Effect of Detergents on Morphology, Size Distribution, and Concentration of Copolymer-Based Polymersomes.

Polymersomes made of amphiphilic diblock copolymers are generally regarded as having higher physical and chemical stability than liposomes composed of phospholipids. This enhanced stability arises from the higher molecular weight of polymer constituents. Despite their increased stability, polymer bilayers are solubilized by detergents in a similar manner to lipid bilayers. In this work, we evaluated the stability of poly(ethylene glycol)-block-poly(ε-caprolactone) (PEG-PCL)-based polymersomes exposed to three different detergents: N-octyl-ß-d-glucopyranoside (OG), lauryldimethylamine N-oxide (LDAO), and Triton X-100 (TX-100). Changes in morphology, particle size distribution, and concentrations of the polymersomes were evaluated during the titration of the detergents into the polymersome solutions. Furthermore, we discussed the effect of detergent features on the solubilization of the polymeric bilayer and compared it to the results reported in the literature for liposomes and polymersomes. This information can be used for tuning the properties of PEG-PCL polymersomes for use in applications such as drug delivery or protein reconstitution studies.

Purification and initial characterization of Plasmodium falciparum K+ channels, PfKch1 and PfKch2 produced in Saccharomyces cerevisiae.

Resistance towards known antimalarial drugs poses a significant problem, urging for novel drugs that target vital proteins in the malaria parasite Plasmodium falciparum. However, recombinant production of malaria proteins is notoriously difficult. To address this, we have investigated two putative K+ channels, PfKch1 and PfKch2, identified in the P. falciparum genome. We show that PfKch1 and PfKch2 and a C-terminally truncated version of PfKch1 (PfKch11-1094) could indeed be functionally expressed in vivo, since a K+-uptake deficient Saccharomyces cerevisiae strain was complemented by the P. falciparum cDNAs. PfKch11-1094-GFP and GFP-PfKch2 fusion proteins were overexpressed in yeast, purified and reconstituted in lipid bilayers to determine their electrophysiological activity. Single channel conductance amounted to 16 ± 1 pS for PfKch11-1094-GFP and 28 ± 2 pS for GFP-PfKch2. We predicted regulator of K+-conductance (RCK) domains in the C-terminals of both channels, and we accordingly measured channel activity in the presence of Ca2+.

Stationary photocurrent generation from bacteriorhodopsin-loaded lipo-polymersomes in polyelectrolyte multilayer assembly on polyethersulfone membrane.

Vesicles constructed of either synthetic polymers alone (polymersomes) or a combination of polymers and lipids (lipo-polymersomes) demonstrate excellent long-term stability and ability to integrate membrane proteins. Applications using lipo-polymersomes with integrated membrane proteins require suitable supports to maintain protein functionality. Using lipo-polymersomes loaded with the light-driven proton pump bacteriorhodopsin (BR), we demonstrate here how the photocurrent is influenced by a chosen support. In our study, we deposited BR-loaded lipo-polymersomes in a cross-linked polyelectrolyte multilayer assembly either directly physisorbed on gold electrode microchips or cross-linked on an intermediary polyethersulfone (PES) membrane covalently grafted using a hydrogel cushion. In both cases, electrochemical impedance spectroscopic characterization demonstrated successful polyelectrolyte assembly with BR-loaded lipo-polymersomes. Light-induced proton pumping by BR-loaded lipo-polymersomes in the different support constructs was characterized by amperometric recording of the generated photocurrent. Application of the hydrogel/PES membrane support together with the polyelectrolyte assembly decreased the transient current response upon light activation of BR, while enhancing the generated stationary current to over 700 nA/cm2. On the other hand, the current response from BR-loaded lipo-polymersomes in a polyelectrolyte assembly without the hydrogel/PES membrane support was primarily a transient peak combined with a low-nanoampere-level stationary photocurrent. Hence, the obtained results demonstrated that by using a hydrogel/PES support it was feasible to monitor continuously light-induced proton flux in biomimetic applications of lipo-polymersomes. Graphical abstract.

Forward osmosis: dyeing draw solutions for water reclamation from feed water resources.

