Supported Phospholipid Membranes Research Articles

Temperature dependence of the short-range repulsion between hydrated phospholipid membranes: A computer simulation study

The temperature dependence of the short-range water-mediated repulsive pressure between supported phospholipid membranes is calculated at two intermembrane separations using the grand canonical Monte Carlo technique. At both separations, the simulated pressure tends to decrease with temperature, in qualitative agreement with the experimental measurements by Simon and co-workers [Simon et al., Biophys. J. 69, 1473 (1995)]. The decrease in pressure originates, at least in part, from a slight dehydration of the membranes and the associated reduction in the hydration component of the pressure.

GM1 clustering inhibits cholera toxin binding in supported phospholipid membranes.

The present studies explore multivalent ligand-receptor interactions between pentameric cholera toxin B subunits (CTB) and the corresponding membrane ligand, ganglioside GM1. CTB binding was monitored on supported phospholipid bilayers coated on the walls and floors of microfluidic channels. Measurements were made by total internal reflection fluorescence microscopy (TIRFM). Apparent dissociation constants were extracted by fitting the binding data to both the Hill-Waud and Langmuir adsorption isotherm equations. Studies of the effect of ligand density on multivalent CTB-GM1 interactions revealed that binding weakened with increasing GM1 density from 0.02 mol % to 10.0 mol %. Such a result could be explained by the clustering of GM1 on the supported phospholipid membranes, which in turn inhibited the binding of CTB. Atomic force microscopy (AFM) experiments directly verified GM1 clustering within the supported POPC bilayers.

Characterization of Physical Properties of Supported Phospholipid Membranes Using Imaging Ellipsometry at Optical Wavelengths

Subnanometer-scale vertical z-resolution coupled with large lateral area imaging, label-free, noncontact, and in situ advantages make the technique of optical imaging ellipsometry (IE) highly suitable for quantitative characterization of lipid bilayers supported on oxide substrates and submerged in aqueous phases. This article demonstrates the versatility of IE in quantitative characterization of structural and functional properties of supported phospholipid membranes using previously well-characterized examples. These include 1), a single-step determination of bilayer thickness to 0.2 nm accuracy and large-area lateral uniformity using photochemically patterned single 1,2-dimyristoyl- sn-glycero-3-phosphocholine bilayers; 2), hydration-induced spreading kinetics of single-fluid 1-palmitoyl-2-oleoyl- sn-glycero-3-phosphocholine bilayers to illustrate the in situ capability and image acquisition speed; 3), a large-area morphological characterization of phase-separating binary mixtures of 1,2-dilauroyl- sn-glycero-3-phosphocholine and galactosylceramide; and 4), binding of cholera-toxin B subunits to GM 1-incorporating bilayers. Additional insights derived from these ellipsometric measurements are also discussed for each of these applications. Agreement with previous studies confirms that IE provides a simple and convenient tool for a routine, quantitative characterization of these membrane properties. Our results also suggest that IE complements more widely used fluorescence and scanning probe microscopies by combining large-area measurements with high vertical resolution without the use of labeled lipids.

Bio-interface Detection by Surface Plasmon-field Enhanced Fluorescence Spectroscopy (SPFS)

Surface plasmon resonance (SPR) has recently been one of the powerful tools to study surface or interface in the field of biology, since it was applied to biochip array. The advantage of SPR is that the binding or adsorption of a small amount of sample to the interface can be sensitively detected without labeling a fluorescent probe. Surface plasmon-field enhanced fluorescence spectroscopy (SPFS) is based on SPR principle in which SPR field is used as an excited field for fluorescent probe. Therefore, it is available to the interface detection due to the advantages of higher sensitivity and lower background (noise). The author introduces here results of the research projects studied by SPR-SPFS, i.e., affinity of double strands in DNA-DNA hybridization and formation process of substrate supported phospholipid membranes.

