Investigation of selected parameters of capillary zone electrophoresis method for analysis of isolates of outer membrane vesicles

The capillary zone electrophoresis (CZE) has recently been proposed by our group as a novel technique for outer membrane vesicles (OMVs) characterization (J. Chromatography 1621 (2020) 461047). In present work the impact of selected parameters of CZE method on OMVs isolates analysis was assessed. It was shown that the extension of sample injection plug length significantly improves the detectability of macromolecular aggregates in CZE. Moreover, a negligible adsorption of OMVs to both uncoated and polymer-modified (poly(DMA-GMA-MAPS)) capillary walls was proven. Finally, the relaxation effect as well as deformation/polarization of vesicles were demonstrated to affect OMVs electrophoretic mobility. The significance of these findings was discussed.     

Double‐Shell Vesicles,Strings of Vesicles and Filaments Found in Crosslinked Micellar Solutions of Poly(1,2‐butadiene)‐block‐poly(ethylene oxide) Diblock Copolymers

Micellar structures of amphiphilic poly(1,2‐butadiene)‐block‐poly(ethylene oxide) diblock copolymers have been crosslinked in aqueous solution by γ‐irradiation. By transmission electron microscopy (TEM) of negatively stained specimens it is shown that the predominant structures present are copolymer vesicles (which appear to be double‐layered). These fixed vesicles are stable with respect to their shape and can be transferred from water into a good solvent for both blocks, such as tetrahydrofuran, thus demonstrating the effectiveness of the crosslinking. In addition to the vesicles, a small number of flexible cylindrical/filamentous structures, sequentially fused vesicles/strings of vesicles and giant sheet‐like vesicles are also visible after cross‐linking. The vesicle chains seem to be formed by fusion of the double‐layered vesicles; the outer layer of the vesicles apparently fuses sequentially, whereas the inner shell remains intact, creating periodic linear thickenings.     

Solid‐state NMR Study of the YadA Membrane‐Anchor Domain in the Bacterial Outer Membrane

MAS‐NMR was used to study the structure and dynamics at ambient temperatures of the membrane‐anchor domain of YadA (YadA‐M) in a pellet of the outer membrane of E. coli in which it was expressed. YadA is an adhesin from the pathogen Yersinia enterocolitica that is involved in interactions with the host cell, and it is a model protein for studying the autotransport process. Existing assignments were sucessfully transferred to a large part of the YadA‐M protein in the E. coli lipid environment by using ¹³C‐¹³C DARR and PDSD spectra at different mixing times. The chemical shifts in most regions of YadA‐M are unchanged relative to those in microcrystalline YadA‐M preparations from which a structure has previously been solved, including the ASSA region that is proposed to be involved in transition‐state hairpin formation for transport of the soluble domain. Comparisons of the dynamics between the microcrystalline and membrane‐embedded samples indicate greater flexibility of the ASSA region in the outer‐membrane preparation at physiological temperatures. This study will pave the way towards MAS‐NMR structure determination of membrane proteins, and a better understanding of functionally important dynamic residues in native membrane environments.     

Conformation and Topology of Diacylglycerol Kinase in E.coli Membranes Revealed by Solid‐state NMR Spectroscopy

Solid‐state NMR is a powerful tool for studying membrane proteins in a native‐like lipid environment. 3D magic angle spinning (MAS) NMR was employed to characterize the structure of E.coli diacylglycerol kinase (DAGK) reconstituted into its native E.coli lipid membranes. The secondary structure and topology of DAGK revealed by solid‐state NMR are different from those determined by solution‐state NMR and X‐ray crystallography. This study provides a good example for demonstrating the influence of membrane environments on the structure of membrane proteins.     

Point‐to‐Plane Nonhomogeneous Electric‐Field‐Induced Simultaneous Formation of Giant Unilamellar Vesicles (GUVs) and Lipid Tubes

It is well‐known that homogeneous electric fields can be used to generate giant unilamellar vesicles (GUVs). Herein we report an interesting phenomenon of formation of GUVs and lipid tubes simultaneously using a nonhomogeneous electric field generated by point‐to‐plane electrodes. The underlying mechanism was analyzed using finite element analysis. The two forces play main roles, that is, the pulling force (F) to drag GUVs into lipid tubes induced by fluid flow, and the critical force (Fc) to prevent GUVs from deforming into lipid tubes induced by electric fields. In the center area underneath the needle electrode, the GUVs were found because F is less than Fc in that region, whereas in the edge area the lipid tubes were obtained because F is larger than Fc. The diffusion coefficient of lipid in the tubes was found to be 4.45 μm² s^?1 using a fluorescence recovery after photobleaching (FRAP) technique. The method demonstrated here is superior to conventional GUV or lipid tube fabrication methods, and has great potential in cell mimic or hollow material fabrication using GUVs and tubes as templates.     

