Native β-barrel substrates pass through two shared intermediates during folding on the BAM complex

The assembly of β-barrel proteins into membranes is mediated by the evolutionarily conserved BAM complex. In Escherichia coli , BAM folds numerous substrates which vary considerably in size and shape. How BAM is able to efficiently fold such a diverse array of β-barrel substrates is not clear. Here, we develop a disulfide crosslinking method to trap native substrates in vivo as they fold on BAM. By placing a cysteine within the luminal wall of the BamA barrel as well as in the substrate β-strands, we can compare the residence time of each substrate strand within the BamA lumen. We validated this method using two defective, slow-folding substrates. We used this method to characterize stable intermediates which occur during folding of two structurally different native substrates. Strikingly, these intermediates occur during identical stages of folding for both substrates: soon after folding has begun, and just before folding is completed. We suggest that these intermediates arise due to barriers to folding that are common between β-barrel substrates, and that the BAM catalyst is able to fold so many different substrates because it addresses these common challenges. Significance Statement The outer membrane of Gram-negative bacteria is a barrier which protects these organisms from many antimicrobial agents. Here, we study the machine responsible for folding and inserting integral β-barrel proteins into the membrane: BAM. Outer membrane integrity and cell viability is dependent on the proper function of BAM. Here we show that stable intermediates exist on the folding pathway of native substrates. We also show that mutant substrates that increase the stability of these native intermediates can stall during folding. This creates permeability defects that can be exploited by antibiotics that normally do not cross the outer membrane. These observations could enable the design of strategies to combat Gram-negative pathogens.