An intrinsic FRET sensor of protein-ligand interactions.

We describe an approach for the development of fluorescent sensors of metabolite binding in which a genetically encoded fluorescent non-canonical amino acid (fNCAA) containing a 7-hydroxycoumarin moiety (7-HCAA) forms a FRET pair with native tryptophan residues. Although previous studies demonstrated the potential for using 7-HCAA as an acceptor for tryptophan, this approach has not yet been explored within a single protein containing multiple tryptophan residues. A structure-based analysis of a hexokinase enzyme with multiple native tryptophan residues identified glutamate 50 as a potential site of 7-HCAA incorporation; Glu50 moves closer to the native tryptophans upon substrate binding.