The contact interface of a 120 kD CheA-CheW complex by methyl TROSY interaction spectroscopy

During bacterial chemotaxis, the histidine autokinase CheA interacts with the chemotaxis receptors with the help of the coupling protein CheW. This interaction is typical of many macromolecular complexes where protein-protein interactions play an important role. In this case, a relatively small protein, CheW, becomes part of a much larger complex. Here we describe a new method to map the residues at a protein-protein interface for macromolecular complexes of molecular weight greater than 100 kD.     

Analysis of human erythrocyte membrane vesicles produced by shearing

Shearing of ghosts in a French pressure cell produces three classes of microvesicles that differ from endocytic vacuoles, exocytic vacuoles, and inside-out vesicles. It was thought that an analysis of these vesicles might provide some clues about the assembly of proteins within the human erythrocyte membrane. The microvesicles were separated into three visible bands, labeled top, middle, and bottom, and assayed for activity of Mg++-ATPase, Na+,K+-ATPase, acetylcholinesterase, glyceraldehyde-phosphate dehydrogenase, and NADH oxidoreductase. Their proteins were also characterized by polyacrylamide gel electrophoresis with both Coomassie blue staining, to assess total protein content and distribution, and PAS-staining, to characterize sialoglycopeptides. In order to minimize problems inherent in ghost preparation, Dodge or hypotonic ghosts and glycol or isotonic ghosts were used in all studies. Middle membrane vesicles most resembled intact ghosts. Top vesicles had reduced levels of NADH oxidoreductase and more PAS-2 at the expense of PAS-1. The bottom vesicle class was very much enriched with PAS-1 at the expense of PAS-2, and PAS-3 was completely absent. In addition bottom vesicles had highest NADH oxidoreductase activity but lowest activity of all the other enzymes measured. These vesicle classes could not have been produced by tangential shearing through the membrane, nor could radial shearing through a membrane in which all proteins were free to move laterally have accounted for the three discrete vesicle classes or for their different patterns of enzymes and proteins. The analysis of the microvesicles produced by shearing is most consistent with radial shearing through membranes where there may be fixed domains superimposed on the basic fluid-mosaic structure.     

Intracellular distribution of fluorescent probes delivered by vesicles of different lipidic composition

In order to study mechanisms involved in liposome–cell interaction, this work attempted to assess the influence of vesicle composition on the delivery of liposomal content to Hela cells. In particular, to evaluate pH-sensitive properties and cell interaction of the prepared liposomes, the lipid formulations contained cholesterol (Chol) and they were varied by using phosphatidylcholines with different purity degree: soy lecithin (SL; 80% phosphatidylcholine), a commercial mixture of soy phosphatidylcholine (P90; 90% phosphatidylcholine) or dipalmitoylphosphatidylcholine (DPPC; 99% of purity). A second series of liposomes also contained stearylamine (SA). Dehydration-rehydration vesicles (DRV) were prepared and then sonicated to decrease vesicle size. Vesicle–cell interactions and liposomal uptake were examined by fluorescence microscopy using carboxyfluorescein (CF) and phosphatidylethanolamine-dioleoyl-sulforhodamine B (Rho-PE) as fluorescent markers. Fluorescence dequenching assay was used to study the influence of pH on CF release from the liposomal formulations. Liposome adhesion on the cell surface and internalization were strongly dependent on vesicle bilayer composition. SA vesicles were not endocytosed. DPPC/Chol liposomes were endocytosed but did not release their fluorescent content into the cytosol. SL/Chol and P90/Chol formulations displayed a diffuse cytoplasmic fluorescence of liposomal marker.     

Characterizing assembly morphology changes during solubilization process of dimyristoyl phosphocholine vesicles by n-dodecyl triethylammonium bromide

In the present work, the assembly morphology changes during the solubilization process of the sonicated unilamellar vesicles from dimyristoyl phosphocholine (DMPC) by a cationic surfactant, n-dodecyl triethylammonium bromide (DTEAB) were well characterized with DSC, FF-TEM and DLS and fluorescence probes technique. Based on an analysis on the above results, a primary multi-stage model was brought forward to sketch the assembly morphology changes during the DMPC vesicle solubilization by DTEAB. In comparison with classical models, vesicles division, tubule-like structure formation and fission to vesicle were found in the middle stages of this model. Additionally, it is the first time that the transversally-cut profiles of tubule-like structures were observed during vesicle solubilization process.     

