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Protein synthesis in natural cells involves intricate interactions between chemical environments, protein-protein interactions, and protein machinery. Replicating such interactions in artificial and cell-free environments can control the precision of protein synthesis, elucidate complex cellular mechanisms, create synthetic cells, and discover new therapeutics. Yet, creating artificial synthesis environments, particularly for membrane proteins, is challenging due to the poorly defined chemical-protein-lipid interactions. Here, we introduce MEMPLEX (Membrane Protein Learning and Expression), which utilizes machine learning and a fluorescent reporter to rapidly design artificial synthesis environments of membrane proteins. MEMPLEX generates over 20,000 different artificial chemical-protein environments spanning 28 membrane proteins. It captures the interdependent impact of lipid types, chemical environments, chaperone proteins, and protein structures on membrane protein synthesis. As a result, MEMPLEX creates new artificial environments that successfully synthesize membrane proteins of broad interest but previously intractable. In addition, we identify a quantitative metric, based on the hydrophobicity of the membrane-contacting amino acids, that predicts membrane protein synthesis in artificial environments. Our work allows others to rapidly study and resolve the “dark” proteome using predictive generation of artificial chemical-protein environments. Furthermore, the results represent a new frontier in artificial intelligence-guided approaches to creating synthetic environments for protein synthesis. Membrane proteins are notoriously difficult to synthesize. Here, authors introduce MEMPLEX, a high-throughput experimentation platform guided by machine learning that designs artificial cell-free environments to synthesize membrane proteins.

Synthetic biology has focused on engineering genetic modules that operate orthogonally from the host cells. A synthetic biological module, however, can be designed to reprogram the host proteome, which in turn enhances the function of the synthetic module. Here, we apply this holistic synthetic biology concept to the engineering of cell-free systems by exploiting the crosstalk between metabolic networks in cells, leading to a protein environment more favorable for protein synthesis. Specifically, we show that local modules expressing translation machinery can reprogram the bacterial proteome, changing the expression levels of more than 700 proteins. The resultant feedback generates a cell-free system that can synthesize fluorescent reporters, protein nanocages, and the gene-editing nuclease Cas9, with up to 5-fold higher expression level than classical cell-free systems. Our work demonstrates a holistic approach that integrates synthetic and systems biology concepts to achieve outcomes not possible by only local, orthogonal circuits. Synthetic biological modules can be used to reprogram host proteomes, which in turn enhance the function of the synthetic modules. The authors use this holistic synthetic biology approach to engineer a more favorable environment for cell-free protein synthesis.

We used micropipette aspiration to directly measure the area compressibility modulus, bending modulus, lysis tension, lysis strain, and area expansion of fluid phase 1-stearoyl, 2-oleoyl phosphatidylcholine (SOPC) lipid bilayers exposed to aqueous solutions of short-chain alcohols at alcohol concentrations ranging from 0.1 to 9.8 M. The order of effectiveness in decreasing mechanical properties and increasing area per molecule was butanol> propanol> ethanol> methanol, although the lysis strain was invariant to alcohol chain-length. Quantitatively, the trend in area compressibility modulus follows Traube’s rule of interfacial tension reduction, i.e., for each additional alcohol CH 2 group, the concentration required to reach the same area compressibility modulus was reduced roughly by a factor of 3. We convert our area compressibility data into interfacial tension values to: confirm that Traube’s rule is followed for bilayers; show that alcohols decrease the interfacial tension of bilayer-water interfaces less effectively than oil-water interfaces; determine the partition coefficients and standard Gibbs adsorption energy per CH 2 group for adsorption of alcohol into the lipid headgroup region; and predict the increase in area per headgroup as well as the critical radius and line tension of a membrane pore for each concentration and chain-length of alcohol. The area expansion predictions were confirmed by direct measurements of the area expansion of vesicles exposed to flowing alcohol solutions. These measurements were fitted to a membrane kinetic model to find membrane permeability coefficients of short-chain alcohols. Taken together, the evidence presented here supports a view that alcohol partitioning into the bilayer headgroup region, with enhanced partitioning as the chain-length of the alcohol increases, results in chain-length-dependent interfacial tension reduction with concomitant chain-length-dependent reduction in mechanical moduli and membrane thickness.

A fundamental attribute of cell membranes is transmembrane asymmetry, specifically the formation of ordered phase domains in one leaflet that are compositionally different from the opposing leaflet of the bilayer. Using model membrane systems, many previous studies have demonstrated the formation of ordered phase domains that display complete transmembrane symmetry; but there have been few reports on the more biologically relevant asymmetric membrane structures. Here we report on a combined atomic force microscopy and fluorescence microscopy study whereby we observe three different states of transmembrane symmetry in phase-separated supported lipid bilayers formed by vesicle fusion. We find that if the leaflets differ in gel-phase area fraction, then the smaller domains in one leaflet are in registry with the larger domains in the other leaflet and the system is dynamic. In a presumed lipid flip-flop process similar to Ostwald ripening, the smaller domains in one leaflet erode away whereas the large domains in the other leaflet grow until complete compositional asymmetry is reached and remains stable. We have quantified this evolution and determined that the lipid flip-flop event happens most frequently at the interface between symmetric and asymmetric DSPC domains. If both leaflets have identical area fraction of gel-phase, gel-phase domains are in registry and are static in comparison to the first state. The stability of these three DSPC domain distributions, the degree of registry observed, and the domain immobility have biological significance with regards to maintenance of lipid asymmetry in living cell membranes, communication between inner leaflet and outer leaflet, membrane adhesion, and raft mobility.

