We have studied the folding pathway of a ␤-barrel membrane protein using outer membrane protein A (OmpA) of Escherichia coli as an example. The deletion of the gene of periplasmic Skp impairs the assembly of outer membrane proteins of bacteria. We investigated how Skp facilitates the insertion and folding of completely unfolded OmpA into phospholipid membranes and which are the biochemical and biophysical requirements of a possible Skp-assisted folding pathway. In refolding experiments, Skp alone was not sufficient to facilitate membrane insertion and folding of OmpA. In addition, lipopolysaccharide (LPS) was required. OmpA remained unfolded when bound to Skp and LPS in solution. From this complex, OmpA folded spontaneously into lipid bilayers as determined by electrophoretic mobility measurements, fluorescence spectroscopy, and circular dichroism spectroscopy. The folding of OmpA into lipid bilayers was inhibited when one of the periplasmic components, either Skp or LPS, was absent. Membrane insertion and folding of OmpA was most efficient at specific molar ratios of OmpA, Skp, and LPS. Unfolded OmpA in complex with Skp and LPS folded faster into phospholipid bilayers than urea-unfolded OmpA. Together, these results describe a first assisted folding pathway of an integral membrane protein on the example of OmpA.

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Across bacteria, the protein that makes the flagellar hook, FlgE, has a high variability in amino acid residue composition and sequence length. We hereby present the structure of two fragments of FlgE protein from Campylobacter jejuni and from Caulobacter crescentus, which were obtained by X-ray crystallography, and a high-resolution model of the hook from Caulobacter. By comparing these new structures of FlgE proteins, we show that bacterial hook can be divided in two distinct parts. The first part comprises domains that are found in all FlgE proteins and that will make the basic structure of the hook that is common to all flagellated bacteria. The second part, hyper-variable both in size and structure, will be bacteria dependent. To have a better understanding of the C. jejuni hook, we show that a special strain of Salmonella enterica, which was designed to encode a gene of flgE that has the extra domains found in FlgE from C. jejuni, is fully motile. It seems that no matter the size of the hook protein, the hook will always have a structure made of 11 protofilaments.

Evolution of a nano-machine consisting of multiple parts, each with a specific function, is a complex process. A change in one part should eventually result in changes in other parts, if the overall function is to be conserved. In bacterial flagella, the filament and the hook have distinct functions and their respective proteins, FliC and FlgE, have different three-dimensional structures. The filament functions as a helical propeller and the hook as a flexible universal joint. Two proteins, FlgK and FlgL, assure a smooth connectivity between the hook and the filament. Here we show that, in Campylobacter, the 3D structure of FlgK differs from that of its orthologs in Salmonella and Burkholderia, whose structures have previously been solved. Docking the model of the FlgK junction onto the structure of the Campylobacter hook provides some clues about its divergence. These data suggest how evolutionary pressure to adapt to structural constraints, due to the structure of Campylobacter hook, causes divergence of one element of a supra-molecular complex in order to maintain the function of the entire flagellar assembly.

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