isopropyl-thiogalactoside and thiodigalactoside

By using a lactose permease mutant containing a single Cys residue in place of Val 331 (helix X), conformational changes induced by ligand binding were studied. With right-side-out membrane vesicles containing Val 331-->Cys permease, lactose transport is inactivated by either N-ethylmaleimide (NEM) or 7-diethylamino-3-(4'-maleimidylphenyl)-4-methylcoumarin (CPM). Remarkably, beta,D-galactopyranosyl 1-thio-beta,D-galactopyranoside (TDG) enhances the rate of inactivation by CPM, a hydrophobic sulfhydryl reagent, whereas NEM inactivation is attenuated by the ligand. Val 331-->Cys permease was then purified and studied in dodecyl-beta,D-maltoside by site-directed fluorescence spectroscopy. The reactivity of Val 331-->Cys permease with 2-(4'-maleimidylanilino)-naphthalene-6-sulfonic acid (MIANS) is not changed over a low range of TDG concentrations (< 0.8 mM), but the fluorescence of the MIANS-labeled protein is quenched in a saturable manner (apparent Kd approximately equal to 0.12 mM) without a change in emission maximum. In contrast, over a higher range of TDG concentrations (1-10 mM), the reactivity of Val 331-->Cys permease with MIANS is enhanced and the emission maximum of MIANS-labeled permease is blue shifted by 3-7 nm. Furthermore, the fluorescence of MIANS-labeled Val 331 -->Cys permease is quenched by both acrylamide and iodide, but the former is considerably more effective. A low concentration of TDG (0.2 mM) does not alter quenching by either compound, whereas a higher concentration of ligand (10 mM) decreases the quenching constant for iodide by about 50% and for acrylamide by about 20%.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Amino Acid Sequence; Anilino Naphthalenesulfonates; Bacterial Proteins; Binding Sites; Coumarins; Cysteine; Electron Spin Resonance Spectroscopy; Escherichia coli; Escherichia coli Proteins; Ethylmaleimide; Isopropyl Thiogalactoside; Lactose; Ligands; Membrane Transport Modulators; Membrane Transport Proteins; Molecular Sequence Data; Monosaccharide Transport Proteins; Mutagenesis, Site-Directed; Protein Conformation; Recombinant Fusion Proteins; Spectrometry, Fluorescence; Spin Labels; Symporters; Thiogalactosides; Valine

The effects of competing alternative substrates on the rate of uptake by galactoside/proton symport were investigated. These experiments produced a decrease in apparent maximum velocity with increased alternative-substrate concentration that cannot be accounted for by a simple ordered mechanism. This, together with non-linearities in the variation of the apparent kinetic constants with alternative-substrate concentration, can be accounted for by a random mechanism for galactoside and proton binding.

Topics: Binding, Competitive; Biological Transport; Escherichia coli; Galactosides; Glycosides; Hydrolysis; Isopropyl Thiogalactoside; Kinetics; Nitrophenylgalactosides; Protons; Thiogalactosides