Diphytanoyl Phosphatidylcholine Research Articles

Enzyme Activity to Augment the Characterization of Tethered Bilayer Membranes

The rate of Ca2+ -triggered phospholipase A2 (PLA2) degradation of tethered bilayer membranes (tBLMs), composed of a synthetic lipid, beta-mercaptoethanol, and palmitoyloleoylphosphatidylcholine (POPC), is approximately 80 times greater than for those prepared with diphytanoylphosphatidylcholine (DPhyPC). Electrochemical impedance spectroscopy (EIS) and neutron reflectivity (NR) data indicate complete, water-free tBLMs that exhibit near ideal capacitive behavior and the presence of a water reservoir in the bilayer subspace proximal to the substrate (Au) surface for both tBLMs. Together these data indicate that the POPC and the DPhyPC tBLMs are structurally similar along the surface normal but markedly different at the outer leaflet/solution interface and that PLA2 is a sensitive probe of short length scale structural differences not revealed by EIS and NR.

On measurements of the higher harmonics of transmembrane current

Measurements of higher harmonics of transmembrane current in bilayer lipid membranes from diphytanoyl phosphatidylcholine (DPhPC) in n-decane and n-tetradecane, caused by alternating voltage applied to the membrane, have been conducted. A universal relation between the amplitudes of harmonics was suggested and experimentally checked. This allowed one to calculate the coefficients of expansion of membrane capacity in series with even powers of membrane voltage and to compare the inhomogeneity of membranes from diphytanoyl phosphatidylcholine in n-decane and n-tetradecane with respect to thickness.

Tethered Bilayer Lipid Membranes Based on Monolayers of Thiolipids Mixed with a Complementary Dilution Molecule. 1. Incorporation of Channel Peptides

Tethered bilayer lipid membranes (tBLMs) are described based on the self-assembly of a monolayer on template stripped gold of an archea analogue thiolipid, 2,3-di-o-phytanyl-sn-glycerol-1-tetraethylene glycol-d,l-alpha-lipoic acid ester lipid (DPTL), and a newly designed dilution molecule, tetraethylene glycol-d,l-alpha-lipoic acid ester (TEGL). The tBLM is completed by fusion of liposomes made from a mixture of diphytanoylphosphatidyl choline (DPhyPC), cholesterol, and 1,2-diphytanoyl-sn-glycero-3-phosphate (DPhyPG) in a molar ratio of 6:3:1. Melittin and gramicidin are incorporated into these tBLMs as shown by surface plasmon resonance (SPR) and electrochemical impedance spectroscopy (EIS) studies. Ionic conductivity at 0 V vs Ag|AgCl, 3 M KCl, measured by EIS measurements are comparable to the results obtained by other research groups. Admittance plots as a function of potential are discussed on a qualitative basis in terms of the kinetics of ion transport through the channels.

Whole-cell Voltage Clamp Measurements of Anthrax Toxin Pore Current

Protective antigen (PA) of anthrax toxin binds cellular receptors and forms pores in target cell membranes, through which catalytic lethal factor (LF) and edema factor (EF) are believed to translocate to the cytoplasm. Using patch clamp electrophysiological techniques, we assayed pore formation by PA in real time on the surface of cultured cells. The membranes of CHO-K1 cells treated with activated PA had little to no electrical conductivity at neutral pH (7.3) but exhibited robust mixed ionic currents in response to voltage stimuli at pH 5.3. Pore formation depended on specific cellular receptors and exhibited voltage-dependent inactivation at large potentials (>60 mV). The pH requirement for pore formation was receptor-specific as membrane insertion occurs at significantly different pH values when measured in cells specifically expressing tumor endothelial marker 8 (TEM8) or capillary morphogenesis protein 2 (CMG2), the two known cellular receptors for anthrax toxin. Pores were inhibited by an N-terminal fragment of LF and by micromolar concentrations of tetrabutylammonium ions. These studies demonstrated basic biophysical properties of PA pores in cell membranes and served as a foundation for the study of LF and EF translocation in vivo.

