ObjectivesTo determine the binding sites for l-phenylalanine in TyrR protein via a rational mutation analysis combining biosensors and computer-aided simulation.ResultsTyrR protein of Escherichia coli is the chief transcriptional regulator of several genes essential for the biosynthesis and transport of aromatic amino acids. The identification of ligand-binding sites is often the starting point for protein function annotation and structure-based protein design. Here we combined computer-aided prediction methods and biosensors to identify the ligand-binding sites for l-Phe in TyrR protein.ConclusionsResidues at positions 160, 173 and 184 of TyrR protein are important for transcriptional activation of target genes tyrP induced by l-Phe, which indicates that they are the bona fide l-Phe binding sites of TyrR protein.