Molecular engineering of UnaG: Insights into fluorescence dynamics and biosensor potential.

UnaG represents the first ligand-inducible (unconjugated bilirubin) fluorescent protein to be discovered in vertebrates. It was isolated from the muscles of the Japanese eel (Anguilla japonica) and has been shown to emit green fluorescence exclusively when bound to unconjugated bilirubin (UC-BR). This distinctive property of UnaG renders it a promising biosensor candidate for the detection of UC-BR. The sensitive and specific detection of UC-BR is of great importance for clinical diagnosis and treatment. It is imperative to enhance the binding affinity and thermal stability of wild type UnaG for successful clinical applications These proteins may serve as promising candidates for future diagnostic applications, pending validation in biological samples. In this study, Arginine at position R112 and R132 in the UnaG-UC-BR binding site was replaced with Methionine. This was done to regulate the binding affinity of wild-type UnaG protein to UC-BR to a level that could be measured in a physiological context, and to improve its thermal stability. In this study, the positive charge in the relevant binding sites was eliminated and the biophysical changes of these mutations on the UnaG protein were investigated. The secondary structures and thermal melting temperatures of the proteins were analysed by circular dichroism spectroscopy. Excitation and emission wavelengths were determined by fluorescence spectroscopy, while dissociation constants (Kd) were determined by titration studies with UC-BR. The findings indicate that R112M, R132M and R112&132 M mutants exhibited high sensitivity, reaching fluorescence saturation at UC-BR concentrations of ∼10 nM, ∼12 nM and ∼10 nM, respectively. Thermal stability analyses revealed that the R112M&R132M double mutant was the most stable variant, with a melting temperature of 72°C. The results of this study indicate that engineered UnaG variants have the potential to function as high-affinity and stable biosensors for the detection of UC-BR.