Formation of inverted lipid micelles in aqueous dispersions of mixed sn-3-galactosyldiacylglycerols induced by heat and ethylene glycol

The formation of ‘lipidic’ particles corresponding to inverted lipid micelles in freeze-fracture replicas of aqueous dispersions of mono- and digalactosyldiacylglycerols can be greatly enhanced either by increasing the temperature from which the samples are thermally quenched or by the addition of cryoprotectants such as ethylene glycol. In the case of the heated samples, the lipids tend to form quasi-crystalline structures consisting of sheets of 8–9 nm diameter particles organised on an orthorhombic lattice. The orientation of alternate sheets varies giving rise to a characteristic herring-bone pattern. Ethylene glycol-treated samples, in contrast, form more regular structures consisting of 13–16 nm diameter particles. Lowering the temperature from which the samples are quenched and/or decreasing the concentration of ethylene glycol reduces the frequency of formation of such structures. A number of intermediate states associated with the reincorporation of the lipid molecules of the inverted micelles into the lamella phase are also identified. The factors influencing particle formation are briefly discussed. It is concluded that the destabilisation of lipid-water interactions play a major role in this process.     

Effects of reconstitution method on the structural organization of isolated chloroplast membrane lipids

Plant galactolipids were isolated from spinach thylakoids and reconstituted by (1) hydration in water or buffer, (2) solubilization in Triton X-100 and subsequent slow detergent removal, and (3) reverse phase evaporation using Freon 11 (trichlorofluoromethane, b.p. 23°C). Digalactosyldiacylglycerol (DG) formed bilayer liposomes when reconstituted by any of these methods. Monogalactosyldiacylglycerol (MG) was very difficult to transfer quantitatively to the aqueous phase. Reverse phase evaporation was the most successful method, and conventional hydration in water or buffer the least efficient, for reconstituting MG quantitatively. Freeze-fracture electron microscopy of pure MG showed arrays of hexagonal II tubes as well as packed inverted micelles (7–9 nm diameter in both cases) covered by a monolayer of lipid. Reconstitution of binary mixtures of MG and DG using the various methods produced the same structures. However, the concentration of MG at which various structural changes occurred depended on the method used for reconstitution. Some of the differences between buffer-hydrated and reverse phase evaporated-reconstituted DG/MG mixtures were traced to the conventional hydration method leaving MG selectively behind on the glass. Reverse phase evaporation allowed the most MG to be incorporated into vesicular structures (about 50% vs. about 30–40% by the detergent method). Irregularities in the bilayer vesicles, ‘lipidic particles’ and ‘fusion pores’, varied proportionally with the amount of MG in the mixture. The transition from vesicular structures to packed tubes and particles had occured by approx. 66% MG using reverse phase evaporation and by approx. 50% MG using detergent solubilization. Aggregates of packed inverted micelles were present in several DG/MG mixtures. The diameters of the inverted micelles varied from 7–9 nm (pure MG) to 20–21 nm (60:40, DG/MG). A model is presented that relates this variation in diameter geometrically to the overall ‘cone’ shape of an MG molecule and the cylindrical shape of DG. In contrast to a previous report, glycerol had no effect on the type of structures observed in replicas of mixed DG/MG samples. However, all structures were more clearly defined in freeze-fracture replicate from glycerinated samples.     

Artificial biomembrane morphology: a dissipative particle dynamics study

Artificial membranes mimicking biological structures are rapidly breaking new ground in the areas of medicine and soft-matter physics. In this endeavor, we use dissipative particle dynamics simulation to investigate the morphology and behavior of lipid-based biomembranes under conditions of varied lipid density and self-interaction. Our results show that a less-than-normal initial lipid density does not create the traditional membrane; but instead results in the formation of a ‘net’, or at very low densities, a series of disparate ‘clumps’ similar to the micelles formed by lipids in nature. When the initial lipid density is high, a membrane forms, but due to the large number of lipids, the naturally formed membrane would be larger than the simulation box, leading to ‘rippling’ behavior as the excess repulsive force of the membrane interior overcomes the bending energy of the membrane. Once the density reaches a certain point however, ‘bubbles’ appear inside the membrane, reducing the rippling behavior and eventually generating a relatively flat, but thick, structure with micelles of water inside the membrane itself. Our simulations also demonstrate that the interaction parameter between individual lipids plays a significant role in the formation and behavior of lipid membrane assemblies, creating similar structures as the initial lipid density distribution. This work provides a comprehensive approach to the intricacies of lipid membranes, and offers a guideline to design biological or polymeric membranes through self-assembly processes as well as develop novel cellular manipulation and destruction techniques.     

