One major objective of synthetic biology is the bottom-up assembly of minimalistic nanocells consisting of lipid or polymer vesicles as architectural scaffolds and of membrane and soluble proteins as functional elements. However, there is no reliable method to orient membrane proteins reconstituted into vesicles. Here, we introduce a simple approach to orient the insertion of the light-driven proton pump proteorhodopsin (PR) into liposomes. To this end, we engineered red or green fluorescent proteins to the N- or C-terminus of PR, respectively. The fluorescent proteins optically identified the PR constructs and guided the insertion of PR into liposomes with the unoccupied terminal end facing inward. Using the PR constructs, we generated proton gradients across the vesicle membrane along predefined directions such as are required to power (bio)chemical processes in nanocells. Our approach may be adapted to direct the insertion of other membrane proteins into vesicles.

Light-driven proton pumps, such as proteorhodopsin, have been proposed as an energy source in the field of synthetic biology. Energy is required to power biochemical reactions within artificially created reaction compartments like proto-or nanocells, which are typically based on either lipid or polymer membranes. The insertion of membrane proteins into these membranes is delicate and quantitative studies comparing these two systems are needed. Here we present a detailed analysis of the formation of proteoliposomes and proteopolymersomes and the requirements for a successful reconstitution of the membrane protein proteorhodopsin. To this end, we apply design of experiments to provide a mathematical framework for the reconstitution process. Mathematical optimization identifies suitable reconstitution conditions for lipid and polymer membranes and the obtained data fits well to the predictions. Altogether, our approach provides experimental and modeling evidence for different reconstitution mechanisms depending on the membrane type which resulted in a surprisingly similar performance.

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.