Reactive Black 5 and Basic Blue 41 GRL dyeing solutions (dye-to-salt mixture in a 1:10 dye-to-salt mass ratio) were investigated as draw solutions (DS) in a forward osmosis (FO) system with a biomimetic membrane. Synthetic seawater (SSW) and textile wastewater (TWW1 and TWW2) were evaluated as feed solutions (FS) for water reclamation. Reactive Black 5 and Basic Blue 41 GRL were diluted from 0.02 M to concentrations of 0.002 and 0.004 M, respectively. With Reactive Black 5 as DS and SSW as FS, an initial flux of 20.24 L/m2 h and water recovery of 75% was achieved. Using TWW1 and TWW2, initial water fluxes of 19.51 and 13.43 L/m2 h were achieved, respectively, with a 30% water recovery. Using Basic Blue 41 GRL, initial water fluxes of 18.72, 15.13 and 13.42 L/m2 h were achieved with SSW, TWW1, and TWW2 as FS with water recoveries of 50%, 20% and 20%, respectively. The average reverse solute fluxes for Reactive Black 5 and Basic Blue 41 GRL were 0.06 to 0.34 g/m2 h, respectively. Diluted dyeing solutions were produced, with simultaneous water reclamation from SSW and TWW resulting in similar or higher water fluxes and lower reverse solute fluxes compared with other commercially available membranes.

From channel proteins to industrial biomimetic membrane technology.

Biomimetic membrane technology, based on the use of nano-scale functional additives in the form of channel proteins or artificially made channel structures, represents an attractive way of optimizing membrane separation technology. However, the nano-scale nature of the additives inherently points to the challenge in up-scaling the membranes to square meter areas. Thus, the ability to up-scale the processes involved in manufacturing will be crucial for translating the protein/nano-science into technology. Here we discuss how highly selective aquaporin proteins can be used to enhance the performance of the classical thin film composite membrane, and how this can be used in relevant membrane elements and module form factors. A particular up-scaling challenge lies in securing large scale membrane protein production. We demonstrate our framework for making batch amounts which are compatible with the large scale production of biomimetic membranes for water purification based on the use of the E. coli expression system.

Forward Osmosis in Wastewater Treatment Processes.

In recent years, membrane technology has been widely used in wastewater treatment and water purification. Membrane technology is simple to operate and produces very high quality water for human consumption and industrial purposes. One of the promising technologies for water and wastewater treatment is the application of forward osmosis. Essentially, forward osmosis is a process in which water is driven through a semipermeable membrane from a feed solution to a draw solution due to the osmotic pressure gradient across the membrane. The immediate advantage over existing pressure driven membrane technologies is that the forward osmosis process per se eliminates the need for operation with high hydraulic pressure and forward osmosis has low fouling tendency. Hence, it provides an opportunity for saving energy and membrane replacement cost. However, there are many limitations that still need to be addressed. Here we briefly review some of the applications within water purification and new developments in forward osmosis membrane fabrication.

A reusable device for electrochemical applications of hydrogel supported black lipid membranes.

Black lipid membranes (BLMs) are significant in studies of membrane transport, incorporated proteins/ion transporters, and hence in construction of biosensor devices. Although BLMs provide an accepted mimic of cellular membranes, they are inherently fragile. Techniques are developed to stabilize them, such as hydrogel supports. In this paper, we present a reusable device for studies on hydrogel supported (hs) BLMs. These are formed across an ethylene tetrafluoroethylene (ETFE) aperture array supported by the hydrogel, which is during in situ polymerization covalently "sandwiched" between the ETFE substrate and a gold electrode microchip, thus allowing direct electrochemical studies with the integrated working electrodes. Using electrochemical impedance spectroscopy (EIS), X-ray photoelectron spectroscopy and contact angle measurements, we demonstrate the optimized chemical modifications of the gold electrode microchips and plasma modification of the ETFE aperture arrays facilitating covalent "sandwiching" of the hydrogel. Both fluorescence microscopy and EIS were used to demonstrate the induced spontaneous thinning of a deposited lipid solution, leading to formation of stabilized hsBLMs on average in 10 min. The determined specific membrane capacitance and resistance were shown to vary in the range 0.31-0.49 µF/cm(2) and 45-65 kΩ cm(2), respectively, corresponding to partially solvent containing BLMs with an average life time of 60-80 min. The characterized hsBLM formation and devised equivalent circuit models lead to a schematic model to illustrate lipid molecule distribution in hydrogel-supported apertures. The functionality of stabilized hsBLMs and detection sensitivity of the platform were verified by monitoring the effect of the ion transporter valinomycin.