Ultrastructural organization of cytoplasmatic membrane of Anaerobacter polyendosporus studied by electron microscopic cryofractography]

Anaerobacter polyendosporus cells do not have typical mesosomes. However, the analysis of this anaerobic multispore bacterium by electron microscopic cryofractography showed that its cytoplasmic membrane contains specific intramembrane structures in the form of flat lamellar inverted lipid membranes tenths of nanometers to several microns in size. It was found that these structures are located in the hydrophobic interior between the outer and inner leaflets of the cytoplasmic membrane and do not contain intramembrane particles that are commonly present on freeze-fracture replicas. The flat inverted lipid membranes were revealed in bacterial cells cultivated under normal growth conditions, indicating the existence of a complex-type compartmentalization in biological membranes, which manifests itself in the formation of intramembrane compartments having the appearance of vesicles and inverted lipid membranes.     

Low pH and phospholipase A2 treatment induce the phase-separation of non-bilayer lipids within pea chloroplast membranes

Exposure of chloroplasts to pH < 4.5, or incubation in the presence of phospholipase A₂, leads to membrane lipid phase separations and the irreversible formation of non-bilayer lipid structures. Freeze-fracture replicas of the thylakoid membranes of treated chloroplasts are characterized by the presence of aggregates of cylindrical inverted lipid micelles. These structural changes are accompanied by an inhibition of photosystem II-mediated electron transport and a stimulation of photosystem I-mediated transport. These data have important implications both with respect to the factors governing the stability of thylakoid membranes and the use of lipases as probes of chloroplast structure.MembranelipidHexagonalphaseFreeze-fractureChloroplast     

Ultrastructural Organization of the Cytoplasmic Membrane of Anaerobacter polyendosporusas Evidenced by Electron Microscopic Cryofractography

Anaerobacter polyendosporuscells do not have typical mesosomes. However, the analysis of this anaerobic multispore bacterium by electron microscopic cryofractography showed that its cytoplasmic membrane contains specific intramembrane structures in the form of flat lamellar inverted lipid membranes tenths of nanometers to several microns in size. It was found that these structures are located in the hydrophobic interior between the outer and inner leaflets of the cytoplasmic membrane and do not contain intramembrane particles that are commonly present on freeze-fracture replicas. The flat inverted lipid membranes were revealed in bacterial cells cultivated under normal growth conditions, indicating the existence of a complex-type compartmentalization in biological membranes, which manifests itself in the formation of intramembrane compartments having the appearance of vesicles and inverted lipid membranes.     

Hyperfine shift NMR studies of hydrated phospholipid inverted micelles

The ³¹P nuclear magnetic resonance (NMR) spectra of benzene solutions of hydrated dipalmitoyl lecithin (DPL) inverted micelles, with and without incorporated paramagnetic lanthanide ions, have been recorded. Individual resonances for micelles containing none, one, and two ions can be resolved and observed in the presence of one another. The relative intensities of these peaks yield some information on the state of aggregation of lipid inverted micelles prepared by ultrasonic irradiation. The relative intensities and chemical shifts of resonances of unsonicated mixtures of preformed micelles containing different numbers of ions per micelle indicate that some kind of equilibration occurs. The data are consistent with a selective fusion of multi-ion micelles with ion-free micelles. The NMR spectra place constraints on the lifetimes of metal ions and lipid and water molecules within a micelle before transfer to another.     

Formation of Flat Lamellar Intramembrane Lipid Structures in Microorganisms

Analysis of freeze-fracture replicas and thin sections of cells of the bacteria Sulfobacillus thermosulfidooxidans and Anaerobacter polyendosporus showed that their cytoplasmic membranes contain some regions in the form of flat lamellar inverted lipid membranes a few tenths of nanometers to a few microns in size. The specific features of these membrane structures are as follows: (i) they contain no familiar intramembrane particles commonly present on freeze-fracture replicas; (ii) in cross thin sections, intramembrane structures are bifurcate on the periphery and look like thylakoids; and (iii) the leaflets of intramembrane structures in S. thermosulfidooxidans cells are corrugated. These structures were revealed in bacterial cells cultivated under normal growth conditions. The data obtained suggest the occurrence of a complex type of compartmentalization in biological membranes. Received: 17 July 2000/Revised: 22 November 2000     

The formation of non-bilayer structures in total polar lipid extracts of chloroplast membranes

The structural organisation of aqueous dispersions of total membrane lipid extracts of broad bean (Vicia faba) chloroplasts is dependent on pH and the presence of cations. In the absence of inorganic salts, sonicated dispersions of lipid extract in distilled water form smooth, single-shell vesicles approximately 30–50 nm in diameter. Reducing the pH of the dispersions, to neutralise the acidic lipids present in the extract, or the addition of low concentrations of metal cations, leads to the fusion of the vesicles and a partial phase-separation of the non-bilayer forming lipid monogalactosyldiacylglycerol to form spherical inverted micelles similar to those previously reported for binary mixtures of monogalactosyl and digalactosyldiacylglycerol (Biochim. Biophys. Acta 685, 297–306). Increasing concentrations of polyvalent, but not monovalent, cations lead to further structural rearrangements involving the formation of para-crystalline arrays of tubular and spherical inverted micelles. The factors determining the formation of these different structures, and their possible relevance to the structural organisation of the native chloroplast membrane, are discussed.     

