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 membrane-attached 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.
Highlights
The cell envelope of Gram-negative bacteria is an elaborate cellular environment, consisting of two lipid membranes separated by the aqueous periplasm
In a pioneering study using atomic force microscopy (AFM), we demonstrated that proteins enriched in this way persisted in the membrane of outer membrane vesicles (OMVs) in their native conformation.[9]
To obtain OmpA-enriched OMVs uniformly labeled with stable isotopes (2H and 15N) to perform solution-state nuclear magnetic resonance (NMR) experiments, culture conditions were optimized for growth in D2O-based M9 minimal medium
Summary
The cell envelope of Gram-negative bacteria is an elaborate cellular environment, consisting of two lipid membranes separated by the aqueous periplasm. The latter eliminates possible background signals originating from OmpA-CTD and results in an increased level of vesiculation compared to that of the parental BL21 strain.[18] To test the possibility of studying periplasmic proteins, we prepared OMVs luminally enriched in CpxP (Figure S1B).
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