Rapid fabrication of precise high-throughput filters from membrane protein nanosheets.

Biological membranes are ideal for separations as they provide high permeability while maintaining high solute selectivity due to the presence of specialized membrane protein (MP) channels. However, successful integration of MPs into manufactured membranes has remained a significant challenge. Here, we demonstrate a two-hour organic solvent method to develop 2D crystals and nanosheets of highly packed pore-forming MPs in block copolymers (BCPs). We then integrate these hybrid materials into scalable MP-BCP biomimetic membranes. These MP-BCP nanosheet membranes maintain the molecular selectivity of the three types of ß-barrel MP channels used, with pore sizes of 0.8 nm, 1.3 nm, and 1.5 nm. These biomimetic membranes demonstrate water permeability that is 20-1,000 times greater than that of commercial membranes and 1.5-45 times greater than that of the latest research membranes with comparable molecular exclusion ratings. This approach could provide high performance alternatives in the challenging sub-nanometre to few-nanometre size range.

Concentrating membrane proteins using ultrafiltration without concentrating detergents.

Membrane proteins (MPs) are of rapidly growing interest in the design of pharmaceutical products, novel sensors, and synthetic membranes. Ultrafiltration (UF) using commercially available centrifugal concentrators is typically employed for laboratory-scale concentration of low-yield MPs, but its use is accompanied by a concomitant increase in concentration of detergent micelles. We present a detailed analysis of the hydrodynamic processes that control detergent passage during ultrafiltration of MPs and propose methods to optimize detergent passage during protein concentration in larger-scale membrane processes. Experiments were conducted using nonionic detergents, octyl-ß-D glucoside (OG), and decyl-ß-D maltoside (DM) with the bacterial water channel protein, Aquaporin Z (AqpZ) and the light driven chloride pump, halorhodopsin (HR), respectively. The observed sieving coefficient (So ), a measure of detergent passage, was evaluated in both stirred cell and centrifugal systems. So for DM and OG increased with increasing filtrate flux and decreasing shear rates in the stirred cell, that is, with increasing concentration polarization (CP). Similar effects were observed during filtration of MP-detergent (MPD) micelles. However, lower transmission was observed in the centrifugal system for both detergent and MPD systems. This is attributed to free convection-induced shear and hence reduced CP along the membrane surface during centrifugal UF. Thus to concentrate MPs without retention of detergent, design of UF systems that promote CP is required. Biotechnol. Bioeng. 2016;113: 2122-2130. © 2016 Wiley Periodicals, Inc.

Highly permeable polymeric membranes based on the incorporation of the functional water channel protein Aquaporin Z.

The permeability and solute transport characteristics of amphiphilic triblock-polymer vesicles containing the bacterial water-channel protein Aquaporin Z (AqpZ) were investigated. The vesicles were made of a block copolymer with symmetric poly-(2-methyloxazoline)-poly-(dimethylsiloxane)-poly-(2-methyloxazoline) (PMOXA(15)-PDMS(110)-PMOXA(15)) repeat units. Light-scattering measurements on pure polymer vesicles subject to an outwardly directed salt gradient in a stopped-flow apparatus indicated that the polymer vesicles were highly impermeable. However, a large enhancement in water productivity (permeability per unit driving force) of up to approximately 800 times that of pure polymer was observed when AqpZ was incorporated. The activation energy (E(a)) of water transport for the protein-polymer vesicles (3.4 kcal/mol) corresponded to that reported for water-channel-mediated water transport in lipid membranes. The solute reflection coefficients of glucose, glycerol, salt, and urea were also calculated, and indicated that these solutes are completely rejected. The productivity of AqpZ-incorporated polymer membranes was at least an order of magnitude larger than values for existing salt-rejecting polymeric membranes. The approach followed here may lead to more productive and sustainable water treatment membranes, whereas the variable levels of permeability obtained with different concentrations of AqpZ may provide a key property for drug delivery applications.

Observing proteins as single molecules encapsulated in surface-tethered polymeric nanocontainers.

Immobilizing biomolecules provides the advantage of observing them individually for extended time periods, which is impossible to accomplish for freely diffusing molecules in solution. In order to immobilize individual protein molecules, we encapsulated them in polymeric vesicles made of amphiphilic triblock copolymers and tethered the vesicles to a cover slide surface. A major goal of this study is to investigate polymeric vesicles with respect to their suitability for protein-folding studies. The fact that polymeric vesicles possess an extreme stability under various chemical conditions is supported by our observation that harsh unfolding conditions do not perturb the structural integrity of the vesicles. Moreover, polymerosomes prove to be permeable to GdnHCl and, thereby, ideally suited for unfolding and refolding studies with encapsulated proteins. We demonstrate this with encapsulated phosphoglycerate kinase, which was fluorescently labeled with Atto655, a dye that exhibits pronounced photoinduced electron transfer (PET) to a nearby tryptophan residue in the native state. Under unfolding conditions, PET was reduced, and we monitored alternating unfolding and refolding conditions for individual encapsulated proteins.

Membrane Protein Insertion into and Compatibility with Biomimetic Membranes.

Membrane protein and membrane protein-mimic functionalized materials are rapidly gaining interest across a wide range of applications, including drug screening, DNA sequencing, drug delivery, sensors, water desalination, and bioelectronics. In these applications, material performance is highly dependent on activity-per-protein and protein packing density in bilayer and bilayer-like structures collectively known as biomimetic membranes. However, a clear understanding of, and accurate tools to study these properties of biomimetic membranes does not exist. This paper presents methods to evaluate membrane protein compatibility with biomimetic membrane materials. The methods utilized provide average single protein activity, and for the first time, provide experimentally quantifiable measures of the chemical and physical compatibility between proteins (and their mimics) and membrane materials. Water transport proteins, rhodopsins, and artificial water channels are reconstituted into the full range of current biomimetic membrane matrices to evaluate the proposed platform. Compatibility measurement results show that both biological and artificial water channels tested largely preserve their single protein water transport rates in biomimetic membranes, while their reconstitution density is variable, leading to different overall membrane permeabilities. It is also shown that membrane protein insertion efficiency inversely correlates with both chemical and physical hydrophobicity mismatch between membrane protein and the membrane matrix.

