Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:2.7.1.1 (hexokinase)
 5,274 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

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. Substitution of 7-HCAA at residue 50 led to an increase in FRET efficiency in the presence of the substrate; this effect was not observed in a control protein where no change in distance between 7-HCAA and the native tryptophans occurs on substrate binding. This system was then used to directly observe differences in binding affinity of the hexokinase that occur at a number of pH values. Our approach builds on previous research in that it eliminates the need for the incorporation of multiple fNCAAs or fluorescent labels within a target protein and can be used to study substrate binding with native ligands. As such, it serves to expand the versatility of FRET-based techniques.
 ...
PMID:An intrinsic FRET sensor of protein-ligand interactions. 3242 52


<< Previous 1 2 3