Project 02

Mechanism of bacterial transenvelope protein and lipid transport

Principle Investigator: Prof. Dr. Benesh Joseph

Research Areas: Membrane Biophysics, EPR Spectroscopy

Summary

The cell envelope of Gram-negative bacteria consists of an inner membrane (IM) and an outer membrane (OM) that protects cells from harsh conditions. The OM is an asymmetric bilayer made up of phospholipids (PL) and lipopolysaccharides (LPS) and harbors numerous β-barrel proteins (outer membrane proteins, OMPs). The LptDE translocon mediates the final step of LPS insertion into the OM. Folding and insertion of unfolded OMPs from the periplasm into the OM is mediated by the β-barrel assembly machinery (BAM) with the assistance of periplasmic chaperones, primarily SurA. We will use in situ pulsed electron spin resonance (ESR) spectroscopy combined with other biophysical approaches to elucidate the structural and dynamical basis for outer membrane biogenesis. For SurA, its substrate-dependent conformational rearrangements and interaction with BAM complex will be investigated. For LptDE, conformational dynamics/heterogeneity of its key structural elements will be characterized in the native membrane environment in presence of LPS or the periplasmic LPS binding protein LptA. The binding and inhibition mode for the peptidomimetic antibiotic thanatin on LptDE will be investigated in a similar manner. Finally, we will develop new ESR approaches to study heterooligomeric OMP complexes in the native membranes employing different spin labels and unnatural amino acids.