Probing the interaction of the potassium channel modulating KCNE1 in lipid bilayers via solid‐state NMR spectroscopy

KCNE1 is known to modulate the voltage‐gated potassium channel α subunit KCNQ1 to generate slowly activating potassium currents. This potassium channel is essential for the cardiac action potential that mediates a heartbeat as well as the potassium ion homeostasis in the inner ear. Therefore, it is important to know the structure and dynamics of KCNE1 to better understand its modulatory role. Previously, the Sanders group solved the three‐dimensional structure of KCNE1 in LMPG micelles, which yielded a better understanding of this KCNQ1/KCNE1 channel activity. However, research in the Lorigan group showed different structural properties of KCNE1 when incorporated into POPC/POPG lipid bilayers as opposed to LMPG micelles. It is hence necessary to study the structure of KCNE1 in a more native‐like environment such as multi‐lamellar vesicles. In this study, the dynamics of lipid bilayers upon incorporation of the membrane protein KCNE1 were investigated using ³¹P solid‐state nuclear magnetic resonance (NMR) spectroscopy. Specifically, the protein/lipid interaction was studied at varying molar ratios of protein to lipid content. The static ³¹P NMR and T₁ relaxation time were investigated. The ³¹P NMR powder spectra indicated significant perturbations of KCNE1 on the phospholipid headgroups of multi‐lamellar vesicles as shown from the changes in the ³¹P spectral line shape and the chemical shift anisotropy line width. ³¹P T₁ relaxation times were shown to be reversely proportional to the molar ratios of KCNE1 incorporated. The ³¹P NMR data clearly indicate that KCNE1 interacts with the membrane. Copyright © 2017 John Wiley & Sons, Ltd.     

CHARMM‐GUI Martini Maker for modeling and simulation of complex bacterial membranes with lipopolysaccharides

A complex cell envelope, composed of a mixture of lipid types including lipopolysaccharides, protects bacteria from the external environment. Clearly, the proteins embedded within the various components of the cell envelope have an intricate relationship with their local environment. Therefore, to obtain meaningful results, molecular simulations need to mimic as far as possible this chemically heterogeneous system. However, setting up such systems for computational studies is far from trivial, and consequently the vast majority of simulations of outer membrane proteins still rely on oversimplified phospholipid membrane models. This work presents an update of CHARMM‐GUI Martini Maker for coarse‐grained modeling and simulation of complex bacterial membranes with lipopolysaccharides. The qualities of the outer membrane systems generated by Martini Maker are validated by simulating them in bilayer, vesicle, nanodisc, and micelle environments (with and without outer membrane proteins) using the Martini force field. We expect this new feature in Martini Maker to be a useful tool for modeling large, complicated bacterial outer membrane systems in a user‐friendly manner. © 2017 Wiley Periodicals, Inc.     

Ion Channel Behavior of a Supported Bilayer Lipid Membrane Composed of 5,5－Ditetradecyl－2－ （2－trimethyl－ammonioethyl）－l, 3－dioxane Bromide Modified Glassy Carbon Electrode

A synthetic cationic surfactant, 5,5-ditetradecyl-2-(2-trimethyl-ammonioethyl)-l,3-dioxane bromide (DTDB), was used to construct a supported bilayer lipid membrane (s-BLM) coatedon an underlying glassy carbon electrode (GCE). Electrochemical impedance spectroscopy (EIS), small-angle X-ray diffraction (SAXD) and cyclic voltammetry were used to characterize the s-BLM. Both EIS and SAXD data indicated that the synthetic lipid exists as a well-oriented bilayer in the membrane.The voltammetric study showed that the lipid membrane can open ion channels in the presence of ClO4^- stimulant with Ru(bpy)3^2 as marker ions and give distinct channel currents.The channels can be dosed and open up again many times by removing or introducing ClO4^- anions.     

