Bilayer Disks Research Articles

Hybrid Nanoparticles of Poly (Methyl Methacrylate) and Antimicrobial Quaternary Ammonium Surfactants.

Quaternary ammonium surfactants (QACs) are microbicides, whereas poly (acrylates) are biocompatible polymers. Here, the physical and antimicrobial properties of two QACs, cetyl trimethyl ammonium bromide (CTAB) or dioctadecyl dimethyl ammonium bromide (DODAB) in poly (methyl methacrylate) (PMMA) nanoparticles (NPs) are compared to those of QACs alone. Methyl methacrylate (MMA) polymerization using DODAB or CTAB as emulsifiers and initiator azobisisobutyronitrile (AIBN) yielded cationic, nanometric, homodisperse, and stable NPs. NPs’ physical and antimicrobial properties were assessed from dynamic light scattering (DLS), scanning electron microscopy, and viability curves of Escherichia coli, Staphylococcus aureus, or Candida albicans determined as log(colony-forming unities counting) over a range of [QACs]. NPs were spherical and homodisperse but activity for free QACs was higher than those for QACs in NPs. Inhibition halos against bacteria and yeast were observed only for free or incorporated CTAB in NPs because PMMA/CTAB NPs controlled the CTAB release. DODAB displayed fungicidal activity against C. albicans since DODAB bilayer disks could penetrate the outer glycoproteins fungus layer. The physical properties and stability of the cationic NPs highlighted their potential to combine with other bioactive molecules for further applications in drug and vaccine delivery.

Fabrication of consolidated layered samples by high-pressure torsion processing of rapidly solidified Al-based ribbons with amorphous and crystalline structures

The fully consolidated bilayer (consisting of two amorphous Al86Ni9Gd5 ribbons) and trilayer (composed by the Al95.1Cr2.5Mo1.4Ti0.4Zr0.3V0.3 crystalline melt-spun ribbon placed between two amorphous ribbons) disks of 5 mm in diameter were fabricated by unconstrained high pressure torsion (HPT) processing up to four turns at applied stress of 2 GPA. The structure of the cross sections of the trilayer disk was analyzed by scanning electron microscopy and the critical strains required for partial and full mixing of the amorphous and crystalline ribbons were estimated. HPT processing of amorphous ribbons resulted in formation of Al nanocrystals about 8–9 nm in size and essential enhancement in microhardness from 2.7 to 4.1 GPa after one turn. The averaged surface microhardness of the firstly consolidated trilayer amorphous/crystalline disks increased from 2.3 to 4.7 GPa with increasing of up to 4 turns. The strain-hardening of these disks was caused by a simultaneous increase of the volume fraction of Al nanocrystals up to 0.41 and a decrease in their sizes from 41 to 14 nm. The internal structure of the HPT processed trilayer disks was strongly inhomogeneous and its microhardness varied in the range from 2.86 GPa in the central part to 6.45 GPa in the zone of partial mixing which was an exceptionally high value for Al-based alloys.

Homogeneous nanodiscs of native membranes formed by stilbene-maleic-acid copolymers.

Methylstilbene-alt-maleic acid copolymers spontaneously convert biological membranes into bilayer discs with ∼20 nm diameters. This readily functionalizable class of copolymers has the compositional homogeneity, hydrophobicity, dynamics, and charge that may help to achieve optimal structural resolution, membrane dissolution, stability, and broad utility.

Aescin-Induced Conversion of Gel-Phase Lipid Membranes into Bicelle-like Lipid Nanoparticles.

Mixtures of the phospholipid 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) and the saponin β-aescin spontaneously form monodisperse, bilayered discoidal micelles (also known as "bicelles" or "nanodisks") in aqueous solution. Such bicelles form below the melting temperature of DMPC when the phospholipids are in the rigid Lβ' state and are precursors of spontaneously formed vesicles. The aescin concentration must be far above the cmcaescin (≈0.3-0.4 mM). It was found that the shape and size of the bicelles are tunable by composition. High amounts of aescin decrease the size of the bicelles from diameters of ∼300 Å at 7 mol % to ∼120 Å at 30 mol % β-aescin. The structures are scrutinized by complementary small-angle X-ray and neutron scattering experiments. The scattering curves are subsequently analyzed by a model-independent (indirect Fourier transform analysis) and a model-based approach where bicelles are described as polydisperse bilayer disks encircled by a β-aescin rim. Moreover, the monomodal distribution and low polydispersity of the samples were confirmed by photon correlation spectroscopy. The discoidal structures were visualized by transmission electron microscopy.