Advancements in understanding and engineering of virus-based nanomaterials (VBNs) for biomedical applications motivate a need to explore the interfaces between VBNs and other biomedically-relevant chemistries and materials. While several strategies have been used to investigate some of these interfaces with promising initial results, including VBN-containing slow-release implants and VBN-activated bioceramic bone scaffolds, there remains a need to establish VBN-immobilized three dimensional materials that exhibit improved stability and diffusion characteristics for biosensing and other analyte-capture applications. Silica sol–gel chemistries have been researched for biomedical applications over several decades and are well understood; various cellular organisms and biomolecules (e.g., bacteria, algae, enzymes) have been immobilized in silica sol-gels to improve viability, activity, and form factor (i.e., ease of use). Here we present the immobilization of an antibody-binding VBN in silica sol–gel by pore confinement. We have shown that the resulting system is sufficiently diffuse to allow antibodies to migrate in and out of the matrix. We also show that the immobilized VBN is capable of antibody binding and elution functionality under different buffer conditions for multiple use cycles. The promising results of the VBN and silica sol–gel interface indicate a general applicability for VBN-based bioseparations and biosensing applications. Graphical Abstract

Giant vesicles formed of 1,2-dipalmitoylphosphatidylcholine (DPPC) and sterols (cholesterol or ergosterol) in water and water/ethanol solutions have been used to examine the effect of sterol composition and ethanol concentration on the area compressibility modulus ( K a), overall mechanical behavior, vesicle morphology, and induction of lipid alkyl chain interdigitation. Our results from micropipette aspiration suggest that cholesterol and ergosterol impact the order and microstructure of the gel (L β ′) phase DPPC membrane. At low concentration (10–15 mol%) these sterols disrupt the long-range lateral order and fluidize the membrane ( K a ∼ 300 mN/m). Then at 18 mol%, these sterols participate in the formation of a continuous cohesive liquid-ordered (L o) phase with a sterol-dependent membrane density ( K a ∼ 750 for DPPC/ergosterol and K a ∼ 1100 mN/m for DPPC/cholesterol). Finally at ∼40 mol% both cholesterol and ergosterol impart similar condensation to the membrane ( K a ∼ 1200 mN/m). Introduction of ethanol (5–25 vol.) results in drops in the magnitude of K a, which can be substantial, and sometimes individual vesicles with lowered K a reveal two slopes of tension versus apparent area strain. We postulate that this behavior represents disruption of lipid-sterol intermolecular interactions and therefore the membrane becomes interdigitation prone. We find that for DPPC vesicles with sterol concentrations of 20–25 mol%, significantly more ethanol is required to induce interdigitation compared to pure DPPC vesicles; ∼7 vol. more for ergosterol and ∼10 vol. more for cholesterol. For lower sterol concentrations (10–15 mol%), interdigitation is offset, but by <5 vol.. These data support the idea that ergosterol and cholesterol do enhance survivability for cells exposed to high concentrations of ethanol and provide evidence that the appearance of the interdigitated (L β I) phase bilayer is a major factor in the disruption of cellular activity, which typically occurs between ∼12 and ∼16 vol. ethanol in yeast fermentations. We summarize our findings by producing, for the first time, “elasticity/phase diagrams” over a wide range of sterol (cholesterol and ergosterol) and ethanol concentrations.

Proteins and other macromolecules are believed to hinder molecular lateral diffusion in cellular membranes. We have constructed a well-characterized model system to better understand how obstacles in lipid bilayers obstruct diffusion. Fluorescence recovery after photobleaching was used to measure the lateral diffusion coefficient in single supported bilayers composed of mixtures of 1,2-dilauroylphosphotidylcholine (DLPC) and 1,2-distearoylphosphotidylcholine (DSPC). Because these lipids are immiscible and phase separate at room temperature, a novel quenching technique allowed us to construct fluid DLPC bilayers containing small disk-shaped gel-phase DSPC domains that acted as obstacles to lateral diffusion. Our experimental setup enabled us to analyze the same samples with atomic force microscopy and exactly characterize the size, shape, and number of gel-phase domains before measuring the obstacle-dependent diffusion coefficient. Lateral obstructed diffusion was found to be dependent on obstacle area fraction, size, and geometry. Analysis of our results using a free area diffusion model shows the possibility of unexpected long-range ordering of fluid-phase lipids around the gel-phase obstacles. This lipid ordering has implications for lipid-mediated protein interactions in cellular membranes.

