Protein-enriched outer membrane vesicles as a native platform for outer membrane protein studies

Abstract

Most studies characterizing the folding, structure, and function of membrane proteins rely on solubilized or reconstituted samples. Whereas solubilized membrane proteins lack the functionally important lipid membrane, reconstitution embeds them into artificial lipid bilayers, which lack characteristic features of cellular membranes including lipid diversity, composition and asymmetry. Here, we utilize outer membrane vesicles (OMVs) released from Escherichia coli to study outer membrane proteins (Omps) in the native membrane environment. Enriched in the native membrane of the OMV we characterize the assembly, folding, and structure of OmpG, FhuA, Tsx, and BamA. Comparing Omps in OMVs to those reconstituted into artificial lipid membranes, we observe different unfolding pathways for some Omps. This observation highlights the importance of the native membrane environment to maintain the native structure and function relationship of Omps. Our fast and easy approach paves the way for functional and structural studies of Omps in the native membrane.

Antibiotic uptake across gram-negative outer membranes: better predictions towards better antibiotics

“…In contrast, osmotic swelling of bacterial outer membrane vesicles provides a means to measure outer membrane permeability simply and directly in a bacterial membrane. Procedures for production and isolation of such OMVs have been well described [28,29], and they offer the advantage of a more physiological outer membrane (including LPS composition) than proteoliposomes [27]. Measuring osmotic swelling also permits the detection of a broader range of compounds than enzymatic detection approaches [8,30] and with less specialized apparatus than electrophysiological ones [24].…”

Unfolding and identification of membrane proteins in situ

“…However, the information that is possible to extrapolate from the F-D curves (e.g. mechanical stability 9,10 , structural heterogeneity 11 ) depends on the physical and chemical properties of the cell membrane 12,13 , therefore it is desirable to unfold membrane proteins in their original membrane.…”

Electrophysiological Characterization of Transport Across Outer‐Membrane Channels from Gram‐Negative Bacteria in Presence of Lipopolysaccharides

“…Other conditions resulted in 2D crystals, which in all likeliness are not readily able to fuse with a lipid bilayer. [25] Single-channel current traces resulted in somewhat lower conductance and with a broad standard deviation compared to purified and reconstituted single membrane protein. We compared single channel translocation data of OmpF-OMV with purified OmpF-WT and identified a barrier effect: lower event rates and slower translocation times.…”

“…[24] As they also contain outer membrane channels in their natural environment, we utilized them for channel characterization. [25] Within the current urgent need for novel antibiotics, the key questions concerning the contribution of the so-called porins to drug uptake are: why do some molecules permeate rapidly whereas others do not; which channels are involved; and what are the flux-limiting interactions between antibiotics and channels? Recently established methods such as whole cell uptake assays and mass spectrometry provide an answer to the total penetration, which is the relevant parameter for the survival of a cell.…”

“…The purification process yielded samples containing the overexpressed porins as their major protein component (Figure 1 A) as previously shown mass spectrometry analysis. [25,28] In order to determine OmpF/C concentration in the samples, direct measurement of protein concentration was not possible since other proteins are also present. To circumvent this issue, we used densitometry on Coomassie-stained SDS-PAGE to determine the porins concentration (Figure 1 B).…”