Optical determination of glutamine using a genetically engineered protein

We have developed a reagentless optical biosensor for glutamine based on the Escherichia coli glutamine binding protein (GlnBP). Site-directed mutagenesis was performed to engineer single cysteine mutants which were covalently modified with environmentally sensitive sulfhydryl-reactive probes. The fluorescence emission of acrylodan and 2-(4 '-(iodoacetamido)anilino)naphthalene-6-sulfonic acid (IAANS) attached to GlnBP mutant S179C was shown to decrease 65 and 35%, respectively, upon titration with increasing amounts of glutamine (0 to 6.4 muM; K-Dapp 160 nM). No significant changes in the fluorescence intensity were observed for the structurally similar amino acids glutamate, asparagine, and arginine. Time-resolved intensity decays showed a 2.4-fold decrease in mean lifetime for GlnBP Sl79C-acrylodan upon the addition of glutamine, indicating the possibility of a lifetime-based assay. Anisotropy decay measurements for GLnBP S179C-acrylodan showed a 13-ns rotational correlation time in the ligand-free state, whereas multiple correlation times were assigned in the glutamine-bound conformation. The decrease in fluorescence intensity of S179C-acrylodan was adapted to polarization sensing of glutamine. The engineered GLnBP is a first step toward the development of a nonenzymatic biosensor capable of determining glutamine concentrations in cell cultures. a zool Academic Press.