Shape transitions in lipid membranes and protein mediated vesicle fusion and fission

In the cell, the plasma membrane is often densely decorated by transmembrane proteins. The morphology and dynamics of the membrane are strongly influenced by the presence of proteins. In this paper, we use a coarse-grained model to explore the composite membrane-protein system and develop a simulation methodology based on thermodynamic integration to examine free energy changes during membrane shape transitions. The authors show that a critical concentration of conical membrane proteins or proteins with nonzero spontaneous curvature can drive the formation of small vesicles. The driving force of vesicle budding stems from the preference of proteins to gather in regions of high curvature. A sufficiently high concentration of proteins therefore can influence the topology of the membrane. The biological significance of our results is discussed.     

Vesicle shapes from molecular dynamics simulations

Lipid bilayer membranes are known to form various structures such as large sheets or vesicles. When the two leaflets of the bilayer have an equal composition, the membrane preferentially forms a flat sheet or a spherical vesicle. However, a difference in the composition of the two leaflets may result in a curved bilayer or in a wide variety of vesicle shapes. Vesicles with different shapes have already been shown in experiments and diverse vesicle shapes have been predicted theoretically from energy minimization of continuous curves. Here we present a molecular dynamics study of the effect of small changes in the phospholipid headgroups on the spontaneous curvature of the bilayer and on the resulting vesicle shape transformations. Small asymmetries in the bilayers already result in high spontaneous curvature and large vesicle deformations. Vesicle shapes that are formed include ellipsoids, discoids, pear-shaped vesicles, cup-shaped vesicles, as well as budded vesicles. Comparison of these vesicles with theoretically derived vesicle shapes shows both resemblances and differences.     

Shape transformation of giant phospholipid vesicles at high concentrations of C12E8

Giant unilamellar phospholipid vesicles were prepared by the method of electroformation from 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylcholine (POPC). We studied the influence of different concentrations of the surfactant octaethyleneglycol dodecylether (C(12)E(8)) on the spontaneous shape transformations of POPC vesicles at room temperature. In accordance with previous results, we observed that low concentration of C(12)E(8) increased the speed of the characteristic vesicle shape transformation, starting from the initial shape with thin tubular protrusion, through beaded protrusion where the number of beads gradually decreased, to final spherical shapes with invagination, whereby the average mean curvature of the vesicle membrane monotonously decreased. In contrast, higher concentration of C(12)E(8) initially induced the shape transformation in the "opposite direction": in the protrusion, the number of beads gradually increased and eventually a tube was formed whereby the average mean curvature of the vesicle membrane gradually increased. However, at a certain point, an abrupt shape change took place to yield the vesicle with invagination. In this transition, the average mean curvature of the vesicle membrane discontinuously decreased. After this transition, the vesicle began to shrink and finally disappeared. We discuss possible mechanisms involved in the observed transformations.     

Engineering bio-mimicking functional vesicles with multiple compartments for quantifying molecular transport

Controlled design of giant unilamellar vesicles under defined conditions has vast applications in the field of membrane and synthetic biology. Here, we bio-engineer bacterial-membrane mimicking models of controlled size under defined salt conditions over a range of pH. A complex bacterial lipid extract is used for construction of physiologically relevant Gram-negative membrane mimicking vesicles whereas a ternary mixture of charged lipids (DOPG, cardiolipin and lysyl-PG) is used for building Gram-positive bacterial-membrane vesicles. Furthermore, we construct stable multi-compartment biomimicking vesicles using the gel-assisted swelling method. Importantly, we validate the bio-application of the bacterial vesicle models by quantifying diffusion of chemically synthetic amphoteric antibiotics. The transport rate is pH-responsive and depends on the lipid composition, based on which a permeation model is proposed. The permeability properties of antimicrobial peptides reveal pH dependent pore-forming activity in the model vesicles. Finally, we demonstrate the functionality of the vesicles by quantifying the uptake of membrane-impermeable molecules facilitated by embedded pore-forming proteins. We suggest that the bacterial vesicle models developed here can be used to understand fundamental biological processes like the peptide assembly mechanism or bacterial cell division and will have a multitude of applications in the bottom-up assembly of a protocell.

Giant vesicle functional models mimicking a bacterial membrane under physiological conditions are constructed.     

