Design of oscillatory dynamics in numerical simulations of compartment-based enzyme systems.

Enzymatic reactions that yield non-neutral products are known to involve feedback due to the bell-shaped pH-rate curve of the enzyme. Compartmentalizing the reaction has been shown to lead to transport-driven oscillations in theory; however, there have been few reproducible experimental examples. Our objective was to determine how the conditions could be optimized to achieve pH oscillations. We employed numerical simulations to investigate the hydrolysis of ethyl acetate in a confined esterase enzyme system, examining the influence of key factors on its behavior. Specific parameter ranges that lead to bistability and self-sustained pH oscillations and the importance of fast base transport for oscillations in this acid-producing system are highlighted. Suggestions are made to expand the parameter space for the occurrence of oscillations, including modifying the maximum of the enzyme pH-rate curve and increasing the negative feedback rate. This research not only sheds light on the programmable nature of enzyme-driven pH regulation but also furthers knowledge on the optimal design of such feedback systems for experimentalists.

Unraveling the Phase Behavior, Mechanical Stability, and Protein Reconstitution Properties of Polymer-Lipid Hybrid Vesicles.

Hybrid vesicles consisting of natural phospholipids and synthetic amphiphilic copolymers have shown remarkable material properties and potential for biotechnology, combining the robustness of polymers with the biocompatibility of phospholipid membranes. To predict and optimize the mixing behavior of lipids and copolymers, as well as understand the interaction between the hybrid membrane and macromolecules like membrane proteins, a comprehensive understanding at the molecular level is essential. This can be achieved by a combination of molecular dynamics simulations and experiments. Here, simulations of POPC and PBD22-b-PEO14 hybrid membranes are shown, uncovering different copolymer configurations depending on the polymer-to-lipid ratio. High polymer concentrations created thicker membranes with an extended polymer conformation, while high lipid content led to the collapse of the polymer chain. High concentrations of polymer were further correlated with a decreased area compression modulus and altered lateral pressure profiles, hypothesized to result in the experimentally observed improvement in membrane protein reconstitution and resistance toward destabilization by detergents. Finally, simulations of a WALP peptide embedded in the bilayer showed that only membranes with up to 50% polymer content favored a transmembrane configuration. These simulations correlate with previous and new experimental results and provide a deeper understanding of the properties of lipid-copolymer hybrid membranes.

Controlling the Self-Assembly and Material Properties of ß-Sheet Peptide Hydrogels by Modulating Intermolecular Interactions.

Self-assembling peptides are a promising biomaterial with potential applications in medical devices and drug delivery. In the right combination of conditions, self-assembling peptides can form self-supporting hydrogels. Here, we describe how balancing attractive and repulsive intermolecular forces is critical for successful hydrogel formation. Electrostatic repulsion is tuned by altering the peptide's net charge, and intermolecular attractions are controlled through the degree of hydrogen bonding between specific amino acid residues. We find that an overall net peptide charge of +/-2 is optimal to facilitate the assembly of self-supporting hydrogels. If the net peptide charge is too low then dense aggregates form, while a high molecular charge inhibits the formation of larger structures. At a constant charge, altering the terminal amino acids from glutamine to serine decreases the degree of hydrogen bonding within the assembling network. This tunes the viscoelastic properties of the gel, reducing the elastic modulus by two to three orders of magnitude. Finally, hydrogels could be formed from glutamine-rich, highly charged peptides by mixing the peptides in combinations with a resultant net charge of +/-2. These results illustrate how understanding and controlling self-assembly mechanisms through modulating intermolecular interactions can be exploited to derive a range of structures with tuneable properties.

High Resolution Membrane Structures within Hybrid Lipid-Polymer Vesicles Revealed by Combining X-Ray Scattering and Electron Microscopy.

