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.     

Translational Diffusion and Interaction of a Photoreceptor and Its Cognate Transducer Observed in Giant Unilamellar Vesicles by Using Dual‐Focus FCS

In order to monitor membrane–protein binding in lipid bilayers at physiological protein concentrations, we employed the recently developed dual‐focus fluorescence correlation spectroscopy (2fFCS) technique. In a case study on a photoreceptor consisting of seven transmembrane helices and its cognate transducer (two transmembrane helices), the lateral diffusion for these integral membrane proteins was analyzed in giant unilamellar vesicles (GUVs). The two‐dimensional diffusion coefficients of both separately diffusing proteins differ significantly, with D=2.2×10^?8 cm² s^?1 for the photoreceptor and with D=4.1×10^?8 cm² s^?1 for the transducer. In GUVs with both membrane proteins present together, we observed significantly smaller diffusion coefficients for labelled transducer molecules; this indicates the presence of larger diffusing units and therefore intermolecular protein binding. Based on the phenomenological dependence of diffusion coefficients on the molecule's cylindrical radius, we are able to estimate the degree of membrane protein binding on a quantitative level.     

Observations of Membrane Domain Reorganization in Mechanically Compressed Artificial Cells

Giant unilamellar vesicles (GUVs) are considered to be the gold standard for assembling artificial cells from the bottom up. In this study, we investigated the behavior of such biomimetic vesicles as they were subjected to mechanical compression. A microfluidic device is presented that comprises a trap to capture GUVs and a microstamp that is deflected downwards to mechanically compress the trapped vesicle. After characterization of the device, we show that single-phase GUVs can be controllably compressed to a high degree of deformation (D=0.40) depending on the pressure applied to the microstamp. A permeation assay was implemented to show that vesicle bursting is prevented by water efflux. Next, we mechanically compressed GUVs with co-existing liquid-ordered and liquid-disordered membrane phases. Upon compression, we observed that the normally stable lipid domains reorganized themselves across the surface and fused into larger domains. This phenomenon, observed here in a model membrane system, not only gives us insights into how the multicomponent membranes of artificial cells behave, but might also have interesting consequences for the role of lipid rafts in biological cells that are subjected to compressive forces in a natural environment.     

Single-Step Reconstitution of Apo-Hemoproteins at the Disruption Stage of Escherichia coli Cells

Hemoproteins on their metal: We report a novel strategy for the reconstitution of hemoproteins with non-natural metal complexes; simple addition of manganese and ruthenium porphyrin to E. coli cells immediately prior to homogenization yields the reconstituted proteins. We believe that this simple approach could become a standard reconstitution method for hemoproteins.     

Surfactant-Assisted Purification of Hydrophobic DNA Nanostructures

Purification of functional DNA nanostructures is an essential step in achieving intended functions because misfolded structures and the remaining free DNA strands in a solution can interact and affect their behavior. However, due to hydrophobicity-mediated aggregation, it is difficult to purify DNA nanostructures modified with hydrophobic molecules by conventional methods. Herein, we report the purification of cholesterol-modified DNA nanostructures by using a novel surfactant-assisted gel extraction. The addition of sodium cholate (SC) to the sample solution before structure folding prevented aggregation; this was confirmed by gel electrophoresis. We also found that adding sodium dodecyl sulfate (SDS) to the sample inhibited structural folding. The cholesterol-modified DNA nanostructures prepared with SC were successfully purified by gel extraction, and their ability to bind to the lipid membrane surfaces was maintained. This method will facilitate the purification of DNA nanostructures modified with hydrophobic molecules and expand their applicability in the construction of artificial cell-like systems.     

Optimization of the Inverted Emulsion Method for High-Yield Production of Biomimetic Giant Unilamellar Vesicles

In the field of bottom-up synthetic biology, lipid vesicles provide an important role in the construction of artificial cells. Giant unilamellar vesicles (GUVs), due to their membrane's similarity to natural biomembranes, have been widely used as cellular mimics. So far, several methods exist for the production of GUVs with the possibility to encapsulate biological macromolecules. The inverted emulsion-based method is one such technique, which has great potential for rapid production of GUVs with high encapsulation efficiencies for large biomolecules. However, the lack of understanding of various parameters that affect production yields has resulted in sparse adaptation within the membrane and bottom-up synthetic biology research communities. Here, we optimize various parameters of the inverted emulsion-based method to maximize the production of GUVs. We demonstrate that the density difference between the emulsion droplets, oil phase, and the outer aqueous phase plays a crucial role in vesicle formation. We also investigated the impact that centrifugation speed/time, lipid concentration, pH, temperature, and emulsion droplet volume has on vesicle yield and size. Compared to conventional electroformation, our preparation method was not found to significantly alter the membrane mechanical properties. Finally, we optimize the parameters to minimize the time from workbench to microscope and in this way open up the possibility of time-sensitive experiments. In conclusion, our findings will promote the usage of the inverted emulsion method for basic membrane biophysics studies as well as the development of GUVs for use as future artificial cells.     

