Supported Phospholipid Membranes Research Articles

Interaction of monomeric and self-assembled aromatic amino acids with model membranes: self-reproduction phenomena.

The spontaneous formation of amyloid structures of proteins is responsible for several major human neurodegenerative diseases. Here, we demonstrate that the formation of amyloid aggregates of the amino acids results in the formation of supported phospholipid membrane and aggregated vesicles via fusion and self-reproduction of the lipid vesicles. Importantly, during the vesicle growth, we found the formation of large "mother vesicles" containing small "daughter vesicles". This observation is significant for interpreting the protein-membrane interaction and mimicking the origin of cellular life.

Biomimetic Phospholipid Membrane Organization on Graphene and Graphene Oxide Surfaces: A Molecular Dynamics Simulation Study

Supported phospholipid membrane patches stabilized on graphene surfaces have shown potential in sensor device functionalization, including biosensors and biocatalysis. Lipid dip-pen nanolithography (L-DPN) is a method useful in generating supported membrane structures that maintain lipid functionality, such as exhibiting specific interactions with protein molecules. Here, we have integrated L-DPN, atomic force microscopy, and coarse-grained molecular dynamics simulation methods to characterize the molecular properties of supported lipid membranes (SLMs) on graphene and graphene oxide supports. We observed substantial differences in the topologies of the stabilized lipid structures depending on the nature of the surface (polar graphene oxide vs nonpolar graphene). Furthermore, the addition of water to SLM systems resulted in large-scale reorganization of the lipid structures, with measurable effects on lipid lateral mobility within the supported membranes. We also observed reduced lipid ordering within the supported structures relative to free-standing lipid bilayers, attributed to the strong hydrophobic interactions between the lipids and support. Together, our results provide insight into the molecular effects of graphene and graphene oxide surfaces on lipid bilayer membranes. This will be important in the design of these surfaces for applications such as biosensor devices.

Surface-Enhanced Infrared Spectroscopy and Neutron Reflectivity Studies of Ubiquinone in Hybrid Bilayer Membranes under Potential Control.

Surface-enhanced infrared adsorption spectroscopy (SEIRAS) and neutron reflectometry (NR) were employed to characterize ubiquinone (UQ) containing hybrid bilayer membranes. The biomimetic membrane was prepared by fusing phospholipid vesicles on a hydrophobic octadecanethiol monolayer self-assembled on a thin gold film. Using SEIRAS, the assembly of the membrane is monitored in situ. The presence of ubiquinone is verified by the characteristic carbonyl peaks from the quinone ester. A well-ordered distal lipid leaflet results from fusion of vesicles with and without the addition of ubiquinone. With applied potential, the hybrid bilayer membrane in the absence of UQ behaves in the same way as previously reported solid supported phospholipid membranes. When ubiquinone is incorporated in the membrane, electric field induced changes in the distal leaflet are suppressed. Changes in the infrared vibrations of the ubiquinone due to applied potential indicate the head groups are located in both polar and nonpolar environments. The spectroscopic data reveal that the isoprenoid unit of the ubiquinone is likely lying in the midplane of the lipid bilayer while the head has some freedom to move within the hydrophobic core. The SEIRAS experiments show redox behavior of UQ incorporated in a model lipid membrane that are otherwise inaccessible with traditional electrochemistry techniques.

Configurable Lipid Membrane Gradients Quantify Diffusion, Phase Separations and Binding Densities

Systematically testing the composition of supported phospholipid membranes typically requires many separate lipid depositions in distinct experiments or bins. Alternatively, we have developed a simple, low-cost method to create phospholipid gradients that exhibit a broad range of spatially resolvable compositions. We image these gradients as they relax to equilibrium to reveal diffusion coefficients, phase separation parameters, and protein binding densities as a function of localized lipid mixtures, in single experiments. Compositional gradients are formed by directed self-assembly where rapid-prototyping techniques (i.e., 3D printing or laser-cutting) prescribe supported lipid geometries that self-spread, heal and mix by diffusion. We are currently applying this platform to explore mechanisms of intra-bilayer cholesterol transport.http://dx.doi.org/10.1039/C5SM02013A, Soft Matter (2015).

