Dissecting the Effects of Periplasmic Chaperones on the In Vitro Folding of the Outer Membrane Protein PagP

Abstract

Although many periplasmic folding factors have been identified, the mechanisms by which they interact with unfolded outer membrane proteins (OMPs) to promote correct folding and membrane insertion remain poorly understood. Here, we have investigated the effect of two chaperones, Skp and SurA, on the folding kinetics of the OMP, PagP. Folding kinetics of PagP into both zwitterionic diC12:0PC (1,2-dilauroyl-sn-glycero-3-phosphocholine) liposomes and negatively charged 80:20 diC12:0PC:diC12:0PG [1,2-dilauroyl-sn-glycero-3-phospho-(1′-rac-glycerol)] liposomes were investigated using a combination of spectroscopic and SDS-PAGE assays. The results indicate that Skp modulates the observed rate of PagP folding in a manner that is dependent on the composition of the membrane and the ionic strength of the buffer used. These data suggest that electrostatic interactions play an important role in Skp-assisted substrate delivery to the membrane. In contrast, SurA showed no effect on the observed folding rates of PagP, consistent with the view that these chaperones act by distinct mechanisms in partially redundant parallel chaperone pathways that facilitate OMP assembly. In addition to delivery of the substrate protein to the membrane, the ability of Skp to prevent OMP aggregation was investigated. The results show that folding and membrane insertion of PagP can be restored, in part, by Skp in conditions that strongly favour PagP aggregation. These results illustrate the utility of in vitro systems for dissecting the complex folding environment encountered by OMPs in the periplasm and demonstrate the key role of Skp in holding aggregation-prone OMPs prior to their direct or indirect delivery to the membrane.

Sequential Steps in the Assembly of the Multimeric Outer Membrane Secretin PulD

“…A second unresolved question is raised by the discrepancy between the requirement for highly fluid membranes for the folding of outer membrane proteins in vitro (56,57) and the presumed low fluidity of the outer membrane (58). Recent observations indicate that at least certain chaperones can help overcome this barrier (59,60). PulS might play such a role.…”

Envelope Stress Responses: An Interconnected Safety Net

“…While the Bam complex catalyzes OMP folding in vivo , direct folding of OMPs into membranes in vitro is well established [89]. Skp, unlike the major chaperone SurA, can promote direct OMP insertion into membranes [90,91]. Defects in the primary SurA-Bam OMP pathway activate σ E in order to maintain OMPs in folding-competent states within the periplasm, including by up-regulating Skp [53].…”

Outer membrane protein biogenesis in Gram-negative bacteria

“…Crystallographic analysis of constructs of SurA bound to two types of artificial peptides of differing lengths [51] indicated SurA interacts preferentially with aromatic residues independently of whether the peptide binds in an extended or a helical arrangement. A change in SurA conformation coupled with dimerization of the P1 domains was observed when SurA was bound to the longer peptide, suggesting SurA is able to bind a variety of aromatic sequences within OMPs by altering its overall domain structure (figure 2b [52]. SurA is the only soluble chaperone that has been cross-linked in vivo to BamA [23], and addition of SurA increases the rate of OmpT folding in an in vitro reconstituted BAM system [53].…”