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Hybrid membranes assembled from biological lipids and synthetic polymers are a promising scaffold for the reconstitution and utilization of membrane proteins. Recent observations indicate that inclusion of small fractions of polymer in lipid membranes can improve protein folding and function, but the exact structural and physical changes a given polymer sequence imparts on a membrane often remain unclear. Here, we use all-atom molecular dynamics simulations to study the structure of hybrid membranes assembled from DOPC phospholipids and PEO

Cell-free gene expression (CFE) systems are powerful tools for transcribing and translating genes outside of a living cell. Given their diverse roles in nature, synthesis of membrane proteins is of particular interest, but their yield in CFE is substantially lower than for soluble protein. In this paper, we study factors that affect the cell-free synthesis of membrane proteins and develop a quantitative kinetic model of their production. We identify that stalling of membrane protein translation on the ribosome is a strong predictor of membrane protein synthesis and creates a negative feedback loop in which stalled peptide sequences quench ribosome activity through aggregation between the ribosome nascent chains. Synthesis can be improved by the addition of lipid membranes which incorporate protein nascent chains and, therefore, kinetically competes with aggregation. Using both quantitative modeling and experiment, we show that the balance between peptide-membrane association and peptide aggregation rates determines the total yield of synthesized membrane protein. We then demonstrate that this balance can be shifted by altering membrane composition or the protein N-terminal domain sequence. Based on these findings, we define a membrane protein expression score that can be used to rationalize the engineering of N-terminal domain sequences both of a native and computationally designed membrane proteins produced through CFE.

Supported lipid bilayers (SLBs) hold tremendous promise as cellular-mimetic structures that can be readily interfaced with analytical and screening tools. The incorporation of transmembrane proteins, a key component in biological membranes, is a significant challenge that has limited the capacity of SLBs to be used for a variety of biotechnological applications. Here, we report an approach using a cell-free expression system for the cotranslational insertion of membrane proteins into hybrid-supported lipid bilayers (HSLBs) containing phospholipids and diblock copolymers. We use cell-free expression techniques and a model transmembrane protein, the large conductance mechanosensitive channel (MscL), to demonstrate two routes to integrate a channel protein into a HSLB. We show that HSLBs can be assembled with integrated membrane proteins by either cotranslational integration of protein into hybrid vesicles, followed by fusion of these proteoliposomes to form a HSLB, or preformation of a HSLB followed by the cell-free synthesis of the protein directly into the HSLB. Both approaches lead to the assembly of HSLBs with oriented proteins. Notably, using single-particle tracking, we find that the presence of diblock copolymers facilitates membrane protein mobility in the HSLBs, a critical feature that has been difficult to achieve in pure lipid SLBs. The approach presented here to integrate membrane proteins directly into preformed HSLBs using cell-free cotranslational insertion is an important step toward enabling many biotechnology applications, including biosensing, drug screening, and material platforms requiring cell membrane-like interfaces that bring together the abiotic and biotic worlds and rely on transmembrane proteins as transduction elements.

Hybrid membranes comprised of diblock copolymers, and phospholipids have gained interest due to their unique properties that result from blending natural and synthetic components. The integration of membrane proteins into these synthetic membranes is an important step towards creating biomembrane systems for uses such as artificial cellular systems, biosensors, and drug delivery vehicles. Here, we outline a technique to create hybrid membranes composed of phospholipids and diblock copolymers. Next, we describe how membrane proteins can be co-translationally integrated into hybrid lipid/polymer membranes using a cell-free reaction. We then outline a method to monitor insertion and folding of a membrane-embedded channel protein into the hybrid membrane using a fluorescent-protein reporter and dye release assay, respectively. This method is expected to be applicable for a wide range of membrane proteins that do not require chaperones for co-translational integration into vesicles and provides a generalized protocol for expressing a membrane protein into a membrane mimetic.

The assembly of channel proteins into vesicle membranes is a useful strategy to control activities of vesicle-based systems. Here, we developed a membrane AND gate that responds to both a fatty acid and a pore-forming channel protein to induce the release of encapsulated cargo. We explored how membrane composition affects the functional assembly of α-hemolysin into phospholipid vesicles as a function of oleic acid content and α-hemolysin concentration. We then showed that we could induce α-hemolysin assembly when we added oleic acid micelles to a specific composition of phospholipid vesicles. Finally, we demonstrated that our membrane AND gate could be coupled to a gene expression system. Our study provides a new method to control the temporal dynamics of vesicle permeability by controlling when the functional assembly of a channel protein into synthetic vesicles occurs. Furthermore, a membrane AND gate that utilizes membrane-associating biomolecules introduces a new way to implement Boolean logic that should complement genetic logic circuits and ultimately enhance the capabilities of artificial cellular systems.

