title: Development of a fluorescent nanosensor for ribose.
abstract: To analyze ribose uptake and metabolism in living cells, nanosensors were engineered by flanking the Escherichia coli periplasmic ribose binding protein with two green fluorescent protein variants. Following binding of ribose, fluorescence resonance energy transfer decreased with increasing ribose concentration. Five affinity mutants were generated covering binding constants between 400 nM and 11.7 mM. Analysis of nanosensor response in COS-7 cells showed that free ribose accumulates in the cell and is slowly metabolized. Inhibitor studies suggest that uptake is mediated by a monosaccharide transporter of the GLUT family, however, ribose taken up into the cell was not or only slowly released, indicating irreversibility of uptake.
authors.affiliation: ZMBP Tübingen, Plant Physiology, Tübingen, Germany.
authors.forename: Ida
authors.initials: I
authors.lastname: Lager
authors.forename: Marcus
authors.initials: M
authors.lastname: Fehr
authors.forename: Wolf B
authors.initials: WB
authors.lastname: Frommer
authors.forename: Sylvie
authors.initials: S
authors.lastname: Lalonde
mesh_terms: D000818:Animals; D001692:Biological Transport; D015374:Biosensing Techniques / Q000379:methods*; D019556:COS Cells; D003600:Cytosol / Q000378:metabolism; D004926:Escherichia coli; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000378:metabolism; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D009154:Mutation; D036103:Nanotechnology / Q000379:methods*; D033902:Periplasmic Binding Proteins / Q000737:chemistry / Q000378:metabolism; D012266:Ribose / Q000032:analysis* / Q000737:chemistry / Q000378:metabolism; D013379:Substrate Specificity; D013816:Thermodynamics
chemical_list: D029968:Escherichia coli Proteins; D033902:Periplasmic Binding Proteins; C028003:RbsB protein, E coli; D012266:Ribose