Resolving the Structural Basis of Bacterial Envelope Homeostasis In Situ

Proteins and their functional states are directly influenced by their surrounding molecular environment.

This poses an imminent problem for the characterization of proteins using biophysical methods, which demand proteins in a highly pure form and isolated from their cellular environment.

Here, I will address this discrepancy and for the first time structurally characterize key proteins involved in bacterial envelope homeostasis within the native environment.

To this end I will exploit a unique approach utilizing bacterial outer membrane vesicles (OMVs) with a controlled protein composition to characterize how envelope proteins are impacted by the native environment: I will determine how the crowded periplasmic environment impacts the structures and dynamics controlling the function of folding factors Skp and SurA, which play a crucial role in delivering proteins to the outer membrane, using solution NMR spectroscopy.

In parallel, I will resolve how the membrane protein PagP interacts with the asymmetric outer membrane, where it enzymatically modifies bacterial lipopolysaccharides, and how the β-barrel assembly machinery BAM complex facilitates protein insertion into the native membrane using solid-state NMR spectroscopy, aided by cryo-electron tomography.

The project has the unique potential to address questions, which cannot be answered on the basis of existing methods, and to provide a methodological milestone in the emerging era of structural biology in situ.