Hydrolysis of fluid supported membrane islands by phospholipase A 2: Time-lapse imaging and kinetic analysis

The activity of phospholipase A 2 (PLA 2) which catalyzes the hydrolysis of phospholipids into free fatty acids and lysolipids, depends on the structure and thermodynamic state of the membrane. To further understand how the substrate conformation correlates with enzyme activity, model systems that are based on time-resolved membrane microscopy are needed. We demonstrate a methodology for preparing and investigating the dynamics of fluid supported phospholipid membranes hydrolyzed by snake venom PLA 2. The method uses quantitative analysis of time-lapse fluorescence images recording the evolution of fluid bilayer islands during hydrolysis. In order to minimize interactions with the support surface, we use double bilayer islands situated on top of a complete primary supported membrane prepared by hydration of spincoated lipid films. Our minimal kinetic analysis describes adsorption of enzyme to the membrane in terms of the Langmuir isotherm as well as enzyme kinetics. We use two related models assuming hydrolysis to occur either at the perimeter or at the surface of the membrane island. We find that the adsorption constant is similar for the two cases, while the estimated turnover rate is markedly different. The PLA 2 concentration series is measured in the absence and presence of β-cyclodextrin which forms water soluble complexes with the reaction products. The results demonstrate the versatility of double bilayer islands as a membrane model system and introduces a new method for quantifying the kinetics of lipase activity on membranes by directly monitoring the evolution in substrate morphology.

Computer simulation of short-range repulsion between supported phospholipid membranes

The grand canonical Monte Carlo technique is used to calculate the water-mediated pressure between two supported 1,2-dilauroyl-DL-phosphatidylethanolamine (DLPE) membranes in the short separation range. The intra- and intermolecular interactions in the system are described with a combination of a united-atom AMBER-based force field for DLPE and a TIP4P model for water. The total pressure is analyzed in terms of its hydration component and the component due to the direct interaction between the membranes. The latter is, in addition, partitioned into the electrostatic, dispersion, and steric repulsion contributions to give an idea of their relative significance in the water-mediated intermembrane interaction. It is found that the force field used exaggerates the water affinity of the membranes, resulting in an overestimated hydration level and intermembrane pressure. The simulations of the hydrated membranes with damped water-lipid interaction potentials show that both the hydration and pressure are extremely sensitive to the strength of the water-lipid interactions. Moreover, the damping of the mixed interactions by only 10%-20% changes significantly the relative contribution of the individual pressure components to the intermembrane repulsion.

Evidence for the Regulatory Role of the N-terminal Helix of Secretory Phospholipase A2 from Studies on Native and Chimeric Proteins

The phospholipase A(2) (PLA(2)) enzymes are activated by binding to phospholipid membranes. Although the N-terminal alpha-helix of group I/II PLA(2)s plays an important role in the productive mode membrane binding of the enzymes, its role in the structural aspects of membrane-induced activation of PLA(2)s is not well understood. In order to elucidate membrane-induced conformational changes in the N-terminal helix and in the rest of the PLA(2), we have created semisynthetic human group IB PLA(2) in which the N-terminal decapeptide is joined with the (13)C-labeled fragment, as well as a chimeric protein containing the N-terminal decapeptide from human group IIA PLA(2) joined with a (13)C-labeled fragment of group IB PLA(2). Infrared spectral resolution of the unlabeled and (13)C-labeled segments suggests that the N-terminal helix of membrane-bound IB PLA(2) has a more rigid structure than the other helices. On the other hand, the overall structure of the chimeric PLA(2) is more rigid than that of the IB PLA(2), but the N-terminal helix is more flexible. A combination of homology modeling and polarized infrared spectroscopy provides the structure of membrane-bound chimeric PLA(2), which demonstrates remarkable similarity but also distinct differences compared with that of IB PLA(2). Correlation is delineated between structural and membrane binding properties of PLA(2)s and their N-terminal helices. Altogether, the data provide evidence that the N-terminal helix of group I/II PLA(2)s acts as a regulatory domain that mediates interfacial activation of these enzymes.

Preparation and applications of biofunctionalized, supported lipid bilayers and vesicles

Biofunctional surfaces require advanced design and preparation to match the (bio)recognition ability of biological systems [1]. This requires combined topographic, chemical and visco-elastic surface patterns to match proteins at the nm scale and cells at the micrometer scale. One example of biochemical functionalization, presented here, and which is of both fundamental and application interest, is supported biomimectic (cell)membranes. Specifically we describe preparation and applications of supported phospholipid membranes, which can be made on certain surfaces from unilamellar, 25–200 nm vesicles. On SiO2 at normal pH and with neutral lipids, the vesicles first adsorb intact, and then undergo a phase transformation to a supported bilayer. We have studied the coverage-, vesicle size-, and T-dependence of this process [2], using QCM-D [3], AFM, and SPR. When SiO2 is replaced by TiO2, vesicles adsorb intact. A surface pre-covered with intact vesicles, can be AFM patterned into areas with bilayer, vesicles, and empty surface patches [4]. The results depend critically on AFM tip interaction with vesicle and bilayer, which has been modeled by Monte Carlo simulations [5]. These biomembranes are inert towards protein adsorption [6] and cell attachement [7], which opens up for various applications. Addition of functional molecules, allows sensor functions [8]. Another application is functionalized membranes for surface-specific (stem) cell interactions [9].