Lipid and Protein Transfer between Nanolipoprotein Particles and Supported Lipid Bilayers.

A nanolipoprotein particle (NLP) is a lipid bilayer disc stabilized by two amphipathic "scaffold" apolipoproteins. It has been most notably utilized as a tool for solubilizing a variety of membrane proteins while preserving structural and functional properties. Transfer of functional proteins from NLPs into model membrane systems such as supported lipid bilayers (SLBs) would enable new opportunities, for example, two-dimensional protein crystallization and studies on protein-protein interactions. This work used fluorescence microscopy and atomic force microscopy to investigate the interaction between NLPs and SLBs. When incubated with SLBs, NLPs were found to spontaneously deliver lipid and protein cargo. The impact of membrane composition on lipid exchange was explored, revealing a positive correlation between the magnitude of lipid transfer and concentration of defects in the target SLB. Incorporation of lipids capable of binding specifically to polyhistidine tags encoded into the apolipoproteins also boosted transfer of NLP cargo. Optimal conditions for lipid and protein delivery from NLPs to SLBs are proposed based on interaction mechanisms.

Reconstitution of the Rhodopsin-Transducin Complex into Lipid Nanodiscs.

Transmembrane proteins, such as G protein-coupled receptors (GPCR), require solubilization in detergents prior to purification. The recent development of novel detergents has allowed for the stabilization of GPCRs, which typically have a high degree of structural flexibility and are otherwise subject to denaturation. However, the detergent micelle environment is still very different from the native lipid membrane and the activity of GPCRs can be profoundly affected by interactions with annular lipid molecules. Moreover, GPCRs are often palmitoylated at their intracellular side, and a lipid bilayer environment would allow for proper orientation of these lipid modifications. Therefore, a reconstituted lipid bilayer environment would best mimic the physiological receptor microenvironment for biophysical studies of GPCRs and nanodiscs provide a methodology to address this aim. Nanodiscs are lipid bilayer discs stabilized by amphipathic membrane scaffolding proteins (MSP) where detergent-solubilized transmembrane proteins can be incorporated into them through a self-assembly process. Here we present a method for reconstituting the purified detergent-solubilized rhodopsin-transducin complex, the GPCR-G protein complex in visual phototransduction, into nanodiscs. The resulting complex incorporated into lipid nanodiscs can be used in biophysical studies including small-angle X-ray scattering and electron microscopy. This method is applicable to integral membrane proteins that mediate protein lipidation, including the zDHHC-family of S-acyltransferases and membrane-bound O-acyltransferases.

Single-Unit Imaging of Membrane Protein-Embedded Nanodiscs from Two Oriented Sides by High-Speed Atomic Force Microscopy

Membrane proteins play important roles in various cellular functions. To analyze membrane proteins, nanodisc technology using membrane scaffold proteins allows single membrane protein units to be embedded into the lipid bilayer disc without detergents. Recent advancements in high-speed atomic force microscopy (HS-AFM) have enabled us to monitor the real-time dynamics of proteins in solution at the nanometer scale. In this study, we report HS-AFM imaging of membrane proteins reconstituted into nanodiscs using two membrane protein complexes, SecYEG complex and MgtE dimer. The observed images showed single particles of membrane protein-embedded nanodiscs in an end-up orientation whereby the membrane was fixed parallel to the supporting solid surface and in a side-on orientation whereby the membrane plane was vertically fixed to the solid surface, enabling the elucidation of domain fluctuations in membrane proteins. This technique provides a basic method for the high-resolution imaging of single membrane proteins by HS-AFM.

