Proteoliposome-Incorporated Seawater Reverse Osmosis Polyamide Membrane: Is the Aquaporin Water Channel Effect in Improving Membrane Performance Overestimated?

The water channel feature of the aquaporin (AQP) is considered to be the key in improving the permselectivity of AQP-based thin-film composite (TFC) polyamide (PA) membranes, yet much less attention has been paid to the physicochemical property changes of the PA layer induced by AQP-reconstituted proteoliposomes. This study systematically investigated the roles of proteoliposome constituents (liposome/detergent/AQP) in affecting the physicochemical properties and performance of the membranes. For the first time, we demonstrated that the constituents in the proteoliposome could facilitate the formation of a PA layer with enlarged protuberances and thinner crumples, resulting in a 79% increase in effective surface area and lowering of hydraulic resistance for filtration. These PA structural changes of the AQP-based membrane were found to contribute over 70% to the water permeability increase via comparing the separation performance of the membranes prepared with liposome, detergent, and proteoliposome, respectively, and one proteoliposome-ruptured membrane. The contribution from the AQP water channel feature was about 27% of water permeability increase in the current study, attributed to only ∼20% vesicle coverage in the PA matrix, and this contribution may be easily lost as a result of vesicle rupture during the real seawater reverse osmosis process. This study reveals that the changed morphology dominates the performance improvement of the AQP-based PA membrane and well explains why the actual AQP-based PA membranes cannot acquire the theoretical water/salt selectivity of a biomimetic AQP membrane, deepening our understanding of the AQP-based membranes.

Reproducible Preparation of Proteopolymersomes via Sequential Polymer Film Hydration and Membrane Protein Reconstitution.

Film rehydration method is commonly used for membrane protein (MP) reconstitution into block copolymer (BCP), but the lack of control in the rehydration step formed a heterogeneous population of proteopolymersomes that interferes with the characterization and performance of devices incorporating them. To improve the self-assembly of polymersomes with simultaneous MP reconstitution, the study reported herein aimed to understand the effects of different variants of the rehydration procedure on the MP reconstitution into BCP membranes. The model MP used in this study was AquaporinZ (AqpZ), an α-helical MP that has been shown to have a high permeation rate exclusive to water molecules. Comparing four rehydration methods differing in the hydration time (i.e., brief wetting or full hydration) and medium (i.e., in buffer or AqpZ stock solution), prehydration with buffer prior to adding AqpZ was found to be most desirable and reproducible reconstitution method because it gave rise to the highest proportion of well-formed vesicles with intact AqpZ functionality as evidenced by the transmission electron microscopy images, dynamic light scattering, and stopped-flow analyses. The mechanisms by which effective AqpZ reconstitution takes place were also investigated and discussed. Small-angle X-ray scattering analysis shows that hydrating the initially dry multilamellar BCP films allows the separation of lamellae. This is anticipated to increase the membrane fluidity that facilitates a fast and spontaneous integration of AqpZ as the detergent concentration is considerably lowered below its critical micelle concentration. Dilution of detergent can result in precipitation of proteins in the absence of well-fluidized membranes for protein integration that underscores the importance of membrane fluidity in MP reconstitution.

Fragment Screening of Human Aquaporin 1.

Aquaporins (AQPs) are membrane proteins that enable water transport across cellular plasma membranes in response to osmotic gradients. Phenotypic analyses have revealed important physiological roles for AQPs, and the potential for AQP water channel modulators in various disease states has been proposed. For example, AQP1 is overexpressed in tumor microvessels, and this correlates with higher metastatic potential and aggressiveness of the malignancy. Chemical modulators would help in identifying the precise contribution of water channel activity in these disease states. These inhibitors would also be important therapeutically, e.g., in anti-cancer treatment. This perceived importance contrasts with the lack of success of high-throughput screens (HTS) to identify effective and specific inhibitors of aquaporins. In this paper, we have screened a library of 1500 "fragments", i.e., smaller than molecules used in HTS, against human aquaporin (hAQP1) using a thermal shift assay and surface plasmon resonance. Although these fragments may not inhibit their protein target, they bound to and stabilized hAQP1 (sub mM binding affinities (KD), with an temperature of aggregation shift ΔTagg of +4 to +50 °C) in a concentration-dependent fashion. Chemically expanded versions of these fragments should follow the determination of their binding site on the aquaporin surface.

Flavivirus Zika NS4A protein forms large oligomers in liposomes and in mild detergent.

In flaviviruses such as Dengue or Zika, non-structural (NS) NS4A protein forms homo-oligomers, participates in membrane remodelling and is critical for virulence. In both viruses, mature NS4A has the same length and three predicted hydrophobic domains. The oligomers formed by Dengue NS4A are reported to be small (n = 2, 3), based on denaturing SDS gels, but no high-resolution structure of a flavivirus NS4A protein is available, and the size of the oligomer in lipid membranes is not known. Herein we show that crosslinking Zika NS4A protein in lipid membranes results in oligomers at least up to hexamers. Further, sedimentation velocity shows that NS4A in mild detergent C14-betaine appears to be in fast equilibrium between at least two species, where one is smaller, and the other larger, than a trimer or a tetramer. Consistently, sedimentation equilibrium data was best fitted to a model involving an equilibrium between dimers (n = 2) and hexamers (n = 6). Overall, the large, at least hexameric, oligomers obtained herein in liposomes and in mild detergent are more likely to represent the forms of NS4A present in cell membranes.

