Lipid Bilayer Assemblies Research Articles

Specific phospholipid recognition by human immunodeficiency virus type-1 neutralizing anti-gp41 2F5 antibody

HIV-1 neutralizing monoclonal antibody (Mab) 2F5 recognizes a membrane-partitioning gp41 sequence. Just recently its capacity to react with cardiolipin has been demonstrated. Here, we have studied the specificity of Mab2F5-phospholipid interactions comparing partitioning into lipid bilayers with recognition of molecular species dispersed in solution. Using a liposome-based ELISA we demonstrate a preferential association with cardiolipin bilayers. When different soluble lysoderivatives were compared in their capacity to inhibit Mab2F5 binding to immobilized HIV-1 peptide epitope, only dilysocardiolipin resulted effective in blocking the process. Dilyso-cardiolipin also competed with native-functional gp41 for 2F5 recognition. Thus, our data support specific cardiolipin recognition by 2F5 that is not dependent on lipid bilayer assembly and involves the epitope-binding site. These findings might be of relevance for understanding the molecular basis of HIV-1 immune evasion.

Directed Assembly of Surface-Supported Bilayers with Transmembrane Helices

The lateral assembly of transmembrane (TM) helices gives rise to membrane proteins with complex folds, which play important roles in biochemical processes. Therefore, the assembly of surface-supported bilayers containing TM helices is the first step toward the development of functional biomembrane mimetics. Here we report novel directed assembly of surface-supported lipid bilayers with laterally mobile TM helices. The TM helices were incorporated into lipid monolayers at the air/water interface, and the monolayers were then transferred onto glass substrates using Langmuir-Blodgett (LB) deposition. Finally, bilayers were assembled using lipid vesicle fusion on top of the LB monolayers. The novelty is the incorporation of the peptides into the monolayer at the first step of bilayer assembly, which allows control over the peptide concentration and orientation. The transmembrane orientation of the peptides was confirmed using oriented circular dichroism (OCD), lateral mobility was assessed using fluorescence recovery after photobleaching (FRAP), and diffusion coefficients were determined using a novel boundary profile evolution (BPE) method. The described directed-assembly approach can be used to develop versatile bilayer platforms for studying membrane proteins interactions in native bilayer environments.

Reorganizational dynamics of multilamellar lipid bilayer assemblies using continuously scanning Fourier transform infrared spectroscopic imaging

We employ an implementation of rapid-scan Fourier transform infrared (FT-IR) microspectroscopic imaging to acquire time-resolved images for assessing the non-repetitive reorganizational dynamics of aqueous dispersions of multilamellar lipid vesicles (MLVs) derived from distearoylphosphatidylcholine (DSPC). The spatially and temporally resolved images allow direct and simultaneous determinations of various physical and chemical properties of the MLVs, including the main thermal gel to liquid crystalline phase transition, comparisons of vesicle diffusion rates in both phases and the variation in lipid bilayer packing properties between the inner and outer lamellae defining the vesicle. Specifically, in the lipid liquid crystalline phase, the inner bilayers of the MLVs are more intermolecularly ordered than the outer regions, while the intramolecular acyl chain order/disorder parameters, reflecting the overall characteristics of the fluid phase, remain uniform across the vesicle diameter. In contrast, the lipid vesicle gel phase displays no intermolecular or intramolecular dependence as a function of distance from the MLV center.

Phase Dependent Electrochemical Properties of Polar Self-Assembled Monolayers (SAMs) Modified via the Fusion of Mixed Phospholipid Vesicles

Unilamellar vesicles of 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) and varying quantities of either 1,2-dimyristoyl-sn-glycero-3-[phospho-rac-(1-glycerol) (sodium salt) (DMPG) or 1,2-dimyristoyl-3-trimethylammonium-propane (chloride salt) (DMTAP) were used to deposit lipid bilayer assemblies on self-assembled monolayers (SAMs) on gold. The supporting SAMs in turn were composed of ferrocene-functionalized hexadecanethiol chains (FcC16SH) diluted to low coverage in 1-hydroxylhexadecanethiol (HOC16SH) or a single-component monolayer phase of the latter. The mass coverages of the DMPC/DMPG layers deposited in this way were measured using surface plasmon resonance (SPR) and found to decrease with an increasing content of DMPG in the vesicles. The SPR data show that the lipid assembly, while stable with respect to gentle rinsing in aqueous buffer, is reversible and the lipid adlayer is removable by immersion in a solvent such as ethanol. The effects of the adsorbed lipid layer on the electrochemical interactions of the hybrid lipid/SAM with several redox probes [e.g., K4Fe(CN)6, Ru(NH3)6Cl3, and CsHsFe-[(C5H4CH2N+H(CH3)2] were characterized using cyclic voltammetry (CV). At a composition of 5% DMPG in DMPC, the permeabilities of the probes through the lipid layer were affected significantly relative to that observed with a pure DMPC layer. These effects include a striking observation of an enhanced, ionic-charge-specific molecular discrimination of the electrochemical probes. At higher concentrations of the DMPG, significant permeation of the lipid adlayer was seen for all the probes. These latter changes are also attended by a significant increase in the capacitive currents measured in CV experiments as compared to those observed for either a pure SAM or one modified by only DMPC. This effect likely results from the influence of the charged lipid on the diffuse Gouy-Chapman electrolyte layer at the SAM interface. In contrast to the behaviors seen with DMPG, the incorporation of DMTAP into the adsorbed DMPC had no impact on the permeation of the adlayer by soluble redox probes as judged by the observed electrochemistry, a result that appears to correlate with a less ideal mixing of lipids in the DMPC/DMTAP system relative to that of a DMPC/DMPG mixture.