Effects of Amphipathic Polypeptides on Membrane Organization Inferred from Studies Using Bicellar Lipid Mixtures

SP-B63-78, a lung surfactant protein fragment, and magainin 2, an antimicrobial peptide, are amphipathic peptides with the same overall charge but different biological functions. Deuterium nuclear magnetic resonance has been used to compare the interactions of these peptides with dispersions of 1,2-dimyristoyl- sn-glycero-3-phophocholine (DMPC)/1,2-dihexanoyl- sn-glycero-3-phophocholine (DHPC) (4:1) and DMPC/1,2-dimyristoyl- sn-glycero-3-phopho-(1'-rac-glycerol) (DMPG)/DHPC (3:1:1), two mixtures of long-chain and short-chain lipids that display bicellar behavior. This study exploited the sensitivity of a bicellar system structural organization to factors that modify partitioning of their lipid components between different environments. In small bicelle particles formed at low temperatures, short-chain components preferentially occupy curved rim environments around bilayer disks of the long-chain components. Changes in chain order and lipid mixing, on heating, can drive transitions to more extended assemblies including a magnetically orientable phase at intermediate temperature. In this work, neither peptide had a substantial effect on the behavior of the zwitterionic DMPC/DHPC mixture. For bicellar mixtures containing the anionic lipid DMPG, the peptide SP-B63-78 lowered the temperature at which magnetically orientable particles coalesced into more extended lamellar structures. SP-B63-78 did not promote partitioning of the zwitterionic and anionic long-chain lipid components into different environments. Magainin 2, on the other hand, was found to promote separation of the anionic lipid, DMPG, and the zwitterionic lipid, DMPC, into different environments for temperatures above 34 °C. The contrast between the effects of these two peptides on the lipid mixtures studied appears to be consistent with their functional roles in biological systems.

Macrodiscs Comprising SMALPs for Oriented Sample Solid-State NMR Spectroscopy of Membrane Proteins

Macrodiscs, which are magnetically alignable lipid bilayer discs with diameters of >30 nm, were obtained by solubilizing protein-containing liposomes with styrene-maleic acid copolymers. Macrodiscs provide a detergent-free phospholipid bilayer environment for biophysical and functional studies of membrane proteins under physiological conditions. The narrow resonance linewidths observed from membrane proteins in styrene-maleic acid macrodiscs advance structure determination by oriented sample solid-state NMR spectroscopy.

Magnetically Oriented Phospholipid Bilayer Discs for Membrane Protein NMR

Magnetically Oriented Phospholipid Bilayer Discs for Membrane Protein NMR

The ER morphology-regulating lunapark protein induces the formation of stacked bilayer discs.

Lunapark (Lnp) is a conserved membrane protein that localizes to and stabilizes three-way junctions of the tubular ER network. In higher eukaryotes, phosphorylation of Lnp may contribute to the conversion of the ER from tubules to sheets during mitosis. Here, we report on the reconstitution of purified Lnp with phospholipids. Surprisingly, Lnp induces the formation of stacked membrane discs. Each disc is a bicelle, with Lnp sitting in the bilayer facing both directions. The interaction between bicelles is mediated by the cytosolic domains of Lnp, resulting in a constant distance between the discs. A phosphomimetic Lnp mutant shows reduced bicelle stacking. Based on these results, we propose that Lnp tethers ER membranes in vivo in a cell cycle-dependent manner. Lnp appears to be the first membrane protein that induces the formation of stacked bicelles.

Antigenic characterization of the human immunodeficiency virus (HIV-1) envelope glycoprotein precursor incorporated into nanodiscs.

