Sugar transport by the bacterial phosphotransferase system. Preparation of a fluorescein derivative of the glucose-specific phosphocarrier protein IIIGlc and its binding to the phosphocarrier protein HPr.
In diauxic growth, the bacterial phosphoenolpyruvate: glycose phosphotransferase system (PTS) regulates the utilization of certain compounds which are not PTS substrates. It has recently been shown that this PTS regulation is mediated via one of the PTS phosphocarrier proteins, IIIGlc. In the present studies, IIIGlc was derivatized with the fluorescent reagent fluorescein-5-isothiocyanate. One mol of label was incorporated per mol of protein and the label was located at the NH2-terminal amine, as shown by tryptic peptide mapping and one-step Edman-type degradation. The fluorescent moiety was found to be stable and resistant to photodecomposition. The fluorescent IIIGlc was purified and shown to be fully active in its ability to accept phosphate from phospho-HPr (the histidine-containing phosphocarrier protein of the phosphotransferase system), but only 20% active in catalyzing the transfer of the phosphate to methyl alpha-glucoside via the membrane-bound II-BGlc protein. The decay of the fluorescence intensity was dominated by a single component (90%) with a lifetime of 4 ns. The decay of the fluorescence emission anisotropy was determined for excitation in both a negative and positive transition of fluorescein and was best described in terms of a biexponential function, indicating internal motion of the fluorophore and possible anisotropic rotation of the protein as a whole. The formation of a complex between IIIGlc and HPr was demonstrated by using the techniques of time-resolved and steady state fluorescence emission measurements, resonance energy transfer, and equilibrium gel filtration.
D000595:Amino Acid Sequence; D000818:Animals; D001419:Bacteria / Q000201:enzymology*; D001426:Bacterial Proteins; D001693:Biological Transport, Active; D050260:Carbohydrate Metabolism; D002850:Chromatography, Gel; D004735:Energy Transfer; D029968:Escherichia coli Proteins; D005260:Female; D016650:Fluorescein-5-isothiocyanate; D005452:Fluoresceins / Q000378:metabolism; D005453:Fluorescence; D010731:Phosphoenolpyruvate Sugar Phosphotransferase System / Q000378:metabolism*; D011817:Rabbits; D013861:Thiocyanates / Q000378:metabolism; D013997:Time Factors
D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; D005452:Fluoresceins; D013861:Thiocyanates; C032242:crr protein, E coli; D010731:Phosphoenolpyruvate Sugar Phosphotransferase System; C030182:phosphocarrier protein HPr; D016650:Fluorescein-5-isothiocyanate