Ligand-induced conformational changes in the lactose permease of Escherichia coli: Evidence for two binding sites

J. Wu, S. Frillingos, John C Voss, H. R. Kaback

Research output: Contribution to journalArticle

45 Citations (Scopus)

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, β,D-galactopyranosyl 1-thio-β,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-β,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 K(d) ≃ 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%. Finally, the EPR spectrum of nitroxide spin-labeled Val 331 → Cys permease exhibits 2 components with different mobilities, and TDG causes the immobilized component to increase. The results provide evidence for the argument that lac permease has more than a single binding site. TDG binding to a higher affinity site quenches the fluorescence of MIANS-labeled Val 331 → Cys permease, and occupation of a second lower affinity site causes position 331 to become more accessible from a hydrophobic environment.

Original languageEnglish (US)
Pages (from-to)2294-2301
Number of pages8
JournalProtein Science
Volume3
Issue number12
StatePublished - 1994

Fingerprint

Membrane Transport Proteins
Escherichia coli
Galactose
Sulfonic Acids
Binding Sites
Ligands
Ethylmaleimide
Acrylamide
Fluorescence
Iodides
Quenching
Sulfhydryl Reagents
Fluorescence Spectrometry
Fluorescence spectroscopy
lactose permease
Occupations
Paramagnetic resonance
naphthalene
Membranes

ASJC Scopus subject areas

  • Biochemistry

Cite this

Ligand-induced conformational changes in the lactose permease of Escherichia coli : Evidence for two binding sites. / Wu, J.; Frillingos, S.; Voss, John C; Kaback, H. R.

In: Protein Science, Vol. 3, No. 12, 1994, p. 2294-2301.

Research output: Contribution to journalArticle

@article{21601d44862642f3b772630d9e9d7762,
title = "Ligand-induced conformational changes in the lactose permease of Escherichia coli: Evidence for two binding sites",
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, β,D-galactopyranosyl 1-thio-β,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-β,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 K(d) ≃ 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{\%}. Finally, the EPR spectrum of nitroxide spin-labeled Val 331 → Cys permease exhibits 2 components with different mobilities, and TDG causes the immobilized component to increase. The results provide evidence for the argument that lac permease has more than a single binding site. TDG binding to a higher affinity site quenches the fluorescence of MIANS-labeled Val 331 → Cys permease, and occupation of a second lower affinity site causes position 331 to become more accessible from a hydrophobic environment.",
author = "J. Wu and S. Frillingos and Voss, {John C} and Kaback, {H. R.}",
year = "1994",
language = "English (US)",
volume = "3",
pages = "2294--2301",
journal = "Protein Science",
issn = "0961-8368",
publisher = "Cold Spring Harbor Laboratory Press",
number = "12",

}

TY - JOUR

T1 - Ligand-induced conformational changes in the lactose permease of Escherichia coli

T2 - Evidence for two binding sites

AU - Wu, J.

AU - Frillingos, S.

AU - Voss, John C

AU - Kaback, H. R.

PY - 1994

Y1 - 1994

N2 - 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, β,D-galactopyranosyl 1-thio-β,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-β,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 K(d) ≃ 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%. Finally, the EPR spectrum of nitroxide spin-labeled Val 331 → Cys permease exhibits 2 components with different mobilities, and TDG causes the immobilized component to increase. The results provide evidence for the argument that lac permease has more than a single binding site. TDG binding to a higher affinity site quenches the fluorescence of MIANS-labeled Val 331 → Cys permease, and occupation of a second lower affinity site causes position 331 to become more accessible from a hydrophobic environment.

AB - 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, β,D-galactopyranosyl 1-thio-β,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-β,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 K(d) ≃ 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%. Finally, the EPR spectrum of nitroxide spin-labeled Val 331 → Cys permease exhibits 2 components with different mobilities, and TDG causes the immobilized component to increase. The results provide evidence for the argument that lac permease has more than a single binding site. TDG binding to a higher affinity site quenches the fluorescence of MIANS-labeled Val 331 → Cys permease, and occupation of a second lower affinity site causes position 331 to become more accessible from a hydrophobic environment.

UR - http://www.scopus.com/inward/record.url?scp=0028587807&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0028587807&partnerID=8YFLogxK

M3 - Article

C2 - 7756985

AN - SCOPUS:0028587807

VL - 3

SP - 2294

EP - 2301

JO - Protein Science

JF - Protein Science

SN - 0961-8368

IS - 12

ER -