Hybrid vesicles consisting of phospholipids and block-copolymers are increasingly finding applications in science and technology. Herein, small angle X-ray scattering (SAXS) and cryo-electron tomography (cryo-ET) are used to obtain detailed structural information about hybrid vesicles with different ratios of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) and poly(1,2-butadiene-block-ethylene oxide) (PBd22 -PEO14 , Ms  = 1800 g mol-1 ). Using single particle analysis (SPA) the authors are able to further interpret the information gained from SAXS and cryo-ET experiments, showing that increasing PBd22 -PEO14 mole fraction increases the membrane thickness from 52 Å for a pure lipid system to 97 Å for pure PBd22 -PEO14 vesicles. Two vesicle populations with different membrane thicknesses in hybrid vesicle samples are found. As these lipids and polymers are reported to homogeneously mix, bistability is inferred between weak and strong interdigitation regimes of PBd22 -PEO14 within the hybrid membranes. It is hypothesized that membranes of intermediate structure are not energetically favorable. Therefore, each vesicle exists in one of these two membrane structures, which are assumed to have comparable free energies. The authors conclude that, by combining biophysical methods, accurate determination of the influence of composition on the structural properties of hybrid membranes is achieved, revealing that two distinct membranes structures can coexist in homogeneously mixed lipid-polymer hybrid vesicles.

Dynamics of asymmetric membranes and interleaflet coupling as intermediates in membrane fusion.

Membrane fusion is a tool to increase the complexity of model membrane systems. Here, we use silica nanoparticles to fuse liquid-disordered DOPC giant unilamellar vesicles (GUVs) and liquid-ordered DPPC:cholesterol (7:3) GUVs. After fusion, GUVs display large membrane domains as confirmed by fluorescence confocal microscopy. Laurdan spectral imaging of the membrane phases in the fused GUVs shows differences compared with the initial vesicles indicating some lipid redistribution between phase domains as dictated by the tie lines of the phase diagram. Remarkably, using real-time confocal microscopy we were able to record the dynamics of formation of asymmetric membrane domains in hemifused GUVs and detected interleaflet coupling phenomena by which the DOPC-rich liquid-disordered domains in outer monolayer modulates the phase state of the DPPC:cholesterol inner membrane leaflet which transitions from liquid-ordered to liquid-disordered phase. We find that internal membrane stresses generated by membrane asymmetry enhance the efficiency of full fusion compared with our previous studies on symmetric vesicle fusion. Furthermore, under these conditions, the liquid-disordered monolayer dictates the bilayer phase state of asymmetric membrane domains in >90% of observed cases. By comparison to the findings of previous literature, we suggest that the monolayer phase that dominates the bilayer properties could be a mechanoresponsive signaling mechanism sensitive to the local membrane environment.

Protein corona alters the mechanisms of interaction between silica nanoparticles and lipid vesicles.

The use of nanoparticles (NPs) for biomedical applications implies their delivery into the organism where they encounter biological fluids. In such biological fluids, proteins and other biomolecules adhere to the surface of the NPs forming a biomolecular corona that can alter significantly the behaviour of the nanomaterials. Here, we investigate the impact of a bovine serum albumin corona on interactions between silica nanoparticles (SNPs) of two different sizes and giant lipid vesicles. The formation of the protein corona leads to a significant increase of the hydrodynamic size of the SNPs. Confocal microscopy imaging shows that the protein corona alters the morphological response of vesicles to SNPs. In addition, Laurdan spectral imaging show that the protein corona weakens the effect of SNPs on the lipid packing in the GUV membrane. Our results demonstrate that a protein corona can change the interaction mechanism between nanoparticles and lipid membranes.

Detergent-Free Functionalization of Hybrid Vesicles with Membrane Proteins Using SMALPs.

Hybrid vesicles (HVs) that consist of mixtures of block copolymers and lipids are robust biomimetics of liposomes, providing a valuable building block in bionanotechnology, catalysis, and synthetic biology. However, functionalization of HVs with membrane proteins remains laborious and expensive, creating a significant current challenge in the field. Here, using a new approach of extraction with styrene-maleic acid (SMA), we show that a membrane protein (cytochrome bo 3) directly transfers into HVs with an efficiency of 73.9 ± 13.5% without the requirement of detergent, long incubation times, or mechanical disruption. Direct transfer of membrane proteins using this approach was not possible into liposomes, suggesting that HVs are more amenable than liposomes to membrane protein incorporation from a SMA lipid particle system. Finally, we show that this transfer method is not limited to cytochrome bo 3 and can also be performed with complex membrane protein mixtures.

