Mechanistic basis for outer membrane biogenesis investigated under in situ conditions using dipolar EPR spectroscopy

• Applicant: Benesh Joseph, Ph.D.
• Subject Area: Biophysics ; Structural Biology
• Term: since 2018
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 411411450

The outer membrane (OM) of Gram-negative bacteria is an asymmetric bilayer consisting of phospholipids and lipopolysaccharides (LPS). It harbors numerous β-barrel proteins (outer membrane proteins, OMPs) involved in diverse cellular processes. The LPS molecules and the OMPs are synthesized in the cytoplasm. Thus, the biogenesis of the OM involves transport of millions of LPS molecules as well as the folding and insertion of numerous OMPs during each cell cycle. In Escherichia coli, seven essential proteins, LptABCDEFG form the LPS transport (Lpt) system, with the ABC exporter LptB2FG forming the energizing module. The folding and insertion of OMPs is mediated by the β-barrel assembly machinery (BAM) complex, which consists of the BamABCDE subunits. Both BAM and Lpt system are essential and conserved and they are potential targets for developing novel antibacterial drugs. The overall objective of this project is to unravel the mechanistic basis for OM biogenesis by the BAM and the Lpt system. The large heterooligomeric architecture, complex nature of the substrates, and an extremely dynamic nature make these systems challenging targets for high-resolution structural biology. The BAM and the Lpt systems might excurse through a complex energy landscape and undergo large conformational changes to achieve their functions. Thus the mechanistic description of their functions necessitates an understanding of conformational changes and equilibrium dynamics. Moreover, OM is an integral part of these molecular machineries as they insert the substrates directly into the OM. Therefore; investigations under in situ conditions (whole E. coli and native OM) are required for a holistic understanding of their function. We aim to characterize the conformational heterogeneity and dynamics that form the mechanistic basis for BAM and LptB2FGC complex function under in vitro and in situ conditions. For this purpose, we will employ state of the art EPR spectroscopy techniques combined with biochemical and biophysical tools. For BAM, conformational dynamics of the transmembrane β-barrel BamA and its modulation by the lipoproteins (BamB-E) and or the substrate will be characterized in E. coli, native OM and detergent solution to elucidate the mechanistic basis of OMP folding and insertion. For the Lpt system, we will investigate the mechanism for energy transduction in the unusual ABC exporter LptB2FG (which energizes the whole transport system, PDB 5X5Y), in detergent solution and native lipid membranes. The LptB2FGC complex will be modeled using PELDOR (Pulsed Electron-Electron Double Resonance, also known as DEER) constraints and LPS transport by this complex will be elucidated in the native lipid bilayers. Both of these subprojects will further benefit from the third subproject in which we will further develop the in situ EPR for spin labeling and PELDOR in the periplasm of E. coli. New spin labels and different labeling strategies will be employed for this purpose.

• DFG Programme: Independent Junior Research Groups