Voltammetric detection of phytochelatin transported across unmodified and protoplast modified model phospholipid membranes

Possibility of phytochelatin PC2 transport was examined using developed electrochemical cell with two compartments separated by a phospholipid membrane created in pores of polycarbonate support. At first cell penetrating peptides (transportan 10 and mastoparan X) were used to allow the transfer of phytochelatin PC2. The results demonstrated that transportan 10 is several times more efficient than mastoparan X for transferring of phytochelatin across model membrane. Voltammetry with hanging mercury drop electrode or with solid amalgam electrodes was applied for detection of transported species. For determination of PC2, hanging mercury drop electrode and mercury meniscus-modified silver solid amalgam electrode were suitable. Modified model phospholipid membrane was prepared with parts of protoplasts from barley roots or leaves, and transport of phytochelatin PC2 has been confirmed. Electrochemical impedance spectroscopy was utilized to monitor formation, stability, and qualitatively the transporting process across the supported phospholipid membranes.

Monitoring of Single Vesicle Cytochrome-c Release Illuminates BAK as a Novel Target of Aβ Oligomers

Monitoring of Single Vesicle Cytochrome-c Release Illuminates BAK as a Novel Target of Aβ Oligomers

Plasmonic nanoantenna arrays for surface-enhanced Raman spectroscopy of lipid molecules embedded in a bilayer membrane.

We demonstrate a strategy for surface-enhanced Raman spectroscopy (SERS) of supported lipid membranes with arrays of plasmonic nanoantennas. Colloidal lithography refined with plasma etching is used to synthesize arrays of triangular shaped gold nanoparticles. Reducing the separation distance between the triangle tips leads to plasmonic coupling and to a strong enhancement of the electromagnetic field in the nanotriangle gap. As a result, the Raman scattering intensity of molecules that are located at this plasmonic "hot-spot" can be increased by several orders of magnitude. The nanoantenna array is then embedded with a supported phospholipid membrane which is fluid at room temperature and spans the antenna gap. This configuration offers the advantage that molecules that are mobile within the bilayer membrane can enter the "hot-spot" region via diffusion and can therefore be measured by SERS without static entrapment or adsorption of the molecules to the antenna itself.

Gold nanoparticle supported phospholipid membranes as a biomimetic biosensor platform for phosphoinositide signaling detection

Enzyme mediated phosphoinositide signaling plays important regulatory roles in diverse cellular processes and has close implication in human diseases. However, detection of phosphoinositide enzymes remains a challenge because of the difficulty in discriminating the phosphorylation patterns of phosphoinositide. Here we develop a novel enzyme-activated gold nanoparticles (AuNPs) assembly strategy as a homogeneous colorimetric biosensor for activity detection of phosphoinositide kinases and phosphatases. This strategy utilizes a biomimetic mechanism of phosphoinositide signaling, in which AuNP supported phospholipid membranes are constructed to mimic the cellular membrane substrate, and AuNPs modified with the pleckstrin homology (PH) domain of cytosolic proteins are designed for specific, multivalent recognition of phosphorylated phosphoinositides. This biomimetic strategy enables efficient enzymatic reactions of the substrate and highly selective detection of target enzyme. The biosensor is demonstrated for the detection of phosphoinositide 3-kinase (PI3K) and phosphatase with tensin homology (PTEN). The results revealed that it allows sensitive, rapid visual detection of the enzymes with pM detection limits and four-decade wide dynamic ranges, and is capable of detecting enzyme activities in complex cell lysate samples. This biosensor might provide a general biosensor platform for high-throughput detection of phosphoinositide enzymes with high sensitivity and selectivity in biomedical research and clinical diagnostics.