Activity coefficients of CaCl2 and MgCl2 in the presence of dipalmitoylphosphatidylcholine-phosphatidylinositol vesicles in aqueous media

Mean activity coefficients of CaCl₂ and MgCl₂ salts have been measured in the presence of phospholipid vesicles, by use of the EMF method with ion-exchange membrane electrodes, as a novel procedure for studying the direct interactions between electrolytes and phospholipid vesicles. Mixed lipid sonicated vesicles have been prepared from dipalmitoylphosphatidylcholine (DPPC)-phosphatidylinositol (PI) mixtures, covering a range of composition. The CaCl₂ and MgCl₂ concentration range studied was 0.01−0.001 mol l⁻¹. For both salts studied, the activity coefficients decrease linearly as the vesicle concentration in the suspension increases while the concentration of salt was kept constant. Maintaining a constant vesicle concentration, the decrease in the activity coefficients is more pronounced the more dilute the concentration of the salts. Quantitative differences were observed in the behaviour of both salts which are explained on the basis of a higher affinity of the Ca²⁺, compared with that of Mg²⁺ ions, to the vesicle surface. Using a simple model for the vesicle-salt systems, the number of cations specifically bound to the vesicles has been calculated. In view of the results it can be concluded that estimation of the activity coefficients of the salts is a useful method for the study of the interactions taking place in vesicle-salt systems.     

Deformations of lipid vesicles induced by attached spherical particles

Wrapping of a spherical colloidal particle, located inside and outside a lipid vesicle, by the membrane which forms the wall of the vesicle is investigated. The process is studied for vesicles of different geometries: prolate, oblate, stomatocytes. We focus on the bending energy change and shape transformations induced by binding the membrane to the spherical particles. The ground-state shapes of vesicles are calculated within the framework of a Helfrich curvature energy functional.     

Vesicle Tubulation with Self‐Assembling DNA Nanosprings

A major goal of nanotechnology and bioengineering is to build artificial nanomachines capable of generating specific membrane curvatures on demand. Inspired by natural membrane‐deforming proteins, we designed DNA‐origami curls that polymerize into nanosprings and show their efficacy in vesicle deformation. DNA‐coated membrane tubules emerge from spherical vesicles when DNA‐origami polymerization or high membrane‐surface coverage occurs. Unlike many previous methods, the DNA self‐assembly‐mediated membrane tubulation eliminates the need for detergents or top‐down manipulation. The DNA‐origami design and deformation conditions have substantial influence on the tubulation efficiency and tube morphology, underscoring the intricate interplay between lipid bilayers and vesicle‐deforming DNA structures.     

Characterization of micropatterned lipid membranes on a gold surface by surface plasmon resonance imaging and electrochemical signaling of a pore-forming protein

We report the fabrication and characterization of a micropatterned membrane electrode for electrochemical signaling of a bacterial pore-forming toxin, Streptolysin O (SLO) from S. pyogenes. Microcontact printing of an alkylthiol monolayer was used to fabricate an array template, onto which cholesterol-containing DMPC vesicles were fused to form lipid layer structures. The construction of the supported membranes, including pattern transfer and vesicle fusion, was characterized by in-situ surface plasmon resonance (SPR) imaging and electrochemistry. Quantitative analysis of the resulting membrane by using SPR angular shift measurements indicates that the membranes in the hydrophilic pockets have an average thickness of 8.2 +/- 0.4 nm. Together with fluorescence microscopy studies, the results suggest that this could be a mixed lipid assembly that may consist of a bilayer, vesicle fragments, and lipid junctions. The voltammetric response of the redox probe ferrocene carboxylic acid (FCA) was measured to quantify the toxin action on the supported membrane. The electrochemical measurements indicate that fusion of vesicles on the template blocked the access of FCA, whereas the injection of SLO toxin restored the redox response. The anodic peak current of FCA was found to increase with toxin concentration until a plateau was reached at 40 HU/mL. The method is highly sensitive such that 0.1 HU/mL of SLO (1.25 pM) can yield a well-defined response. In addition, it eliminates the need for a highly insulating layer in membrane sensing, which opens up new avenues in developing novel sensing interfaces for membrane-targeting proteins and peptides.     

