High-Resolution In Situ NMR Spectroscopy of Bacterial Envelope Proteins in Outer Membrane Vesicles

Abstract

The cell envelope of Gram-negative bacteria is an elaborate cellular environment, consisting of two lipid membranes separated by the aqueous periplasm. So far, efforts to mimic this environment under laboratory conditions have been limited by the complexity of the asymmetric bacterial outer membrane. To evade this impasse, we recently established a method to modify the protein composition of bacterial outer membrane vesicles (OMVs) released from Escherichia coli as a platform for biophysical studies of outer membrane proteins in their native membrane environment. Here, we apply protein-enriched OMVs to characterize the structure of three envelope proteins from E. coli using nuclear magnetic resonance (NMR) spectroscopy and expand the methodology to soluble periplasmic proteins. We obtain high-resolution in situ NMR spectra of the transmembrane protein OmpA as well as the periplasmic proteins CpxP and MalE. We find that our approach facilitates structural investigations of membraneattached protein domains and is especially suited for soluble proteins within their native periplasmic environment. Thereby, the use of OMVs in solution NMR methods allows in situ analysis of the structure and dynamics of proteins twice the size compared to the current in-cell NMR methodology. We therefore expect our work to pave the way for more complex NMR studies of bacterial envelope proteins in the native environment of OMVs in the future.

Conformational Flexibility of the Protein Insertase BamA in the Native Asymmetric Bilayer Elucidated by ESR Spectroscopy

“…Chemie states.I ti sr eassuring to confirm the lateral gate,L 3-L8, T1-T6, and the T7-POTRA5d istances corresponding to the different conformations of BamA (which were solved in different detergent micelles) in the native membrane.Atthe same time,i ti sa lso shown that the native environment can populate an equilibrium between those states and may further modulate the structure.Complementary to the developments in the in situ solid-state NMR spectroscopy, [57,58] our approach offers agreat opportunity for further investigations of BamA and the BAMc omplex in the native outer membrane environment.…”

Conformational Flexibility of the Protein Insertase BamA in the Native Asymmetric Bilayer Elucidated by ESR Spectroscopy

“…states.I ti sr eassuring to confirm the lateral gate,L 3-L8, T1-T6, and the T7-POTRA5d istances corresponding to the different conformations of BamA (which were solved in different detergent micelles) in the native membrane.Atthe same time,i ti sa lso shown that the native environment can populate an equilibrium between those states and may further modulate the structure.Complementary to the developments in the in situ solid-state NMR spectroscopy, [57,58] our approach offers agreat opportunity for further investigations of BamA and the BAMc omplex in the native outer membrane environment.…”

Role of the lipid bilayer in outer membrane protein folding in Gram-negative bacteria

“…At the same time, modern biophysical and biochemical tools are also needed to make these same measurements directly on OMPs in their native context within bacteria. En route to this, the use of outer membrane vesicles directly derived from bacteria may provide an excellent stepping stone ( 393 – 395 ).…”