β-Amyloid (1–40) Peptide Interactions with Supported Phospholipid Membranes: A Single-Molecule Study

Recent evidence supports the hypothesis that the oligomers formed by the β-amyloid peptide early in its aggregation process are neurotoxic and may feature in Alzheimer’s disease. Although the mechanism underlying this neurotoxicity remains unclear, interactions of these oligomers with neuronal membranes are believed to be involved. Identifying the neurotoxic species is challenging because β-amyloid peptides form oligomers at very low physiological concentrations (nM), and these oligomers are highly heterogeneous and metastable. Here, we report the use of single-molecule imaging techniques to study the interactions between β-amyloid (1–40) peptides and supported synthetic model anionic lipid membranes. The evolution of the β-amyloid species on the membranes was monitored for up to several days, and the results indicate an initial tight, uniform, binding of β-amyloid (1–40) peptides to the lipid membranes, followed by oligomer formation in the membrane. At these low concentrations, the behavior at early times during the formation of small oligomers is interpreted qualitatively in terms of the two-state model proposed by H. W. Huang for the interaction between amphipathic peptides and membranes. However, the rate of oligomer formation in the membrane and their size are highly dependent on the concentrations of β-amyloid (1–40) peptides in aqueous solution, suggesting two different pathways of oligomer formation, which lead to drastically different species in the membrane and a departure from the two-state model as the concentration increases.

Fluorescence microscopic characterization of ionic polymer bead-supported phospholipid bilayer membrane systems

Supported phospholipid membrane structures on cationic organic polymer beads were prepared using mixtures of dioleoylphosphatidylserine (PS) and egg yolk phosphatidylcholine (PC). Confocal fluorescence microscopic observations using a fluorescent membrane probe (N-4-nitrobenzo-2-oxa-1,3-diazole-phosphatidylethanolamine) revealed that the phospholipid molecules in the PS/PC-bead complexes were along the outer surface of the beads, but not inside the beads. The anionic PS on the most outer surface of the PS/PC-bead complexes was responsible for the binding of a positively charged macromolecule, rhodamine isothiocyanate dextran (Mw 70,000) by electrostatic attractive forces. The fluidity of the membranes in the PS/PC-bead complexes was investigated by the fluorescence recovery after a photobleaching technique. The lateral diffusion coefficients (D) for the PS/PC-bead complexes were one-half or less than that for 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine giant unilamellar vesicles without solid supporting materials. Such a constrain of the phospholipid bilayer membrane in the complexes appeared to be due to its immobilization on the cationic polymer bead by electrostatic attractive forces between the PS and ammonium group on the surface of the bead. The D values for the complexes were dependent on the phospholipid composition; the PS(25mol%)/PC(75mol%)-bead complex produced a more fluid membrane than the PS(50mol%)/PC(50mol%)-bead one. Thus, the fluidity of the phospholipid bilayer membranes formed on the cationic polymer beads was significantly affected by the anionic phospholipid fraction used for the preparation of the complexes.

Insertion mechanism of cell-penetrating peptides into supported phospholipid membranes revealed by X-ray and neutron reflection

X-Ray and neutron reflectivity measurements on systems composed of a 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) bilayer and transcription-activating-factor derived peptides (TDPs) have allowed us to determine the mechanism of membrane translocation. By monitoring the structural changes of the bilayers caused by the binding of TDPs while systemically varying temperature and TDP concentration, our results revealed the detailed molecular structures of the stepwise interactions that occurred during the translocation of TDP across the lipid bilayers. While little indication of membrane perturbation was observed at low TDP concentrations, we found that the TDP movement across the membrane induced defect formations in the membrane at higher TDP concentrations.

Surface-grafted zwitterionic polymers as platforms for functional supported phospholipid membranes

Polymer brushes grafted from surfaces using controlled polymerization techniques, most notably surface-initiated atom-transfer radical polymerization (SI-ATRP), provide robust and reproducible platforms with precise control of surface properties. These platforms are especially useful in biologically oriented applications involving the confinement of membrane proteins onto solid supports, including screening of pharmaceuticals and biosensing. Here we investigate a tunable zwitterion-based polymeric interface that can guide the assembly of neutral lipid membranes with high mechanical stability and reproducibility on various synthetic materials. By controlling the polymer architecture using ATRP, we show that phospholipid membranes can be made to self-assemble on thin layers of charge-balanced poly(sulfobetaine methacrylate) from fusion of DOPC vesicles under physiological conditions. The self-assembly kinetics and functionality of the polymer-supported lipid membranes are investigated using various surface sensitive techniques, including surface plasmon resonance, fluorescence microscopy, and atomic force microscopy. The growth of zwitterionic polymer layers with controlled length and grafting density allows for modulation of the adhesion of the lipid bilayers to surfaces, thus offering unique advantages for the design and synthesis of bioactive surfaces.

