Importance of phospholipid bilayer integrity in the analysis of protein–lipid interactions

The integrity of supported phospholipid bilayer membranes is of crucial importance for the investigation of lipid–protein interactions. Therefore we recorded the formation of supported membranes on SiO₂ and mica by quartz crystal microbalance and controlled the integrity by atomic force microscopy. This study aims to analyze how membrane defects affect protein–lipid interactions. The experiments focused on a lipid mixture of POPC/DOPC/Chol/POPS/PI(4,5)P₂ (37:20:20:20:3) and the binding of the peripheral membrane associated protein annexin A2. We found that formation of a continuous undisturbed bilayer is an indispensable precondition for a reliable determination and quantification of lipid–protein-interactions. If membrane defects were present, protein adsorption causes membrane disruption and lipid detachment on a support thus leading to false determination of binding constants. Our results obtained for PI(4,5)P₂ and cholesterol containing supported membranes yield new knowledge to construct functional surfaces that may cover nanoporous substrates, form free standing membranes or may be used for lab-on-a-chip applications.     

Asymmetric dipping of bacteriophage M13 coat protein with increasing lipid bilayer thickness

Knowledge about the vertical movement of a protein with respect to the lipid bilayer plane is important to understand protein functionality in the biological membrane. In this work, the vertical displacement of bacteriophage M13 major coat protein in a lipid bilayer is used as a model system to study the molecular details of its anchoring mechanism in a homologue series of lipids with the same polar head group but different hydrophobic chain length. The major coat proteins were reconstituted into 14:1PC, 16:1PC, 18:1PC, 20:1PC, and 22:1PC bilayers, and the fluorescence spectra were measured of the intrinsic tryptophan at position 26 and BADAN attached to an introduced cysteine at position 46, located at the opposite ends of the transmembrane helix. The fluorescence maximum of tryptophan shifted for 700 cm^-1 on going from 14:1PC to 22:1PC, the corresponding shift of the fluorescence maximum of BADAN at position 46 was approximately 10 times less (∼ 70 cm^-1). Quenching of fluorescence with the spin label CAT 1 indicates that the tryptophan is becoming progressively inaccessible for the quencher with increasing bilayer thickness, whereas quenching of BADAN attached to the T46C mutant remained approximately unchanged. This supports the idea that the BADAN probe at position 46 remains at the same depth in the bilayer irrespective of its thickness and clearly indicates an asymmetrical nature of the protein dipping in the lipid bilayer. The anchoring strength at the C-terminal domain of the protein (provided by two phenylalanine residues together with four lysine residues) was estimated to be roughly 5 times larger than the anchoring strength of the N-terminal domain.     

Super-resolution Imaging of the Natural Killer Cell Immunological Synapse on a Glass-supported Planar Lipid Bilayer

The glass-supported planar lipid bilayer system has been utilized in a variety of disciplines. One of the most useful applications of this technique has been in the study of immunological synapse formation, due to the ability of the glass-supported planar lipid bilayers to mimic the surface of a target cell while forming a horizontal interface. The recent advances in super-resolution imaging have further allowed scientists to better view the fine details of synapse structure. In this study, one of these advanced techniques, stimulated emission depletion (STED), is utilized to study the structure of natural killer (NK) cell synapses on the supported lipid bilayer. Provided herein is an easy-to-follow protocol detailing: how to prepare raw synthetic phospholipids for use in synthesizing glass-supported bilayers; how to determine how densely protein of a given concentration occupies the bilayer''s attachment sites; how to construct a supported lipid bilayer containing antibodies against NK cell activating receptor CD16; and finally, how to image human NK cells on this bilayer using STED super-resolution microscopy, with a focus on distribution of perforin positive lytic granules and filamentous actin at NK synapses. Thus, combining the glass-supported planar lipid bilayer system with STED technique, we demonstrate the feasibility and application of this combined technique, as well as intracellular structures at NK immunological synapse with super-resolution.     

Biologically active amphotericin B-calix[4]arene conjugates

In order to provide tools for investigations of amphotericin B ion channels, new conjugates bearing a calix[4]arene scaffold covalently linked to four amphotericin B molecules were synthesized. These macromolecules adopt a cone conformation that mimics the structure of a transmembrane pore. The antifungal activity of the conjugates 3 and 4 was superior or similar to that of native amphotericin B, with minimal inhibitory concentration values of 0.10 and 0.25 microM, respectively. Furthermore, the hemotoxicity of the new conjugates was considerably lower (at least 10 times) than the hemotoxicity of monomeric amphotericin B. Finally, the formation of ion channels in the lipid bilayer by the amphotericin B tetramer was monitored by measuring the K+ efflux from various liposomes.     

