Characterization of in vitro interactions between a truncated TonB protein from Escherichia coli and the outer membrane receptors FhuA and FepA.
High-affinity iron uptake in gram-negative bacteria depends upon TonB, a protein which couples the proton motive force in the cytoplasmic membrane to iron chelate receptors in the outer membrane. To advance studies on TonB structure and function, we expressed a recombinant form of Escherichia coli TonB lacking the N-terminal cytoplasmic membrane anchor. This protein (H(6)-'TonB; M(r), 24,880) was isolated in a soluble fraction of lysed cells and was purified by virtue of a hexahistidine tag located at its N terminus. Sedimentation experiments indicated that the H(6)-'TonB preparation was almost monodisperse and the protein was essentially monomeric. The value found for the Stokes radius (3.8 nm) is in good agreement with the value calculated by size exclusion chromatography. The frictional ratio (2.0) suggested that H(6)-'TonB adopts a highly asymmetrical form with an axial ratio of 15. H(6)-'TonB captured both the ferrichrome-iron receptor FhuA and the ferric enterobactin receptor FepA from detergent-solubilized outer membranes in vitro. Capture was enhanced by preincubation of the receptors with their cognate ligands. Cross-linking assays with the purified proteins in vitro demonstrated that there was preferential interaction between TonB and ligand-loaded FhuA. Purified H(6)-'TonB was found to be stable and thus shows promise for high-resolution structural studies.
Moeck
G S
GS
Institut de Biochimie et Biophysique Moléculaire et Cellulaire, UMR CNRS 8619, Université de Paris-Sud, F-91405, Orsay cedex, France.
Letellier
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L
D001425:Bacterial Outer Membrane Proteins / Q000737:chemistry* / Q000378:metabolism; D001426:Bacterial Proteins / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics; D002352:Carrier Proteins / Q000737:chemistry* / Q000378:metabolism; D002850:Chromatography, Gel; D003432:Cross-Linking Reagents; D004926:Escherichia coli / Q000235:genetics; D029968:Escherichia coli Proteins; D005291:Ferrichrome / Q000031:analogs & derivatives; D005557:Formaldehyde; D005822:Genetic Vectors; D066298:In Vitro Techniques; D008565:Membrane Proteins / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics; D011485:Protein Binding; D011956:Receptors, Cell Surface; D011991:Receptors, Virus / Q000737:chemistry* / Q000378:metabolism; D011994:Recombinant Proteins / Q000096:biosynthesis; D013329:Structure-Activity Relationship
D001425:Bacterial Outer Membrane Proteins; D001426:Bacterial Proteins; D002352:Carrier Proteins; D003432:Cross-Linking Reagents; D029968:Escherichia coli Proteins; C050900:FhuA protein, E coli; D008565:Membrane Proteins; D011956:Receptors, Cell Surface; D011991:Receptors, Virus; D011994:Recombinant Proteins; C029489:enterobactin receptor; C057616:tonB protein, Bacteria; C489181:tonB protein, E coli; D005291:Ferrichrome; D005557:Formaldehyde; C021678:ferricrocin