FMN-Based Fluorescent Proteins as Heavy Metal Sensors Against Mercury Ions

Abstract

Bacterial light-oxygen-voltage-sensing photoreceptor-derived flavin mononucleotide (FMN)- based fluorescent proteins act as a promising distinct class of fluorescent proteins utilized for various biomedical and biotechnological applications. The key property of its independency towards oxygen for its chromophore maturation has greatly helped this protein to outperform the other fluorescent proteins such as GFP and DsRed for anaerobic applications. Here, we describe the feasibility of FMN-containing fluorescent protein FbFP as a metal-sensing probe by measuring the fluorescence emission changes of a protein with respect to the concentration of metal ions. In the present study, we demonstrated the mercury-sensing ability of FbFP protein and the possible amino acids responsible for metal binding. A ratiometric approach was employed here in order to exploit the fluorescence changes observed at two different emission maxima with respect to Hg(2+) at micromolar concentration. The engineered variant FbFPC56I showed high sensitivity towards Hg(2+) and followed a good linear relationship from 0.1 to 3 µM of Hg(2+). Thus, further engineering with a rational approach would enable the FbFP to be developed as a novel and highly selective and sensitive biosensor for other toxic heavy metal ions as well.

MerR‐fluorescent protein chimera biosensor for fast and sensitive detection of Hg2+ in drinking water

“…As summarized in Table , MerR:EYFP chimera biosensor showed higher sensitivity when compared with four of the previously reported single fluorescent protein biosensors . Although the LOD values of GFP mutant biosensor and MerR:EYFP chimera biosensor were same, the highest detectable Hg 2+ concentration was more than 10‐fold higher for MerR:EYFP.…”

Flavin Mononucleotide-Binding Fluorescent Proteins

“…2). Another fluorescence-based FbFP application is the in-cell sensing of heavy metals ions, such as mercury (Ravikumar et al 2016) and arsenic (Ravikumar et al 2017).…”

Characterization of flavin binding in oxygen‐independent fluorescent reporters

“…19 We have also discovered brighter LOV reporters from algae by implementing a genome mining-based approach, 20 which was recently extended to identify a new and highly thermostable LOV reporter from thermophilic bacteria. 21 Taken together with additional benefits such as small size (~12 kDa), acid tolerance, 22 and metal-responsive fluorescence, [23][24][25] fluorescence imaging to several anaerobes, including gut bacteria, oral flora, parasitic protists, and pathogenic fungi. [26][27][28][29][30][31][32] As the fluorescence properties of LOV reporters are determined by protein-bound FMN, it would be useful to develop a method for separating FMN from the protein and reconstituting the apo protein with FMN (or other flavins) in order to characterize the equilibrium dissociation constant (that is, K d ), specificity, reaction kinetics, binding energetics, and related thermodynamic properties.…”