High yield purification of full-length functional hERG K+ channels produced in Saccharomyces cerevisiae.

The hERG potassium channel is essential for repolarization of the cardiac action potential. Due to this vital function, absence of unintended and potentially life-threatening interactions with hERG is required for approval of new drugs. The structure of hERG is therefore one of the most sought-after. To provide purified hERG for structural studies and new hERG biomimetic platforms for detection of undesirable interactions, we have developed a hERG expression platform generating unprecedented amounts of purified and functional hERG channels. Full-length hERG, with or without a C-terminally fused green fluorescent protein (GFP) His 8-tag was produced from a codon-optimized hERG cDNA in Saccharomyces cerevisiae. Both constructs complemented the high potassium requirement of a knock-out Saccharomyces cerevisiae strain, indicating correct tetramer assembly in vivo. Functionality was further demonstrated by Astemizole binding to membrane embedded hERG-GFP-His 8 with a stoichiometry corresponding to tetramer assembly. The 156 kDa hERG-GFP protein accumulated to a membrane density of 1.6%. Fluorescence size exclusion chromatography of hERG-GFP-His 8 solubilized in Fos-Choline-12 supplemented with cholesteryl-hemisuccinate and Astemizole resulted in a monodisperse elution profile demonstrating a high quality of the hERG channels. hERG-GFP-His 8 purified by Ni-affinity chromatography maintained the ability to bind Astemizole with the correct stoichiometry indicating that the native, tetrameric structure was preserved. To our knowledge this is the first reported high-yield production and purification of full length, tetrameric and functional hERG. This significant breakthrough will be paramount in obtaining hERG crystal structures, and in establishment of new high-throughput hERG drug safety screening assays.

Computational Design of Biomimetic Phosphate Scavengers.

Phosphorus has long been the target of much research, but in recent years the focus has shifted from being limited only to reducing its detrimental environmental impact, to also looking at how it is linked to the global food security. Therefore, the interest in finding novel techniques for phosphorus recovery, as well as improving existing techniques, has increased. In this study we apply a hybrid simulation approach of molecular dynamics and quantum mechanics to investigate the binding modes of phosphate anions by a small intrinsically disordered peptide. Our results confirm that the conformational ensemble of the peptide is significantly changed, or stabilized, by the binding of phosphate anions and that binding does not take place purely as a result of a stable P-loop binding nest, but rather that multiple binding modes may be involved. Such small synthetic peptides capable of binding phosphate could be the starting point of new novel technological approaches toward phosphorus recovery, and they represent an excellent model system for investigating the nature and dynamics of functional de novo designed intrinsically disordered proteins.

Lipid directed intrinsic membrane protein segregation.

We demonstrate a new approach for direct reconstitution of membrane proteins during giant vesicle formation. We show that it is straightforward to create a tissue-like giant vesicle film swelled with membrane protein using aquaporin SoPIP2;1 as an illustration. These vesicles can also be easily harvested for individual study. By controlling the lipid composition we are able to direct the aquaporin into specific immiscible liquid domains in giant vesicles. The oligomeric α-helical protein cosegregates with the cholesterol-poor domains in phase separating ternary mixtures.

Solution plasma synthesis of Pt-decorated Bi12O17Cl2 photocatalysts for efficient upcycling of plastics.