Effect of Amyloid-β Monomers on Lipid Membrane Mechanical Parameters–Potential Implications for Mechanically Driven Neurodegeneration in Alzheimer’s Disease

Alzheimer’s disease (AD) is a neurodegenerative disease that results in memory loss and the impairment of cognitive skills. Several mechanisms of AD’s pathogenesis were proposed, such as the progressive accumulation of amyloid-β (Aβ) and τ pathology. Nevertheless, the exact neurodegenerative mechanism of the Aβ remains complex and not fully understood. This paper proposes an alternative hypothesis of the mechanism based on maintaining the neuron membrane’s mechanical balance. The incorporation of Aβ decreases the lipid membrane’s elastic properties, which eventually leads to the impairment of membrane clustering, disruption of mechanical wave propagation, and change in gamma oscillations. The first two disrupt the neuron’s ability to function correctly while the last one decreases sensory encoding and perception enabling. To begin discussing this mechanical-balance hypothesis, we measured the effect of two selected peptides, Aβ-40 and Aβ-42, as well as their fluorescently labeled modification, on membrane mechanical properties. The decrease of bending rigidity, consistent for all investigated peptides, was observed using molecular dynamic studies and experimental flicker-noise techniques. Additionally, wave propagation was investigated with molecular dynamic studies in membranes with and without incorporated neurodegenerative peptides. A change in membrane behavior was observed in the membrane system with incorporated Aβ.     

课程重组与模块教学模式的探讨

从职业教育的培养方向、模式要求等方面，阐述了课程重组，即课程整合与模块化教学在高等职业教育中的地位、作用。对机电技术应用专业的课程整合、模块化教学模式从整合的依据、整合的要求、模块的划分、专业能力要求的设定、师资、设备配备、教材的编写等方面进行了探讨。     

重组蛹虫草超氧化物岐化酶脱辅基蛋白的表达、纯化及其活性重建

目的表达并纯化重组蛹虫草超氧化物歧化酶脱辅基蛋白,并考查各种金属离子对其活性重建的作用。方法用不含Cu2+和Zn2+的培养基培养重组菌株,并表达重组蛋白,采用DEAE-FF和CM-52进行纯化;在脱辅基蛋白溶液中加入Cu2+、Mn2+、Mg2+、Ni2+或Ag+,测定SOD酶活力的变化。结果蛹虫草脱辅基Cu,Zn-SOD在不含Cu2+和Zn2+的培养基中得到表达,纯化后的样品经SDS-PAGE分析,在相对分子质量17000处出现单一条带,经活性电泳分析无酶活力;脱辅基蛋白活性重建的最适宜Cu2+浓度为0.005mmol/L,但其紫外吸收图谱与天然SOD不同;0.1mmol/L的Mn2+、Mg2+、Ni2+、Ag+重建的酶活力远低于Cu2+重建的cm-SOD的酶活力。结论已成功表达并纯化了蛹虫草脱辅基Cu,Zn-SOD,各种金属离子只能有限地重建脱辅基Cu,Zn-SOD的活性。     

Self-Assembling Properties and Recovery Effects on Damaged Skin Cells of Chemically Synthesized Mannosylerythritol Lipids

Mannosylerythritol lipids (MELs), which are one of the representative sugar-based biosurfactants (BSs) produced by microorganisms, have attracted much attention in various fields in the sustainable development goals (SDGs) era. However, they are inseparable mixtures with respect to the chain length of the fatty acids. In this study, self-assembling properties and structure-activity relationship (SAR) studies of recovery effects on damaged skin cells using chemically synthesized MELs were investigated. It was revealed, for the first time, that synthetic and homogeneous MELs exhibited significant self-assembling properties to form droplets or giant vesicles. In addition, a small difference in the length of the fatty acid chains of the MELs significantly affected their recovery effects on the damaged skin cells. MELs with medium or longer length alkyl chains exhibited much higher recovery effects than that of C18-ceramide NP.     