Engineering asymmetric vesicles.

Vesicles are bilayers of lipid molecules enclosing a fixed volume of aqueous solution. Ubiquitous in cells, they can be produced in vitro to study the physical properties of biological membranes and for use in drug delivery and cosmetics. Biological membranes are, in fact, a fluid mosaic of lipids and other molecules; the richness of their chemical and mechanical properties in vivo is often dictated by an asymmetric distribution of these molecules. Techniques for vesicle preparation have been based on the spontaneous assembly of lipid bilayers, precluding the formation of such asymmetric structures. Partial asymmetry has been achieved only with chemical methods greatly restricting the study of the physical and chemical properties of asymmetric vesicles and their use in potential applications for drug delivery. Here we describe the systematic engineering of unilamellar vesicles assembled with two independently prepared monolayers; this process produces asymmetries as high as 95%. We demonstrate the versatility of our method by investigating the stability of the asymmetry. We also use it to engineer hybrid structures comprised of an inner leaflet of diblock copolymer and an independent lipid outer leaflet.

Cryo-Em of Large Unilamellar Phospholipid Vesicles That Self- Assemble At a Critical Temperature

The mechanism for assembly of membrane lipid bilayers in vivo is generally considered to occur on existing membranes which act as templates for the assembly process. Not presently understood is the source of the membrane template. A thermodynamic theory describing a spontaneous process for assembly of unilamellar vesicles with properties of a critical state (1,2) has been tested in this study. The spontaneous formation of large unilamellar vesicles (LUV’s) from phospholipid components of cell membranes at a critical temperature T* has now been documented by cryo-electron microscope studies using aqueous dispersions of dimyrystoylphosphatidylcholine (T* = 29°C), and the total lipid extracts of E. coli membranes cultured at 32°C (T* = 32°C). The lipids were incubated at temperatures below, at and above the critical temperatures for LUV assembly. in conformity with thermodynamic theory, and other physical measurements that indicate unique bilayer properties exist at T* (3-5), LUV’s are seen to form only at T*.

Lipid bilayer electrostatic energy, curvature stress, and assembly of gramicidin channels.

Hydrophobic interactions between lipid bilayers and imbedded membrane proteins couple protein conformation to the mechanical properties of the bilayer. This coupling is widely assumed to account for the regulation of membrane protein function by the membrane lipids' propensity to form nonbilayer phases, which will produce a curvature stress in the bilayer. Nevertheless, there is only limited experimental evidence for an effect of bilayer curvature stress on membrane protein structure. We show that alterations in curvature stress, due to alterations in the electrostatic energy of dioleoylphosphatidylserine bilayers, modulate the structurally well-defined gramicidin A monomer <--> dimer reaction. Maneuvers that decrease the electrostatic energy of the unperturbed bilayer promote channel dissociation; we measure the change in interaction energy. The bilayer electrostatic energy thus can affect membrane protein structure by a mechanism that does not involve the electrostatic field across the bilayer, but rather electrostatic interactions among the phospholipid head groups in each monolayer which affect the bilayer curvature stress. These results provide further evidence for the importance of mechanical interactions between a bilayer and its imbedded proteins for protein structure and function.

Assembly and molecular organization of self-assembled lipid bilayers on solid substrates monitored by surface plasmon resonance spectroscopy

The structural properties of lipid films, made from a squalene/butanol solution containing varying amounts (0–15 mg/ml) of egg phosphatidylcholine and deposited on a thin metallic silver layer, were investigated using surface plasmon resonance (SPR) spectroscopy. Optical parameters (thickness, refractive index and extinction coefficient) of such supported self-assembled lipid membranes were obtained from a theoretical analysis of the experimental SPR curves. The mass of the lipid membrane and the area and volume occupied by one lipid molecule were also calculated. The results were consistent with the formation of durable and homogeneous lipid bilayers on the solid substrate, and indicated similarities in structural properties between the present lipid bilayers and freely suspended and Langmuir-Blodgett bilayer membranes. Such bilayers represent a simple model for biological membranes, as well as providing a means of immobilizing proteins for various practical applications, including receptor-based sensors and molecular devices. The results confirm the value of the SPR technique for investigating the properties of thin biomolecular dielectric films deposited on a metal surface.