Bipolar Tetraether Lipids: Chain Flexibility and Membrane Polarity Gradients from Spin-Label Electron Spin Resonance

Membranes of thermophilic Archaea are composed of unique tetraether lipids in which C40, saturated, methyl-branched biphytanyl chains are linked at both ends to polar groups. In this paper, membranes composed of bipolar lipids P2 extracted from the acidothermophile archaeon Sulfolobus solfataricus are studied. The biophysical basis for the membrane formation and thermal stability is investigated by using electron spin resonance (ESR) of spin-labeled lipids. Spectral anisotropy and isotropic hyperfine couplings are used to determine the chain flexibility and polarity gradients, respectively. For comparison, similar measurements have been carried out on aqueous dispersions of diacyl reference lipid dipalmitoyl phosphatidylcholine and also of diphytanoyl phosphatidylcholine, which has methyl-branched chains. At a given temperature, the bolaform lipid chains are more ordered and less flexible than in normal bilayer membranes. Only at elevated temperatures (80 degrees C) does the flexibility of the chain environment in tetraether lipid assemblies approach that of fluid bilayer membranes. The height of the hydrophobic barrier formed by a monolayer of archaebacterial lipids is similar to that in conventional fluid bilayer membranes, and the permeability barrier width is comparable to that formed by a bilayer of C16 lipid chains. At a mole ratio of 1:2, the tetraether P2 lipids mix well with dipalmitoyl phosphatidylcholine lipids and stabilize conventional bilayer membranes. The biological as well as the biotechnological relevance of the results is discussed.

Alamethicin influence on the membrane bending elasticity

We investigate the bending elasticity of lipid membranes with the increase of the alamethicin concentrations in the membrane via analysis of the thermally induced shape fluctuations of quasi-spherical giant vesicles. Our experimental results prove the strong influence of alamethicin molecules on the bending elasticity of diphytanoyl phosphatidylcholine and dilauroyl phosphatidylcholine membranes even in the range of very low peptide concentrations (less than 10(-3) mol/mol in the membrane). The results presented in this work, testify to the peripheral orientation of alamethicin molecules at low peptide concentrations in the membrane for both types of lipid bilayers. An upper limit of the concentration of the peptide in the membrane is determined below which the system behaves as an ideal two-dimensional solution and the peptide molecules have a planar orientation in the membrane.

Kinetics of Folding ofEscherichia coliOmpA from Narrow to Large Pore Conformation in a Planar Bilayer

The outer membrane protein of Escherichia coli, OmpA, is currently alleged to adopt two native conformations: a major two-domain conformer in which 171 N-terminal residues form a narrow eight beta-barrel pore and 154 C-terminal residues are in the periplasm and a minor one-domain conformer in which all 325 residues create a large pore. However, recent studies in planar bilayers indicate the conformation of OmpA is temperature-sensitive and that increasing temperature converts narrow pores to large pores. Here we examine the reversibility and kinetics of this transition for single OmpA molecules in planar bilayers of diphytanoylphosphatidylcholine (DPhPC). We find that the transition is irreversible. When temperatures are decreased, large pores close down, and when temperatures are stabilized they reopen in the large pore conformation, with gradually increasing open time. Large pores are converted to narrow pores only by denaturing agents. The transition from narrow to large pores requires temperatures >or= 26 degrees C and is a biphasic process with rates that rise steeply with temperature. The first phase, a flickering stepwise transition from a low-conductance to a high-conductance state requires approximately 7 h at 26 degrees C but only approximately 13 min at 42 degrees C, signifying an activation energy of 139 +/- 12 kJ/mol. This is followed by a gradual increase in conductance and open probability, interpreted as optimization of the large pore structure. The results indicate that the two-domain structure is a partially folded intermediate that is kinetically stable at lower temperatures and that mature fully folded OmpA is a large pore.

Comparative molecular dynamics study of ether- and ester-linked phospholipid bilayers

The lipid membranes found in archaea have high bilayer stability and low permeability. The molecular structure of their constituent lipids is characterized by ether-linked, branched hydrophobic chains, whereas the conventional lipids obtained from eukaryotic or eubacterial sources have ester linked straight chains. In order to elucidate the influence of the ether linkage, instead of an ester one, on the physical properties of the lipid bilayers, we have carried out comparative 10 ns molecular dynamics simulations of diphytanyl phosphatidylcholine (ether-DPhPC) and diphytanoyl phosphatidylcholine (ester-DPhPC) bilayers in water, respectively. We analyze bilayer structures, hydration of the lipids, membrane dipole potentials, and free energy profiles of water and oxygen across the bilayers. We observe that the membrane dipole potential for the ether-DPhPC bilayer, which arises mainly from the ether linkage, is about half of that of the ester-DPhPC. The calculated free energy barrier for a water molecule in the ether-DPhPC bilayer system is slightly higher than that in the ester-DPhPC counterpart, which is in accord with experimental data.

Diffraction techniques for nonlamellar phases of phospholipids.

