Folding and Insertion of the Outer Membrane Protein OmpA Is Assisted by the Chaperone Skp and by Lipopolysaccharide

Paula V Bulieris,Susanne Behrens,Otto Holst,Jörg H Kleinschmidt

doi:10.1074/jbc.m211177200

Abstract

We have studied the folding pathway of a beta-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.

Highlights

We have studied the folding pathway of a ␤-barrel membrane protein using outer membrane protein A (OmpA) of Escherichia coli as an example
Upon investigating the roles of the periplasmic components Skp and LPS on the insertion and folding of OmpA into phospholipid bilayers in detail, we found a first assisted folding pathway for the integral membrane protein OmpA that is described by three major stages
Skp binding to OmpA alone is sufficient to keep OmpA unfolded in solution, effectively replacing the denaturant urea, it is inhibiting the membrane insertion and folding of OmpA

Summary

The abbreviations used are

OmpA, outer membrane protein A; OMPs, outer membrane proteins; DOPC, 1,2-dioleoyl-sn-glycero-3phosphocholine; DOPE, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine; DOPG, 1,2-dioleoyl-sn-glycero-3-phosphoglycerol; Mcps, million counts per second, mdeg, millidegrees; LPS, lipopolysaccharide; Skp, 17-kDa protein; SUV, small unilamellar vesicle. It was further demonstrated that secondary and tertiary structure formation in OmpA are synchronized and coupled to membrane insertion [6] In these previous studies, the OmpA folding kinetics were relatively slow, suggesting that folding in vivo might be facilitated by folding catalysts. Pulse-labeling and biochemical reconstitution experiments suggested that LPS is required for efficient assembly of OMPs such as trimeric PhoE [33] and monomeric OmpA [34] into outer membranes In these previous studies, refolding was performed with micelles of LPS and Triton X-100 instead of phospholipid bilayers. It is not clear whether the LPS concentration in the periplasm is above the CMC, but OMPs would not fold in the presence of monomers [7]. We have addressed the following questions. 1) How do Skp and LPS (either separately or in combination) affect unfolded OmpA in solution? 2) Are there effects of Skp (or LPS) on the folding of OmpA into lipid bilayers? 3) At which stoichiometries and how strong does Skp (or LPS) bind to OmpA? 4) Does Skp (or LPS) lead to secondary structure formation of OmpA in solution?

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION