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[摘要]：The basic biochemical and biophysical principles by which chaperone-bound membrane proteins are targeted to the outer membrane of Gram-negative bacteria for insertion and folding are unknown. Here we compare spontaneous folding of outer membrane protein A (OmpA) of Escherichia coli from its urea-unfolded form and from the complex with its periplasmic chaperone Skp into lipid bilayers. Skp facilitated folding of OmpA into negatively charged membranes containing dioleoylphosphatidylglycerol (DOPG). In contrast, Skp strongly inhibited folding of OmpA when bilayers were composed of dioleoylphosphatidylethanolamine and diolcoylphosphatidylcholine (DOPC). These results indicate that the positively charged Skp targets OmpA to a negatively charged membrane, which facilitates the release of OmpA from its complex with Skp for subsequent folding and membrane insertion. The dual functionality of Skp as a chaperone and as a targeting protein is ideal to mediate the transport of OmpA and other outer membrane proteins across the periplasm in a folding-competent form to the outer membrane, which is negatively charged on its periplasmic side. OmpA (pI 5.5) folded most efficiently above its isoelectric point. In the absence of Skp and in contrast to folding into DOPC bilayers, insertion and folding of OmpA were retarded for membranes containing DOPG at neutral or basic pH because of electrostatic repulsion. When folding of OmpA was performed near its isoelectric point, urea dilution led to a more compact aqueous form of OmpA previously characterized by fluorescence, which folded at a Much slower rate. Under conditions where two different aqueous conformations of OmpA coexisted, e.g., in the titration region of OmpA, the last step of OmpA folding could be well described by two parallel pseudo-first-order kinetic phases. In this kinetic model, the contribution of the faster folding process, but not the changes in the rate constants, determined the folding yields obtained at different pH. The Faster phase dominated when the experimental conditions favored the less compact form of aqueous OmpA. |
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