Effects of proteoliposome composition and draw solution types on separation performance of aquaporin-based proteoliposomes: implications for seawater desalination using aquaporin-based biomimetic membranes.

Aquaporins are a large family of water transport proteins in cell membranes. Their high water permeability and solute rejection make them potential building blocks for high-performance biomimetic membranes for desalination. In the current study, proteoliposomes were prepared using AquaporinZ from Escherichia coli cells, and their separation properties were characterized by stopped-flow measurements. The current study systematically investigated the effect of proteoliposome composition (lipid type, protein-to-lipid ratio (PLR), and the addition of cholesterol) on water permeability and NaCl retention. Among the various lipids investigated, 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC)-based proteoliposomes were found to have excellent osmotic water permeability and NaCl reflection coefficient values. Increasing the PLR of DOPC proteoliposomes up to 1:200 increased their osmotic water permeability. However, further increase in the PLR reduced the osmotic water permeability probably due to the occurrence of defects in the proteoliposomes, whereas the addition of cholesterol improved their osmotic water permeation likely due to defects sealing. The current study also investigated the effect of major dissolved ions in seawater (e.g., Mg(2+) and SO(4)(2-)) on the stability of proteoliposomes, and design criteria for aquaporin-based biomimetic membranes are proposed in the context of desalination.

Fusion behaviour of aquaporin Z incorporated proteoliposomes investigated by quartz crystal microbalance with dissipation (QCM-D).

Aquaporin-based biomimetic membranes have potential as promising membranes for water purification and desalination due to the exceptionally high water permeability and selectivity of aquaporins. However, the design and preparation of such membranes for practical applications are very challenging as the relevant fundamental research is rather limited to provide guidance. Here we investigated the basic characteristics and fusion behaviour of proteoliposomes incorporated with aquaporin Z (AqpZ) on to solid surfaces. This study is expected to offer a better understanding of the properties of proteoliposomes and the potential of the vesicle fusion technique. Our results show that after incorporation of AqpZ, the size and surface charge density of the proteoliposomes change significantly compared with those of liposomes. Although the liposome could easily form a supported lipid bilayer on silica via vesicle rupture, it is much more difficult for proteoliposomes to fuse completely into a bilayer on the same substrate. In addition, the fusion of proteoliposomes is further hindered as the density of incorporated AqpZ is increased, suggesting that proteoliposome with more proteins become more robust. However, both the liposome and proteoliposome have difficulty forming supported lipid bilayers on the surface of a polyelectrolyte layer even though it carries an opposite charge, indicating that the polymer may play an important role in stabilising vesicles. It was also observed that a high concentration of AqpZ could be incorporated into the 1,2-diphytanoyl-sn-glycero-3-phosphocholine (DPhPC) liposome even though its permeability decreased. These findings may provide some useful guidance for preparing such biomimetic membranes.

C-deuterated alanine: a new label to study membrane protein structure using site-specific infrared dichroism.

The helix tilt and rotational orientation of the transmembrane segment of M2, a 97-residue protein from the Influenza A virus that forms H(+)-selective ion channels, have been determined by attenuated total reflection site-specific infrared dichroism using a novel labeling approach. Triple C-deuteration of the methyl group of alanine in the transmembrane domain of M2 was used, as such modification shifts the asymmetric and symmetric stretching vibrations of the methyl group to a transparent region of the infrared spectrum. Structural information can then be obtained from the dichroic ratios corresponding to these two vibrations. Two consecutive alanine residues were labeled to enhance signal intensity. The results obtained herein are entirely consistent with previous site-specific infrared dichroism and solid-state nuclear magnetic resonance experiments, validating C-deuterated alanine as an infrared structural probe that can be used in membrane proteins. This new label adds to the previously reported (13)C [double bond] (18)O and C-deuterated glycine as a tool to analyze the structure of simple transmembrane segments and will also increase the feasibility of the study of polytopic membrane proteins with site-specific infrared dichroism.

Topology of the Salmonella invasion protein SipB in a model bilayer.

A critical early event in Salmonella infection is entry into intestinal epithelial cells. The Salmonella invasion protein SipB is required for the delivery of bacterial effector proteins into target eukaryotic cells, which subvert signal transduction pathways and cytoskeletal dynamics. SipB inserts into the host plasma membrane during infection, and the purified protein has membrane affinity and heterotypic membrane fusion activity in vitro. We used complementary biochemical and biophysical techniques to investigate the topology of purified SipB in a model membrane. We show that the 593 residue SipB is predominantly alpha-helical in aqueous solution, and that no significant change in secondary structural content accompanies lipid interaction. SipB contains two -helical transmembrane domains (residues 320-353 and 409-427), which insert deeply into the bilayer. Their integration allowed the hydrophilic region between the hydrophobic domains (354-408) to cross the bilayer. SipB membrane integration required both the hydrophobic domains and an additional helical C-terminal region (428-593). Further spectroscopic analysis of these domains in isolation showed that the hydrophobic regions insert obliquely into the bilayer, whereas the C-terminal domain associates with the bilayer surface, tilted parallel to the membrane. The combined data suggest a topological model for membrane-inserted SipB.