RESUMEN
Phospholipid bilayers are dynamic cellular components that undergo constant changes in their topology, facilitating a broad diversity of physiological functions including endo- and exocytosis, cell division, and intracellular trafficking. These shape transformations consume energy, supplied invariably by the activity of proteins. Here, we show that cycles of oppositely directed osmotic stressesâunassisted by any protein activityâcan induce well-defined remodeling of giant unilamellar vesicles, minimally recapitulating the phenomenologies of surface area homeostasis and macropinocytosis. We find that a stress cycle consisting of deflationary hypertonic stress followed by an inflationary hypotonic one prompts an elaborate sequence of membrane shape changes ultimately transporting molecular cargo from the outside into the intravesicular milieu. The initial osmotic deflation produces microscopic spherical invaginations. During the subsequent inflation, the first subpopulation contributes area to the swelling membrane, thereby providing a means for surface area regulation and tensional homeostasis. The second subpopulation vesiculates into the lumens of the mother vesicles, producing pinocytic vesicles. Remarkably, the gradients of solute concentrations between the GUV and the daughter pinocytic vesicles create cascades of water current, inducing pulsatory transient poration that enable solute exchange between the buds and the GUV interior. This results in an efficient water-flux-mediated delivery of molecular cargo across the membrane boundary. Our findings suggest a primitive physical mechanism for communication and transport across protocellular compartments driven only by osmotic stresses. They also suggest plausible physical routes for intravesicular, and possibly intracellular, delivery of ions, solutes, and molecular cargo stimulated simply by cycles of osmotic currents of water.
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Fosfolípidos , Liposomas Unilamelares , Presión Osmótica , Liposomas Unilamelares/metabolismo , Ósmosis , AguaRESUMEN
A variety of cellular processes use liquid-liquid phase separation (LLPS) to create functional levels of organization, but the kinetic pathways by which it proceeds remain incompletely understood. Here in real time, we monitor the dynamics of LLPS of mixtures of segregatively phase-separating polymers inside all-synthetic, giant unilamellar vesicles. After dynamically triggering phase separation, we find that the ensuing relaxation-en route to the new equilibrium-is non-trivially modulated by a dynamic interplay between the coarsening of the evolving droplet phase and the interactive membrane boundary. The membrane boundary is preferentially wetted by one of the incipient phases, dynamically arresting the progression of coarsening and deforming the membrane. When the vesicles are composed of phase-separating mixtures of common lipids, LLPS within the vesicular interior becomes coupled to the membrane's compositional degrees of freedom, producing microphase-separated membrane textures. This coupling of bulk and surface phase-separation processes suggests a physical principle by which LLPS inside living cells might be dynamically regulated and communicated to the cellular boundaries.
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Separación de Fases , Liposomas UnilamelaresRESUMEN
The hydrophobic effect, a ubiquitous process in biology, is a primary thermodynamic driver of amphiphilic self-assembly. It leads to the formation of unique morphologies including two highly important classes of lamellar and micellar mesophases. The interactions between these two types of structures and their involved components have garnered significant interest because of their importance in key biochemical technologies related to the isolation, purification, and reconstitution of membrane proteins. This work investigates the structural organization of mixtures of the lamellar-forming phospholipid 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) and two zwitterionic micelle-forming surfactants, being n-dodecyl-N,N-dimethyl-3-ammonio-1-propanesulfonate (Zwittergent 3-12 or DDAPS) and 1-oleoyl-2-hydroxy-sn-glycero-3-phosphocholine (O-Lyso-PC), when assembled by water vapor hydration with X-ray diffraction measurements, brightfield optical microscopy, wide-field fluorescence microscopy, and atomic force microscopy. The results reveal that multilamellar mesophases of these mixtures can be assembled across a wide range of POPC to surfactant (POPC:surfactant) concentration ratios, including ratios far surpassing the classical detergent-saturation limit of POPC bilayers without significant morphological disruptions to the lamellar motif. The mixed mesophases generally decreased in lamellar spacing (D) and headgroup-to-headgroup distance (Dhh) with a higher concentration of the doped surfactant, but trends in water layer thickness (Dw) between each bilayer in the stack are highly variable. Further structural characteristics including mesophase topography, bilayer thickness, and lamellar rupture force were revealed by atomic force microscopy (AFM), exhibiting homogeneous multilamellar stacks with no significant physical differences with changes in the surfactant concentration within the mesophases. Taken together, the outcomes present the assembly of unanticipated and highly unique mixed mesophases with varied structural trends from the involved surfactant and lipidic components. Modulations in their structural properties can be attributed to the surfactant's chemical specificity in relation to POPC, such as the headgroup hydration and the hydrophobic chain tail mismatch. Taken together, our results illustrate how specific chemical complexities of surfactant-lipid interactions can alter the morphologies of mixed mesophases and thereby alter the kinetic pathways by which surfactants dissolve lipid mesophases in bulk aqueous solutions.