Direct computer simulation of water-mediated force between supported phospholipid membranes

The grand canonical Monte Carlo technique is used to calculate the water-mediated force operating between two supported 1,2-dilauroyl-DL-phosphatidylethanolamine (DLPE) membranes in the short separation range. The intra- and intermolecular interactions in the system are described with a combination of an AMBER-based force field for DLPE and a TIP4P model for water. The long range contributions to the electrostatic interaction energy are treated in the dipole-dipole group-based approximation. The total water-mediated force is analyzed in terms of its hydration component and the component due to the direct interaction between the membranes. The latter is, in addition, partitioned into the electrostatic, van der Waals, and steric repulsion contributions to give an idea of their relative significance in the water-mediated interaction of the membranes.

3P238 表面プラズモン分光法(SPR)と表面プラズモン励起蛍光分光法(SPFS)を用いた固体基板上におけるリン脂質二分子膜吸着挙動の解析(生体膜・人工膜 A) 構造・物性))

3P238 表面プラズモン分光法(SPR)と表面プラズモン励起蛍光分光法(SPFS)を用いた固体基板上におけるリン脂質二分子膜吸着挙動の解析(生体膜・人工膜 A) 構造・物性))

Investigating the interaction of the toxin ricin and its B-chain with immobilised glycolipids in supported phospholipid membranes by surface plasmon resonance

The affinity of the plant toxin ricin to glycolipid receptor molecules is important in mediating toxicity to the cell. The interactions of ricin and its B-chain with glycolipids in artificial lipid membranes were monitored using surface plasmon resonance (SPR). Liposomes containing GM1, GM2, GD1b, asialo-GM1, globotriaosylceramide, lactosylceramide and galactosylceramide glycolipids were fused to the sensor surface in order to form lipid films. The affinity of intact ricin and its B-chain differed markedly and was also pH dependent. The binding of the intact toxin was low at neutral pH and increased upon acidification whereas the B-chain had greater and more specific binding at both pH tested. The discrepancy in affinity between the intact toxin and the B-chain may depend on the overall structure of the toxin, giving low availability of the binding sites in the intact toxin. The apparent kinetic constants were determined for GM1, GD1b and asialo-GM1 where the binding between the B-chain and the gangliosides was significant. The association constants were in the order of 0.3×10 6–1.0×10 7 per M. The chain length of the carbohydrate moiety also influenced the binding. A higher affinity was observed with increasing chain length and this was likely due to the availability of the carbohydrate residues. Longer chains are available for binding to the ligand, whereas shorter chains may not sufficiently extend from the lipid layer.

The interaction of trichosanthin with supported phospholipid membranes studied by surface plasmon resonance

Trichosanthin (TCS) is a toxic protein isolated from a Chinese herbal medicine, the root tuber of Trichosanthes kirilowii Maximowicz of the Curcurbitaceae family. It is now used in China to terminate early and mid-trimester pregnancies. The ribosome inactivating property is thought to be account for its toxicity; it can inactivate the eukaryotic ribosome through its RNA N-glycosidase activity. The interactions of TCS with biological membrane is thought to be essential for its physiological effect, for it must get across the membrane before it can enter the cytoplasm and exert its RIP function. In the present work, the interaction of TCS with supported phospholipid monolayers is studied by surface plasmon resonance. The results show that electrostatic forces dominate the interaction between TCS and negatively charged phospholipid containing membranes under acid condition and that both the pH value and the ionic strength can influence its binding. It is proposed that, besides electrostatic forces, hydrophobic interaction may also be involved in the binding process.

