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We report on the fabrication of AqpZ immobilized flat sheet membranes. The effects of interfacial polymerization conditions as well as proteoliposome concentration were evaluated. Commercial AqpZ were used as positive control for cloned AqpZ. Specific permeate flux of membranes at higher proteoliposome concentrations increased up to 25 times higher than thin film composite membranes; however; MgSO4 rejection is lowered almost to 1.5%. FTIR and SEM confirm immobilization of proteoliposomes. Thermal analysis showed that increasing proteoliposome concentration has no positive effect on the incorporation of proteoliposomes into polyamide structures. On the contrary, at lower proteoliposome concentrations, incorporation of proteoliposomes was found better. When combined membrane performances were compared in terms of specific permeate flux; MgSO4 and humic rejection and flux recovery after humic acid filtration, the performance of cloned AqpZ incorporated membranes (having 0.1 mg/mL proteoliposome concentration and polyamide formed with 2 min piperazine reaction time) improved 1.7 times regarding TFC membranes. According to the results, increasing proteoliposome concentration did not improve nanofiltration membrane performance. On the contrary, lower proteoliposome concentrations were found to be more effective in increasing membrane performance.

Native mass spectrometry (native-MS) of membrane proteins typically requires a detergent screening protocol, protein solubilization in the preferred detergent, followed by protein liberation from the micelle by collisional activation. Here, submicrometer nano-ESI emitter tips are used for native-MS of membrane proteins solubilized in both nonionic and ionic detergent solutions. With the submicrometer nano-ESI emitter tips, resolved charge-state distributions of membrane protein ions are obtained from a 150 mM NaCl, 25 mM Tris-HCl with 1.1% octyl glucoside solution. The relative abundances of NaCl and detergent cluster ions at high m /z are significantly reduced with the submicrometer emitters compared with larger nano-ESI emitters that are commonly used. This technique is beneficial for significantly decreasing the abundances (by two to three orders of magnitude compared with the larger tip size: 1.6 μm) of detergent cluster ions formed from aqueous ammonium acetate solutions containing detergents that can overlap with the membrane protein ion signal. Resolved charge-state distributions of membrane protein ions from aqueous ammonium acetate solutions containing ionic detergents were obtained with the submicrometer nano-ESI emitters; this is the first report of native-MS of membrane proteins solubilized by ionic detergents. Graphical Abstract.

Aquaporins are integral membrane channel proteins found in all kingdoms of life. The Escherichia coli aquaporin Z (AqpZ) has been shown to solely conduct water at high permeability. Functional AqpZ is generally purified from the membrane fraction. However, the quantity of the purified protein is limited. In this study, a new method is developed to achieve high yield of bioactive AqpZ protein. A mild detergent n-dodecyl-β-D-maltopyranoside (DDM) was used to solubilize the over-expressed insoluble AqpZ from inclusion bodies without a refolding process. The recovered AqpZ protein showed high water permeability comparable with AqpZ obtained from the membrane fraction. In this way, the total yield of bioactive AqpZ has been increased greatly, which will facilitate the structural and functional characterization and future applications of AqpZ.

BACKGROUND: The discovery of stable, yet functional, protein mutants is a limiting factor in the development of biotechnological applications, structural studies or in drug discovery. Rapid detection of functional mutants is especially challenging for water channel aquaporins, as they do not have a directly measurable enzymatic or binding activity. Current methods available are time consuming and only applicable to specific aquaporins.
METHODS: Herein we describe an assay based on the protective effect of aquaporins on yeast S. cerevisiae in response to rapid freezing.
RESULTS: Yeast overexpressing a functional water-permeable aquaporin of choice are rescued after the challenge, while inactive or blocked aquaporins confer no protection and lead to cell death. The potential of this assay is shown by screening a small number of E. coli aquaporin Z (AQPZ) mutants. Additionally, a library of ~10,000 drug-like compounds was tested against human AQP1 (hAQP1).
CONCLUSIONS: Since rescue is only dependent on transmembrane water flux, the assay is applicable to water-permeable aquaporins of any origin.
CONCLUSIONS: Mapping of permissive mutations on the aquaporin structure can help delineate the minimal requirements for effective water transport. Alternatively, the assay can be potentially used to discover compounds that inhibit aquaporin water transport. When additionally screened for thermostability, functional aquaporin mutants can be useful in the development of biomimetic membranes for water purification, or to improve the likelihood of producing well-diffracting crystals, enabling rational design of much needed aquaporin inhibitors.

Aquaporin Z (AqpZ) is a water channel protein from Escherichia coli and has attracted many attentions to develop the biomimetic water filtration technology. Cell-free protein synthesis (CFPS) system, one of the most complex multi-enzymatic systems, has the ability of producing the integral membrane protein in vitro. To enhance the synthesis of AqpZ in E. coli cell-free system, several natural leader peptides were respectively fused at the N-terminus and were verified to enhance the expression level significantly. Moreover, the supplementation of detergents or liposome could activate leader peptidase from the cell-free extract and provide hydrophobic environment for proper folding of AqpZ. Thus, the release of mature AqpZ via the in situ removal of leader peptide was achieved, with a specific water transport activity of (2.1 ± 0.1) × 10⁻¹⁴ cm³ s⁻¹ monomer⁻¹. Using this in situ removable leader peptide strategy, the transcription-translation, leader sequence cleavage and membrane protein folding were integrated into a simple process in the cell-free system, providing a convenient approach to enhance the expression of target proteins, especially those membrane proteins difficult to achieve.

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.