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Membrana Dobles de Lípidos , Surfactantes Pulmonares , Membrana Dobles de Lípidos/química , Fosfatidilcolinas/química , Tensoactivos , Fosfolípidos/química , LipoproteínasRESUMEN
The occurrence of modular peptide repeats in load-bearing (structural) proteins is common in nature, with distinctive peptide sequences that often remain conserved across different phylogenetic lineages. These highly conserved peptide sequences endow specific mechanical properties to the material, such as toughness or elasticity. Here, using bioinformatic tools and phylogenetic analysis, we have identified the GX8 peptide with the sequence GLYGGYGX (where X can be any residue) in a wide range of organisms. By simple mutation of the X residue, we demonstrate that GX8 can be self-assembled into various supramolecular structures, exhibiting vastly different physicochemical and viscoelastic properties, from liquid-like coacervate microdroplets to hydrogels to stiff solid materials. A combination of spectroscopic, electron microscopy, mechanical, and molecular dynamics studies is employed to obtain insights into molecular scale interactions driving self-assembly of GX8 peptides, underscoring that π-π stacking and hydrophobic interactions are the drivers of peptide self-assembly, whereas the X residue determines the extent of hydrogen bonding that regulates the macroscopic mechanical response. This study highlights the ability of single amino-acid polymorphism to tune the supramolecular assembly and bulk material properties of GX8 peptides, enabling us to cover a broad range of potential biomedical applications such as hydrogels for tissue engineering or coacervates for drug delivery.
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Aminoácidos , Péptidos , Filogenia , Péptidos/química , Hidrogeles/química , MutaciónRESUMEN
Atomic force microscopy (AFM) in conjunction with microfluidic delivery was utilized to produce three-dimensional (3D) lipid structures following a custom design. While AFM is well-known for its spatial precision in imaging and 2D nanolithography, the development of AFM-based nanotechnology into 3D nanoprinting requires overcoming the technical challenges of controlling material delivery and interlayer registry. This work demonstrates the concept of 3D nanoprinting of amphiphilic molecules such as 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC). Various formulations of POPC solutions were tested to achieve point, line, and layer-by-layer material delivery. The produced structures include nanometer-thick disks, long linear spherical caps, stacking grids, and organizational chiral architectures. The POPC molecules formed stacking bilayers in these constructions, as revealed by high-resolution structural characterizations. The 3D printing reached nanometer spatial precision over a range of 0.5 mm. The outcomes reveal the promising potential of our designed technology and methodology in the production of 3D structures from nanometer to continuum, opening opportunities in biomaterial sciences and engineering, such as in the production of 3D nanodevices, chiral nanosensors, and scaffolds for tissue engineering and regeneration.
