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 microM; 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 S179C-acrylodan upon the addition of glutamine, indicating the possibility of a lifetime-based assay. Anisotropy decay measurements for GlnBPS179C-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.
Department of Biochemistry and Molecular Biology, Center for Fluorescence Spectroscopy, University of Maryland, School of Medicine, 725 West Lombard Street, Baltimore, MD 21201, USA.
J D
JD
Dattelbaum
J R
JR
Lakowicz
D015374:Biosensing Techniques; D002352:Carrier Proteins / Q000032:analysis* / Q000737:chemistry*; D004926:Escherichia coli; D005456:Fluorescent Dyes / Q000032:analysis; D005973:Glutamine / Q000032:analysis* / Q000737:chemistry; D016297:Mutagenesis, Site-Directed; D015202:Protein Engineering
D002352:Carrier Proteins; D005456:Fluorescent Dyes; D005973:Glutamine