Mechanisms of outer membrane protein folding: effects of the lipid environment and periplasmic chaperones

In contrast with the wealth of information on the folding of soluble, cytosolic proteins, little is known about the folding of integral membrane proteins. The outer membrane proteins (OMPs) of Gram-negative bacteria have a β-barrel structure and are essential for cell survival. The mechanisms of OMP transport across the periplasm and how these proteins subsequently fold and insert into the outer membrane remain to be elucidated. The work presented herein examines the folding and membrane insertion of four different OMP constructs. Two homologous bacterial OMPs, OmpT and OmpP, were cloned, over-expressed and purified before biochemical and biophysical methods were employed to examine their folding properties. This work demonstrates that small differences in primary sequence can have large effects on folding efficiency and stability. In spite of both OmpT and OmpP being able to fold under a variety of conditions, it was not possible to establish conditions under which folding was completely reversible. Examination of the origins of irreversible OMP folding was carried out using OmpT, as well as both hexa-histidine tagged and untagged constructs of the outer membrane acyltransferase enzyme, PagP. This study revealed evidence that lipid adhesion of the protein in the unfolded state may be important in preventing aggregation and promoting reversibility. Finally, conditions were established to promote the folding of untagged PagP in low urea concentrations to allow the study of OMPs in the presence of the periplasmic chaperones, SurA and Skp. SurA was shown to have little effect on the folding of PagP into liposomes with zwitterionic or negatively charged membrane surfaces, while Skp was shown to exhibit holdase activity and to modulate PagP folding rate, dependent on the lipid composition. The results present the first detailed insights into the mechanism by which Skp and SurA act to facilitate PagP folding in vitro.