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Lipid based nanoparticulate formulations have been widely used for the encapsulation and sustain release of hydrophilic drugs, but they still face challenges such as high initial burst release. Nanolipogel (NLG) emerges as a potential system to encapsulate and deliver hydrophilic drug while suppressing its initial burst release. However, there is a lack of characterization of the drug release mechanism from NLGs. In this work, we present a study on the release mechanism of hydrophilic Dextran-Fluorescein Isothiocyanate (DFITC) from Poly (ethylene glycol) Diacrylate (PEGDA) NLGs by using different molecular weights of PEGDA to vary the mesh size of the nanogel core, drawing inspiration from the macromolecular crowding effect in cells, which can be viewed as a mesh network of undefined sizes. The effect is then further characterized and validated by studying the diffusion of DFITC within the nanogel core using Fluorescence Recovery after Photobleaching (FRAP), on our newly developed cell derived microlipogels (MLG). This is in contrast to conventional FRAP works on cells or bulk hydrogels, which is limited in our application. Our work showed that the mesh size of the NLGs can be controlled by using different Mw of PEGDA, such as using a smaller MW to achieve higher crosslinking density, which will lead to having smaller mesh size for the crosslinked nanogel, and the release of hydrophilic DFITC can be sustained while suppressing the initial burst release, up to 10-fold more for crosslinked PEGDA 575 NLGs. This is further validated by FRAP which showed that the diffusion of DFITC is hindered by the decreasing mesh sizes in the NLGs, as a result of lower mobile fractions. These findings will be useful for guiding the design of PEGDA NLGs to have different degree of suppression of the initial burst release as well as the cumulative release, for a wide array of applications. This can also be extended to other different types of nanogel cores and other nanogel core-based nanoparticles for encapsulation and release of hydrophilic biomolecules.
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Lamellar mesophases of insoluble lipids are readily solubilized by the micellar mesophases of soluble surfactants. This simple process underscores a broad array of biochemical methodologies, including purification, reconstitution, and crystallization of membrane proteins, as well as the isolation of detergent-resistant membrane fractions. Although much is now known about the thermodynamic driving forces of membrane solubilization, the kinetic pathways by which the surfactant alters vesicular mesophases are only beginning to be appreciated. Little is known about how these interactions affect the solubilization of more complex, multilamellar mesophases. Here, we investigate how a common zwitterionic detergent affects the solubilization of a smectic, multilamellar, cylindrical mesophase of lipids, called the myelin figure. Our results reveal that myelin solubilization occurs in a multistep manner, producing a well-defined sequence of morphologically distinct intermediates en route to complete solubilization. The kinetic processes producing these intermediates include (1) coiling, which encompasses the formation, propagation, and tightening of extended helices; (2) thinning, which reflects the unbinding of lamellae in the smectic stacks; and (3) detachment or retraction, which either dissociates the myelinic protrusion from the source lipid mass or returns the myelinic protrusion to the source lipid massâall in transit toward complete solubilization. These occasionally overlapping steps are most pronounced in single-lipid component myelins, while compositionally graded multicomponent myelins inhibit the coiling step and detach more frequently. Taken together, the appearance of these intermediates during the solubilization of myelins suggests a complex free-energy landscape characterizing myelin solubilization populated by metastable, morphological intermediates correlated with locally minimized changes in energy dependent upon the mesophase's composition. This then predicts the accessibility of structurally distinct, kinetic intermediatesâsuch as loose and tight coiled helices, peeled myelins, retracted tubes, and detached protrusionsâbefore reaching the stable ground state corresponding to a dissolved suspension of mixed surfactant-lipid micelles.
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Surfactantes Pulmonares , Tensoactivos , Detergentes/química , Excipientes , Lípidos , Micelas , Vaina de Mielina , Solubilidad , Tensoactivos/químicaRESUMEN
Molecular dynamics (MD) simulations in the MARTINI model are used to study the assembly of 1-palmitoyl-2-oleoylphosphatidylcholine (POPC) molecules under spatial confinement, such as during solvent evaporation from ultrasmall (femtoliter quantity) droplets. The impact of surface polarity on molecular assembly is discussed in detail. To the best of our knowledge, this work represents the first of its kind. Our results reveal that solvent evaporation gives rise to the formation of well-defined stacks of lipid bilayers in a smectic alignment. These smectic mesophases form on both polar and nonpolar surfaces but with a notable distinction. On polar surfaces, the director of the stack is oriented perpendicular to the support surface. By contrast, the stacks orient at an angle on the nonpolar surfaces. The packing of head groups on surfaces and lipid molecular mobility exhibits significant differences as surface polarity changes. The role of glycerol in the assembly and stability is also revealed. The insights revealed from the simulation have a significant impact on additive manufacturing, biomaterials, model membranes, and engineering protocells. For example, POPC assemblies via evaporation of ultrasmall droplets were produced and characterized. The trends compare well with the bilayer stack models. The surface polarity influences the local morphology and structures at the interfaces, which could be rationalized via the molecule-surface interactions observed from simulations.