Investigating cellular signaling reactions in single attoliter vesicles

Understanding cellular signaling mediated by cell surface receptors is key to modern biomedical research and drug development. The discovery of a growing number of potential molecular targets and therapeutic compounds requires downscaling and accelerated functional screening. Receptor-mediated cellular responses are typically investigated on single cells or cell populations. Here, we show how to monitor cellular signaling reactions at a yet unreached miniaturization level. On the basis of our observations, cytochalasin induces mammalian cells to extrude from their plasma membrane submicrometer-sized native vesicles. They comprise functional cell surface receptors correctly exposing their extracellular ligand binding sites on the outer vesicle surface and retaining cytosolic proteins in the vesicle interior. As a prototypical example, ligand binding to the ionotropic 5-HT(3) receptor and subsequent transmembrane Ca(2+) signaling were monitored in single attoliter vesicles. Thus, native vesicles are the smallest autonomous containers capable of performing cellular signaling reactions under physiological conditions. Because a single cell delivers about 50 native vesicles, which can be isolated and addressed as individuals, our concept allows multiple functional analyses of individual cells having a limited availability and opens new vistas for miniaturized bioanalytics.     

Experimental evidence to support a theory of lipid membrane fusion

Membrane fusion between two lipid membranes with different curvatures was measured by using a fluorescence fusion assay for lipid vesicle systems and was also obtained by measuring lipid monolayer surface tension upon the fusion of vesicles to monolayer membranes. For such membrane systems, it was found that when lysolipid was incorporated only in the membrane with a greater curvature, membrane fusion was more suppressed than those for the case where the same amount (molar ratio of lysolipid to non-lysolipids) of lysolipid was incorporated only in the membrane with a lower curvature. When lysolipid was incorporated only in a flat membrane (e.g., monolayer) and the fusion of small vesicles (SUV) to the monolayer was measured, suppression of membrane fusion by lysolipid was minimal. It is known that lysolipid lowers the surface energy of curved membranes, which stabilizes energetically such membrane surfaces, and thus suppresses membrane fusion. Our results support our theory of lipid membrane fusion where the membrane fusion occurs through the most curved membrane region at the contact area of two interacting membranes.     

Dynamics of vesicle self-assembly and dissolution

The dynamics of membranes is studied on the basis of a particle-based meshless surface model, which was introduced earlier [Phys. Rev. E 73, 021903 (2006)]. The model describes fluid membranes with bending energy and-in the case of membranes with boundaries-line tension. The effects of hydrodynamic interactions are investigated by comparing Brownian dynamics with a particle-based mesoscale solvent simulation (multiparticle collision dynamics). Particles self-assemble into vesicles via disk-shaped membrane patches. The time evolution of assembly is found to consist of three steps: particle assembly into discoidal clusters, aggregation of clusters into larger membrane patches, and finally vesicle formation. The time dependence of the cluster distribution and the mean cluster size is evaluated and compared with the predictions of Smoluchowski rate equations. On the other hand, when the line tension is suddenly decreased (or the temperature is increased), vesicles dissolve via pore formation in the membrane. Hydrodynamic interactions are found to speed up the dynamics in both cases. Furthermore, hydrodynamics makes vesicle more spherical in the membrane-closure process.     

磁性非对称双链长表面活性剂囊泡凝胶

合成了一系列含磁性反离子的非对称双疏水链长的阳离子表面活性剂,其中三氯一溴铁合十六烷基戊基二甲基铵(C_(16)C_5DMA~+[FeCl_3Br]-~)与偶氮羧酸钠盐(AzoNa_4)在酸性条件水溶液中形成磁性囊泡凝胶。运用cryo-透射电镜(TEM)、冷冻蚀刻TEM(FF-TEM)、流变仪、傅里叶变换红外光谱(FT-IR)和超导量子干涉(SQUID)等表征技术对囊泡凝胶进行了结构和性质研究,结果发现:凝胶含有曲率多变的融合性的双层囊泡,这些双分子层结构模构了自然界中各种物象的结构轮廓,展现了不可预测的多变曲率和良好柔性。聚集体双分子层膜内由长短不对称烷基链采取交错相扣的双分子层排列模式,这种构建模式结构稳定,短烷基链可游离出囊泡双分子层并伸向外部水相介质。两个相邻囊泡间的短链在疏水相互作用下形成非共价的囊泡"补丁",疏水的囊泡"补丁"克服相邻囊泡之间的斥力而融合。磁性反离子[FeCl_3Br]~-不仅赋予囊泡磁性,且在囊泡的形成过程中调控烷基链的组装。这种多形态融合性囊泡为揭示膜曲率的调节机制和构建人工细胞提供实验数据和理论参考。     

