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.
Highlights
Most studies characterizing the folding, structure, and function of membrane proteins rely on solubilized or reconstituted samples
This is because essential properties such as the specific lipid composition and the unique lipid asymmetry of the two leaflets of the bilayer are lost during reconstitution
To investigate outer membrane proteins (Omps) in the native outer membrane we searched for ways to overexpress Omps in E. coli and to purify the outer membrane vesicles (OMVs) released by the bacteria (Fig. 1a and Methods)
Summary
Most studies characterizing the folding, structure, and function of membrane proteins rely on solubilized or reconstituted samples. The majority of functional and structural studies of membrane proteins rely on samples that have been extracted from native membranes by solubilization and reconstituted into artificial lipid bilayers. Membrane proteins reconstituted into artificial lipid bilayers are frequently used for biophysical and biochemical studies, the bilayers can only roughly mimic the native environment of the much more complex cellular membrane. This is because essential properties such as the specific lipid composition and the unique lipid asymmetry of the two leaflets of the bilayer are lost during reconstitution. In an attempt to overcome these problems, we here introduce a fast and easy approach to prepare outer membrane proteins (Omps) in native membranes
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