Conformational heterogeneity of the β-barrel assembly machinery investigated by pulsed electron-electron double resonance spectroscopy
Authors:   , Gopinath Pandarakandy Aathira 
Published: October 2024

With the technological advancements over the past years, structure determination and prediction for membrane proteins have become easier. While those approaches give snapshots of one or more conformational states of the protein, complementary techniques are necessary to elucidate the conformational space and transition between states during function. Electron paramagnetic resonance (EPR) spectroscopy is a powerful tool for addressing these aspects. In this thesis, site-directed spin labeling and pulsed electron-electron double resonance (PELDOR) spectroscopy combined with various biochemical tools were used to explore the conformational heterogeneity of the β-barrel assembly machinery (BAM) complex under in vitro and in situ conditions. The BAM complex present in the outer membrane (OM) of gram-negative bacteria is responsible for the folding and insertion of the outer membrane proteins (OMPs). As the majority of OMPs depend on the BAM complex for their biogenesis, it is one of the most essential components for the cell and hence a potential target for new antibiotics. BAM is a heterooligomeric complex composed of BamA, BamB, BamC, BamD, and BamE subunits. BamA is the central transmembrane protein directly involved in the folding and insertion process. The periplasmic regions of BamA are scaffolded by BamB-E lipoproteins. Available structures of the BAM complex reveal a highly dynamic behaviour. BAM complex is also highly intertwined with the complex membrane environment and is hypothesized to be dependent on the asymmetric bilayer for its function. The functional relevance of the accessory lipoproteins or how BAM recruits and folds diverse OMPs remains elusive. The thesis examines the membrane bilayer dependence of the BAM complex and the role of the lipoproteins in the conformational cycling of BamA. By comparing the conformational states of the central component BamA in detergent micelles and isolated native outer membranes, it is demonstrated that the native bilayer helps BamA attain multiple conformational states. In the native outer membrane environment, BamA exhibits greater flexibility than observed in the detergent micelles. Further, the conformational dynamics of BamA were explored in different subcomplexes in detergent micelles. The binding of BamCDE subcomplex creates specific changes in BamA at the lateral gate, periplasmic regions, and extracellular loops leading to a lateral-open state. BamB alone does not induce any changes in BamA, revealing that it might play an accessory role in the function of the complex. The results demonstrate that BamCDE plays a key regulatory role in the lateral gating mechanism of BamA. Additionally, the spin labeling and PELDOR spectroscopy were optimized for the extracellular loops of the full complex in intact E. coli cells. The data validates the conformational states of the complex observed in the detergent micelles. However, the distance distributions show increased dynamics, especially at the lateral gate region in the cellular environments. The increased heterogeneity might be due to the presence of the asymmetric membrane, lipopolysaccharides, or substrate interactions. Overall, the thesis answers key questions on the conformational dynamics of BamA and delineates the role of lipoproteins in the folding mechanism. It also provides new opportunities to study the functional mechanism of BAM under physiologically relevant conditions by performing experiments in native outer membranes and intact E. coli cells.