Size of supramolecular SNARE complex: membrane-directed self-assembly

Full length v-SNARE protein in lipid vesicles when exposed to t-SNARE-reconstituted lipid membrane results in the self-assembly of a t-/v-SNARE complex in a ring pattern, forming pores, and establishing continuity between the opposing bilayers. It is known that smaller vesicles fuse more efficiently than larger ones, and hence the curvature of secretory vesicles may dictate the potency and efficacy of their fusion at the cell plasma membrane. The diameter of t- and v-SNARE vesicles may, therefore, reflect the size of the t-/v-SNARE complex formed. In the present study, this hypothesis was tested, and results from the study demonstrate that the size of the t-/v-SNARE complex is directly proportional to the vesicle diameter (R2 = 0.9725).     

Incomplete lipid chain freezing of sonicated vesicular dispersions of double-tailed ionic surfactants

Lipid freezing in dilute sonicated vesicular dispersions was studied using differential scanning calorimetry (DSC) and 1H NMR. For charged, anionic, or cationic lipids, approximately half of the lipids remain in a fluid state when cooled 20 degrees C below the main chain melting temperature. With a zwitterionic phospholipid, on the other hand, essentially no supercooling of the liquid state was observed. The observations are analyzed in terms of the nucleation and growth of flat solid domains in originally fluid spherical vesicles. As the solid domains grow, the remaining fluid domain is deformed, resulting in a curvature stress. Depending on the vesicle size and the bilayer bending rigidity, the solid domain growth may terminate as the gain in cohesive free energy is balanced by the curvature stress of the remaining fluid domain. It is argued that high bending rigidities are required for having a significant supercooling, which is why it is only observed for charged lipids.     

Shape of fluid vesicles anchored by rigid rod

A method combined the self-consistent field theory (SCFT) for the rigid rod with the Helfrich curvature elasticity theory for the vesicle has been developed for studying the shape of vesicles anchored by rigid rod. Both the deformation of the vesicle and the density distribution of rod segments can be obtained. Because of the vesicle's impenetrability for the rod segments and the decrease of the available space for the rod orientational configurations, the anchored rod segments exert the inhomogeneous entropic pressure on the vesicle and induce the change of vesicle shape. The interaction between the rod segments and the vesicle membrane exerts an extra tension to the membrane. Thus the interaction between the vesicle membrane and the rod segments, the rod length, and the bending rigidity of vesicle are investigated as the important factors to the shape transformation of the vesicle and the density distribution of rod segments. This method can be extended to more complicated and real biological systems, such as polymers with different topological architectures/vesicle, multiple chains/vesicle, protein inclusions, etc.     

Selective assembly of photosynthetic antenna proteins into a domain-structured lipid bilayer for the construction of artificial photosynthetic antenna systems: structural analysis of the assembly using surface plasmon resonance and atomic force microscopy

Two types of photosynthetic membrane proteins, the peripheral antenna complex (LH2) and the core antenna/reaction center complex (LH1-RC), play an essential role in the primary process of purple bacterial photosynthesis, that is, capturing light energy, transferring it to the RC where it is used in subsequent charge separation. Establishment of experimental platforms is required to understand the function of the supramolecular assembly of LH2 and LH1-RC molecules into arrays. In this study, we assembled LH2 and LH1-RC arrays into domain-structured planar lipid bilayers placed on a coverglass using stepwise combinations of vesicle-to-planar membrane formation and vesicle fusion methods. First, it was shown that assembly of LH2 and LH1-RC in planar lipid bilayers, through vesicle-to-planar membrane formation, could be confirmed by absorption spectroscopy and high resolution atomic force microscopy (AFM). Second, formation of a planar membrane incorporating LH2 molecules made by the vesicle fusion method was corroborated by AFM together with quantitative analysis by surface plasmon resonance (SPR). By combining planar membrane formation and vesicle fusion, in a stepwise manner, LH2 and LH1-RC were successfully organized in the domain-structured planar lipid membrane. This methodology for construction of LH2/LH1-RC assemblies will be a useful experimental platform with which to investigate energy transfer from LH2 to LH1-RC where the relative arrangement of these two complexes can be controlled.