Reconstitution Of Tonb-dependent Transporters In Planar Lipid Bilayers

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 to open a space wide enough for siderophores to diffuse through.Attempts to reconstitute these plugged channels in an ion-conducting state in lipid bilayer membranes have so far been unsuccessful. Recently, however, we have discovered that if the cis solution contains 4 M urea, then macroscopic conductances and single channel events could be observed. These results were obtained with FhuA, Cir and BtuB, and in the case of the former two, the channels were closed by removing the 4 M urea. The basic native structure of these proteins appears to be preserved by our reconstitution procedure. With FhuA, for example, addition of ferrichrome (its siderophore) to the trans side reversibly inhibits 4 M urea-induced channel opening and blocks the channels with a Kd of ∼0.5 nM, the same as that found biologically. In the case of Cir, as another example, addition of colicin Ia (the microbial toxin that targets Cir) to the trans side prevents 4 M urea-induced channel opening. Our working hypothesis is that the 4 M urea reversibly denatures the plug, causing it to either rearrange itself or to come completely out of the barrel, thereby providing a conduction pathway. Whether the 4 M urea action on the plug mimics to some extent that of TonB remains for future investigation.