Development of a fluorescent nanosensor for ribose.
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.
Lager
Ida
I
ZMBP Tübingen, Plant Physiology, Tübingen, Germany.
Fehr
Marcus
M
Frommer
Wolf B
WB
Lalonde
Sylvie
S
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
D029968:Escherichia coli Proteins; D033902:Periplasmic Binding Proteins; C028003:RbsB protein, E coli; D012266:Ribose