Photocatalytic upcycling of plastic waste is a promising approach to relieving pressure caused by solid waste, but the rational design of novel efficient photocatalysts remains a challenge. Herein, we utilize subnano-sized platinum (Pt)-based photocatalysts for plastic upcycling. A solution plasma strategy is developed to fabricate Pt-decorated Bi12O17Cl2 (SP-BOC). The Pt in an oxidant state and oxygen vacancies optimize the electronic structure for fast charge transfer. As a result, SP-BOC displays high performance for upcycling polyvinyl chloride (PVC) and polylactic acid (PLA) into acetic acid and formic acid, with yield rate and selectivity of 6.07 mg g-1cat. h-1 and 94 %, and 47.43 mg g-1cat. h-1 and 55.1 %, respectively. In addition, the dichlorination efficiency of PVC reaches 78.1 % within 10 h reaction, effectively reducing the environmental hazards associated with PVC waste disposal treatments. This research provides insight into the effective conversion of plastics into high-value chemicals, contributing to the reduction of carbon and toxic emissions in a practical and meaningful way, and offering a useful way for solving challenges of waste management and environmental sustainability.

Microwave Synthesis of Poly(Acrylic) Acid-Coated Magnetic Nanoparticles as Draw Solutes in Forward Osmosis.

Polyacrylic acid (PAA)-coated magnetic nanoparticles (MNP@PAA) were synthesized and evaluated as draw solutes in the forward osmosis (FO) process. MNP@PAA were synthesized by microwave irradiation and chemical co-precipitation from aqueous solutions of Fe2+ and Fe3+ salts. The results showed that the synthesized MNPs have spherical shapes of maghemite Fe2O3 and superparamagnetic properties, which allow draw solution (DS) recovery using an external magnetic field. Synthesized MNP, coated with PAA, yielded an osmotic pressure of ~12.8 bar at a 0.7% concentration, resulting in an initial water flux of 8.1 LMH. The MNP@PAA particles were captured by an external magnetic field, rinsed in ethanol, and re-concentrated as DS in repetitive FO experiments with deionized water as a feed solution (FS). The osmotic pressure of the re-concentrated DS was 4.1 bar at a 0.35% concentration, resulting in an initial water flux of 2.1 LMH. Taken together, the results show the feasibility of using MNP@PAA particles as draw solutes.

Population shift between the open and closed states changes the water permeability of an Aquaporin Z mutant.

Aquaporins are tetrameric transmembrane channels permeable to water and other small solutes. Wild-type (WT) and mutant Aquaporin Z (AqpZ) have been widely studied and multiple factors have been found to affect their water permeability. In this study, molecular dynamics simulations have been performed for the tetrameric AqpZ F43W/H174G/T183F mutant. It displayed ∼10% average water permeability compared to WT AqpZ, which had been attributed to the increased channel lumen hydrophobicity. Our simulations, however, show a ring stacking between W43 and F183 acting as a secondary steric gate in the triple mutant with R189 as the primary steric gate in both mutant and WT AqpZ. The double gates (R189 and W43-F183) result in a high population of the closed conformation in the mutant. Occasionally an open state, with diffusive water permeability very close to that of WT AqpZ, was observed. Taken together, our results show that the double-gate mechanism is sufficient to explain the reduced water permeability in the mutant without invoking effects arising from increased hydrophobicity of the channel lumen. Our findings provide insights into how aquaporin-mediated water transport can be modulated and may further point to how aquaporin function can be optimized for biomimetic membrane applications.

Interaction between sodium dodecyl sulfate and membrane reconstituted aquaporins: a comparative study of spinach SoPIP2;1 and E. coli AqpZ.

This study describes the interaction between sodium dodecyl sulfate (SDS) and membrane proteins reconstituted into large unilamellar lipid vesicles and detergent micelles studied by circular dichroism (CD) and polarity sensitive probe labeling. Specifically, we carried out a comparative study of two aquaporins with high structural homology SoPIP2;1 and AqpZ using identical reconstitution conditions. Our CD results indicate that SDS, when added to membrane-reconstituted aquaporins in concentrations below the SDS critical micelle concentration (CMC, ~8mM), causes helical rearrangements of both aquaporins. However, we do not find compelling evidence for unfolding. In contrast when SDS is added to detergent stabilized aquaporins, SoPIP2;1 partly unfolds, while AqpZ secondary structure is unaffected. Using a fluorescent polarity sensitive probe (Badan) we show that SDS action on membrane reconstituted SoPIP2;1 as well as AqpZ is associated with initial increased hydrophobic interactions in protein transmembrane (TM) spanning regions up to a concentration of 0.1× CMC. At higher SDS concentrations TM hydrophobic interactions, as reported by Badan, decrease and reach a plateau from SDS CMC up to 12.5× CMC. Combined, our results show that SDS does not unfold neither SoPIP2;1 nor AqpZ during transition from a membrane reconstituted form to a detergent stabilized state albeit the native folds are changed.