1,2-Dielaidoylphosphocholine/1,2-dimyristoylphosphoglycerol supported phospholipid bilayer formation in calcium and calcium-free buffer

Phospholipid membranes are useful in the field of biocatalysis because a supported phospholipid membrane can create a biomimetic platform where biocatalytic processes can readily occur. In this work, supported bilayer formation from sonicated phospholipid vesicles containing 1,2-dielaidoyl-sn-glycero-3-phosphocholine and 1,2-dimyristoyl-sn-glycero-3-[phospho-rac-(1-glycerol)] was studied using a quartz crystal microbalance with dissipation monitoring and an atomic force microscope. The molar percentages of DEPC and DMPG were varied to determine the effect of overall lipid composition on supported bilayer formation. This work also explored the effect that calcium ion concentration had on supported bilayer formation. Results show that vesicles with up to 50mol% dimyristoylphosphoglycerol can form a supported bilayer without the presence of calcium ions; however, supported bilayer formation in calcium buffer was inhibited as the anionic (negatively charged) lipid concentration increased. Data suggest that supported phospholipid bilayer formation in the absence of Ca2+ from vesicles containing negatively charged lipids is specific to phosphatidylglycerol.

Supported Phospholipid Membranes Formation at a Gel Electrode and Transport of Divalent Cations across them

Supported Phospholipid Membranes Formation at a Gel Electrode and Transport of Divalent Cations across them

Nanoscale structural and mechanical effects of beta-amyloid (1–42) on polymer cushioned membranes: A combined study by neutron reflectometry and AFM Force Spectroscopy

The interaction of beta-amyloid peptides with lipid membranes is widely studied as trigger agents in Alzheimer's disease. Their mechanism of action at the molecular level is unknown and their interaction with the neural membrane is crucial to elucidate the onset of the disease. In this study we have investigated the interaction of water soluble forms of beta-amyloid Aβ(1–42) with lipid bilayers supported by polymer cushion. A reproducible protocol for the preparation of a supported phospholipid membrane with composition mimicking the neural membrane and in physiological condition (PBS buffer, pH=7.4) was refined by neutron reflectivity. The change in structure and local mechanical properties of the membrane in the presence of Aβ(1–42) was investigated by neutron reflectivity and Atomic Force Microscopy (AFM) Force Spectroscopy. Neutron reflectivity evidenced that Aβ(1–42) interacts strongly with the supported membrane, causing a change in the scattering length density profile of the lipid bilayer, and penetrates into the membrane. Concomitantly, the local mechanical properties of the bilayer are deeply modified by the interaction with the peptide as seen by AFM Force Spectroscopy. These results may be of great importance for the onset of the Alzheimer's disease, since a simultaneous change in the structural and mechanical properties of the lipid matrix could influence all membrane based signal cascades.

Effects of single amino acid substitutions on peptide interaction with lipid membranes and bacteria–variants of GKE21, an internal sequence from human LL-37

Effects of helix destabilization on lipid membrane interaction, liposome rupture, and bacterial killing was investigated for variants of the antimicrobial peptide GKE21 (GKEFKRI VQRIKDFLRNL VPR), an internal sequence of human cathelicidin LL-37, by ellipsometry, circular dichroism, fluorescence spectroscopy, and bacterial radial diffusion assay. GKE21 displayed moderate helix induction in buffer, which increased on interaction with phospholipid membranes. Substituting either of the two valines (V) in GKE21 with either proline (P) or d-valine (dV) resulted in helix destabilization, while peptide isoelectric point, net charge at pH 7.4, and mean hydrophobicity remained unchanged. The decreased tendency for helix formation in GKE21 (V → P, V → dV) resulted in a lower induced (helix-related) amphiphilicity, and correlated to a lower peptide adsorption at supported phospholipid membranes, as well as to decreased peptide-induced liposome leakage, particularly at high electrolyte concentration where conformation-invariant electrostatic interactions are screened. In addition, bacterial killing was reduced for the substituted peptides, indicating that even minor changes in induced peptide amphiphilicity may be of relevance for the bactericidal properties of this type of antimicrobial peptides.