A neutron diffraction method applicable to nonlamellar phases of substrate-supported lipid membranes is described and validated. When prepared on a flat substrate, the resulting nonlamellar phases have layered symmetry which provides some advantages over powder diffraction for detailed structure determination. This approach recently led to the detection of a rhombohedral phase and a distorted hexagonal phase of lipids. Here the determination of intensity and phase information for such phases is demonstrated by application to the hexagonal phase of diphytanoyl phosphatidylcholine (DPhPC). The hexagonal symmetry is used to verify the data reduction procedure for the intensities of the diffraction peaks. Diffraction intensities measured while varying the D2O/H2O ratio in the relative humidity was used to solve the phase problem. The neutron scattering length density distribution of the hexagonal phase was constructed and analyzed to elucidate the packing of the lipid molecules. The structure of DPhPC in the hexagonal phase is of interest in connection with its stalk structure in the rhombohedral phase. We also found that the incorporation of tetradecane into the DPhPC hexagonal phase is limited, similar to the case for dioleoyl phosphatidylethanolamine.

Molecular Dynamics Study on the Effects of Chain Branching on the Physical Properties of Lipid Bilayers: 2. Permeability

We studied the effects of chain branching on the water and nonionic (neutral) solute permeability of lipid bilayers in a molecular dynamics simulation comparing two bilayers: dipalmitoylphosphatidylcholine (DPPC) and diphytanoylphosphatidylcholine (DPhPC). The calculated free energy profiles of several neutral solute and water molecules across the lipid membranes showed that chain branching caused no significant changes in the solubility of these molecules inside the membrane core. However, an analysis of the cavity distribution in each of these bilayer systems demonstrated that the branch-chained DPhPC bilayer had, compared with the straight-chained DPPC bilayer, a relatively small and discrete free volume distribution in the hydrophobic part. This suggests that small penetrants have a lower rate of diffusion inside branch-chained lipid bilayers. Actually, water molecules showed lower local diffusion coefficients inside the DPhPC membrane than inside the DPPC membrane. The low penetrant mobility of the former must correlate with the slower dynamics of the branched DPhPC chains. Thus, we conclude that chain branching effects on the permeability are, as far as neutral small penetrants are concerned, attributable mainly to the reduction of chain dynamics. The effects of chain branching on proton permeability are also discussed in the context of the proton-wire hypothesis.

Dynamics of a highly branched lipid bilayer: a molecular dynamics study

A comparison of branch-chained diphytanoylphosphatidylcholine (DPhPC) and straight-chained dipalmitoylphosphatidylcholine (DPPC) for their dynamics in the bilayers is made using 10 ns molecular dynamics simulations in the microcanonical ensemble. It is demonstrated that DPhPC molecules, compared with DPPC, indeed have slower wobbling, rotational, and translational motions by a factor of 2–3, whereas slightly faster headgroup rotation. The revision of the lipid dynamics due to chain branching is similar to that observed by the addition of cholesterol.

Ion channel-like activity of the antimicrobial peptide tritrpticin in planar lipid bilayers

The cationic peptide tritrpticin (VRRFPWWWPFLRR, Trp3) has a broad action spectrum, acting against Gram-positive and Gram-negative bacteria, as well as some fungi, while also displaying hemolytic activity. We have studied the behavior of Trp3 in planar lipid bilayers (or black lipid membrane – BLM) and were able to demonstrate its ion channel-like activity. Channel-like activity was observed in negatively charged azolectin BLM as a sudden appearance of discrete current fluctuations upon application of a constant voltage across the membrane. Trp3 formed large conductance channels (500–2000 pS) both at positive and negative potentials. In azolectin bilayers, the predominant ion-channel activity was characterized by very regular and discrete current steps (corresponding to openings) of uniform amplitude, which exhibited relatively long residence times (of the order of seconds). Occasionally, multiple conductance steps were observed, indicating the simultaneous presence of more than one open pore. In bilayers of zwitterionic diphytanoylphosphatidyl choline (DPhPC) Trp3 also showed ion-channel activity, but in a much less frequent and less prominent way. Studies of ion selectivity indicated that Trp3 forms a cation-selective channel. These results should contribute to the understanding of the molecular interactions and mechanism of action of Trp3 in lipid bilayers and biological membranes.

