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

Abstract

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.

Membrane protein folding on the example of outer membrane protein A of Escherichia coli

“…In contrast, only 3 molecules of Skp bind to OmpA with a much larger binding constant of K Skp = 46 ± 30 mM -1 (i.e. with DG = -43 ± 2 kJ/mol) [32]. The 8-150-fold greater OmpA binding constant of Skp explains that Skp prevents the folding of OmpA upon addition of LPS micelles.…”

“…Therefore, ~1.5-5 mol LPS bind to Skp-OmpA complexes (i.e. much lower amounts than observed in the absence of Skp) to catalyze membrane insertion and folding [32]. Binding of LPS to Skp-OmpA complexes is quite specific to promote subsequent insertion into membranes.…”

“…[20]). However, the negative charge of SDS only contributes to this effect of the SDS headgroup and cannot be the only cause of inhibition of OmpA folding in the presence of SDS, since OmpA can fold partially into micelles of negatively charged LPS [32] and also into bilayers containing negatively charged phosphatidylglycerol, which are both components of the bacterial outer membrane [32,33]. A range of detergents have also been used for refolding other b-barrel membrane proteins for subsequent membrane protein crystallization (for an overview, see e.g.…”

“…Obviously, these chaperones are more efficient in preventing OMP aggregation compared with the denaturant urea that has been used in folding studies in vitro and that must be diluted before OMPs can insert and fold. Bulieris et al [32] demonstrated a first chaperone-assisted pathway of OmpA folding that consists of three major stages. The periplasmic chaperone Skp is sufficient to keep OmpA soluble in vitro at pH 7 in an unfolded form even after strong dilution of the denaturant urea.…”

“…Binding of LPS to Skp-OmpA complexes is quite specific to promote subsequent insertion into membranes. CD spectroscopy and KTSE assays indicated that large amounts of secondary and tertiary structure in OmpA only form in the third stage of the assembly pathway, upon addition of phospholipid bilayers [32]. The interaction of the OmpA/Skp/LPS complex with the lipid bilayer is apparently the most important event to initiate folding of OmpA in the presence of chaperones and LPS as a folding catalyst.…”