Use of designed metal-binding sites to study helix proximity in the lactose permease of Escherichia coli. 1. Proximity of helix VII (Asp237 and Asp240) with helices X (Lys319) and XI (Lys358)

Molly M. He, John C Voss, Wayne L. Hubbell, H. Ronald Kaback

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Abstract

The lactose permease of Escherichia coli contains two pairs of oppositely charged residues that interact functionally, Asp240 (helix VII)/Lys319 (helix X) and Asp237 (helix VII)/Lys358 (helix XI). Single- and double-His replacement mutants at these positions have been constructed and characterized with respect to transport activity and Mn2+ binding. The following results confirm the functional interactions between both sets of residues: (i) At pH 7.5, where the imidazole is likely to be unprotonated, the double-His mutants Asp237→His/Lys358→His and Asp240→His/Lys319→His exhibit significant transport activity while the single-His mutants Lys319→His and Lys358-His are inactive, (ii) At pH 5.5, where the imidazole is likely to be protonated, the double-His mutants Asp240→His/Lys319→His and Asp237→His/ Lys358→→His are inactive; however, the single-His mutant Lys319→His exhibits significant activity, (iii) The single-His mutant Asp237→His or Asp240→His is inactive at all pH values tested. In addition, a pH titration of Asp237→His/Lys→His permease activity exhibits a midpoint at about 6.2. Finally, the purified mutant proteins Asp237→His/Lys358→His and Asp240→His/Lys319→His were assayed for Mn2+ binding by electron paramagnetic resonance spectroscopy. Asp237→His/Lys358→His permease binds Mn2+ with a stoichiometry of unity at pH 7.5, but much less binding is observed at pH 5.5, demonstrating directly that helix VII (Asp237) is in close proximity to helix XI (Lys358). In contrast, Asp240→His/Lys319→His permease does not bind Mn2+, indicating that these two residues interact over a longer distance. In the following paper, the same approach is used to confirm the close proximity of helices IX (Arg302) and X (His322 and Glu325).

Original languageEnglish (US)
Pages (from-to)15661-15666
Number of pages6
JournalBiochemistry
Volume34
Issue number48
StatePublished - 1995

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Membrane Transport Proteins
Escherichia coli
Metals
Binding Sites
Mutant Proteins
Titration
Stoichiometry
Paramagnetic resonance
Spectroscopy
Electron Spin Resonance Spectroscopy
lactose permease
Spectrum Analysis
imidazole

ASJC Scopus subject areas

  • Biochemistry

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Use of designed metal-binding sites to study helix proximity in the lactose permease of Escherichia coli. 1. Proximity of helix VII (Asp237 and Asp240) with helices X (Lys319) and XI (Lys358). / He, Molly M.; Voss, John C; Hubbell, Wayne L.; Ronald Kaback, H.

In: Biochemistry, Vol. 34, No. 48, 1995, p. 15661-15666.

Research output: Contribution to journalArticle

@article{996cc61a4f034dadadc5765c35a491bb,
title = "Use of designed metal-binding sites to study helix proximity in the lactose permease of Escherichia coli. 1. Proximity of helix VII (Asp237 and Asp240) with helices X (Lys319) and XI (Lys358)",
abstract = "The lactose permease of Escherichia coli contains two pairs of oppositely charged residues that interact functionally, Asp240 (helix VII)/Lys319 (helix X) and Asp237 (helix VII)/Lys358 (helix XI). Single- and double-His replacement mutants at these positions have been constructed and characterized with respect to transport activity and Mn2+ binding. The following results confirm the functional interactions between both sets of residues: (i) At pH 7.5, where the imidazole is likely to be unprotonated, the double-His mutants Asp237→His/Lys358→His and Asp240→His/Lys319→His exhibit significant transport activity while the single-His mutants Lys319→His and Lys358-His are inactive, (ii) At pH 5.5, where the imidazole is likely to be protonated, the double-His mutants Asp240→His/Lys319→His and Asp237→His/ Lys358→→His are inactive; however, the single-His mutant Lys319→His exhibits significant activity, (iii) The single-His mutant Asp237→His or Asp240→His is inactive at all pH values tested. In addition, a pH titration of Asp237→His/Lys→His permease activity exhibits a midpoint at about 6.2. Finally, the purified mutant proteins Asp237→His/Lys358→His and Asp240→His/Lys319→His were assayed for Mn2+ binding by electron paramagnetic resonance spectroscopy. Asp237→His/Lys358→His permease binds Mn2+ with a stoichiometry of unity at pH 7.5, but much less binding is observed at pH 5.5, demonstrating directly that helix VII (Asp237) is in close proximity to helix XI (Lys358). In contrast, Asp240→His/Lys319→His permease does not bind Mn2+, indicating that these two residues interact over a longer distance. In the following paper, the same approach is used to confirm the close proximity of helices IX (Arg302) and X (His322 and Glu325).",
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T1 - Use of designed metal-binding sites to study helix proximity in the lactose permease of Escherichia coli. 1. Proximity of helix VII (Asp237 and Asp240) with helices X (Lys319) and XI (Lys358)