两种品质类型花生品质形成与子叶细胞超微结构的关系

在大田条件下研究了两种品质类型花生(Arachis hypogaea)品质形成的动态差异及其子叶细胞超微结构的差异。结果表明, 高蛋白品种‘XB023’的蛋白质含量在籽仁发育前期较高油品种‘鲁花9号’低, 后期显著高于‘鲁花9号’, 且成熟期籽仁8种必需氨基酸组分含量均高于‘鲁花9号’, 其中谷氨酸、赖氨酸和亮氨酸含量差异极显著; ‘XB023’脂肪含量在籽仁发育期一直低于‘鲁花9号’。‘XB023’各时期的籽仁可溶性糖含量和油酸/亚油酸(O/L)值均显著低于‘鲁花9号’。两品种在果针入土10天时子叶细胞即形成淀粉粒、脂体和蛋白体, 随后脂体、蛋白体的数量不断增加, 淀粉粒先增大后逐渐缩小解体。‘XB023’的脂体达到最大的时间早于‘鲁花9号’, 而‘鲁花9号’的脂体快速积累的时间比‘XB023’长。两品种蛋白体大小都在果针入土40天时达到最大值, ‘XB023’的蛋白体在籽仁发育后期数量增加较快。高蛋白品种较高的蛋白质含量由其子叶细胞中较大蛋白体的大小和较多的蛋白体数量决定, 而高油品种较高的脂肪含量是由其较多的脂体数量决定。     

The nature of lipidic particles and their roles polymorphic transitions

The structural transition between bilayer (L_α), inverted hexagonal (H_II and inverted cubic (C_II) phases in mixtures of unsaturated phosphatidylethanolamines (PE) and phosphatidylcholines (PC) were investigated. Freeze fracture electron micrographs of intermediate stages of phase transitions showed that C_II was a stable intermediate form between the L_α-H_II transition. The electron microscopic observation was supported by X-ray diffraction and ³¹P-NMR results. Detailed morphology revealed that during the L_α-C_II transition, interlamellae attachment points (conical lipidic particles) connect adjacent bilayers to form arrays of entrapped water pockets (inverted micelles). These water-containg spherical units were packed in a cubic lattice. In the C_II to H_II transition, these spherical units were linearly connected to form tubes. During the L_α-H_II transition, a ripple pattern was observed across the otherwise smooth lamellar. The troughs of the ripples were transformed into linear connections between adjacent bilayers, thereby converting multilayer structures into parallel tubes. No lipidic particles were involved in this type of transition. We show that there are different mechanisms involved in the L_α, H_II, C_II polymorphic transitions, and that different types of ‘lipidic particles’ representing different molecular organizations may be involved in each case. Models of these transitions are proposed.     

Phospholipid-based reverse micelles

Physicochemical investigations on the aggregation of phospholipids (mainly phosphatidylcholines) in organic solvents are reviewed and compared with the aggregation behaviour of phospholipids in aqueous medium. In particular we review the data showing that phosphatidylcholines (lecithins) form reverse micellar structures in certain apolar solvents. In these systems not only low molecular weight compounds but also catalytically active enzymes and entire cells can be solubilized. In addition, highly viscous phosphatidylcholine gels can be obtained in organic solvents upon solubilizing a critical amount of water. Generally, phospholipid-based reverse micelles can be regarded as thermodynamically stable models for inverted micellar lipid structures possibly occurring in biological membranes.     

The organisation of cholesterol and ergosterol in lipid bilayers based on studies using non-perturbing fluorescent sterol probes.

The fluorescence properties of dehydroergosterol and cholesta-5,7,9-trien-3 beta-ol have been studied in organic solution, in aqueous dispersions and incorporated into aqueous lipid dispersions. The absorption spectra of aqueous dispersions of the probes are very different to those in organic solution, and aqueous dispersions are non-fluorescent. This can be attributed to micelle formation with dimerisation and/or aggregation in the micelles. Concentration quenching also occurs when sterols are incorporated into lipid bilayers, but relatively high fluorescence is observed even at a 1 : 1 steroid:lipid molar ratio. Further, the fluorescence is still polarized at these high molar ratios. We attribute this to the formation of ordered arrays of sterol molecules in the lipid bilayers. In these arrays the sterol molecules are organised in an end-to-end fashion, and face-to-face overlap of the sterols is prevented by the lipid molecules. Possible structures for 1 : 1 mixtures are presented.     