Galactosylceramide (GalCer), a glycosphingolipid, is believed to exist in the extracellular leaflet of cell membranes in nanometer-sized domains or rafts. The local clustering of GalCer within rafts is thought to facilitate the initial adhesion of certain viruses, including HIV-1, and bacteria to cells through multivalent interactions between receptor proteins (gp120 for HIV-1) and GalCer. Here we use atomic force microscopy (AFM) to study the effects of cholesterol on solid-phase GalCer domain microstructure and miscibility with a fluid lipid 1,2-dilauroyl- sn-glycero-3-phosphocholine (DLPC) in supported lipid bilayers. Using “slow-cooled vesicle fusion” to prepare the supported lipid bilayers, we were able to overcome the nonequilibrium effects of the substrate (verified by comparison to results for giant unilamellar vesicles) and accurately quantify the dramatic effect of cholesterol on the GalCer domain surface area/perimeter ratio ( A D/ P) and DLPC-GalCer miscibility. We compare these results to a supported lipid bilayer system in which the bilayer is rapidly cooled (nonequilibrium conditions), “quenched vesicle fusion”, and find that the microstructures are remarkably similar above a cholesterol mol fraction of ∼0.06. We determined that GalCer domains were contained in one leaflet distal to the mica substrate through qualitative binding experiments with Trichosanthes kirilowii agglutinin (TKA), a galactose-specific lectin, and AFM of Langmuir-Blodgett deposited GalCer/DLPC supported lipid bilayers. In addition, GalCer domains in bilayers containing cholesterol rearranged upon tip-sample contact. Our results further serve to clarify why discrepancies exist between different model membrane systems and between model membranes and cell membranes. In addition, these results offer new insight into the effect of cholesterol and surrounding lipid on domain microstructure and behavior. Finally, our observations may be pertinent to cell membrane structure, dynamics, and HIV infection.

We report the microstructure and phase behavior of three ternary mixtures each containing a long-chain saturated glycosphingolipid, galactosylceramide (GalCer), and cholesterol at room temperature. The unsaturation level of the fluid-phase component was varied by lipid choice, i.e., saturated 1,2-dilauroyl- sn-glycero-3-phosphocholine (DLPC), singly unsaturated 1-palmitoyl-2-oleoyl- sn-glycero-3-phosphocholine (POPC), or doubly unsaturated 1,2-dioleoyl- sn-glycero-3-phosphocholine (DOPC). GalCer was used because of its biological significance, for example, as a ligand in the sexual transmission of HIV and stimulator of natural killer T-cells. Supported lipid bilayers of the ternary mixtures were imaged by atomic force microscopy and GalCer-rich domains were characterized by area/perimeter ratios (A/P). GalCer domain phase transitions from solid (S) to liquid (L) phase were verified by domain behavior in giant unilamellar vesicles, which displayed two-dimensional microstructure similar to that of supported lipid bilayers. As cholesterol concentration was increased, we observed ∼2.5, ∼10, and ∼20-fold decreases in GalCer domain A/P for bilayers in L-S phase coexistence containing DOPC, POPC, and DLPC, respectively. The transition to L-L phase coexistence occurred at ∼10 mol % cholesterol for bilayers containing DOPC or POPC and was accompanied by maintenance of a constant A/P. L-L phase coexistence did not occur for bilayers containing DLPC. We systematically relate our results to the impact of chain unsaturation on the interaction of the fluid-phase lipid and cholesterol. Physiologically, these observations may give insight into the interplay of fatty acid chain unsaturation, sterol concentration, and lipid hydrophobic mismatch in membrane phenomena.

Domains within the plane of the plasma membrane, referred to as membrane rafts, have been a topic of considerable interest in the field of membrane biophysics. Although model membrane systems have been used extensively to study lipid phase behavior as it relates to the existence of rafts, very little work has focused on either the initial stage of lipid domain nucleation, or the relevant physical parameters such as temperature and interfacial line tension which control nucleation. In this work, we utilize a method in which the kinetic process of lipid domain nucleation is imaged by atomic force microscopy and modeled using classical theory of nucleation to map interfacial line tension in ternary lipid mixtures. These mixtures consist of a fluid phase lipid component (1,2-dilauroyl-sn-glycero-3-phosphocholine, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine, or 1,2-dioleoyl-sn-glycero-3-phosphocholine), a solid phase component (galactosylceramide), and cholesterol. Interfacial line tension measurements of galactosylceramide-rich domains track with our previously measured area/perimeter ratios and height mismatches measured here. Line tension also follows known trends in cholesterol interactions and partitioning, as we observed previously with area/perimeter ratios. Our line tension measurements are discussed in combination with recent line tension measurements to address line tension regulation by cholesterol and the dynamic nature of membrane rafts.