Single-Molecule Anisotropy Imaging

A novel method, single-molecule anisotropy imaging, has been employed to simultaneously study lateral and rotational diffusion of fluorescence-labeled lipids on supported phospholipid membranes. In a fluid membrane composed of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine, in which the rotational diffusion time is on the order of the excited-state lifetime of the fluorophore rhodamine, a rotational diffusion constant, D rot = 7 × 10 7 rad 2/s, was determined. The lateral diffusion constant, measured by direct analysis of single-molecule trajectories, was D lat = 3.5 × 10 −8 cm 2/s. As predicted from the free-volume model for diffusion, the results exhibit a significantly enhanced mobility on the nanosecond time scale. For membranes of DPPC lipids in the L β gel phase, the slow rotational mobility permitted the direct observation of the rotation of individual molecules characterized by D rot = 1.2 rad 2/s. The latter data were evaluated by a mean square angular displacement analysis. The technique developed here should prove itself profitable for imaging of conformational motions of individual proteins on the time scale of milliseconds to seconds.

Glucose minisensor based on self-assembled biotinylated phospholipid membrane on a solid support and its physical properties

We have developed a glucose minisensor based on a biotinylated, supported phospholipid membrane (s-BLM). We immobilized glucose oxidase (GOX) by coupling an avidin-GOX complex to a phospholipid bilayer formed from biotinylated crude ox brain extract (COB). The bilayer was supported on the free metal tip of a Teflon-coated stainless steel wire (diameter, 0.33 mm). The determination of glucose was based on detection of enzymatically generated hydrogen peroxide at a potential of +670 mV. We found an almost linear increase of membrane current with increasing glucose concentration up to 10 mM and a saturation effect above 30 mM glucose. A lower voltage for detection of glucose was made possible by modification of the membrane by the electron carrier TCNQ. Several methods have been used to study the physical properties of native and modified s-BLM and conventional BLM. With the electrostriction method we showed that addition of avidin-GOX complex to the electrolyte in which the biotinylated s-BLM was formed resulted in a decrease of membrane capacitance and a decrease of membrane compressibility perpendicular to the bilayer surface. The capacitance relaxation method was used to determine the changes of dielectric relaxation times (reorientation of dipole moments of polar groups of individual lipid molecules or lipid clusters) following addition of avidin-GOX. Native BLM formed from COB extract (2% solution in n-decane: butanol (8:1 v/v) exhibited one relaxation time of (5 ± 1) μs. Additional relaxation components (115 ± 27 μs and 26 ± 1 μs) appeared in BLM modified by biotin. Addition of the avidin-GOX complex to the biotinylated BLM resulted in the appearance of a slow component 505 ± 16 m ̊ s . These results clearly document the interaction of the strongly immobilized enzyme with the bilayer.

Supported phospholipid membranes: comparison among different deposition methods for a phospholipid monolayer

Supported lipid membranes consisting of self-assembled alkanethiol and lipid monolayers on gold substrates could be produced by three different deposition methods: the Langmuir-Blodgett (L-B) technique, the painted method, and the paint-freeze method. By using cyclic voltammetry, chronoamperometry/chronocoulometry and a.c. impedance measurements, we demonstrated that lipid membranes prepared by these three deposition methods had obvious differences in specific capacitance, resistance and thickness. The specific capacitance of lipid membranes prepared by depositing an L-B monolayer on the alkanethiol alkylated surfaces was 0.53 μF cm −2, 0.44 μFcm −2 by the painted method and 0.68 μFcm −2 by the paint-freeze method. The specific conductivity of lipid membranes prepared by the L-B method was over three times lower than that of the painted lipid membranes, while that of the paint-freeze method was the lowest. The difference among the three types of lipid membranes was ascribed to the influence of the organic solvent in lipid films and the changes in density of the films. The lipid membranes prepared by the usual painted method contained a trace amount of the organic solvent. The organic solvent existing in the hydrocarbon core of the membrane reduced the density of the membrane and increased the thickness of the membrane. The membrane prepared by depositing an L-B monolayer containing no solvent had higher density and the lowest fluidity, and the thickness of the membrane was smaller. The lipid membrane prepared by the paint-freeze method changed its structure sharply at the lower temperature. The organic solvent was frozen out of the membrane while the density of the membrane increased greatly. All these caused the membrane to exist in a “tilted” state and the thickness of this membrane was the smallest. The lipid membrane produced by the paint-freeze method was a membrane not containing organic solvent. This method was easier in manipulation and had better reproducibility than that of the usual painting method and the method of forming free-standing lipid film. The solvent-free membrane had a long lifetime and a higher mechanical stability. This model membrane would be useful in many areas of scientific research.

Experimental observation of chemically modulated admittance of supported phospholipid membranes

Experimental observation of chemically modulated admittance of supported phospholipid membranes