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Membrana Dobles de Lípidos , Fosfatidilcolinas , Membrana Dobles de Lípidos/química , Simulación de Dinámica Molecular , Fosfatidilcolinas/química , SolventesRESUMEN
When a dry mass of certain amphiphiles encounters water, a spectacular interfacial instability ensues: It gives rise to the formation of ensembles of fingerlike tubular protrusions called myelin figuresâtens of micrometers wide and tens to hundreds of micrometers longârepresenting a novel class of nonequilibrium higher-order self-organization. Here, we report that when phase-separating mixtures of unsaturated lipid, cholesterol, and sphingomyelin are hydrated, the resulting myelins break symmetry and couple their compositional degrees of freedom with the extended myelinic morphology: They produce complementary, interlamellar radial gradients of concentrations of cholesterol (and sphingomyelin) and unsaturated lipid, which stands in stark contrast to interlamellar, lateral phase separation in equilibrated morphologies. Furthermore, the corresponding gradients of molecule-specific chemistries (i.e., cholesterol extraction by methyl-ß-cyclodextrin and GM1 binding by cholera toxin) produce unusual morphologies comprising compositionally graded vesicles and buckled tubes. We propose that kinetic differences in the information processing of hydration characteristics of individual molecules while expending energy dictate this novel behavior of lipid mixtures undergoing hydration.
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Membrana Dobles de Lípidos , Esfingomielinas , Fenómenos Biofísicos , ColesterolRESUMEN
The role of an amphiphilic environment in the functional regulation of integral membrane proteins is well appreciated but how specific amphiphilic surrounding influences the conformational plasticity and function of a protein is less obvious. We focus on the Salmonella phosphoglycerate transport system (pgt)-encoded outer membrane protease E (PgtE), which plays an important role in tissue infiltration and survival of Salmonella enterica. Despite our understanding of its physiological functions, elucidation of its enzymatic behavior in response to the immediate amphiphilic surrounding is lacking. We monitor the proteolytic activity of PgtE reconstituted in Zwittergent 3-12 detergent micelles or a 1-palmitoyl-2-oleoyl-glycero-3-phosphocholine (POPC) bilayer and examine factors that influence its activity. We find, to our surprise, that PgtE, which is thought to elicit a rapid response toward various substrates, showed hysteretic enzymatic behavior, characterized by a prominent lag phase prior to achieving the exponential steady state in its detergent-stabilized form as well as in the outer membrane embedded native state in live bacteria. The lag phase was abolished under three conditions: preformation of an inactive detergent-stabilized PgtE-substrate complex without lipopolysaccharide (LPS), LPS-bound detergent-stabilized PgtE that had reached steady state velocity, or PgtE reconstituted into a POPC bilayer environment. Interestingly, detergent- and bilayer-stabilized PgtE showed comparable steady-state activity. And strikingly, lipopolysaccharide (LPS) becomes nonessential for the activation of PgtE when the protein is reconstituted in the phospholipid bilayer, contrasting a long-standing notion that LPS is required for proteases belonging to the omptin family to be proteolytically active. These findings suggest intriguing biological nuances for the proteolytic function of PgtE that were not well appreciated previously and offer new perspectives that may generally be applicable for omptins.
