Engineering an FMN-based iLOV protein for the detection of arsenic ions

Over the past few decades, genetically encoded fluorescent proteins have been widely used as efficient probes to explore and investigate the roles of metal ions in biological processes. The discovery of small FMN-based fluorescent proteins, such as iLOV and FbFP, has enabled researchers to exploit these fluorescent reporter proteins for metal-sensing applications. In this study, we report the inherent binding properties of iLOV towards arsenic ions. The fluorescence quenching of iLOV was linearly related to the concentration of arsenic ions, and engineered proteins showed better sensitivity than the wild-type protein. Engineering key residues around the chromophore converted the iLOV protein into a highly sensitive sensor for As³⁺ ions. iLOV_N468S exhibited an improved binding affinity with a dissociation constant of 1.5 μM. Furthermore, the circular dichroism spectra indicated that the fluorescence quenching mechanism might be related to arsenic-protein complex formation. Thus, the reagentless sensing of arsenic can potentially be exploited to determine intracellular or environmental arsenic using a genetically encoded biosensing approach.