Synthetic and Mycobacterial Trehalose Glycolipids Confer Dehydration Resistance to Supported Phospholipid Monolayers

We have recently demonstrated that the glycolipid trehalose dimycolate (TDM), a major outer membrane component of dehydration resistant Mycobacterium tuberculosis (MTb), can impart significant dehydration resistance to supported phospholipid membranes. We now report studies of other, related glycolipids both natural and synthetic that exhibit behavior similar to TDM, conferring protection from desiccation to membranes of which they are constituents. We examined solid-supported lipid monolayers, characterizing membrane integrity and two-dimensional fluidity before and after de- and re-hydration with fluorescence imaging and fluorescence recovery after photobleaching (FRAP). As with TDM, the degree of protection is dependent on the fraction of synthetic lipid in the monolayer and there is a distinct minimum fraction needed for protection by each glycolipid. We can control the synthetic lipid fluidity and minimum protecting fraction by designing and synthesizing lipids with particular hydrophobic chain lengths, saturation, and branching, thereby illuminating the role of molecular structure in biophysical function. The advent of these synthetic, protective glycolipids opens the door to the creation of lipid bilayers and liposomes since the relevant hydrophobic and hydrophilic domain sizes can be controlled. These new structures allow investigations of the physical origins of well-known mycobacaterial properties beyond dehydration resistance in controlled experimental contexts, such as the inhibition of membrane fusion.

Formation and Dynamics of Supported Phospholipid Membranes on a Periodic Nanotextured Substrate

We have studied and modeled the morphology and dynamics of fluid planar lipid bilayer membranes supported on a textured silicon substrate. The substrate is fabricated to have channels on its surface that are a few hundred nanometers across, with a channel depth of a few hundred nanometers perpendicular to the plane of observation. Using atomic force microscopy and quantitative fluorescence microscopy, we have shown that the bilayer assemblies conform to the underlying nanostructured substrate. As far as dynamics is concerned, when observed over length scales exceeding the dimensions of the nanostructured features, the macroscopic diffusion is anisotropic. However, the macroscopic anisotropy is well simulated using models of diffusion on the nanostructured surface that consider the lipids to diffuse homogeneously and isotropically on the supporting substrate. Consistent with previous observations on less well characterized or less periodic nanostructures, we find that the nanostructured substrate produces an effective anisotropy in macroscopic diffusion of the conformal membrane. More importantly, we demonstrate how quantitative analysis of dynamics probed by larger-scale fluorescence imaging can yield information on nanoscale thin-film morphology.

3TA3-07 脂質・界面活性剤混合系を用いた固体基板表面への脂質二分子膜固定化手法の開発(生体膜・人工膜-構造・物性,第47回日本生物物理学会年会)

3TA3-07 脂質・界面活性剤混合系を用いた固体基板表面への脂質二分子膜固定化手法の開発(生体膜・人工膜-構造・物性,第47回日本生物物理学会年会)

Supported Phospholipid Membrane Interactions with 1-Butyl-3-methylimidazolium Chloride

Quartz crystal microbalance with dissipation measurements were conducted for a 98/2 mole ratio of 1,2-dielaidoylphosphocholine (DEPC) and 1,2-dimyristoylphosphoglycerol (DMPG) on silica, gold, and a self-assembled monolayer of 11-mercapto-1-undecanol (11MU) and 11-mercaptoundecanoic acid (11MUA) at 50 mol % each. This study demonstrates that vesicles composed of DEPC and DMPG at 98 and 2 mol %, respectively, formed a supported bilayer with unruptured vesicles present when adsorbed onto the self-assembled monolayer. Also, the partially formed supported bilayer apparently deadsorbed in the presence of 1-butyl-3-methylimidazolium chloride, suggesting that surface-bilayer interactions are weaker on a hydrophilic modified gold surface composed of 50/50 11MU/11MUA than the surface-bilayer interactions on silica.