Solution‐NMR Characterization of Outer‐Membrane Protein A from E. coli in Lipid Bilayer Nanodiscs and Detergent Micelles

X‐ray crystallography and solution NMR of detergent‐reconstituted OmpA (outer membrane protein A from E. coli) had shown that this protein forms an eight‐stranded transmembrane β‐barrel, but only limited information was obtained for the extracellular loops. In NMR studies of OmpA in two different detergent micelles, “NMR‐invisible” amino acid residues in‐between the extracellular loops and the β‐barrel prevented complete structural characterization. Here, we show that this NMR‐invisible ring around the β‐barrel of OmpA is also present in lipid bilayer nanodiscs and in mixed micelles with a third detergent, thus suggesting that the implicated rate processes have a functional role rather than representing an artifact of the protein reconstitution. In addition to sequence‐specific NMR assignments for OmpA in the nanodiscs, the present results are based on a protocol of micro‐coil TROSY‐ and CRINEPT‐type NMR diffusion measurements for studying the hydrodynamic properties and the foldedness of [²H,¹⁵N]‐labeled membrane proteins in nanodiscs. This protocol can be applied under conditions closely similar to those used for NMR structure determinations or crystallization trials.     

Formation kinetics and stability of surfactant vesicles

A three-component phase diagram of a system containing water, dimethyl isosorbide and Laureth 4 (Brij^® 30), a commercial surfactant, was determined, and the kinetics of vesicle formation from dilution with water of the hydrotrope solution was studied using a stop-flow process in conjunction with light-scattering determinations. The phase diagram consisted of a large microemulsion phase and a lamellar liquid crystalline region. Results from the stop-flow/light-scattering determinations were tentatively interpreted using the Aniansson-Kahlweit-Zana theory of micellar relaxation for a system close to equilibrium. The interpretation indicated the vesicles to be formed by monomolecular buildup for surfactant concentrations less than 5%, while for vesicles formed at greater concentrations an agglomeration of vesicle fractions appeared more reasonable.     

Interaction of vesicles: adhesion, fusion and multicompartment systems formation

In this review, interactions of selected vesicles, induced either by molecular recognition or by electrostatic interactions, for the simulation of cell-cell and cell-drug interaction processes are discussed. In order of increasing complexity, examples of vesicles adhesion are presented at first, followed by recognition experiments in which fusion takes place. This differentiation in behavior was primarily attributed to the structural features of interacting vesicular pairs primarily affected by concentration and lateral phase separation of the anchored recognizable groups. In certain cases, fusion is accompanied by multicompartmentalization of the obtained aggregates. In connection with the formation of these multicompartment systems, it was proposed that an analogous mechanism could be operating in the evolution of eukaryotes during the symbiosis of prokaryotes.     

Evaluation of in vitro stability of small unilamellar vesicles coated with collagen and chitosan

To improve the in vitro stability of small unilamellar vesicles (SUV), the permeability of SUV, made from soyabean phosphatidylcholine (PC) and coated with collagen and chitosan, was studied using 5(6)-carboxyfluorescein (5(6)-CF) as a fluorescence probe. The results showed that the coating with a collagen/PC weight ratio of 2, or a chitosan/PC weight ratio of 8, significantly decreased the permeability of liposomal membranes. In addition, the fluorescence polarization method was used to study the influence of a coating with collagen and chitosan at the above ratios on the fluidity of liposomal membranes, employing an intramolecular charge transfer compound, a 3-methoxy-4′-N ,N-dimethylaminoflavone derivative (DMMF), as a fluorescence probe. The fluidity of a liposomal membrane coated with either of the two macromolecules showed no obvious changes, indicating that SUV coated with collagen and chitosan at appropriate weight ratios, can significantly improve the in vitro stability of liposomal membranes without disturbing their fluidity. © 1999 Society of Chemical Industry     

Manufacture of Multilayered Artificial Cell Membranes through Sequential Bilayer Deposition on Emulsion Templates

Efforts to manufacture artificial cells that replicate the architectures, processes and behaviours of biological cells are rapidly increasing. Perhaps the most commonly reconstructed cellular structure is the membrane, through the use of unilamellar vesicles as models. However, many cellular membranes, including bacterial double membranes, nuclear envelopes, and organelle membranes, are multilamellar. Due to a lack of technologies available for their controlled construction, multilayered membranes are not part of the repertoire of cell-mimetic motifs used in bottom-up synthetic biology. To address this, we developed emulsion-based technologies that allow cell-sized multilayered vesicles to be produced layer-by-layer, with compositional control over each layer, thus enabling studies that would otherwise remain inaccessible. We discovered that bending rigidities scale with the number of layers and demonstrate inter-bilayer registration between coexisting liquid–liquid domains. These technologies will contribute to the exploitation of multilayered membrane structures, paving the way for incorporating protein complexes that span multiple bilayers.