Mapping the Folding Pathway of an Essential Outer Membrane Protein by the β-Barrel Assembly Machine

Abstract

Membrane β-barrel proteins within the outer membranes of Gram-negative bacteria play important roles in maintaining the integrity of the cell envelope and protecting the cell from the outside world. In E. coli, the β-barrel assembly machine (Bam) complex accelerates their insertion into the membrane. This complex is conserved across Gram-negative bacteria and key components are also conserved in chloroplasts and mitochondria. Outer membrane protein assembly by the Bam complex is a complex process that involves substrate recognition, barrel folding, and membrane insertion. Whether folding and insertion occur in a defined sequence or are coupled together in one concerted step remains an open question largely because of difficulties in characterizing intermediates in the process of folding on the Bam complex. This thesis describes a series of biochemical and genetic analyses that interrogate how structure emerges within an essential substrate as it interacts with the Bam complex to identify the requirements that permit membrane integration. Capture and characterization of assembly intermediates demonstrate that barrel folding begins in the periplasm and that during insertion, substrates must traverse the interior of the central component of the Bam complex, the β-barrel BamA. The entirety of the substrate barrel forms prior to completion of membrane insertion with a large portion of its N-terminus residing within the lumen BamA. Barrel closure triggered by extracellular loop interactions within the substrate then controls release into the membrane. The work presented suggests a comprehensive physical model for the insertion path of substrates into the membrane by the Bam complex. A detailed mechanistic understanding of how these outer membrane proteins are assembled may allow us to identify antimicrobial strategies to interfere with outer membrane biogenesis.