1-2-oleoylphosphatidylcholine and 1-palmitoyl-2-oleoylglycero-3-phosphoglycerol

An important advance in biosensor research is the extension and application of laboratory-developed methodologies toward clinical diagnostics, though the propensity toward nonspecific binding of materials in clinically relevant matrices, such as human blood serum and plasma, frequently leads to compromised assays. Several surface chemistries have been developed to minimize nonspecific interactions of proteins and other biological components found within blood and serum samples, though these often exhibit substantially variable outcomes. Herein we report a surface chemistry consisting of a charged-matched supported lipid membrane that has been tailored to form over a gold surface functionalized with protein A. Fine tuning of the interfacial charge of this membrane, along with rational selection of a backfilling self-assembled monolayer, allows for high surface coverage with retention of orientation-controlled capture antibody attachment. We demonstrate using surface-plasmon resonance (SPR) that this highly charged lipid membrane is antifouling, allowing for complete removal of nonspecific human serum and plasma components using only a mild buffer rinse, which we attribute to unique steric interactions with the underlying surface. Furthermore, this surface chemistry is successfully applied for specific detection of IgG and cholera toxin in undiluted human biofluids with negligible sacrifice of SPR signal compared to buffered analysis. This novel lipid membrane interface over protein A may open new avenues for direct biosensing of disease markers within clinical samples.

Topics: Animals; Antibodies, Immobilized; Cholera Toxin; Gold; Humans; Immunoassay; Immunoglobulin Fab Fragments; Immunoglobulin G; Membranes, Artificial; Mice; Phosphatidylcholines; Phosphatidylglycerols; Proof of Concept Study; Staphylococcal Protein A

Lipids and proteins are organized in cellular membranes in clusters, often called 'lipid rafts'. Although raft-constituent ordered lipid domains are thought to be energetically unfavourable for membrane fusion, rafts have long been implicated in many biological fusion processes. For the case of HIV gp41-mediated membrane fusion, this apparent contradiction can be resolved by recognizing that the interfaces between ordered and disordered lipid domains are the predominant sites of fusion. Here we show that line tension at lipid domain boundaries contributes significant energy to drive gp41-fusion peptide-mediated fusion. This energy, which depends on the hydrophobic mismatch between ordered and disordered lipid domains, may contribute tens of kBT to fusion, that is, it is comparable to the energy required to form a lipid stalk intermediate. Line-active compounds such as vitamin E lower line tension in inhomogeneous membranes, thereby inhibit membrane fusion, and thus may be useful natural viral entry inhibitors.

Topics: Cholesterol; HIV Envelope Protein gp41; HIV-1; Humans; Lipid Bilayers; Membrane Fusion; Membrane Microdomains; Peptides; Phosphatidylcholines; Phosphatidylethanolamines; Phosphatidylglycerols; Phosphatidylserines; Thermodynamics; Virus Internalization; Vitamin E

We present a new atom density profile (ADP) model and a statistical approach for extracting structural characteristics of lipid bilayers from X-ray and neutron scattering data. Models for five lipids with varying head and tail chemical composition in the fluid phase, 1,2-dioleoyl-sn-glycero-3-phosphatidylcholine (DOPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylcholine (POPC), 1,2-dipalmitoyl-sn-glycero-3-phosphatidylcholine (DPPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylserine (POPS), and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylglycerol (POPG), are optimized using a simplex based method to simultaneously reproduce both neutron and X-ray scattering data. Structural properties are determined using statistical analysis of multiple optimal model structures. The method and models presented make minimal assumptions regarding the atomic configuration, while taking into account the underlying physical properties of the system. The more general model and statistical approach yield data with well defined uncertainties, indicating the precision in determining density profiles, atomic locations, and bilayer structural characteristics. Resulting bilayer structures include regions exhibiting large conformational variation. Due to the increased detail in the model, the results demonstrate the possibility of a distinct hydration layer within the interfacial (backbone) region.

Topics: Lipid Bilayers; Models, Chemical; Neutron Diffraction; Phosphatidylcholines; Phosphatidylglycerols; Phosphatidylserines; Quantum Theory; Scattering, Radiation; X-Ray Diffraction

Planar lipid bilayers suspended in apertures provide a controlled environment for ion channel studies. However, short lifetimes and poor mechanical stability of suspended bilayers limit the experimental throughput of bilayer electrophysiology experiments. Although bilayers are more stable in smaller apertures, ion channel incorporation through vesicle fusion with the suspended bilayer becomes increasingly difficult. In an alternative bilayer stabilization approach, we have developed shaped apertures in SU8 photoresist that have tapered sidewalls and a minimum diameter between 60 and 100 μm. Bilayers formed at the thin tip of these shaped apertures, either with the painting or the folding method, display drastically increased lifetimes, typically >20 h, and mechanical stability, being able to withstand extensive perturbation of the buffer solution. Single-channel electrical recordings of the peptide alamethicin and of the proteoliposome-delivered potassium channel KcsA demonstrate channel conductance with low noise, made possible by the small capacitance of the 50 μm thick SU8 septum, which is only thinned around the aperture, and unimpeded proteoliposome fusion, enabled by the large aperture diameter. We anticipate that these shaped apertures with micrometer edge thickness can substantially enhance the throughput of channel characterization by bilayer lipid membrane electrophysiology, especially in combination with automated parallel bilayer platforms.