Collective Behavior of Urease pH Clocks in Nano- and Microvesicles Controlled by Fast Ammonia Transport.

The transmission of chemical signals via an extracellular solution plays a vital role in collective behavior in cellular biological systems and may be exploited in applications of lipid vesicles such as drug delivery. Here, we investigated chemical communication in synthetic micro- and nanovesicles containing urease in a solution of urea and acid. We combined experiments with simulations to demonstrate that the fast transport of ammonia to the external solution governs the pH-time profile and synchronizes the timing of the pH clock reaction in a heterogeneous population of vesicles. This study shows how the rate of production and emission of a small basic product controls pH changes in active vesicles with a distribution of sizes and enzyme amounts, which may be useful in bioreactor or healthcare applications.

Membrane mixing and dynamics in hybrid POPC/poly(1,2-butadiene-block-ethylene oxide) (PBd-b-PEO) lipid/block co-polymer giant vesicles.

Lipids and block copolymers can individually self-assemble into vesicles, each with their own particular benefits and limitations. Combining polymers with lipids allows for further optimisation of the vesicle membranes for bionanotechnology applications. Here, POPC lipid is mixed with poly(1,2-butadiene-block-ethylene oxide) of two different molecular weights (PBd22-PEO14, Mr = 1800 g mol-1 and PBd12-PEO11, Mr = 1150 g mol-1) in order to investigate how increasing the polymer fraction affects membrane mixing, hydration and fluidity. Intensity contributions of fluorescently labelled lipid and polymer within mixed GUV membranes confirm membrane homogeneity within the hybrids. General polarisation measurements of Laurdan in GUVs showed little change in membrane hydration as polymer fraction is increased, which suggests good structural compatibility between lipids and polymers that gives rise to well-mixed vesicles. Membrane fluidity in hybrid GUVs was found to decrease non-linearly with increasing polymer fraction. However, the diffusion coefficients for the fluorescent polymer in hybrid membranes did not change significantly with increasing polymer content. While increasing the polymer fraction does reduce the movement of lipids through a polymer-rich matrix, insignificant difference in diffusion coefficients of the polymer suggests that its diffusion is minimally affected by increasing lipid composition in the range studied. These results lay further foundations for the wider development of hybrid vesicles with controlled properties for advanced biotechnologies.

Evaluation of injectable nucleus augmentation materials for the treatment of intervertebral disc degeneration.

Back pain affects a person's health and mobility as well as being associated with large health and social costs. Lower back pain is frequently caused by degeneration of the intervertebral disc. Current operative and non-operative treatments are often ineffective and expensive. Nucleus augmentation is designed to be a minimally invasive method of restoring the disc to its native healthy state by restoring the disc height, and mechanical and/or biological properties. The majority of the candidate materials for nucleus augmentation are injectable hydrogels. In this review, we examine the materials that are currently under investigation for nucleus augmentation, and compare their ability to meet the design requirements for this application. Specifically, the delivery of the material into the disc, the mechanical properties of the material and the biological compatibility are examined. Recommendations for future testing are also made.

Biomimetic Curvature and Tension-Driven Membrane Fusion Induced by Silica Nanoparticles.

Fusion events in living cells are intricate phenomena that require the coordinate action of multicomponent protein complexes. However, simpler synthetic tools to control membrane fusion in artificial cells are highly desirable. Native membrane fusion machinery mediates fusion, driving a delicate balance of membrane curvature and tension between two closely apposed membranes. Here, we show that silica nanoparticles (SiO2 NPs) at a size close to the cross-over between tension-driven and curvature-driven interaction regimes initiate efficient fusion of biomimetic model membranes. Fusion efficiency and mechanisms are studied by Förster resonance energy transfer and confocal fluorescence microscopy. SiO2 NPs induce a slight increase in lipid packing likely to increase the lateral tension of the membrane. We observe a connection between membrane tension and fusion efficiency. Finally, real-time confocal fluorescence microscopy reveals three distinct mechanistic pathways for membrane fusion. SiO2 NPs show significant potential for inclusion in the synthetic biology toolkit for membrane remodeling and fusion in artificial cells.

