# Reconstitution of the E. Coli Membrane $$\beta$$-Barrel Assembly Machine from Purified Components

 Title: Reconstitution of the E. Coli Membrane $$\beta$$-Barrel Assembly Machine from Purified Components Author: Hagan, Christine Lepicier Citation: Hagan, Christine Lepicier. 2012. Reconstitution of the E. Coli Membrane $$\beta$$-Barrel Assembly Machine from Purified Components. Doctoral dissertation, Harvard University. Full Text & Related Files: Hagan_gsas.harvard_0084L_10202.pdf (20.92Mb; PDF) Abstract: $$\beta$$-barrel membrane proteins perform important functions in the outer membranes of Gram-negative bacteria and in the mitochondria and chloroplasts of eukaryotes. Cellular machines that have been conserved from bacteria to humans assemble these proteins by an unknown mechanism. The components of the $$\beta$$-barrel assembly machine (Bam) in E. coli have been identified, but it has been difficult to study their function in vivo because they catalyze an essential process; mutations in proteins involved in the assembly pathway are often lethal or produce pleiotropic phenotypes that do not reveal the specific roles of the individual proteins. This study describes an in vitro reconstitution of the activity of the Bam complex and the use of this assay to determine how the Bam proteins contribute to the assembly of the complex itself. A sensitive assay for $$\beta$$-barrel assembly was developed using a substrate protein that has protease activity when it is folded. A peptide bond cleavage thereby reports on the conformational change the Bam complex catalyzes. This assay demonstrates that the Bam proteins dramatically increase the rate of $$\beta$$-barrel assembly without any external energy source. The structures of these proteins must inherently facilitate the folding and insertion process. The in vitro reconstitution was then adapted to study the assembly of the central component of the Bam complex, BamA. These studies reveal that the conserved domains of BamA catalyze the steps in the assembly process that are common in all organisms. The accessory components of the Bam complex adapt the mechanism of BamA to improve its efficiency and to allow it to handle a diverse set of substrates. The assembly of the Bam complex thus demonstrates how a cellular machine evolves to achieve generality and high efficiency. A structure of the Bam complex will be required to understand the molecular details of how substrate proteins are bound, folded into $$\beta$$-barrel structures, and inserted into the membrane. Initial efforts indicate that it will be possible to obtain such a structure. By combining structural and biochemical information garnered from the in vitro reconstitution, the general principles that guide the assembly of membrane $$\beta$$-barrels may be determined. Terms of Use: This article is made available under the terms and conditions applicable to Other Posted Material, as set forth at http://nrs.harvard.edu/urn-3:HUL.InstRepos:dash.current.terms-of-use#LAA Citable link to this page: http://nrs.harvard.edu/urn-3:HUL.InstRepos:9556126 Downloads of this work: