Reconstitution of bacterial outer membrane TonB-dependent transporters in planar lipid bilayer membranes

Eshwar Udho, Karen S. Jakes, Susan K. Buchanan, Karron J. James, Xiaoxu Jiang, Phillip E. Klebba, Alan Finkelstein

Research output: Contribution to journalArticle

20 Citations (Scopus)

Abstract

Micronutrients such as siderophore-bound iron and vitamin B12 cross the outer membrane of Gram-negative bacteria through a group of 22-stranded β-barrel proteins. They share the unusual feature that their N-terminal end inserts from the periplasmic side into the β-barrel and plugs the lumen. Transport results from energy-driven movement of TonB protein, which either pulls the plug out of the barrel or causes it to rearrange within the barrel. Attempts to reconstitute native plugged channels in an ion-conducting state in lipid bilayer membranes have so far been unsuccessful. We, however, have discovered that if the cis solution contained 4 M urea, then, with the periplasmic side of the channel facing that solution, macroscopic conductances and single channel events could be observed. These results were obtained with FhuA, Cir, and BtuB; for the former two, the channels were closed by removing the 4 M urea. Channels generated by 4 M urea exposure were not a consequence of general protein denaturation, as their ligand-binding properties were preserved. Thus, with FhuA, addition of ferrichrome (its siderophore) to the trans, extracellular-facing side reversibly inhibited 4 M urea-induced channel opening and blocked the channels. With Cir, addition of colicin Ia (the microbial toxin that targets Cir) to the trans, extracellular-facing side prevented 4 M urea-induced channel opening. We hypothesize that 4 M urea reversibly unfolds the FhuA and Cir plugs, thereby opening an ion-conducting pathway through these channels, and that this mimics to some extent the in vivo action of TonB on these plugs.

Original languageEnglish (US)
Pages (from-to)21990-21995
Number of pages6
JournalProceedings of the National Academy of Sciences of the United States of America
Volume106
Issue number51
DOIs
StatePublished - Dec 22 2009

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Lipid Bilayers
Urea
Membranes
Siderophores
Ferrichrome
Colicins
Protein Denaturation
Micronutrients
Vitamin B 12
Gram-Negative Bacteria
Ion Channels
Proteins
Iron
Ions
Ligands

Keywords

  • BtuB
  • Cir
  • FhuA
  • Urea-denaturation

ASJC Scopus subject areas

  • General

Cite this

Reconstitution of bacterial outer membrane TonB-dependent transporters in planar lipid bilayer membranes. / Udho, Eshwar; Jakes, Karen S.; Buchanan, Susan K.; James, Karron J.; Jiang, Xiaoxu; Klebba, Phillip E.; Finkelstein, Alan.

In: Proceedings of the National Academy of Sciences of the United States of America, Vol. 106, No. 51, 22.12.2009, p. 21990-21995.

Research output: Contribution to journalArticle

Udho, Eshwar ; Jakes, Karen S. ; Buchanan, Susan K. ; James, Karron J. ; Jiang, Xiaoxu ; Klebba, Phillip E. ; Finkelstein, Alan. / Reconstitution of bacterial outer membrane TonB-dependent transporters in planar lipid bilayer membranes. In: Proceedings of the National Academy of Sciences of the United States of America. 2009 ; Vol. 106, No. 51. pp. 21990-21995.
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