Addition of Mercury Causes Quenching of NIR Fluorescence Emission Spectra of a Photoactivatable PAiRFP1 Protein

Background: Biliverdin (BV) containing far-red light photoactivatable near-infrared fluorescent protein (NIR-FP) named PAiRFP1 has been developed by directed molecular evolution from one bathy bacteriophytochrome of Agrobacterium tumefaciens C58 called Agp2 or AtBphP2. Usually, the fluorescence intensity of the NIR emission spectra of PAiRFP1 tends to increase upon repeated excitation by far-red light. Objective: This study aimed at exploring the role of PAiRFP1 and its mutants, such as V386A, V480A, and Y498H, as NIR biosensors for the detection of Hg²⁺ ions in the buffer solutions. Methods: In this study, we used PCR-based site-directed reverse mutagenesis, fluorescence spectroscopy, and molecular modeling approaches on PAiRFP1 and its mutants. Results: It was found that PAiRFP1 and its mutants experienced strong quenching of NIR fluorescence emission spectra upon the addition of different concentrations (0-3μM) of mercuric chloride (HgCl₂). Conclusion: We hypothesized that PAiRFP1 and its variants have some potential to be used as NIR biosensors for the in vitro detection of Hg²⁺ ions in biological media. Moreover, we also hypothesized that PAiRFP1 would be the best tool to use as a NIR biosensor to detect Hg²⁺ ions in living organisms because of its higher signal-to-noise (SNR) ratio than other infra-red fluorescent proteins.