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Single giant unilamellar vesicles (GUVs) rupture spontaneously from their salt-laden suspension onto solid surfaces. At hydrophobic surfaces, the GUVs rupture via a recurrent, bouncing ball rhythm. During each contact, the GUVs, rendered tense by the substrate interactions, porate, and spread a molecularly transformed motif of a monomolecular layer on the hydrophobic surface from the point of contact in a symmetric manner. The competition from pore closure, however, limits the spreading and produces a daughter vesicle, which re-engages with the substrate. At solid hydrophilic surfaces, by contrast, GUVs rupture via a distinctly different recurrent burst-heal dynamics; during burst, single pores nucleate at the contact boundary of the adhering vesicles, facilitating asymmetric spreading and producing a "heart"-shaped membrane patch. During the healing phase, the competing pore closure produces a daughter vesicle. In both cases, the pattern of burst-reseal events repeats multiple times, splashing and spreading the vesicular fragments as bilayer patches at the solid surface in a pulsatory manner. These remarkable recurrent dynamics arise, not because of the elastic properties of the solid surface, but because the competition between membrane spreading and pore healing, prompted by the surface-energy-dependent adhesion, determine the course of the topological transition.
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Lípidos , Liposomas Unilamelares , Fenómenos Biofísicos , Interacciones Hidrofóbicas e Hidrofílicas , Membrana Dobles de LípidosRESUMEN
Lipid miscibility phase separation has long been considered to be a central element of cell membrane organization. More recently, protein condensation phase transitions, into three-dimensional droplets or in two-dimensional lattices on membrane surfaces, have emerged as another important organizational principle within cells. Here, we reconstitute the linker for activation of T cells (LAT):growth-factor-receptor-bound protein 2 (Grb2):son of sevenless (SOS) protein condensation on the surface of giant unilamellar vesicles capable of undergoing lipid phase separations. Our results indicate that the assembly of the protein condensate on the membrane surface can drive lipid phase separation. This phase transition occurs isothermally and is governed by tyrosine phosphorylation on LAT. Furthermore, we observe that the induced lipid phase separation drives localization of the SOS substrate, K-Ras, into the LAT:Grb2:SOS protein condensate.
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Lípidos de la Membrana , Proteínas de la Membrana , Proteína Adaptadora GRB2/metabolismo , Proteínas de la Membrana/metabolismo , Fosforilación , Fosfotirosina , Proteínas Son Of Sevenless/metabolismoRESUMEN
To better understand how lipoproteins interact and enter endothelium and participate in cellular processes, we investigated preferential lipid partitioning of triglyceride rich lipoproteins (TGRL), chylomicrons (CM), low density lipoproteins (LDL), very low density lipoproteins (VLDL) and their lipolysis products using supported phospholipid raft membrane (SPRM) patterns. We prepared SPRM patterns with Texas red labeled phospholipid patterns and Marina blue labeled raft patterns and added Atto-520 labeled lipoproteins (TGRL, CM, VLDL, LDL) and their lipolysis products in separate experiments and characterized these interactions using fluorescence microscopy. We observed that VLDL and LDL preferentially interacted with raft patterns. In contrast the TGRL and lipolysed products of TGRL interacted with both the patterns, slightly elevated preference for raft patterns and CM and its lipolysis products showed greater affinity to phospholipid patterns. The clear preference of VLDL and LDL for raft patterns suggests that these lipoproteins associate with cholesterol and sphingomyelin rich lipid micro-domains during their early interactions with endothelial cells, leading to atherosclerosis.