Creating and Modulating Microdomains in Pore‐Spanning Membranes

The architecture of the plasma membrane is not only determined by the lipid and protein composition, but is also influenced by its attachment to the underlying cytoskeleton. Herein, we show that microscopic phase separation of “raft‐like” lipid mixtures in pore‐spanning bilayers is strongly determined by the underlying highly ordered porous substrate. In detail, lipid membranes composed of DOPC/sphingomyelin/cholesterol/Gb₃ were prepared on ordered pore arrays in silicon with pore diameters of 0.8, 1.2 and 2 μm, respectively, by spreading and fusion of giant unilamellar vesicles. The upper part of the silicon substrate was first coated with gold and then functionalized with a thiol‐bearing cholesterol derivative rendering the surface hydrophobic, which is prerequisite for membrane formation. Confocal laser scanning fluorescence microscopy was used to investigate the phase behavior of the obtained pore‐spanning membranes. Coexisting liquid‐ordered‐ (l_o) and liquid‐disordered (l_d) domains were visualized for DOPC/sphingomyelin/cholesterol/Gb₃ (40:35:20:5) membranes. The size of the l_o‐phase domains was strongly affected by the underlying pore size of the silicon substrate and could be controlled by temperature, and the cholesterol content in the membrane, which was modulated by the addition of methyl‐β‐cyclodextrin. Binding of Shiga toxin B‐pentamers to the Gb₃‐doped membranes increased the l_o‐phase considerably and even induced l_o‐phase domains in non‐phase separated bilayers composed of DOPC/sphingomyelin/cholesterol/Gb₃ (65:10:20:5).     

Heavy‐Atom Labeled Transmembrane β‐Peptides: Synthesis,CD‐Spectroscopy,and X‐ray Diffraction Studies in Model Lipid Multilayer

Transmembrane β‐peptides are promising candidates for the design of well‐controlled membrane anchors in lipid membranes. Here, we present the synthesis of transmembrane β‐peptides with and without tryptophan anchors, as well as a novel iodine‐labeled d ‐β³‐amino acid. By using one or more of the heavy‐atom labeled amino acids as markers, the orientation of the helical peptide was inferred based on the electron‐density profile determined by X‐ray reflectivity. The β‐peptides were synthesized through manual Fmoc‐based solid‐phase peptide synthesis (SPPS) and reconstituted in unilamellar vesicles forming a right‐handed 3₁₄‐helix secondary structure, as shown by circular dichroism spectroscopy. We then integrated the β‐peptide into solid‐supported membrane stacks and carried out X‐ray reflectivity and grazing incidence small‐angle X‐ray scattering to determine the β‐peptide orientation and its effect on the membrane bilayers. These β‐peptides adopt a well‐ordered transmembrane motif in the solid‐supported model membrane, maintaining the basic structure of the original bilayer with some distinct alterations. Notably, the helical tilt angle, which accommodates the positive hydrophobic mismatch, induces a tilt of the acyl chains. The tilted chains, in turn, lead to a membrane thinning effect.     

Accelerating SNARE‐Mediated Membrane Fusion by DNA–Lipid Tethers

SNARE proteins are the core machinery to drive fusion of a vesicle with its target membrane. Inspired by the tethering proteins that bridge the membranes and thus prepare SNAREs for docking and fusion, we developed a lipid‐conjugated ssDNA mimic that is capable of regulating SNARE function, in situ. The DNA–lipid tethers consist of a 21 base pairs binding segment at the membrane distal end that can bridge two liposomes via specific base‐pair hybridization. A linker at the membrane proximal end is used to control the separation distance between the liposomes. In the presence of these artificial tethers, SNARE‐mediated lipid mixing is significantly accelerated, and the maximum fusion rate is obtained with the linker shorter than 40 nucleotides. As a programmable tool orthogonal to any native proteins, the DNA–lipid tethers can be further applied to regulate other biological processes where capturing and bridging of two membranes are the prerequisites for the subsequent protein function.     