The entry of human immunodeficiency virus (HIV-1) into host cells is mediated by the viral envelope glycoproteins (Envs), which are derived by the proteolytic cleavage of a trimeric gp160 Env precursor. The mature Env trimer is a major target for entry inhibitors and vaccine-induced neutralizing antibodies. Env interstrain variability, conformational flexibility and heavy glycosylation contribute to evasion of the host immune response, and create challenges for structural characterization and vaccine development. Here we investigate variables associated with reconstitution of the HIV-1 Env precursor into nanodiscs, nanoscale lipid bilayer discs enclosed by membrane scaffolding proteins. We identified detergents, as well as lipids similar in composition to the viral lipidome, that allowed efficient formation of Env-nanodiscs (Env-NDs). Env-NDs were created with the full-length Env precursor and with an Env precursor with the majority of the cytoplasmic tail intact. The self-association of Env-NDs was decreased by glutaraldehyde crosslinking. The Env-NDs exhibited an antigenic profile expected for the HIV-1 Env precursor. Env-NDs were recognized by broadly neutralizing antibodies. Of note, neutralizing antibody epitopes in the gp41 membrane-proximal external region and in the gp120:gp41 interface were well exposed on Env-NDs compared with Env expressed on cell surfaces. Most Env epitopes recognized by non-neutralizing antibodies were masked on the Env-NDs. This antigenic profile was stable for several days, exhibiting a considerably longer half-life than that of Env solubilized in detergents. Negative selection with weak neutralizing antibodies could be used to improve the antigenic profile of the Env-NDs. Finally, we show that lipid adjuvants can be incorporated into Env-NDs. These results indicate that Env-NDs represent a potentially useful platform for investigating the structural, functional and antigenic properties of the HIV-1 Env trimer in a membrane context.

Aggregation behavior of cyclic rod-coil diblock copolymers in selective solvents

The aggregation behavior of cyclic rod-coil (RC) diblock copolymers in dilute solutions is investigated through dissipative particle dynamics simulation. By varying the rod length and coil length, cyclic RC copolymers in selective solvents exhibit various morphologies, including spherical micelle, vesicle, bilayer disc, and ribbon bundle structure. Compared with the equivalent linear RC copolymer, only spherical micelle and barrel bundle phase are observed. Rod length is the major factor that controls the liquid-crystalline behavior of RC copolymer systems, while the coil length has a secondary effect on the aggregate morphology. The size of rod bundle varies with the coil length, especially for the end-toend ribbon bundle and side-by-side barrel bundle, which are assembled by cyclic and linear RC copolymer solutions. This finding indicates that the ribbon bundle or nanofiber-like structure in cyclic RC copolymers can be obtained by controlling the rod length and coil length, and thus the optical and electrical properties of RC copolymer would be further controlled and optimized. Results illustrate that cyclization of a linear RC copolymer induces remarkable differences in the rod arrangement and aggregation behavior, thereby indicating the competition between interfacial energy, rod orientational entropy, coil stretching entropy, and packing constraints.

Nanostructured Cathodes for Solid Oxide Fuel Cells by a Solution Spray-Coating Process

Composite nano-cathodes containing Sr-doped LaMnO3 (LSM) and Gd2O3-doped CeO2 (GDC) were deposited on yttria-stabilized zirconia (YSZ) discs and YSZ/NiO + YSZ bi-layer discs using an aqueous solution. The samples were heated over a range of temperatures from 700°C to 1200°C to form LSM + GDC porous films. The crystallite size was estimated using X-ray diffraction (XRD) Scherrer formula. The crystallite size was in the ∼3 nm to ∼80 nm range. Polarization resistance was measured by electrochemical impedance spectroscopy (EIS) from 400°C to 700°C. Nano-cathodes of ∼70 nm thickness fired at 700°C exhibited polarization resistance of ∼0.23 Ωcm2 at 700°C. By contrast, LSM + YSZ cathodes made by a conventional process exhibited polarization resistance of ∼2 Ωcm2. Fuel cell tests were conducted at 700°C on cells with nano-cathodes and conventional LSM + YSZ cathode. The maximum power density was ∼0.51 Wcm−2 with nano-cathodes. By contrast, the corresponding power density with conventional LSM + YSZ cathodes was ∼0.11 Wcm−2. The present work shows that very thin cathode functional layers can be deposited on sintered YSZ/NiO + YSZ bi-layer discs by using an aqueous solution. This approach in principle could be extended to large area cells.

