Localization of the galactoside binding site in the lactose carrier of Escherichia coli.
The location of flurophores specifically bound to the lactose/H+ carrier of Escherichia coli was ascertained by the use of various collisional quenchers. The reporter groups were (1) the pyrenyl residue of N-(1-pyrenyl)maleimide attached to the essential cysteine residue 148, which is presumably at or near the galactoside binding site, and (2) the dansyl moieties of a series of fluorescent substrate molecules. The accessibility of these fluorophores from the lipid phase was assessed by nitroxyl-labelled fatty acids and phospholipids. By using a series of nitroxyl-labelled fatty acids carrying the quencher at different positions in the acyl chain, the position of a quenchable fluorophore with respect to the membrane normal can be determined. The accessibility of fluophores from the aqueous phase was assessed by using a water-soluble quencher, the N-methylpicolinium ion. The results of quenching studies suggest that the galactoside binding site is located within the carrier and that this binding site communicates with the aqueous phase through a pore.
D000345:Affinity Labels; D001665:Binding Sites; D004926:Escherichia coli / Q000378:metabolism*; D029968:Escherichia coli Proteins; D005456:Fluorescent Dyes; D005697:Galactosides / Q000378:metabolism*; D006027:Glycosides / Q000378:metabolism*; D007700:Kinetics; D008563:Membrane Lipids; D026901:Membrane Transport Proteins / Q000378:metabolism; D009004:Monosaccharide Transport Proteins; D013050:Spectrometry, Fluorescence; D013329:Structure-Activity Relationship; D027981:Symporters; D014867:Water
D000345:Affinity Labels; D029968:Escherichia coli Proteins; D005456:Fluorescent Dyes; D005697:Galactosides; D006027:Glycosides; C050221:LacY protein, E coli; D008563:Membrane Lipids; D026901:Membrane Transport Proteins; D009004:Monosaccharide Transport Proteins; D027981:Symporters; D014867:Water; C021499:lactose permease