High‐Fidelity Protein Targeting into Membrane Lipid Microdomains in Living Cells

Lipid analogues carrying three nitrilotriacetic acid (tris‐NTA) head groups were developed for the selective targeting of His‐tagged proteins into liquid ordered (l_o) or liquid disordered (l_d) lipid phases. Strong partitioning into the l_o phase of His‐tagged proteins bound to tris‐NTA conjugated to saturated alkyl chains (tris‐NTA DODA) was achieved, while tris‐NTA conjugated to an unsaturated alkyl chain (tris‐NTA SOA) predominantly resided in the l_d phase. Interestingly, His‐tag‐mediated lipid crosslinking turned out to be required for efficient targeting into the l_o phase by tris‐NTA DODA. Robust partitioning into l_o phases was confirmed by using viral lipid mixtures and giant plasma membrane vesicles. Moreover, efficient protein targeting into l_o and l_d domains within the plasma membrane of living cells was demonstrated by single‐molecule tracking, thus establishing a highly generic approach for exploring lipid microdomains in situ.     

Fabrication of Thickness‐Controllable Micropatterned Polyelectrolyte‐Film/Nanoparticle Surfaces by Using the Plasma Oxidation Method

We have demonstrated a novel way to form thickness‐controllable polyelectrolyte‐film/nanoparticle patterns by using a plasma etching technique to form, first, a patterned self‐assembled monolayer surface, followed by layer‐by‐layer assembly of polyelectrolyte‐films/nanoparticles. Octadecyltrimethoxysilane (ODS) and (3‐aminopropyl)triethoxysilane (APTES) self‐assembled monolayers (SAMs) were used for polyelectrolyte‐film and nanoparticle patterning, respectively. The resolution of the proposed patterning method can easily reach approximately 2.5 μm. The height of the groove structure was tunable from approximately 2.5 to 150 nm. The suspended lipid membrane across the grooves was fabricated by incubating the patterned polyelectrolyte groove arrays in solutions of 1,2‐dioleoyl‐sn‐glycero‐3‐phosphocholine (DOPC) giant unilamellar vesicles (GUVs). The method demonstrated here reveals a new path to create patterned 2D or 3D structures.     

Coarse‐grained molecular dynamics simulation study on spherical and tube‐like vesicles formed by amphiphilic copolymers

Molecular‐level understanding of the vesicular structure and formation process is beneficial for potential vesicle applications, especially in drug delivery. In this article, coarse‐grained molecular dynamics simulation was used to study the self‐assembly behavior of amphiphilic poly(acrylic acid)‐b‐polystyrene copolymers in water at different concentrations and PS/PAA block ratios. It was found that various spherical and tube‐like vesicles formed at PS/PAA 3:3 and 4:2. For spherical vesicles, analysis of vesicular structure indicated that the cavity size was influenced by copolymer concentration and wall thickness by the block ratio. Tube‐like vesicle was formed via the fusion of two spherical vesicles, and a key factor for this morphology is polymer movements between inner and outer layer. This simulation study identifies the key factors governing vesicle formation and structure, and provides a guidance to design and prepare various vesicles for wide applications in drug delivery. © 2017 Wiley Periodicals, Inc. J. Polym. Sci., Part B: Polym. Phys. 2017 , 55, 1220–1226     

Photoinduced Fusion of Micro‐Vesicles Self‐Assembled from Azobenzene‐Containing Amphiphilic Diblock Copolymers

Poly(N‐isopropylacrylamide)‐block‐poly{6‐[4‐(4‐methylphenyl‐azo) phenoxy] hexylacrylate} (PNIPAM‐b‐PAzoM) was synthesized by successive reversible addition‐fragmentation chain transfer (RAFT) polymerization. In H₂O/THF mixture, amphiphilic PNIPAM‐b‐PAzoM self‐assembles into giant micro‐vesicles. Upon irradiation of light at 365 nm, fusion of the vesicles was observed directly under an optical microscope. The real‐time fusion process is presented and the derivation is preliminarily due to the perturbation by the photoinduced trans‐to‐cis isomerization of azobenzene units in the vesicles.

