RAS GTPases are important mediators of oncogenesis in humans. However, pharmacological inhibition of RAS has proved challenging. Here, we describe a functionally critical region of RAS located outside the effector lobe that can be targeted for inhibition. We developed a synthetic binding protein (monobody), termed NS1, that bound with high affinity to both GTP- and GDP-bound states of H- and K-RAS but not N-RAS. NS1 potently inhibited growth factor signaling and oncogenic H- and K-RAS-mediated signaling and transformation but did not block oncogenic N-RAS, BRAF or MEK1. NS1 bound the α4-β6-α5 region of RAS disrupting RAS dimerization/nanoclustering, which in turn blocked CRAF:BRAF heterodimerization and activation. These results establish the importance of the α4-β6-α5 interface in RAS-mediated signaling and define a previously unrecognized site in RAS for inhibiting RAS function.

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.

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.

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. Membrane phase‐segregation provides an energetic driver for membrane fusion that increases the efficiency of DNA‐mediated fusion events. The orthogonality provided by DNA tethers allows us to direct fusion and delivery of DNA cargo to specific vesicle populations. Vesicle fusion between DNA‐tethered vesicles can be used to initiate in vitro protein expression to produce model soluble and membrane proteins. Engineering orthogonal fusion events between DNA‐tethered vesicles provides a new strategy to control the spatiotemporal dynamics of cell‐free reactions, expanding opportunities to engineer artificial cellular systems.

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 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 allatom molecular dynamics simulations to study the structure of hybrid membranes assembled from DOPC phospholipids and PEO-b-PBD diblock copolymers. We verified our computational model using new and existing experimental data and obtained a detailed picture of the polymer conformations in the lipid membrane that we can relate to changes in membrane elastic properties. We find that inclusion of low polymer fractions induces transient packing defects into the membrane. These packing defects act as insertion sites for two model peptides, and in this way, small amounts of polymer content in lipid membranes can lead to large increases in peptide insertion rates. Additionally, we report the peptide conformational space in both pure lipid and hybrid membranes. Both membranes support similar alpha helical peptide structures, exemplifying the biocompatibility of hybrid membranes.

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 (K a ), 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 K a has not been measured previously. Through a series of micropipette aspiration studies, we measured the apparent K a (K app ) of phospholipid model membranes containing nonesterified fatty acids. First, we measured membrane K app as a function of the location of the unsaturated bonds and degree of unsaturation in the incorporated fatty acids and found that K app generally decreases in the presence of fatty acids with three or more unsaturated bonds. Next, we assessed how select u-3 PUFAs, eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), affect the K app 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 K app in comparison to EPA. We also measured how these u-3 PUFAs affect membrane fluidity and bending rigidity to determine how membrane K app changes in relation to these other physical properties. Our study shows that PUFAs generally decrease the K app of membranes and that EPA and DHA have differential effects on K app 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. Membrane phase‐segregation provides an energetic driver for membrane fusion that increases the efficiency of DNA‐mediated fusion events. The orthogonality provided by DNA tethers allows us to direct fusion and delivery of DNA cargo to specific vesicle populations. Vesicle fusion between DNA‐tethered vesicles can be used to initiate in vitro protein expression to produce model soluble and membrane proteins. Engineering orthogonal fusion events between DNA‐tethered vesicles provides a new strategy to control the spatiotemporal dynamics of cell‐free reactions, expanding opportunities to engineer artificial cellular systems.