Membrane Surface Dynamics of DNA-Threaded Nanopores Revealed by Simultaneous Single-Molecule Optical and Ensemble Electrical Recording

We describe a method for simultaneous single-molecule optical and electrical characterization of membrane-based sensors that contain ion-channel nanopores. The technique is used to study the specific and nonspecific interactions of streptavidin-capped DNA polymers with lipid bilayers composed of diphytanoyl phosphatidylcholine and diphytanoyl phosphatidylglycerol. Biotinylated DNA that is bound to fluorescently labeled streptavidin is electrophoretically driven into, or away from, the lumen of alpha hemolysin (alphaHL) ion channels by an external electric field. Confocal microscopy simultaneously captures single-molecule fluorescence dynamics from the membrane interface at different applied potentials. Fluorescence correlation analysis is used to determine the surface number density and diffusion constant of membrane-associated complexes. The dual optical and electrical approach can detect membrane-associated species at a surface coverage below 10(-5) monolayers of streptavidin, a sensitivity that surpasses most other in vitro surface analysis techniques. By comparing the change in transmembrane current to the number of fluorescent molecules leaving the bilayer when the electrical potential is reversed, we demonstrate the general utility of the approach within the context of nanopore-based sensing and discuss a mechanism by which DNA-streptavidin complexes can be nonspecifically retained at the membrane interface.

Tethered Lipid Bilayers on Ultraflat Gold Surfaces

Tethered lipid bilayers (tBLMs) were obtained by the fusion of liposomes from diphytanoylphosphatidylcholine (DPhyPC) with self-assembled monolayers (SAMs) of a newly designed archaea analogue thiolipid, 2,3-di-O-phytanyl-sn-glycerol-1-tetraethylene glycol-d,l-α-lipoic acid ester (DPTL) on template stripped gold (TSG) films from silicon wafer as a template. SAMs, as characterized by reflection absorption infrared spectroscopy (RAIRS), show a mixture of different conformations of the tetraethylene segment in air, which appears to rearrange into the fully extended conformation when the SAM is immersed into an aqueous electrolyte solution, as deduced from thickness measurements by surface plasmon resonance spectroscopy (SPR). The fusion of liposomes was followed by SPR, quartz crystal microbalance (QCM), and fluorescence microscopy. Highly resistive tBLMs were obtained, as demonstrated by electrochemical impedance spectroscopy (EIS) results, which are equivalent to those for the BLM. This large resistivity i...

New Method for Generating Tetraether Lipid Membranes on Porous Supports

The present paper describes a new and reproducible method to generate stable lipid membranes on porous supports. The support was produced by the deposition of a smooth layer of crystalline bacterial cell surface (S-layer) protein lattices on a microfiltration membrane (SUM). The commercially available main phospholipid of Thermoplasma acidophilum (MPL), a membrane-spanning tetraether lipid, but also mixtures of MPL with diphytanoylphosphatidylcholine (DPhPC) at molar ratios of MPL/DPhPC = 1:1 and 5:1 and pure DPhPC were spread at the air/water interface. The monomolecular films were transferred by one (MPL and mixtures) or two (DPhPC) steps on the SUM. In addition, folded membranes were generated with DPhPC and the lipid mixtures. The specific capacitance of the tight SUM-supported membranes increased continuously with increasing MPL to DPhPC ratio from 0.62 μF/cm2 (pure DPhPC) over 0.66 μF/cm2 (equimolar DPhPC/MPL) to 0.76 μF/cm2 for pure MPL membranes. SUM-supported MPL membranes showed a lifetime of 8.3 ± 2.9 h. An additional monomolecular S-layer protein lattice recrystallized on the lipid-faced side increased the lifetime significantly to 21.2 ± 3.1 h. To prove the functionality of the membranes, gramicidin was reconstituted. All folded and SUM-supported membranes allowed reconstitution of gramicidin, and high-resolution measurements on single pores could be performed.

Comparative FTIR-spectroscopic studies of the hydration of diphytanoylphosphatidylcholine and -ethanolamine

Lipids with phytanyl or phytanoyl chains are of basic interest because of their natural occurrence in the membranes of extremophilic archaebacteria and frequent use in biochemical studies and applications as a model for stable bilayers. Since data from physico-chemical studies of phytanoyl lipids in terms of structural and phase properties are rare and partly discrepant, we have studied films of diphytanoylphosphatidylcholine (DPhPC) and its ethanolamine analogue (DPhPE) in dependence on water activity using Fourier-transform infrared spectroscopy. DPhPC imbibes much more water than DPhPE as substantiated by Karl–Fischer-titration and gravimetric data. Marker bands due to the methylene stretching vibrations indicate an extremely high disorder of the acyl chains of both lipids over the whole hydration range. The spectral features of DPhPE are different from those of DPhPC in many respects but similar to those of DPhPE. This similarity encourages us to conclude a predominance of the inverted hexagonal H II phase in DPhPE. Hydration-induced structural changes of DPhPC are indicated by discontinuous wavenumber shifts at water activities of 0.6–0.8, and towards very low water activities for DPhPE, suggesting nonlamellar–lamellar and nonlamellar–nonlamellar phase transitions, respectively, to proceed. Thus, nonlamellar phases may play an important role also in DPhPC.