Electromechanical Displacement of Soft/Hard PZT Bi-Layer Composite Actuator

PZTs can be classified into two types, i.e., soft and hard PZTs, which are categorized by the piezoelectric and ferroelectric properties such as coercive field, piezoelectric strain, mechanical quality factor etc. It is known that the combination effect of the soft/hard PZT composites can generate large strain/actuation compared to monolithic PZT ceramics. In this study, soft and hard PZT powders were co-pressed into bi-layer disks with various ratios between soft and hard PZT powders, ranging from 0:100~100:0 vol. % (with 10 % increments) and then they were co-sintered. Due to the difference in the planar shrinkage of the two layers and thermal expansion coefficient mismatch, dome-shaped bi-layer composites with various dome heights were obtained. It was shown that the constrained layer either soft PZT or hard PZT affected various properties including the dome geometry, the strain-E-field response, and the displacement hysteresis loop. The electromechanical properties and actuation performance of such bi-layer composite actuators have been investigated and compared to the soft and hard PZT single layer counterparts.

Substrate binding to cytochrome P450-2J2 in Nanodiscs detected by nanoplasmonic Lycurgus cup arrays.

Cytochrome P450s are the primary enzymes involved in phase I drug metabolism. They are an important target for early drug discovery research. However, high-throughput drug screening of P450s is limited by poor protein stability and lack of consistent measurement of binding events. Here we present the detection of substrate binding to cytochrome P450-2J2 (CYP2J2), the predominant P450 in the human heart, using a combination of Nanodisc technology and a nanohole plasmonic sensor called nanoplasmonic Lycurgus cup array (nanoLCA). The Nanodisc, a nanoscale membrane bilayer disc, is used to stabilize the protein on the metallic plasmonic surface. Absorption spectroscopy of seven different substrates binding to CYP2J2 in solution showed that they are all type I, resulting in shifting of the protein bands to lower wavelengths (blue shift). Detection on the nanoLCA sensor also showed spectral blue shifts of CYP2J2 following substrate binding. Finite Difference Time Domain (FDTD) electromagnetic simulation suggested that the blue shift on the nanoLCA is because of the hybridization of plasmon polariton Bloch wave and the electronic resonance of the heme group of CYP2J2. We found the plasmonic properties of the nanoLCA sensor to be highly reproducible, which allowed comparisons among the different substrates at different concentrations. Further, due to the unique spectral properties of the nanoLCA sensor, including the transmission of a single color, we were able to perform colorimetric detection of the binding events. These results indicate that a resonance plasmonic sensing mechanism can be used to distinguish between different substrates of the same binding type at different concentrations binding to P450s and that the nanoLCA sensor has the potential to provide consistent high-throughput measurements of this system.

Novel polyglycidol-lipid conjugates create a stabilizing hydrogen-bonded layer around cholesterol-containing dipalmitoyl phosphatidylcholine liposomes

Hybrid liposomes resulting from co-assembly of dipalmitoylphosphatidylcholine and polyglycidol-derivatized lipids were prepared. The latter were composed of a lipid-mimetic residue to which a linear polyglycidol chain (degree of polymerization, DP, in the 23–110 range) was conjugated. Formulations with varying copolymer type and content were prepared by film hydration technique followed by extrusion. The hybrid structures were studied by means of dynamic and electrophoretic light scattering, cryogenic transmission electron microscopy, and fluorescence spectroscopy. Cytotoxicity towards OPM-2 (multiple myeloma) and EJ (human urinary bladder carcinoma) cell lines was assessed as well. Predominantly unilamellar liposomes with mean hydrodynamic diameters in the 113–134 nm range and neutral to slightly negative surface potential were prepared. The integrity of liposomes containing copolymers with DP of the polyglycidol chain 23 and 30 was preserved at copolymer contents up to 10 mol%. Bilayer disks were observed at somewhat lower contents of the copolymers of the highest DP of the polyglycidol chain. The hybrid structures were less leaky than the plain liposomes, which was attributed to formation of a strongly hydrogen-bonded polyglycidol layer around the bilayer membrane. They exhibited low toxicological potential, favorable physicochemical characteristics, and ability to act as containers for sustained release.

