The iLOV protein belongs to a family of blue-light photoreceptor proteins containing a lightoxygen- voltage sensing domain with a noncovalently bound flavin mononucleotide (FMN) as its chromophore. Owing to advantages such as its small size, oxygen-independent nature, and pH stability, iLOV is an ideal candidate over other reporter fluorescent proteins such as GFP and DsRed. Here, for the first time, we describe the feasibility of applying LOV domain-based fluorescent iLOV as a metal sensor by measuring the fluorescence quenching of a protein with respect to the concentration of metal ions. In the present study, we demonstrated the inherent copper sensing property of the iLOV protein and identified the possible amino acids responsible for metal binding. The fluorescence quenching upon exposure to Cu(2+) was highly sensitive and exhibited reversibility upon the addition of the metal chelator EDTA. The copper binding constant was found to be 4.72 ± 0.84 micrometer. In addition, Cu(2+)-bound iLOV showed high fluorescence quenching at near physiological pH. Further computational analysis yielded a better insight into understanding the possible amino acids responsible for Cu(2+) binding with the iLOV protein.

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.