Topics: Alamethicin; Bacterial Proteins; Electric Capacitance; Epoxy Compounds; Fluorescence; Light; Lipid Bilayers; Liposomes; Membrane Fusion; Microscopy, Electron, Scanning; Phosphatidylcholines; Phosphatidylglycerols; Potassium Channels

Collapse of homogeneous lipid monolayers is known to proceed via wrinkling/buckling, followed by folding into bilayers in water. For heterogeneous monolayers with phase coexistence, the mechanism of collapse remains unclear. Here, we investigated collapse of lipid monolayers with coexisting liquid-liquid and liquid-solid domains using molecular dynamics simulations. The MARTINI coarse-grained model was employed to simulate monolayers of ∼80 nm in lateral dimension for 10-25 μs. The monolayer minimum surface tension decreased in the presence of solid domains, especially if they percolated. Liquid-ordered domains facilitated monolayer collapse due to the spontaneous curvature induced at a high cholesterol concentration. Upon collapse, bilayer folds formed in the liquid (disordered) phase; curved domains shifted the nucleation sites toward the phase boundary. The liquid (disordered) phase was preferentially transferred into bilayers, in agreement with the squeeze-out hypothesis. As a result, the composition and phase distribution were altered in the monolayer in equilibrium with bilayers compared to a flat monolayer at the same surface tension. The composition and phase behavior of the bilayers depended on the degree of monolayer compression. The monolayer-bilayer connection region was enriched in unsaturated lipids. Percolation of solid domains slowed down monolayer collapse by several orders of magnitude. These results are important for understanding the mechanism of two-to-three-dimensional transformations in heterogeneous thin films and the role of lateral organization in biological membranes. The study is directly relevant for the function of lung surfactant, and can explain the role of nanodomains in its surface activity and inhibition by an increased cholesterol concentration.

Topics: 1,2-Dipalmitoylphosphatidylcholine; Cholesterol; Lipid Bilayers; Lipids; Molecular Dynamics Simulation; Phosphatidylcholines; Phosphatidylglycerols; Surface Tension

The penetration properties of drug-like molecules on human cell membranes are crucial for understanding the metabolism of xenobiotics and overall drug distribution in the human body. Here, we analyze partitioning of substrates of cytochrome P450s (caffeine, chlorzoxazone, coumarin, ibuprofen, and debrisoquine) and their metabolites (paraxanthine, 6-hydroxychlorzoxazone, 7-hydroxycoumarin, 3-hydroxyibuprofen, and 4-hydroxydebrisoquine) on two model membranes: dioleoylphosphatidylcholine (DOPC) and palmitoyloleoylphophatidylglycerol (POPG). We calculated the free energy profiles of these molecules and the distribution coefficients on the model membranes. The drugs were usually located deeper in the membrane than the corresponding metabolites and also had a higher affinity to the membranes. Moreover, the behavior of the molecules on the membranes differed, as they seemed to have a higher affinity to the DOPC membrane than to POPG, implying they have different modes of action in human (mostly PC) and bacterial (mostly PG) cells. As the xenobiotics need to pass through lipid membranes on their way through the body and the effect of some drugs might depend on their accumulation on membranes, we believe that detailed information of penetration phenomenon is important for understanding the overall metabolism of xenobiotics.

Topics: Caffeine; Cell Membrane; Chlorzoxazone; Coumarins; Cytochrome P-450 Enzyme System; Debrisoquin; Humans; Ibuprofen; Lipid Bilayers; Molecular Dynamics Simulation; Pharmaceutical Preparations; Phosphatidylcholines; Phosphatidylglycerols; Thermodynamics; Xenobiotics

Pulmonary surfactant protein B (SP-B) facilitates the rapid transfer of phospholipids from bilayer stores into air-liquid interfacial films along the breathing cycle, and contributes to the formation of a surface-associated multilayer reservoir of surfactant to optimize the stability of the respiratory interface. To obtain more insights into the mechanisms underlying this transfer and multilayer formation, we established a simple model system that captures different features of SP-B action. We monitored the formation of supported planar bilayers from the collapse of intact phospholipid vesicles on a silica surface using a technique called quartz crystal microbalance with dissipation, which provides information on changes in membrane thickness and viscosity. At physiologically relevant concentrations, SP-B dramatically alters vesicle collapse. This manifests itself as a reduced buildup of intact vesicles on the surface before collapse, and allows the stepwise buildup of multilayered deposits. Accumulation of lipids in these multilayer deposits requires the presence of SP-B in both the receptor and the arriving membranes, surrounded by a comparable phospholipid charge. Thus, the quartz crystal microbalance with dissipation system provides a useful, simplified way to mimic the effect of surfactant protein on vesicle dynamics and permits a detailed characterization of the parameters governing reorganization of surfactant layers.

