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.
Dattelbaum
J D
JD
Department of Biochemistry and Molecular Biology, Center for Fluorescence Spectroscopy, University of Maryland, School of Medicine, 725 West Lombard Street, Baltimore, MD 21201, USA.
Lakowicz
J R
JR
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