Breaking Isolation to Form New Networks: pH-Triggered Changes in Connectivity inside Lipid Nanoparticles.

There is a growing demand to develop smart nanomaterials that are structure-responsive as they have the potential to offer enhanced dose, temporal and spatial control of compounds and chemical processes. The naturally occurring pH gradients found throughout the body make pH an attractive stimulus for guiding the response of a nanocarrier to specific locations or (sub)cellular compartments in the body. Here we have engineered highly sensitive lyotropic liquid crystalline nanoparticles that reversibly respond to changes in pH by altering the connectivity within their structure at physiological temperatures. At pH 7.4, the nanoparticles have an internal structure consisting of discontinuous inverse micellar "aqueous pockets" based on space group Fd3m. When the pH is ≤6, the nanoparticles change from a compartmentalized to an accessible porous internal structure based on a 2D inverse hexagonal phase (plane group p6mm). We validate the internal symmetry of the nanoparticles using small-angle X-ray scattering and cryogenic transmission electron microscopy. The high-resolution electron microscopy images obtained have allowed us for the first time to directly visualize the internal structure of the Fd3m nanoparticles and resolve the two different-sized inverse micelles that make up the structural motif within the Fd3m unit cell, which upon structural analysis reveal excellent agreement with theoretical geometrical models.

The influence of phosphatidylserine localisation and lipid phase on membrane remodelling by the ESCRT-II/ESCRT-III complex.

The endosomal sorting complex required for transport (ESCRT) organises in supramolecular structures on the surface of lipid bilayers to drive membrane invagination and scission of intraluminal vesicles (ILVs), a process also controlled by membrane mechanics. However, ESCRT association with the membrane is also mediated by electrostatic interactions with anionic phospholipids. Phospholipid distribution within natural biomembranes is inhomogeneous due to, for example, the formation of lipid rafts and curvature-driven lipid sorting. Here, we have used phase-separated giant unilamellar vesicles (GUVs) to investigate the link between phosphatidylserine (PS)-rich lipid domains and ESCRT activity. We employ GUVs composed of phase separating lipid mixtures, where unsaturated DOPS and saturated DPPS lipids are incorporated individually or simultaneously to enhance PS localisation in liquid disordered (Ld) and/or liquid ordered (Lo) domains, respectively. PS partitioning between the coexisting phases is confirmed by a fluorescent Annexin V probe. Ultimately, we find that ILV generation promoted by ESCRTs is significantly enhanced when PS lipids localise within Ld domains. However, the ILVs that form are rich in Lo lipids. We interpret this surprising observation as preferential recruitment of the Lo phase beneath the ESCRT complex due to its increased rigidity, where the Ld phase is favoured in the neck of the resultant buds to facilitate the high membrane curvature in these regions of the membrane during the ILV formation process. Ld domains offer lower resistance to membrane bending, demonstrating a mechanism by which the composition and mechanics of membranes can be coupled to regulate the location and efficiency of ESCRT activity.

Hydrodynamic Mixing Tunes the Stiffness of Proteoglycan-Mimicking Physical Hydrogels.

Self-assembling hydrogels are promising materials for regenerative medicine and tissue engineering. However, designing hydrogels that replicate the 3-4 order of magnitude variation in soft tissue mechanics remains a major challenge. Here hybrid hydrogels are investigated formed from short self-assembling ß-fibril peptides, and the glycosaminoglycan chondroitin sulfate (CS), chosen to replicate physical aspects of proteoglycans, specifically natural aggrecan, which provides structural mechanics to soft tissues. Varying the peptide:CS compositional ratio (1:2, 1:10, or 1:20) can tune the mechanics of the gel by one to two orders of magnitude. In addition, it is demonstrated that at any fixed composition, the gel shear modulus can be tuned over approximately two orders of magnitude through varying the initial vortex mixing time. This tuneability arises due to changes in the mesoscale structure of the gel network (fibril width, length, and connectivity), giving rise to both shear-thickening and shear-thinning behavior. The resulting hydrogels range in shear elastic moduli from 0.14 to 220 kPa, mimicking the mechanical variability in a range of soft tissues. The high degree of discrete tuneability of composition and mechanics in these hydrogels makes them particularly promising for matching the chemical and mechanical requirements of different applications in tissue engineering and regenerative medicine.

