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Optical Determination of Glutamine Using a Genetically Engineered Protein by Jonathan D. Dattelbaum; Joseph R. Lakowicz is a Biochemistry, Genetics and Molecular Biology article available to read on EtoBox.

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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 sen

Who reads Optical Determination of Glutamine Using a Genetically Engineered Protein?

Author

Jonathan D. Dattelbaum; Joseph R. Lakowicz

Publisher

Elsevier Science; Elsevier ; Elsevier Inc.; Elsevier BV (ISSN 0003-2697)

Published

2001

Language

EN

Field

Biochemistry, Genetics and Molecular Biology (Life Sciences)