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Colesterol/química , Lipoproteínas/química , Microdominios de Membrana/química , Fosfolípidos/química , Esfingomielinas/química , Colesterol/metabolismo , Humanos , Lipoproteínas/metabolismo , Microdominios de Membrana/metabolismo , Fosfolípidos/metabolismo , Esfingomielinas/metabolismoRESUMEN
Crystallization of membrane-embedded components within phospholipid bilayers represents a distinct class of phase transformation that occurs in structurally organized, molecularly crowded, and dimensionally constrained amphiphilic fluids. Using unstable supported lipid bilayers-transiently assembled via surface-mediated fusion and spreading of bicellar precursors containing supersaturating concentrations of cholesterol-we monitor here the morphological evolution and dynamics of cholesterol crystallization within the membrane media. We find that the three-dimensional (3D) crystallization of cholesterol from an unstable two-dimensional (2D) in-membrane state proceeds via well-defined sequence of intermediates, including filaments, rods, helices, and 2D rectangular plates, before transforming into three-dimensional quadrilateral crystals-characteristic triclinic habit of cholesterol monohydrate. Our observations thus demonstrate that these structurally distinct cholesterol polymorphs are related to one another, contrasting with the notion that they represent disparate crystal habits stabilized by differences in lipid environments. Moreover, these observations indicate that cholesterol crystallization within the membrane media follows nonclassical multistep crystallization governed by the heuristic "Ostwald's rule of stages", which predicts that the crystallization kinetics proceed down the free energy landscape in a multistage process where each successive phase transition incurs the smallest loss of free energy relative to its predecessor. Furthermore, we find that the well-known cholesterol extracting agent, ß-cyclodextrin, acts by catalytically tipping the equilibrium in favor of crystal growth adding cholesterol from the membrane phase to the crystal in a layer-by-layer manner. Taken together, our results provide a new description of in-membrane cholesterol crystallization and may pave for a screening tool for identifying molecular candidates that target cholesterol crystals.
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Membrana Celular/química , Colesterol/química , Fosfolípidos/química , Cristalización , Modelos Moleculares , Conformación Molecular , Agua/químicaRESUMEN
The attachment of foodborne pathogens to leaf surfaces is a complex process that involves multiple physical, chemical, and biological factors. Here, we report the results from a study designed to specifically determine the contribution of spinach leaf surface topography as it relates to leaf axis (abaxial and adaxial) and leaf age (15, 45, and 75 days old) to the ability of Escherichia coli to resist removal by surface wash, to avoid inactivation by chlorine, and to disperse through splash impact. We used fresh spinach leaves, as well as so-called "replicasts" of spinach leaf surfaces in the elastomer polydimethylsiloxane to show that leaf vein density correlated positively with the failure to recover E. coli from surfaces, not only using a simple water wash and rinse, but also a more stringent wash protocol involving a detergent. Such failure was more pronounced when E. coli was surface-incubated at 24°C compared to 4°C, and in the presence, rather than absence, of nutrients. Leaf venation also contributed to the ability of E. coli to survive a 50 ppm available chlorine wash and to laterally disperse by splash impact. Our findings suggest that the topographical properties of the leafy green surface, which vary by leaf age and axis, may need to be taken into consideration when developing prevention or intervention strategies to enhance the microbial safety of leafy greens.
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Membrane active peptides (MAPs) have gained wide interest due to their far reaching applications in drug discovery and drug delivery. The search for new MAPs, however, has been largely skewed with bias selecting for physicochemical parameters believed to be important for membrane activity, such as alpha helicity, cationicity and hydrophobicity. Here we carry out a search-and-find strategy to screen a 100,000-membered one-bead-one-compound (OBOC) combinatorial peptide library for lead compounds, agnostic of those physicochemical constraints. Such a synthetic strategy also permits expansion of our peptide repertoire to include unnatural amino acids. Using this approach, we discovered a structurally unique lead peptide LBF14, a linear 14-mer peptide, that induces gross morphological disruption of membranes, irrespective of membrane composition. Further, we demonstrate that the unique insertion mechanism of the peptide, visualized by spinning disc confocal microscopy and further analyzed by electron paramagnetic resonance measurements, may be the cause of this large scale membrane deformation. We also demonstrate the robustness, reproducibility, and potential application of this technique to discover and characterize new membrane active peptides that display activity by local insertion and subsequent allosteric effects leading to global membrane disruption.
