The impact of conformational dynamics on the function of the BAM complex

The β-barrel assembly machinery complex (BAM) is an essential Gram-negative bacterial protein complex that folds outer-membrane proteins (OMPs) into the outer-membrane (OM). The importance of OMPs in antibiotic resistance, combined with the essential nature of the complex, make BAM a promising antibiotic target. BAM’s mechanism is incompletely understood but seems dependent on conformational dynamics, particularly opening and closing of a lateral-gate in the core subunit, BamA. This thesis further examines the link between BAM’s conformation, and function. In Chapter Three, in vitro folding assays and cryogenic Electron Microscopy (cryoEM) showed that two disulphide lock mutants and a BAM-binding Fab fragment, all previously shown to be lethal in vivo, also inhibit BAM function and alter BAM’s conformational equilibrium in vitro. Additionally, a biophysical assay was used to show a global membrane disordering effect of BAM, dependent on the BamBCDE lipoproteins. Chapter Four investigated whether BAM mutations that potentiate BAM function in vivo also had conformational effects in vitro. In Chapter Five, pulsed Electron Paramagnetic Resonance (EPR) was used to monitor BAM’s conformation within the OM of intact E. coli cells. Overall, these results underscore the importance of conformational dynamics for BAM function, laying the groundwork for development of novel therapeutics which work by disrupting these dynamics.