Autotransporters are the largest family of virulent outer membrane proteins in Gram-negative bacteria, and are defined by their unique modular structure. An autotransporter consists of an N-terminal signal sequence, which translocates the protein across the inner membrane into the periplasm, a 12-stranded β-barrel domain that is embedded in the outer membrane and helps translocate the passenger domain (the module responsible for the protein’s virulence) to the bacterial cell surface. In previous studies of the β-barrel domain, a motif containing a tyrosine interacting with a glycine residue on an adjacent β-strand was found to affect stability and translocation of the autotransporter when removed. This was called the mortise-tenon joint. Using the autotransporter Pet (plasmid encoded toxin) as a model protein, we investigated the role of the mortise-tenon joint on passenger domain translocation rate and stability by adding additional hypothetical mortise-tenon joints to the β-barrel. Using biochemical and biophysical assays it was shown that the mutations introduced had negligible effect on translocation and stability, but a substantial effect on protein folding rates and folding efficiency. This suggests that the mortise-tenon joint is an important motif for the regulation of the folding dynamics in autotransporters and perhaps all β-barrel proteins in general.