Protein-lipid charge interactions control the folding of OMPs into asymmetric membranes

Biological membranes consist of two leaflets of phospholipid molecules that form a bilayer, and typically the composition of lipids in each leaflet is distinct. This asymmetry is created and maintained in vivo by dedicated biochemical pathways, but difficulties in creating stable asymmetric membranes in vitro have restricted our understanding of how bilayer asymmetry modulates the folding, stability and function of membrane proteins. Here we employ cyclodextrin mediated lipid exchange to generate asymmetric liposomes and use these to characterize the stability and folding kinetics of two bacterial outer membrane proteins (OMPs). We show that excess negative charge in the outer leaflet of a liposome impedes the membrane insertion and folding of OmpA and BamA, while excess negative charge in the inner leaflet accelerates their folding, relative to symmetric liposomes with the same membrane composition. Three positively charged residues in the extracellular loops of OmpA that play a critical role in folding are identified using molecular dynamics simulations and mutational analyses. Bioinformatic analysis was then used to identify a conserved patch of positive residues in the extracellular loops of OMPs generally that lies 6-8[A] from the membrane surface. Together, the the results rationalise the well known positive outside rule for OMP sequences and suggest new insights into the mechanisms that drive OMP folding and assembly in vitro and in vivo.