Molecular cloning, overexpression and characterization of a novel water channel protein from Rhodobacter sphaeroides.

Aquaporins are highly selective water channel proteins integrated into plasma membranes of single cell organisms; plant roots and stromae; eye lenses, renal and red blood cells in vertebrates. To date, only a few microbial aquaporins have been characterized and their physiological importance is not well understood. Here we report on the cloning, expression and characterization of a novel aquaporin, RsAqpZ, from a purple photosynthetic bacterium, Rhodobacter sphaeroides ATCC 17023. The protein was expressed homologously at a high yield (∼20 mg/L culture) under anaerobic photoheterotrophic growth conditions. Stopped-flow light scattering experiments demonstrated its high water permeability (0.17±0.05 cm/s) and low energy of activation for water transport (2.93±0.60 kcal/mol) in reconstituted proteoliposomes at a protein to lipid ratio (w/w) of 0.04. We developed a fluorescence correlation spectroscopy based technique and utilized a fluorescent protein fusion of RsAqpZ, to estimate the single channel water permeability of RsAqpZ as 1.24 (±0.41) x 10(-12) cm(3)/s or 4.17 (±1.38)×10(10) H2O molecules/s, which is among the highest single channel permeability reported for aquaporins. Towards application to water purification technologies, we also demonstrated functional incorporation of RsAqpZ in amphiphilic block copolymer membranes.

Membrane protein distribution in composite polymer-lipid thin films.

We present a model system to demonstrate that the positioning of biomolecules (membrane proteins) in a nonnative, complex thin film environment can be regulated by the phase behavior of film components. Partial separation between an amphiphilic polymer and a lipid drives the protein to a fluid phase, mechanically more similar to a cellular bilayer.

SOD antioxidant nanoreactors: influence of block copolymer composition on the nanoreactor efficiency.

The bioavailability limitations of proteins make them difficult to be directly delivered, particularly in diseases caused by insufficient amounts or inactive variants of those proteins. Nanoreactors represent a new promising approach to overcome these limitations because they serve both to protect the protein in their aqueous interior, and simultaneously to allow the protein to act in situ. Here we examine an antioxidant nanoreactor based on SOD encapsulated in amphiphilic block copolymer nanovesicles, and analyze its behavior as a function of the copolymer composition. The membrane of the triblock copolymer nanovesicles plays a double role, both to shield the sensitive protein and selectively to let superoxide and dioxygen penetrate to its inner space. The encapsulation efficiency for different triblock copolymer vesicles was quantified by fluorescence correlation spectroscopy using a fluorescently labeled SOD. Pulse radiolysis experiments and an enzymatic assay were used to compare the permeability of the wall-forming membranes towards superoxide anions. While the encapsulation efficiency mainly depends on the vesicle dimensions, the membrane permeability is mainly affected by the length of the hydrophobic PDMS middle blocks of our polymers. For polymers with very long PDMS chains superoxide anion transport across the membranes was too slow to be detected by our experiments.

Monolayer interactions between lipids and amphiphilic block copolymers.

Interactions in binary mixed monolayers from lipids 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) and amphiphilic poly(2-methyloxazoline)-block-poly(dimethylsiloxane)-block-poly(2-methyloxazoline) block copolymers were studied by using the Langmuir balance technique and Brewster angle microscopy. It is shown that monolayers from the saturated lipid (DPPC) are more sensitive to the presence of polymers in the film, resulting in phase separation and the formation of pure lipid domains at high surface pressure. The morphology and composition of such phase-separated lipid-polymer films were studied by fluorescence microscopy and ToF-SIMS. In contrast, in DOPC-containing monolayers, the polymers tend to phase-separate at low surface pressures only and homogeneous films are obtained upon further compression, due to higher lipid fluidity. The analysis of excess energy of mixing shows that while the separation effect in densely packed DPPC-containing films is strongly dependent on the polymer size (with the larger polymer having a much stronger influence), in the case of monolayers with DOPC much smaller effects are observed. The results are discussed in terms of the monolayer composition, lipid fluidity, and polymer size.

Inhibition of macrophage phagocytotic activity by a receptor-targeted polymer vesicle-based drug delivery formulation of pravastatin.

Ruptures of macrophage-rich atherosclerotic plaques in the coronary arteries are the main reason for heart attack. Targeted therapeutic interventions with an inhibitory effect on the macrophages promise to be beneficial, but currently available drugs such as statins achieve event reductions of only 30%. Dose-limiting adverse effects in remote organs prohibit achieving higher drug levels known to have strong inhibitory effects on macrophages. Receptor-specific targeting using statin-loaded nanometer-sized triblock copolymer vesicles with targeting moieties might allow high-dose treatment for improved efficacy, while minimizing toxicity in other cells. Vesicle uptake by target cells but not other cell types and slow intracellular content release was observed. A major improvement in biologic efficacy was observed for polymer vesicles compared to free drug, whereas no increased cytotoxicity was observed in muscle cells. Such high-dose, targeted therapy of statins through cell-specific polymer vesicles allows novel treatment paradigms not only for atherosclerosis, but appears promising for a wide range of drugs and diseases.