Modulation of bilayer structures derived from diacetylenic phosphocholines containing oxygen linker β to diacetylene

Phospholipids with diacetylenes present in the acyl chains form tubules and helices in aqueous dispersions. In order to modulate the morphology of bilayer structures and to understand the role of diacetylene in lipid-bilayer assembly, two diacetylenic phosphocholines, 1,2-bis(9,16-dioxa-hexacosa-11,13-diynoyl)- sn-3-phosphocholine and 1,2-bis(15-oxa-pentacosa-10,12-diynoyl)- sn-3-phosphocholine, in which the diacetylene is linked to the acyl chain by an oxygen spacer have been synthesized. Lipid dispersions were characterized by calorimetric, film balance and microscopic techniques. Placement of oxygen spacer influences the morphology of the bilayer assemblies formed in aqueous solution. When both ends of the diacetylene were linked to the acyl chain by oxygen atoms, liposomes (diameters ranging from 0.3–3.4 μm) were observed by optical microscopy. Linking only the terminal portion of the acyl chain to the diacetylene with an oxygen atom resulted in a lipid which formed tubular microstructures as well as vesicles. Diameter of the tubular structures ranged from 0.4–4.7 μm. Transmission electron microscopic (TEM) analysis of replicas of a freeze fractured sample of the dispersion revealed that the tubular structures were hollow cylinders consisting of an aqueous core surrounded by a wall of lipid.

Small-angle X-ray diffraction studies on the effects of melittin on lipid bilayer assemblies

The effects of melittin on lipid bilayer assemblies at very low doses, which are important for its toxic action, have been studied. Differential scanning calorimetry indicated that melittin affects the thermotropic behavior of dihexadecylphosphatidylcholine at molar ratios of 10−4. Small-angle X-ray diffraction showed that the structural arrangements are markedly altered. At a melittin/lipid molar ratio of 10−4 the interdigitated structure, which is formed by this lipid below physiological temperatures, is stabilized up to the main-transition temperature (~ 317 K). At higher temperatures a broad continuous transform with superimposed peaks, arising from nonlamellar structures, is observed. At a molar ratio of 10−3 and below the main transition the interdigitated structure represents only a minor fraction. Additionally, the system is very sensitive to swelling. Increasing the water content leads to an increase of the broad continuous transform. Above the main transition a sequence of disordered lamellar→cubic→hexagonal→micellar structure was found in agreement with predictions from general amphiphile/water binary phase diagrams.

Phospholipid surface bilayers at the air-water interface. III. Relation between surface bilayer formation and lipid bilayer assembly in cell membranes

Lipid bilayer assembly in cell membranes has been simulated with total lipid extracts from human red blood cells and from mesophilic and thermophilic bacteria grown at several temperatures. Aqueous dispersions of these natural lipid mixtures form surface bilayers, a single bimolecular lipid state, but only at the growth temperature of the source organism. Thus, a single isolated bilayer state forms spontaneously in vitro from lipids that are available in vivo at the growth temperature of the cell. Surface bilayers form at a specific temperature that is a function of hydrocarbon chain length and degree of fatty acid unsaturation of the phospholipids; this property is proposed as an essential element in the control of membrane lipid composition.

A molecular membrane model of sperm capacitation and the acrosome reaction of mammalian spermatozoa

AbstractThe abundance of data pertaining to the metabolism of lipids in relation to mammalian fertilization has warranted an effort to assemble a molecular membrane model for the comprehensive visualization of the biochemical events involved in sperm capacitation and the acrosome reaction. Derived both from earlier models as well as from current concepts, our membrane model depicts a lipid bilayer assembly of space‐filling molecular models of sterols and phospholipids in dynamic equilibrium with peripheral and integral membrane proteins. A novel feature is the possibility of visualizing individual lipid molecules such as phosphatidylcholine, phosphatidylethanolamine, lysophospholipids, fatty acids, and free or esterified cholesterol. The model illustrates enzymatic reactions which are believed to regulate the permeability and integrity of the plasma membrane overlying the acrosome during interactions between the male gamete and capacitation factors present in fluids of the female genital tract. The use of radioactive lipids as molecular probes for monitoring the metabolism of cholesterol and phosphatidylcholine revealed the presence of (1) steroid sulfatase in hamster cumulus cells, (2) lecithin: cholesterol acyltransferase in human follicular fluid, (3) phospholipase A2, and (4) lysophospholipase in human spermatozoa. These enzymatic reactions can be integrated into a pathway that provides a link between the concepts of lysophospholipid accumulation in the sperm membranes and alteration of the cholesterol/phospholipid ratio as factors involved in the preparation of the membranes for the acrosome reaction. Capacitation is viewed as a reversible phenomenon which, upon completion, results in a decrease in negative surface charge, an efflux of membrane cholesterol, and an influx of calcium between the plasma and outer acrosomal membranes. Triggered by the entry of calcium, the acrosome reaction involves phospholipase A2 activation followed by a transient accumulation of unsaturated fatty acids and lysophospholipids implicated in membrane fusion which occurs during the formation of membrane vesicles in spermatozoa undergoing the acrosome reaction.