Fluorescent Triazolyl unnatural amino acids in protein engineering: Encoding super folder green fluorescent protein and spectroscopic study.

CuAAC-derived fluorescent unnatural amino acids (FTUAAs) have been highly advantageous owing to their enhanced stability, increased resistance to proteolysis, better biocompatibility, and modulated photophysics. However, such a unique class of amino acids has never been previously incorporated into desired protein sequences using the genetic code expansion (GCE) approach. Herein, we have reported our pioneering efforts towards the site-specific incorporation of several novel FTUAAs into the 150th codon position of Super Folder Green Fluorescent Protein (sfGFP), followed by their expression in E. coli cells and the study of their photophysics. A Methanosarcina mazei-derived pyrrolysyl-tRNA synthetase, namely Tet 3.0 aaRS, and its corresponding tRNA pair have been utilised in these studies. The results have indicated the efficient incorporation of all the unnatural amino acids in different experimental settings. Studies of photophysical properties, MALDI TOF mass analysis, fluorescence microscopy, and molecular docking have validated our claims. In fact, the incorporation of FTUAAs into reporter sfGFP using the GCE technique has potentially opened up a gateway for incorporating amino acids containing an integral triazole moiety into any protein sequence to expand their functionalities. Incorporating FTUAAs can also help intrinsically label proteins to understand their roles in various molecular pathways, among other applications.