Comparison of Insertion and Folding of Chaperone-bound Outer Membrane Protein A (OmpA) of E. coli into Phospholipid Bilayers of Various Composition

Abstract

OmpA spontaneously inserts and folds into lipid bilayers from a urea-unfolded state upon urea-dilution. Previous work demonstrated that urea can be replaced effectively by the periplasmic chaperone Skp when lipopolysaccharide (LPS), a component of the outer membrane, is present [1]. Skp was shown to bind outer membrane proteins with nanomolar affinity and to prevent their aggregation [2].Here we investigated folding of Skp-bound OmpA into lipid bilayers of different headgroup composition and chain-length, both in absence and presence of LPS. For urea-denatured OmpA and in absence of Skp and LPS, kinetics of folding into bilayers containing the negatively charged phosphatidylglycerol showed a lag-phase of up to 30 min for dilauroylphospholipid bilayers (LUVs, 100 nm diameter) prior to folding. Skp inhibited folding and prolonged the lag-phase at basic pH when LPS was absent. In presence of LPS, no lag-phase was observed and folding rates increased dramatically.When similar experiments were performed with bilayers composed of the corresponding dioleolylphospholipids (SUVs), a lag-phase was not observed, but Skp inhibited very strongly. LPS again stimulated OmpA insertion and folding, albeit not as much as observed for dilaurylphospholipid bilayers.Skp inhibited folding also in experiments with neutral phosphatidylcholine bilayers, irrespective of lipid chain-length. Here, LPS could also facilitate folding of Skp-bound OmpA, but the effect was less pronounced for dioleoylphosphatidylcholine bilayers. The data suggest, LPS-assisted folding of Skp-bound OmpA depends on both, the surface charge of the membrane and on the lipid-chain composition.