Cryo-EM structures of an insecticidal Bt toxin reveal its mechanism of action on the membrane.

Insect pests are a major cause of crop losses worldwide, with an estimated economic cost of $470 billion annually. Biotechnological tools have been introduced to control such insects without the need for chemical pesticides; for instance, the development of transgenic plants harbouring genes encoding insecticidal proteins. The Vip3 (vegetative insecticidal protein 3) family proteins from Bacillus thuringiensis convey toxicity to species within the Lepidoptera, and have wide potential applications in commercial agriculture. Vip3 proteins are proposed to exert their insecticidal activity through pore formation, though to date there is no mechanistic description of how this occurs on the membrane. Here we present cryo-EM structures of a Vip3 family toxin in both inactive and activated forms in conjunction with structural and functional data on toxin-membrane interactions. Together these data demonstrate that activated Vip3Bc1 complex is able to insert into membranes in a highly efficient manner, indicating that receptor binding is the likely driver of Vip3 specificity.

Tuning stable noble metal nanoparticles dispersions to moderate their interaction with model membranes.

HYPOTHESIS: The properties of stable gold (Au) nanoparticle dispersions can be tuned to alter their activity towards biomembrane models. EXPERIMENTS: Au nanoparticle coating techniques together with rapid electrochemical screens of a phospholipid layer on fabricated mercury (Hg) on platinum (Pt) electrode have been used to moderate the phospholipid layer activity of Au nanoparticle dispersions. Screening results for Au nanoparticle dispersions were intercalibrated with phospholipid large unilamellar vesicle (LUV) interactions using a carboxyfluorescein (CF) leakage assay. All nanoparticle dispersions were characterised for size, by dynamic light scattering (DLS) and transmission electron microscopy (TEM). FINDINGS: Commercial and high quality home synthesised Au nanoparticle dispersions are phospholipid monolayer active whereas Ag nanoparticle dispersions are not. If Au nanoparticles are coated with a thin layer of Ag then the particle/lipid interaction is suppressed. The electrochemical assays of the lipid layer activity of Au nanoparticle dispersions align with LUV leakage assays of the same. Au nanoparticles of decreasing size and increasing dispersion concentration showed a stronger phospholipid monolayer/bilayer interaction. Treating Au nanoparticles with cell culture medium and incubation of Au nanoparticle dispersions in phosphate buffered saline (PBS) solutions removes their phospholipid layer interaction.

Characterisation of Hybrid Polymersome Vesicles Containing the Efflux Pumps NaAtm1 or P-Glycoprotein.

Investigative systems for purified membrane transporters are almost exclusively reliant on the use of phospholipid vesicles or liposomes. Liposomes provide an environment to support protein function; however, they also have numerous drawbacks and should not be considered as a "one-size fits all" system. The use of artificial vesicles comprising block co-polymers (polymersomes) offers considerable advantages in terms of structural stability; provision of sufficient lateral pressure; and low passive permeability, which is a particular issue for transport assays using hydrophobic compounds. The present investigation demonstrates strategies to reconstitute ATP binding cassette (ABC) transporters into hybrid vesicles combining phospholipids and the block co-polymer poly (butadiene)-poly (ethylene oxide). Two efflux pumps were chosen; namely the Novosphingobium aromaticivorans Atm1 protein and human P-glycoprotein (Pgp). Polymersomes were generated with one of two lipid partners, either purified palmitoyl-oleoyl-phosphatidylcholine, or a mixture of crude E. coli lipid extract and cholesterol. Hybrid polymersomes were characterised for size, structural homogeneity, stability to detergents, and permeability. Two transporters, NaAtm1 and P-gp, were successfully reconstituted into pre-formed and surfactant-destabilised hybrid polymersomes using a detergent adsorption strategy. Reconstitution of both proteins was confirmed by density gradient centrifugation and the hybrid polymersomes supported substrate dependent ATPase activity of both transporters. The hybrid polymersomes also displayed low passive permeability to a fluorescent probe (calcein acetomethoxyl-ester (C-AM)) and offer the potential for quantitative measurements of transport activity for hydrophobic compounds.