Mixed micelles and other structures in the solubilization of bilayer lipid membranes by surfactants

The solubilization of lipid bilayers by surfactants is accompanied by morphological changes of the bilayer and the emergence of mixed micelles. From a phase equilibrium perspective, the lipid/surfactant/water system is in a two-phase area during the solubilization: a phase containing mixed micelles is in equilibrium with bilayer structures of the lamellar phase. In some cases three phases are present, the single micelle phase replaced by a concentrated and a dilute solution phase. In the case of non-ionic surfactants, the lipid bilayers reach saturation when mixed micelles, often flexible rod-like or thread-like, start to form in the aqueous solution, at a constant chemical potential of the surfactant. The composition of the bilayers also remains fixed during the dissolution. The phase behavior encountered with many charged surfactants is different. The lamellar phase becomes destabilized at a certain content of surfactant in the membrane, and then disintegrates, forming mixed micelles, or a hexagonal phase, or an intermediate phase. Defective bilayer intermediates, such as perforated vesicles, have been found in several systems, mainly with charged surfactants. The perforated membranes, in some systems, go over into thread-like micelles via lace-like structures, often without a clear two-phase region. Intermediates in the form of disks, either micelles or bilayer fragments, have been observed in several cases. Most noteworthy are the planar and circular disks found in systems containing a large fraction of cholesterol in the bilayer. Bile salts are a special class of surfactants that seem to break down the bilayer at low additions. Originally, disk-like mixed micelles were conjectured, with polar membrane lipids building the disk, and the bile salts covering the hydrophobic rim. Later work has shown that flexible cylinders are the dominant intermediates also in these systems, even if the disk-like structures have been re-established as transients in the transformation from mixed micelles to vesicles.     

Lipid A structural modifications in extreme conditions and identification of unique modifying enzymes to define the Toll-like receptor 4 structure-activity relationship

Strategies utilizing Toll-like receptor 4 (TLR4) agonists for treatment of cancer, infectious diseases, and other targets report promising results. Potent TLR4 antagonists are also gaining attention as therapeutic leads. Though some principles for TLR4 modulation by lipid A have been described, a thorough understanding of the structure-activity relationship (SAR) is lacking. Only through a complete definition of lipid A-TLR4 SAR is it possible to predict TLR4 signaling effects of discrete lipid A structures, rendering them more pharmacologically relevant. A limited ‘toolbox’ of lipid A-modifying enzymes has been defined and is largely composed of enzymes from mesophile human and zoonotic pathogens. Expansion of this ‘toolbox’ will result from extending the search into lipid A biosynthesis and modification by bacteria living at the extremes. Here, we review the fundamentals of lipid A structure, advances in lipid A uses in TLR4 modulation, and the search for novel lipid A-modifying systems in extremophile bacteria. This article is part of a Special Issue entitled: Bacterial Lipids edited by Russell E. Bishop.     

The application of DOSY NMR and molecular dynamics simulations to explore the mechanism(s) of micelle binding of antimicrobial peptides containing unnatural amino acids

Anionic and zwitterionic micelles are often used as simple models for the lipids found in bacterial and mammalian cell membranes to investigate antimicrobial peptide‐lipid interactions. In our laboratory we have employed a variety of 1D, 2D, and diffusion ordered (DOSY) NMR experiments to investigate the interactions of antimicrobial peptides containing unnatural amino acids with SDS and DPC micelles. Complete assignment of the proton spectra of these peptides is prohibited by the incorporation of a high percentage of unnatural amino acids which don't contain amide protons into the backbone. However preliminary assignment of the TOCSY spectra of compound 23 in the presence of both micelles indicated multiple conformers are present as a result of binding to these micelles. Chemical Shift Indexing agreed with previously collected CD spectra that indicated on binding to SDS micelles compound 23 adopts a mixture of α‐helical structures and on binding to DPC micelles this peptide adopts a mixture of helical and β‐turn/sheet like structures. DOSY NMR experiments also indicated that the total positive charge and the relative placement of that charge at the N‐terminus or C‐terminus are important in determining the mole fraction of the peptide that will bind to the different micelles. DOSY and ¹H‐NMR experiments indicated that the length of Spacer #1 plays a major role in defining the binding conformation of these analogs with SDS micelles. Results obtained from molecular simulations studies of the binding of compounds 23 and 36 with SDS micelles were consistent with the observed NMR results. © 2013 Wiley Periodicals, Inc. Biopolymers 99: 548–561, 2013.