Hydration and Molecular Motions in Synthetic Phytanyl-Chained Glycolipid Vesicle Membranes

Proton permeation rates across membranes of a synthetic branch-chained glycolipid, 1,3-di- O-phytanyl-2- O-( β- d-maltotriosyl)glycerol (Mal 3(Phyt) 2) as well as a branch-chained phospholipid, diphytanoylphosphatidylcholine (DPhPC) were lower than those of straight-chained lipids such as egg yolk phosphatidylcholine (EPC) by a factor of ∼4 at pH 7.0 and 25°C. To examine whether degrees of water penetration and molecular motions in Mal 3(Phyt) 2 membranes can account for the lower permeability, nanosecond time-resolved fluorescence spectroscopy was applied to various membranes of branch-chained lipids (Mal 3(Phyt) 2, DPhPC, and a tetraether lipid from an extremely thermoacidophilic archaeon Thermoplasma acidophilum), as well as straight-chained lipids (EPC, 1-palmitoyl-2-oleoyl-phosphatidylcholine (POPC), and digalactosyldiacylglycerol (DGDG)) using several fluorescent lipids. Degrees of hydration of glycolipids, Mal 3(Phyt) 2, and DGDG were lower than those of phospholipids, EPC, POPC, and DPhPC at the membrane-water interfaces. DPhPC showed the highest hydration among the lipids examined. Meanwhile, rotational and lateral diffusive motions of the fluorescent phospholipid in branch-chained lipid membranes were more restricted than those in straight-chained ones. The results suggest that the restricted motion of chain segments rather than the lower hydration accounts for the lower proton permeability of branch-chained lipid membranes.

Noncontact Dipole Effects on Channel Permeation. IV. Kinetic Model of 5F-Trp 13 Gramicidin A Currents

Nonlinear least squares fitting was used to assign rate constants for the three-barrier, two-site, double-occupancy, single-filing kinetic model for previously reported current–voltage relations of (5F-Indole)Trp 13 gramicidin A and gramicidin A channels (Busath et al., Biophys. J., 1998, 75:2830–2844). By judicious coupling of parameters, it was possible to reduce the parameter space from 64 parameters to 24, and a reasonable fit consistent with other experimental data was obtained. The main features of the fit were that fluorination increased the rate constant for translocation by a factor of 2.33, consistent with a free energy change in the translocation barrier of −0.50 kcal/mol, and increased first-ion binding affinity by a factor of 1.13, primarily by decreasing the first-ion exit rate constant. The translocation rate constant was 5.62 times slower in diphytanoyl phosphatidylcholine (DPhPC) bilayers than in monoolein (GMO) bilayers (coupled for the four combinations of peptide and salt), suggesting a 44.2-mV difference in the projection of the interfacial dipole into the channel. Thus fluorination caused increased currents in DPhPC bilayers, where a high interfacial dipole potential makes translocation more rate limiting because the translocation barrier was reduced, and decreased currents in GMO bilayers, where ion exit or entry is rate limiting because these barriers were increased.

Purification and characterization of two voltage-dependent anion channel isoforms from plant seeds.

Mitochondria were isolated from imbibed seeds of lentil (Lens culinaris) and Phaseolus vulgaris. We copurified two voltage-dependent anion channel from detergent solubilized mitochondria in a single purification step using hydroxyapatite. The two isoforms from P. vulgaris were separated by chromatofocusing chromatography in 4 M urea without any loss of channel activity. Channel activity of each isoform was characterized upon reconstitution into diphytanoyl phosphatidylcholine planar lipid bilayers. Both isoforms form large conductance channels that are slightly anion selective and display cation selective substates.

Order–disorder transition in bilayers of diphytanoyl phosphatidylcholine

A comparative study on bilayers of diphytanoyl phosphatidylcholine (DPhPC) and bilayers of dimyristoyl phosphatidylcholine (DMPC) was made by X-ray lamellar diffraction as a function of temperature and the degree of hydration. An order–disorder phase transition of DPhPC reveals an interesting contrast to the standard model of DMPC. Electron density profiles allow us to deduce the conformational changes which occur in the headgroup-glycerol region and in the chain region. The important conclusion is that the lipid headgroup may have different conformational energetics in lipids of different chains. We explain why this is important to protein–membrane interactions.