. Unlike miRFP670 and miRFP709, only Cu2+ quenched ~20% fluorescence intensity of pure (His6)miRFP720, and this quenching could be avoided with removal of the 6x-histidine tag. Comparison of the proposed metal binding sites in miRFP670 and miRFP709 with the same residue positions in miRFP720 revealed a difference where His residues were present in miRFP670/709 and an aspartate residue was present in miRFP720. Therefore, we mutated His22 to Asp for (His6)miRFP709 and found that while the extent of Cu2+ quenching was somewhat reduced, the mutant was more sensitive to Zn2+, Co2+ and Ni2+. It is possible that other residues may join the metal coordination sphere, which is located in a loop region, and additional site-directed mutagenesis studies are needed to better understand transition metal binding and fluorescence quenching in the miRFPs.



Metal quenching, MiRFPs


Portions of this document appear in: Zhao, Haowen, and Melissa L. Zastrow. "Transition metals induce quenching of monomeric near-infrared fluorescent proteins." Biochemistry 61, no. 7 (2022): 494-504.