Binding of spin-labeled galactosides to the lactose permease of Escherichia coli

M. Zhao, T. Kalai, K. Hideg, C. Altenbach, W. L. Hubbell, H. R. Kaback

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Abstract

A series of nitroxide spin-labeled α- or β-galactopyranosides and a nitroxide spin-labeled β-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-β-D-galactopyranosid-1-yl)]aminophe nyl pyrrolidine (NNG(6)/(β)) exhibits the highest affinity for the permease based on the following observations: (a) the analogue inhibits lactose transport with a K(I) about 7 μM; (b) NNG(6)/(β) blocks labeling of single-Cys 148 permease with 2-(4'-maleimidylanilino) naphthalene-6-sulfonic acid (MIANS) with an apparent affinity of about 12 μM; (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 μM and binding stoichiometry is approximately unity. In contrast, the nitroxide spin-labeled glucopyranoside does not inhibit active lactose transport or labeling of single-Cys 148 permease with MIANS. It is concluded that NNG(6)/(β) 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.

Original languageEnglish
Pages (from-to)11381-11388
Number of pages8
JournalBiochemistry
Volume39
Issue number37
DOIs
Publication statusPublished - Sep 19 2000

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ASJC Scopus subject areas

  • Biochemistry

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