Ligand-induced Conformational Changes in the Lactose Permease of Escherichia Coli: Evidence for Two Binding Sites

Overview

Journal Protein Sci

Specialty Biochemistry

Date 1994 Dec 1

PMID 7756985

Citations 13

Authors

J Wu

S Frillingos

J Voss

H R Kaback

Affiliations

Soon will be listed here.

Abstract

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)

Citing Articles

Sugar recognition by CscB and LacY.

Sugihara J, Smirnova I, Kasho V, Kaback H Biochemistry. 2011; 50(51):11009-14.

PMID: 22106930 PMC: 3249425. DOI: 10.1021/bi201592y.

Protonation and sugar binding to LacY.

Smirnova I, Kasho V, Kaback H Proc Natl Acad Sci U S A. 2008; 105(26):8896-901.

PMID: 18567672 PMC: 3021437. DOI: 10.1073/pnas.0803577105.

Sugar binding induces an outward facing conformation of LacY.

Smirnova I, Kasho V, Choe J, Altenbach C, Hubbell W, Kaback H Proc Natl Acad Sci U S A. 2007; 104(42):16504-9.

PMID: 17925435 PMC: 2034228. DOI: 10.1073/pnas.0708258104.

Site-directed alkylation of LacY: effect of the proton electrochemical gradient.

Nie Y, Ermolova N, Kaback H J Mol Biol. 2007; 374(2):356-64.

PMID: 17920075 PMC: 2224895. DOI: 10.1016/j.jmb.2007.09.006.

Monitoring the function of membrane transport proteins in detergent-solubilized form.

Quick M, Javitch J Proc Natl Acad Sci U S A. 2007; 104(9):3603-8.

PMID: 17360689 PMC: 1805550. DOI: 10.1073/pnas.0609573104.

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