TonB/TolA amino-terminal domain modeling

TonB and TolA proteins are energy transducers that couple the ion electrochemical gradient of the cytoplasmic membrane to support energy-dependent processes in the outer membrane of gram-negative bacteria. Energization of these proteins involves specific interactions with multiprotein cytoplasmic membrane energy harvesting complexes. The specific mechanisms by which these energy transfers occur remain unclear, but the evidence to date indicates that the amino-terminally located signal anchors of TonB and TolA play essential roles in the process. Mutant hunts have identified one motif in this region, common to both TonB and TolA, as important for energization. Because TonB and TolA each have a "preferred" energy-harvesting complex, it is clear that additional motifs, not shared between TonB and TolA, are involved in interactions with energy harvesting complexes. We have adopted a strategy of examining derivatives with multiple-residue substitutions to identify such regions. This involves the characterization of specific TonB derivatives generated by two similar approaches: the block substitutions in TonB by alanyl residues and the exchange of short regions between TonB and TolA. The methods by which these derivatives are generated are described, with an illustrative example for each.