AU - He, Molly M.

AU - Voss, John C

AU - Hubbell, Wayne L.

AU - Ronald Kaback, H.

PY - 1995

Y1 - 1995

N2 - The lactose permease of Escherichia coli contains two pairs of oppositely charged residues that interact functionally, Asp240 (helix VII)/Lys319 (helix X) and Asp237 (helix VII)/Lys358 (helix XI). Single- and double-His replacement mutants at these positions have been constructed and characterized with respect to transport activity and Mn2+ binding. The following results confirm the functional interactions between both sets of residues: (i) At pH 7.5, where the imidazole is likely to be unprotonated, the double-His mutants Asp237→His/Lys358→His and Asp240→His/Lys319→His exhibit significant transport activity while the single-His mutants Lys319→His and Lys358-His are inactive, (ii) At pH 5.5, where the imidazole is likely to be protonated, the double-His mutants Asp240→His/Lys319→His and Asp237→His/ Lys358→→His are inactive; however, the single-His mutant Lys319→His exhibits significant activity, (iii) The single-His mutant Asp237→His or Asp240→His is inactive at all pH values tested. In addition, a pH titration of Asp237→His/Lys→His permease activity exhibits a midpoint at about 6.2. Finally, the purified mutant proteins Asp237→His/Lys358→His and Asp240→His/Lys319→His were assayed for Mn2+ binding by electron paramagnetic resonance spectroscopy. Asp237→His/Lys358→His permease binds Mn2+ with a stoichiometry of unity at pH 7.5, but much less binding is observed at pH 5.5, demonstrating directly that helix VII (Asp237) is in close proximity to helix XI (Lys358). In contrast, Asp240→His/Lys319→His permease does not bind Mn2+, indicating that these two residues interact over a longer distance. In the following paper, the same approach is used to confirm the close proximity of helices IX (Arg302) and X (His322 and Glu325).

AB - The lactose permease of Escherichia coli contains two pairs of oppositely charged residues that interact functionally, Asp240 (helix VII)/Lys319 (helix X) and Asp237 (helix VII)/Lys358 (helix XI). Single- and double-His replacement mutants at these positions have been constructed and characterized with respect to transport activity and Mn2+ binding. The following results confirm the functional interactions between both sets of residues: (i) At pH 7.5, where the imidazole is likely to be unprotonated, the double-His mutants Asp237→His/Lys358→His and Asp240→His/Lys319→His exhibit significant transport activity while the single-His mutants Lys319→His and Lys358-His are inactive, (ii) At pH 5.5, where the imidazole is likely to be protonated, the double-His mutants Asp240→His/Lys319→His and Asp237→His/ Lys358→→His are inactive; however, the single-His mutant Lys319→His exhibits significant activity, (iii) The single-His mutant Asp237→His or Asp240→His is inactive at all pH values tested. In addition, a pH titration of Asp237→His/Lys→His permease activity exhibits a midpoint at about 6.2. Finally, the purified mutant proteins Asp237→His/Lys358→His and Asp240→His/Lys319→His were assayed for Mn2+ binding by electron paramagnetic resonance spectroscopy. Asp237→His/Lys358→His permease binds Mn2+ with a stoichiometry of unity at pH 7.5, but much less binding is observed at pH 5.5, demonstrating directly that helix VII (Asp237) is in close proximity to helix XI (Lys358). In contrast, Asp240→His/Lys319→His permease does not bind Mn2+, indicating that these two residues interact over a longer distance. In the following paper, the same approach is used to confirm the close proximity of helices IX (Arg302) and X (His322 and Glu325).

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