Topics: 1,2-Dipalmitoylphosphatidylcholine; Animals; Cytological Techniques; Lipid Bilayers; Phosphatidylcholines; Phosphatidylglycerols; Phospholipids; Pulmonary Surfactant-Associated Protein B; Silicon Dioxide; Swine; Unilamellar Liposomes

Pulmonary surfactant forms a monolayer of lipids and proteins at the alveolar air/liquid interface. Although cholesterol is a natural component of surfactant, its function in surface dynamics is unclear. To further elucidate the role of cholesterol in surfactant, we used a captive bubble surfactometer (CBS) to measure surface activity of spread films containing dipalmitoylphosphatidylcholine/1-palmitoyl-2-oleoylphosphatidylcholine/1-palmitoyl-2-oleoylphosphatidylglycerol (DPPC/POPC/POPG, 50/30/20 molar percentages), surfactant protein B (SP-B, 0.75 mol %), and/or surfactant protein C (SP-C, 3 mol %) with up to 20 mol % cholesterol. A cholesterol concentration of 10 mol % was optimal for reaching and maintaining low surface tensions in SP-B-containing films but led to an increase in maximum surface tension in films containing SP-C. No effect of cholesterol on surface activity was found in films containing both SP-B and SP-C. Atomic force microscopy (AFM) was used, for the first time, to visualize the effect of cholesterol on topography of SP-B- and/or SP-C-containing films compressed to a surface tension of 22 mN/m. The protrusions found in the presence of cholesterol were homogeneously dispersed over the film, whereas in the absence of cholesterol the protrusions tended to be more clustered into network structures. A more homogeneous dispersion of surfactant lipid components may facilitate lipid insertion into the surfactant monolayer. Our data provide additional evidence that natural surfactant, containing SP-B and SP-C, is superior to surfactants lacking one of the components, and furthermore, this raises the possibility that the cholesterol found in surfactant of warm-blooded mammals does not have a function in surface activity.

Topics: Adsorption; Animals; Cattle; Cholesterol; Drug Combinations; Lipid Bilayers; Microscopy, Atomic Force; Phosphatidylcholines; Phosphatidylglycerols; Pulmonary Surfactant-Associated Protein B; Pulmonary Surfactant-Associated Protein C; Surface Properties; Surface Tension; Swine

We measured directly the binding of Lys3, Lys5, and Lys7 to vesicles containing acidic phospholipids. When the vesicles contain 33% acidic lipids and the aqueous solution contains 100 mM monovalent salt, the standard Gibbs free energy for the binding of these peptides is 3, 5, and 7 kcal/mol, respectively. The binding energies decrease as the mol% of acidic lipids in the membrane decreases and/or as the salt concentration increases. Several lines of evidence suggest that these hydrophilic peptides do not penetrate the polar headgroup region of the membrane and that the binding is mainly due to electrostatic interactions. To calculate the binding energies from classical electrostatics, we applied the nonlinear Poisson-Boltzmann equation to atomic models of the phospholipid bilayers and the basic peptides in aqueous solution. The electrostatic free energy of interaction, which arises from both a long-range coulombic attraction between the positively charged peptide and the negatively charged lipid bilayer, and a short-range Born or image charge repulsion, is a minimum when approximately 2.5 A (i.e., one layer of water) exists between the van der Waals surfaces of the peptide and the lipid bilayer. The calculated molar association constants, K, agree well with the measured values: K is typically about 10-fold smaller than the experimental value (i.e., a difference of about 1.5 kcal/mol in the free energy of binding). The predicted dependence of K (or the binding free energies) on the ionic strength of the solution, the mol% of acidic lipids in the membrane, and the number of basic residues in the peptide agree very well with the experimental measurements. These calculations are relevant to the membrane binding of a number of important proteins that contain clusters of basic residues.

Topics: Amino Acid Sequence; Calorimetry; Electrochemistry; Lipid Bilayers; Lysine; Models, Molecular; Models, Theoretical; Molecular Conformation; Peptides; Phosphatidylcholines; Phosphatidylglycerols; Phospholipids; Protein Binding; Thermodynamics