Significance Membrane protein folding is a critical step that underlies proper cellular function as well as the design of technologies like vesicle-based biosensors and artificial cells. Membrane composition is known to play a role in membrane protein folding; however, the specific mechanical properties of membranes that govern protein folding remain unclear. Using a highly elastic nonnatural amphiphile, we highlight the importance of a membrane mechanical property, membrane elasticity, on the spontaneous insertion and folding of a model α-helical membrane protein. Through this study, we gain a deeper understanding of cellular membrane protein folding and offer a potential approach to improve the production of membrane proteins through optimizing the mechanical properties of synthetic scaffolds present in cell-free reactions., The expression and integration of membrane proteins into vesicle membranes is a critical step in the design of cell-mimetic biosensors, bioreactors, and artificial cells. While membrane proteins have been integrated into a variety of nonnatural membranes, the effects of the chemical and physical properties of these vesicle membranes on protein behavior remain largely unknown. Nonnatural amphiphiles, such as diblock copolymers, provide an interface that can be synthetically controlled to better investigate this relationship. Here, we focus on the initial step in a membrane protein’s life cycle: expression and folding. We observe improvements in both the folding and overall production of a model mechanosensitive channel protein, the mechanosensitive channel of large conductance, during cell-free reactions when vesicles containing diblock copolymers are present. By systematically tuning the membrane composition of vesicles through incorporation of a poly(ethylene oxide)-b-poly(butadiene) diblock copolymer, we show that membrane protein folding and production can be improved over that observed in traditional lipid vesicles. We then reproduce this effect with an alternate membrane-elasticizing molecule, C12E8. Our results suggest that global membrane physical properties, specifically available membrane surface area and the membrane area expansion modulus, significantly influence the folding and yield of a membrane protein. Furthermore, our results set the stage for explorations into how nonnatural membrane amphiphiles can be used to both study and enhance the production of biological membrane proteins.

Polyunsaturated fatty acids (PUFAs) modify the activity of a wide range of membrane proteins and are increasingly hypothesized to modulate protein activity by indirectly altering membrane physical properties. Among the various physical properties affected by PUFAs, the membrane area expansion modulus (Ka), which measures membrane strain in response to applied force, is expected to be a significant controller of channel activity. Yet, the impact of PUFAs on membrane Ka has not been measured previously. Through a series of micropipette aspiration studies, we measured the apparent Ka (Kapp) of phospholipid model membranes containing nonesterified fatty acids. First, we measured membrane Kapp as a function of the location of the unsaturated bonds and degree of unsaturation in the incorporated fatty acids and found that Kapp generally decreases in the presence of fatty acids with three or more unsaturated bonds. Next, we assessed how select ω-3 PUFAs, eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), affect the Kapp of membranes containing cholesterol. In vesicles prepared with high amounts of cholesterol, which should increase the propensity of the membrane to phase segregate, we found that inclusion of DHA decreases the Kapp in comparison to EPA. We also measured how these ω-3 PUFAs affect membrane fluidity and bending rigidity to determine how membrane Kapp changes in relation to these other physical properties. Our study shows that PUFAs generally decrease the Kapp of membranes and that EPA and DHA have differential effects on Kapp when membranes contain higher levels of cholesterol. Our results suggest membrane phase behavior and the distribution of membrane-elasticizing amphiphiles impact the ability of a membrane to stretch.

Targeted vesicle fusion is a promising approach to selectively control interactions between vesicle compartments and would enable the initiation of biological reactions in complex aqueous environments. Here, we explore how two features of vesicle membranes, DNA tethers and phase-segregated membranes, promote fusion between specific vesicle populations. We show that membrane phase-segregation provides an energetic driver for membrane fusion that increases the efficiency of DNA-mediated fusion events. Using this system, we show that orthogonality provided by DNA tethers allows us to direct fusion and delivery of DNA cargo to specific vesicle populations. We then demonstrate that vesicle fusion between DNA-tethered vesicles can be used to initiate in vitro protein expression that leads to the synthesis of model soluble and membrane proteins. The ability to engineer orthogonal fusion events between DNA-tethered vesicles will provide a new strategy to control the spatio-temporal dynamics of cell-free reactions, expanding opportunities to engineer artificial cellular systems.

Targeted vesicle fusion is a promising approach to selectively control interactions between vesicle compartments and would enable the initiation of biological reactions in complex aqueous environments. Here, we explore how two features of vesicle membranes, DNA tethers and phase-segregated membranes, promote fusion between specific vesicle populations. We show that membrane phase-segregation provides an energetic driver for membrane fusion that increases the efficiency of DNA-mediated fusion events. Using this system, we show that orthogonality provided by DNA tethers allows us to direct fusion and delivery of DNA cargo to specific vesicle populations. We then demonstrate that vesicle fusion between DNA-tethered vesicles can be used to initiatein vitroprotein expression that leads to the synthesis of model soluble and membrane proteins. The ability to engineer orthogonal fusion events between DNA-tethered vesicles will provide a new strategy to control the spatio-temporal dynamics of cell-free reactions, expanding opportunities to engineer artificial cellular systems.