Reconstitution of bacterial outer membrane TonB-dependent transporters in planar lipid bilayer membranes.
Micronutrients such as siderophore-bound iron and vitamin B(12) cross the outer membrane of gram-negative bacteria through a group of 22-stranded beta-barrel proteins. They share the unusual feature that their N-terminal end inserts from the periplasmic side into the beta-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.
Udho
Eshwar
E
Department of Physiology and Biophysics, Albert Einstein College of Medicine, Bronx, NY 10461, USA.
Jakes
Karen S
KS
Buchanan
Susan K
SK
James
Karron J
KJ
Jiang
Xiaoxu
X
Klebba
Phillip E
PE
Finkelstein
Alan
A
D001426:Bacterial Proteins / Q000502:physiology*; D006090:Gram-Negative Bacteria / Q000187:drug effects / Q000502:physiology; D008051:Lipid Bilayers; D008565:Membrane Proteins / Q000502:physiology*; D014508:Urea / Q000494:pharmacology
D001426:Bacterial Proteins; D008051:Lipid Bilayers; D008565:Membrane Proteins; C057616:tonB protein, Bacteria; D014508:Urea