     

Diversity of non‐stoichiometric substitutions on the lipopolysaccharide of E. coli C demonstrated by electrospray ionization single quadrupole mass spectrometry

The lipopolysaccharide (LPS) of enterobacteria frequently contains various numbers of charged non‐stoichiometric substituents such as phosphate (P) and ethanolamine (EtN) groups and a third residue of 3‐deoxy‐D ‐manno‐2‐octulosonic acid (KDO) on the R‐core polysaccharide backbone. These substituents can modify the biological activities of LPS including varying the stability of the outer membrane, tolerance to cationic antibiotics, pathogenicity, and sensitivity to enterobacteria bacteriophages. These diverse substituents can be clearly detected in degraded samples of LPS from E. coli C using electrospray ionization single quadrupole mass spectrometry (ESI‐Q‐MS) from a 0.1 mg/mL solution in a 50:50 mixture of methanol and 10 mM ammonium acetate (pH 6.8). The O‐deacylated derivative showed multiple peaks of [M–3H]^3? ions which corresponded to species having up to eight phosphates, two ethanolamines, and an additional KDO on the backbone of Hex₅ Hep₃ KDO₂ GlcN₂ C14:0(3‐OH)₂. The major components of the O,N‐deacylated derivative were the species associated with four and five phosphates on Hex₅ Hep₃ KDO₂ GlcN₂. The polysaccharide portion of LPS also revealed species which corresponded to Hex₅ Hep₃ KDO associated with two to four phosphates and an ethanolamine. The present method was proved to be useful to investigate the structural diversity of enterobacterial LPS. Copyright © 2009 John Wiley & Sons, Ltd.     

Blockable Zn10L15 Ion Channels through Subcomponent Self‐Assembly

Metal–organic anion channels based on Zn₁₀L₁₅ pentagonal prisms have been prepared by subcomponent self‐assembly. The insertion of these prisms into lipid membranes was investigated by ion‐current and fluorescence measurements. The channels were found to mediate the transport of Cl⁻ anions through planar lipid bilayers and into vesicles. Tosylate anions were observed to bind and plug the central channels of the prisms in the solid state and in solution. In membranes, dodecyl sulfate blocked chloride transport through the central channel. Our Zn₁₀L₁₅ prism thus inserts into lipid bilayers to turn on anion transport, which can then be turned off through addition of the blocker dodecyl sulfate.     

Supported Membranes with Well‐Defined Polymer Tethers—Incorporation of Cell Receptors

We report the design of supported lipid membranes attached to the surface by tailored lipopolymer tethers. A series of well‐defined lipopolymers were synthesized by means of living cationic polymerization of 2‐methyl‐2‐oxazolines. The polymers were equipped with a silane coupling group on the proximal, and lipid anchors on the distal chain ends. The length of the intermediate hydrophilic polymer tether was varied (n=14, 18, 33) to change the distance between the membrane and the substrate. Supported membranes have been prepared in two‐steps. First, a suitable lipopolymer/lipid mixture was deposited by Langmuir–Blodgett transfer, and annealed to establish the covalent coupling to the surface. On the dry lipopolymer/lipid monolayer, the upper leaflet was deposited by vesicle fusion. Optimization of both preparation steps resulted in the formation of stable and defect‐free membranes. Impacts of the spacer length and the lipopolymer fraction upon the lateral diffusivity of the lipids were systematically compared by fluorescence recovery after photobleaching (FRAP). First experiments on the incorporation of a large transmembrane cell receptor (integrin α_IIbβ₃) into the polymer‐tethered membrane suggested that the length of the polymer tether plays a crucial role in distribution of the proteins on the surface.     

Targeting Bacterial Membranes: NMR Spectroscopy Characterization of Substrate Recognition and Binding Requirements of D‐Arabinose‐5‐Phosphate Isomerase

Lipopolysaccharide (LPS) is an essential component of the outer membrane of Gram‐negative bacteria and consists of three elements: lipid A, the core oligosaccharide, and the O‐antigen. The inner‐core region is highly conserved and contains at least one residue of 3‐deoxy‐D ‐manno‐octulosonate (Kdo). Arabinose‐5‐phosphate isomerase (API) is an aldo–keto isomerase catalyzing the reversible isomerization of D ‐ribulose‐5‐phosphate (Ru5P) to D ‐arabinose‐5‐phosphate (A5P), the first step of Kdo biosynthesis. By exploiting saturation transfer difference (STD) NMR spectroscopy, the structural requirements necessary for API substrate recognition and binding were identified, with the aim of designing new API inhibitors. In addition, simple experimental conditions for the STD experiments to perform a fast, robust, and efficient screening of small libraries of potential API inhibitors, allowing the identification of new potential leads, were set up. Due to the essential role of API enzymes in LPS biosynthesis and Gram‐negative bacteria survival, by exploiting these data, a new generation of potent antibacterial drugs could be developed.