When bacteria grow on glucose, the rate of Glc transport is stringently regulated and is a major determinant of the rate of cell division. The E. coli Glc (or Me alpha-glucoside (MeGlc)) permease is part of the phosphoenolpyruvate:glycose phosphotransferase system (PTS), and catalyzes simultaneous Glc uptake and phosphorylation. The P-transfer sequence is via 4 proteins: PEP <-> Enzyme I (EI) <-> HPr <-> IIAGlc (or IIIGlc) <-> IICBGlc -> Glc. The permease mechanism is not understood or how it is regulated. Regulation is manifested in the uptake of MeGlc by whole cells, where the rate declines virtually immediately until a steady state is attained, unlike in vitro phosphorylation, where the rate remains constant. A mathematical model has now been constructed that explains the initial rate of uptake of MeGlc by whole cells from in vitro data, but does not explain the decline in rate and steady state. One difference between in vivo and in vitro conditions is the protein concentrations. The model predicts that at high in vivo concentrations, most of the proteins exist as complexes with other PTS proteins or boundary metabolites. Since in vivo regulation may be implemented via one or more of these complexes, the principal investigator will study the following interactions: (a) Binding of the membrane Glc receptor, IICBGlc, to Glc (or MeGlc), primarily by flow dialysis and rapid quench kinetics. (b) Binding of IICBGlc to IIAGlc and P-IIAGlc, which may be mediated by the 18 amino acid N-terminal unstructured domain of IIAGlc. Fluorescence anisotropy will be used to determine whether the terminal polypeptide binds to IICBGlc, the bilayer or both. (c) Interaction of El monomer (M) with itself to form dimer (D). Only D is autophosphorylated by PEP, and dimerization of (M) is far slower than catalysis. Because of this slow process, sugar transport would virtually stop if all D were converted to M. The mechanism for the slow dimerization is unknown, and will be studied with the C-terminal domain of El by analytical sedimentation, CD spectroscopy, and fluorescence anisotropy. If successful, these experiments should lead toward a molecular explanation for sugar uptake and its regulation by many pathogens.
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