PMID:10985783
| Citation |
Zhao, M, Kálai, T, Hideg, K, Altenbach, C, Hubbell, WL and Kaback, HR (2000) Binding of spin-labeled galactosides to the lactose permease of Escherichia coli. Biochemistry 39:11381-8 |
|---|---|
| Abstract |
A series of nitroxide spin-labeled alpha- or beta-galactopyranosides and a nitroxide spin-labeled beta-glucopyranoside have been synthesized and examined for binding to the lactose permease of Escherichia coli. Out of the twelve nitroxide spin-labeled galactopyranosides synthesized, 1-oxyl-2, 5, 5-trimethyl-2-[3-nitro-4-N-(hexyl-1-thio-beta-D-galactopyranosid-1 -yl )]aminophenyl pyrrolidine (NN) exhibits the highest affinity for the permease based on the following observations: (a) the analogue inhibits lactose transport with a K(I) about 7 microM; (b) NN blocks labeling of single-Cys148 permease with 2-(4'-maleimidylanilino) naphthalene-6-sulfonic acid (MIANS) with an apparent affinity of about 12 microM; (c) electron paramagnetic resonance demonstrates binding of the spin-labeled sugar by purified wild-type permease in a manner that is reversed by nonspin-labeled ligand. The equilibrium dissociation constant (K(D)) is about 23 microM and binding stoichiometry is approximately unity. In contrast, the nitroxide spin-labeled glucopyranoside does not inhibit active lactose transport or labeling of single-Cys148 permease with MIANS. It is concluded that NN binds specifically to lac permease with an affinity in the low micromolar range. Furthermore, affinity of the permease for the spin-labeled galactopyranosides is directly related to the length, hydrophobicity, and geometry of the linker between the galactoside and the nitroxide spin-label. |
| Links | |
| Keywords |
Alkylating Agents/metabolism; Anilino Naphthalenesulfonates/metabolism; Binding Sites; Biological Transport; Cysteine/metabolism; Electron Spin Resonance Spectroscopy; Escherichia coli/enzymology; Escherichia coli Proteins; Galactose/metabolism; Lactose/metabolism; Membrane Transport Proteins/chemistry; Membrane Transport Proteins/metabolism; Monosaccharide Transport Proteins; Protein Structure, Secondary; Pyrrolidines/chemistry; Pyrrolidines/metabolism; Spin Labels; Spin Trapping; Symporters; Thiogalactosides/chemical synthesis; Thiogalactosides/chemistry; Thiogalactosides/metabolism |
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