Determining the mechanism of β-barrel assembly machinery (BAM) in bacterial outer membranes (360G-Wellcome-105220_Z_14_Z)

Outer membrane proteins (OMPs) in Gram negative bacteria are critical for bacterial survival and virulence 1 . However how these beta-barrels fold in the membrane is not well understood. The in vivo folding of most substrate OMPs relies on the function of the beta -barrel assembly complex (BAM)2, of which the key component is BamA 3, itself a beta-barrel. Here we propose to use a panoply of structural and biophysical methods to dissect the functionality of BamA and the mechanism of OMP folding. Our key aims are: 1) To investigate the hypothesis that BamA functions by lateral gating. Using a combination of disulphide linking and FRET we will determine whether lateral gating is necessary for the folding of different OMPs, and how it may function. 2) To determine how BamA function is affected by liposome size and membrane crowding, by utilising a varied subset of lipid types and comparison of substrate OMPs in folding assays. 3) To determine the importance, and role of the beta -signal of OMPs in the interaction with BamA by dynamic force spectroscopy measurements. The question of how OMPs fold represents a fundamental gap in structural biology understanding. In addition, the OMPs of Gram negative bacteria are key to their pathogenicity, therefore understanding the mechanism of BAM may present new possibilities for drug targets.