The role of water coordination in the pH-dependent gating of hAQP10.

Human aquaporin 10 (hAQP10) is an aquaglyceroporin that assists in maintaining glycerol flux in adipocytes during lipolysis at low pH. Hence, a molecular understanding of the pH-sensitive glycerol conductance may open up for drug development in obesity and metabolically related disorders. Control of hAQP10-mediated glycerol flux has been linked to the cytoplasmic end of the channel, where a unique loop is regulated by the protonation status of histidine 80 (H80). Here, we performed unbiased molecular dynamics simulations of three protonation states of H80 to unravel channel gating. Strikingly, at neutral pH, we identified a water coordination pattern with an inverted orientation of the water molecules in vicinity of the loop. Protonation of H80 results in a more hydrophobic loop conformation, causing loss of water coordination and leaving the pore often dehydrated. Our results indicate that the loss of such water interaction network may be integral for the destabilization of the loop in the closed configuration at low pH. Additionally, a residue unique to hAQP10 (F85) reveals structural importance by flipping into the channel in correlation with loop movements, indicating a loop-stabilizing role in the closed configuration. Taken together, our simulations suggest a unique gating mechanism combining complex interaction networks between water molecules and protein residues at the loop interface. Considering the role of hAQP10 in adipocytes, the detailed molecular insights of pH-regulation presented here will help to understand glycerol pathways in these cells and may assist in drug discovery for better management of human adiposity and obesity.

An epifluorescence microscopy method for generalized polarization imaging.

Generalized polarization (GP) microscopy represents an excellent tool to study lipid-lipid and lipid-protein interactions in situ and in vitro. Here, we present an efficient and cost effective method to perform GP microscopy using a standard light-emitting diode (LED) epifluorescence microscope equipped with a digital color camera.

Assessing the efficacy of vesicle fusion with planar membrane arrays using a mitochondrial porin as reporter.

Reconstitution of functionally active membrane protein into artificially made lipid bilayers is a challenge that must be overcome to create a membrane-based biomimetic sensor and separation device. In this study we address the efficacy of proteoliposome fusion with planar membrane arrays. We establish a protein incorporation efficacy assay using the major non-specific porin of Fusobacterium nucleatum (FomA) as reporter. We use electrical conductance measurements and fluorescence microscopy to characterize proteoliposome fusion with an array of planar membranes. We show that protein reconstitution in biomimetic membrane arrays may be quantified using the developed FomA assay. Specifically, we show that FomA vesicles are inherently fusigenic. Optimal FomA incorporation is obtained with a proteoliposome lipid-to-protein molar ratio (LPR)=50 more than 10(5) FomA proteins could be incorporated in a bilayer array with a total membrane area of 2mm(2) within 20 min. This novel assay for quantifying protein delivery into lipid bilayers may be a useful tool in developing biomimetic membrane applications.

Formation of giant protein vesicles by a lipid cosolvent method.

This paper describes a method to create giant protein vesicles (GPVs) of ≥10 µm by solvent-driven fusion of large vesicles (0.1-0.2 µm) with reconstituted membrane proteins. We found that formation of GPVs proceeded from rotational mixing of protein-reconstituted large unilamellar vesicles (LUVs) with a lipid-containing solvent phase. We made GPVs by using n-decane and squalene as solvents, and applied generalized polarization (GP) imaging to monitor the polarity around the protein transmembrane region of aquaporins labeled with the polarity-sensitive probe Badan. Specifically, we created GPVs of spinach SoPIP2;1 and E. coli AqpZ aquaporins. Our findings show that hydrophobic interactions within the bilayer of formed GPVs are influenced not only by the solvent partitioning propensity, but also by lipid composition and membrane protein isoform.