In vitro Reconstitution to Understand the Mechanism of Bam

Abstract

The outer membrane of Gram-negative bacteria is an essential asymmetric lipid bilayer that serves as a protective barrier and performs required cellular functions, such as nutrient uptake and export. Within the outer membrane are trans-membrane proteins that adopt unique beta-barrel structures. These outer membrane proteins (Omps) are folded and inserted by a multi-protein complex embedded in the membrane called the Bam complex, which performs this function without the use of chemical energy (i.e. ATP or proton motive force). The Bam complex is made of five proteins, BamA-E. BamA is a beta-barrel itself and is considered the central component of the complex. Crystal structures and molecular dynamics simulations suggests that BamA has a destabilized barrel seam that can open and close to potentially nucleate new beta-barrels. Locking the barrel seam in vivo with engineered disulfides is lethal, suggesting that this lateral gate is essential for BamA function. We tested the lateral gate hypothesis in a minimal in vitro system with BamA and a substrate Omp, OmpX in synthetic liposomes. In this system, locking the barrel has no affect on the activity of BamA – locked barrels had the same activity as unlocked barrels. This suggests that either the lateral gate is not important for function or we are not capturing the full activity of BamA. Interestingly, disulfide cross-linking experiments suggest that the barrel seam is highly dynamic, since disulfide pairs separated by up to 4 residues are able to form. To further probe the lateral gate hypothesis, we have moved into a fully reconstituted system with the Bam complex in native E. coli lipids. This system will help us differentiate between these two possibilities. Understanding how the Bam complex assists in protein folding will help answer fundamental questions about membrane protein folding.