Characterization of in vitro interactions between a truncated TonB proteinfrom Escherichia coli and the outer membrane receptors FhuA and FepA

High-affinity iron uptake in gram-negative bacteria depends upon TonB, a pr otein which couples the proton motive force in the cytoplasmic membrane to iron chelate receptors in the outer membrane. To advance studies on TonB st ructure and function, we expressed a recombinant form of Escherichia coli T onB 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 wa s purified by virtue of a hexahistidine tag located at its N terminus. Sedi mentation experiments indicated that the H-6-'TonB preparation was almost m onodisperse and the protein was essentially monomeric. The value found for the Stokes radius (3.8 nm) is in good agreement with the value calculated b y 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 enter obactin receptor FepA from detergent-solubilized outer membranes in vitro. Capture was enhanced by preincubation of the receptors with their cognate l igands. Cross-linking assays with the purified proteins in vitro demonstrat ed that there was preferential interaction between TonB and ligand-loaded F huA. Purified H-6-'TonB was found to be stable and thus shows promise for h igh-resolution structural studies.