Viral Membrane Fusion at Single Pore Resolution

Fusion between membrane-enveloped viruses and their hosts is a critical step in infection, enabling the virus to release its genome and subsequently hijack the host cell's replication machinery. The genomes of enveloped viruses are packaged into multiple bulky ribonucleoprotein (RNP) particles whose release requires the dilation of the fusion pore - the initial, narrow, connection between fusing membranes. Electrophysiological studies of influenza A hemagglutinin (HA)-mediated fusion between HA-expressing fibroblasts and red blood cells and between HA-fibroblasts and voltage-clamped suspended bilayers comprise the most complete information to date about the dynamics of viral fusion pores. Small pores (∼2-5nm) were found to flicker repetitively and/or fluctuate in size prior to terminal dilation. A quantitative understanding of the biophysical driving forces of viral membrane fusion, including membrane contact area, membrane tension and protein fusogen cooperativity, however, remains elusive, and a number of questions - regarding the physical basis of pore flickering and the number of HA trimers required for successful pore dilation - remain open. Here we describe a novel electrophysiological approach, which has been successfully applied to study SNARE-mediated fusion pores, to probe the HA-mediated fusion pore. We voltage-clamped HA-expressing fibroblasts in the cell-attached configuration and included nanodiscs (NDs) in the patch pipette as fusion partners to probe fusion pore nucleation and evolution at single pore resolution. NDs are flat, bilayer discs whose composition and size can be varied. Current through voltage-clamped fusion pores reports pore size with sub-millisecond time resolution. Preliminary data shows that isolated fusion pores flicker, are long-lived (∼10s) and are of diameter ∼1.5nm.

Solubilization of Membranes by Styrene Maleic Acid (SMA) Results in Formation of Nanodiscs with Retention of Native Lipid Composition

Purification of membrane proteins has been a real headache for the scientific community during the last decades. Recently, a new purification method has emerged using the SMA (styrene-maleic acid) co-polymer, which is able to solubilize membranes into small bilayer discs. Its importance lies mostly in the presumed ability of retaining the native lipid environment of the membrane proteins, preventing misfolding and/or loss of activity. However, it cannot be excluded that SMA preferentially solubilizes specific lipids, thereby modifying the extent to which the native lipid environment is indeed retained.To find out whether preferential solubilization by the polymer occurs, we performed an extensive biophysical study using model membranes of synthetic lipids with well-defined compositions. Solubilized and non-solubilized material was obtained by adding limiting amounts of SMA polymer to the phospholipid vesicles. The mixture was centrifuged and lipid analysis was performed on the supernatant, containing the nanodiscs, and the non-solubilized fraction in the pellet. This procedure was performed for mixtures of different phospholipids, in which we varied the acyl chain length, unsaturation, head group charge and lipid phase. The results showed no preferential solubilization in any of these lipid mixtures, the only exception being a raft-like system (DOPC/SM/cholesterol), where it was shown that SMA is incapable of solubilizing SM/cholesterol-rich domains into nanodiscs.The results highlight the potential of SMA polymer as a strong alternative for membrane protein purification, in which the native membrane lipid composition is generally retained. Interestingly, the behavior of the raft-like systems upon addition of SMA resembles the behavior of detergent-resistant membranes (DRMs) when membranes are treated with detergent, paving the way for novel approaches of lipid raft isolation.

Bicelles coming of age: an empirical approach to bicelle crystallization.

Biological membranes represent a unique environment in which integral membrane proteins (MPs) fold to perform diverse biological functions. In many cases, lipids support the native conformation or mediate important interactions between MPs. It is therefore imperative to develop methods that maintain this support for the structural and functional analyses of an exceedingly important class of biological macromolecules. Bicelles are detergent-stabilized phospholipid bilayer discs into which MPs can be reconstituted for biophysical studies. Here, we review recent advances and emerging concepts in employing bicelles for the crystallization and structure determination of MPs. We discuss variations of established procedures as well as alternative approaches, and we present a summary and analysis of the conditions used for bicelle-mediated MP crystallization.