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.

Fluorescent proteins are indispensable for selective, quantitative visualization of localization, dynamics, and interactions of key molecular constituents of live cells. Incorporation of fluorescent proteins into an optical cavity can lead to a significant increase in fluorescence signal levels due to stimulated emission and light amplification in the cavity, forming a laser with biological gain medium. Utilization of lasing emission from fluorescent biological molecules can then greatly enhance the performance of fluorescence-based biosensors benefiting from the high sensitivity of non-linear lasing processes to small perturbations in the cavity and the gain medium. Here we study optofluidic biolasers that exploit active liquid optical resonators formed by surface-supported aqueous microdroplets containing purified yellow fluorescent protein or a suspension of live E. coli bacterial cells expressing the fluorescent protein. We first demonstrate lasing in fluorescent protein solutions at concentrations as low as 49 μM. Subsequently, we show that a single fluorescent bacterial cell of micrometre size confined in a droplet-based cavity can serve as a laser gain medium. Aqueous droplet microcavities allow the maintenance of the bacterial cells under conditions compatible with unimpeded growth. Therefore, our results also suggest a direct route to microscopic sources of laser light with self-regenerating gain media.

Highlights d Mechanical, kinetic, and energetic properties of BamA d Properties change upon binding to an antibiotic d Structural regions change mechanical stability and lifetime d Structural regions change free-energy and mechanical rigidity

Polymyxins are last-resort antibiotics with potent activity against multi-drug resistant pathogens. They interact with lipopolysaccharide (LPS) in bacterial membranes, but mechanistic details at the molecular level remain unclear. Here, we characterize the interaction of polymyxins with native, LPS-containing outer membrane patches of Escherichia coli by high-resolution atomic force microscopy imaging, along with structural and biochemical assays. We find that polymyxins arrange LPS into hexagonal assemblies to form crystalline structures. Formation of the crystalline structures is correlated with the antibiotic activity, and absent in polymyxin-resistant strains. Crystal lattice parameters alter with variations of the LPS and polymyxin molecules. Quantitative measurements show that the crystalline structures decrease membrane thickness and increase membrane area as well as stiffness. Together, these findings suggest the formation of rigid LPS–polymyxin crystals and subsequent membrane disruption as the mechanism of polymyxin action and provide a benchmark for optimization and de novo design of LPS-targeting antimicrobials.

We observe how translocon and insertase insert single polypeptides along remarkably different pathways to fold membrane proteins.

Photochromic fluorescence resonance energy transfer (pcFRET) was used to monitor the redox activity of non-fluorescent heme protein. Venus fluorescent protein was used as a donor where its emission intensity was reversibly modulated by the absorption change of Cytochrome c.

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