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Descubrimiento de Drogas , Proteínas de la Membrana/química , Péptidos/química , Animales , Espectroscopía de Resonancia por Spin del Electrón , Ratones , Ratones Endogámicos BALB C , Microscopía Fluorescente , Conformación ProteicaRESUMEN
Biobutanol production by fermentation is potentially a sustainable alternative to butanol production from fossil fuels. However, the toxicity of butanol to fermentative bacteria, resulting largely from cell membrane fluidization, limits production titers and is a major factor limiting the uptake of the technology. Here, studies were undertaken, in vitro and in silico, on the butanol effects on a representative bacterial (i.e. Escherichia coli) inner cell membrane. A critical butanol : lipid ratio for stability of 2 : 1 was observed, computationally, consistent with complete interdigitation. However, at this ratio the bilayer was â¼20% thicker than for full interdigitation. Furthermore, butanol intercalation induced acyl chain bending and increased disorder, measured as a 27% lateral diffusivity increase experimentally in a supported lipid bilayer. There was also a monophasic Tm reduction in butanol-treated large unilamellar vesicles. Both behaviours are inconsistent with an interdigitated gel. Butanol thus causes only partial interdigitation at physiological temperatures, due to butanol accumulating at the phospholipid headgroups. Acyl tail disordering (i.e. splaying and bending) fills the subsequent voids. Finally, butanol short-circuits the bilayer and creates a coupled system where interdigitated and splayed phospholipids coexist. These findings will inform the design of strategies targeting bilayer stability for increasing biobutanol production titers.
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1-Butanol/química , Membrana Celular/química , Membrana Dobles de Lípidos/química , Escherichia coli/química , Simulación de Dinámica Molecular , Fosfatidiletanolaminas/química , Fosfatidilgliceroles/química , Temperatura de Transición , Liposomas Unilamelares/químicaRESUMEN
A novel conjugated oligoelectrolyte (COE) material, named S6, is designed to have a lipid-bilayer stabilizing topology afforded by an extended oligophenylenevinylene backbone. S6 intercalates biological membranes acting as a hydrophobic support for glycerophospholipid acyl chains. Indeed, Escherichia coli treated with S6 exhibits a twofold improvement in butanol tolerance, a relevant feature to achieve within the general context of modifying microorganisms used in biofuel production. Filamentous growth, a morphological stress response to butanol toxicity in E. coli, is observed in untreated cells after incubation with 0.9% butanol (v/v), but is mitigated by S6 treatment. Real-time fluorescence imaging using giant unilamellar vesicles reveals the extent to which S6 counters membrane instability. Moreover, S6 also reduces butanol-induced lipopolysaccharide release from the outer membrane to further maintain cell integrity. These findings highlight a deliberate effort in the molecular design of a chain-elongated COE to stabilize microbial membranes against environmental challenges.
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Pared Celular/efectos de los fármacos , Electrólitos/farmacología , Compuestos de Vinilo/química , Butanoles/toxicidad , Pared Celular/metabolismo , Electrólitos/química , Escherichia coli/efectos de los fármacos , Escherichia coli/metabolismo , Lipopolisacáridos/química , Pruebas de Sensibilidad Microbiana , Microscopía ConfocalRESUMEN
Positive strand RNA viruses replicate in specialized niches called membranous web within the cytoplasm of host cells. These virus replication organelles sequester viral proteins, RNA, and a variety of host factors within a fluid, amorphous matrix of clusters of endoplasmic reticulum (ER) derived vesicles. They are thought to form by the actions of a nonstructural viral protein NS4B, which remodels the ER and produces dense lipid-protein condensates. Here, we used in vitro reconstitution to identify the minimal components and elucidate physical mechanisms driving the web formation. We found that the N-terminal amphipathic domain of NS4B (peptide 4BAH2) and phospholipid vesicles (â¼100-200 nm in diameter) were sufficient to produce a gel-like, viscoelastic condensate. This condensate coexists with the surrounding aqueous phase and affords rapid exchange of molecules. Together, it recapitulates the essential properties of the virus-induced membranous web. Our data support a novel phase separation mechanism in which phospholipid vesicles provide a supramolecular template spatially organizing multiple self-associating peptides thereby generating programmable multivalency de novo and inducing macroscopic phase separation.