Gravimetric antigen detection utilizing antibody-modified lipid bilayers

Lipid bilayers containing 5% nitrilotriacetic acid (NTA) lipids supported on SiO₂ have been used as a template for immobilization of oligohistidine-tagged single-chained antibody fragments (scFvs) directed against cholera toxin. It was demonstrated that histidine-tagged scFvs could be equally efficiently coupled to an NTA-Ni²⁺-containing lipid bilayer from a purified sample as from an expression supernatant, thereby providing a coupling method that eliminates time-consuming protein prepurification steps. Irrespective of whether the coupling was made from the unpurified or purified antibody preparation, the template proved to be efficient for antigen (cholera toxin) detection, verified using quartz crystal microbalance with dissipation monitoring. In addition, via a secondary amplification step using lipid vesicles containing G_M1 (the natural membrane receptor for cholera toxin), the detection limit of cholera toxin was less than 750 pM. To further strengthen the coupling of scFvs to the lipid bilayer, scFvs containing two histidine tags, instead of just one tag, were also evaluated. The increased coupling strength provided via the bivalent anchoring significantly reduced scFv displacement in complex solutions containing large amounts of histidine-containing proteins, verified via cholera toxin detection in serum.     

A Molecular Dynamics Investigation of Lipid Bilayer Perturbation by PIP2

Phosphoinositides like phosphatidylinositol 4,5-bisphosphate (PIP₂) are negatively charged lipids that play a pivotal role in membrane trafficking, signal transduction, and protein anchoring. We have designed a force field for the PIP₂ headgroup using quantum mechanical methods and characterized its properties inside a lipid bilayer using molecular dynamics simulations. Macroscopic properties such as area/headgroup, density profiles, and lipid order parameters calculated from these simulations agree well with the experimental values. However, microscopically, the PIP₂ introduces a local perturbation of the lipid bilayer. The average PIP₂ headgroup orientation of 45° relative to the bilayer normal induces a unique, distance-dependent organization of the lipids that surround PIP₂. The headgroups of these lipids preferentially orient closer to the bilayer normal. This perturbation creates a PIP₂ lipid microdomain with the neighboring lipids. We propose that the PIP₂ lipid microdomain enables the PIP₂ to function as a membrane-bound anchoring molecule.     

Reconstitution of purified dipeptidyl peptidase IV. A comparison with aminopeptidase N with respect to morphology and influence of anchoring peptide on function

The pig small intestinal dipeptidyl peptidase IV was asymmetrically integrated into egg phosphatidylcholine and microvillar lipid vesicles prepared by a beta-octylglucoside dialysis method. The enzyme molecules appeared dumbell-shaped ((11.0-11.5) X (5.0-5.5)nm) and were separated from the liposomal membrane by a stain-filled gap of about 2.5 nm, representing the 'junctional segment'. The influence of lipid bilayer and detergents on the kinetic parameters of amphiphilic and hydrophilic forms of aminopeptidase N and dipeptidyl peptidase IV was studied. Since the lipid bilayer and detergents, which interact only with the anchoring root, had no crucial effect on the kinetic parameters of the different forms of the enzymes, it is concluded that the anchoring roots exert little effect on the catalytic domain of the stalked integral membrane proteins.     

Investigation of membrane penetration depth and interactions of the amino-terminal domain of huntingtin: refined analysis by tryptophan fluorescence measurement

The membrane-association properties of the amino-terminal domain of huntingtin are accompanied by subcellular redistribution of the protein in cellular compartments. In this study we used tryptophan substitution of amino-acid residues at different positions of the huntingtin 1–17 domain (Htt17) to precisely determine, for the first time, the depth of penetration of the peptides within the lipid bilayer. Initially, secondary structure preferences and membrane association properties were quantitatively determined for several membrane lipid compositions; they were found to be closely related to those of the natural peptide, indicating that changes in the sequence had little effect on these characteristics of the domain. The tryptophan-substituted peptides became inserted into the membranes’ interfacial region, with average tryptophan positions between 7.5 and 11 Å from the bilayer center, in agreement with in-plane orientation of the peptide. Participation of the very-amino terminus of the peptide in the membrane-association process was demonstrated. The results not only revealed the occurrence of association intermediates when the huntingtin 1–17 anchoring sequence became inserted into the membrane but also suggest the formation of aggregates and/or oligomers during membrane association. When inserted, the F11W site was of crucial importance in lipid anchoring and stabilization of the whole peptide, whereas the terminal residues are located close to the membrane surface. The carboxy-terminal tryptophan (F17W), which also constitutes the site of the polyglutamine extension in the natural domain, was found closest to the aqueous environment, accompanied with the highest aqueous quenching constants. These results were used to propose a refined model of lipid interactions of the huntingtin 1–17 domain.     

Template-free self-assembling fullerene and lipopeptide conjugates of alamethicin form voltage-dependent ion channels of remarkable stability and activity.

N- and C-terminally modified with fullerene or lipopeptide alamethicin molecules were designed for the formation of template-free, self-assembling, voltage-dependent ion conducting channels. The automated solid phase synthesis of the alamethicin-F30 sequence was performed by in situ fluoride activation on 2-chlorotritylchloride-polystyrene resin and the conjugation with fullerenes-C60 and -C70 was carried out in solution. Voltage-dependent bilayer experiments revealed preferred channel sizes for C-terminal alamethicin F30-fullerene-C60 and -C70 conjugates and higher activity compared with native alamethicin, whereas N-terminally linked fullerene balls destabilize pore formation. C-terminal alamethicin F30-fullerene-C70 conjugates show pore states with remarkably long lifetimes of seconds. C-terminal lipopeptide conjugates of alamethicin were prepared by coupling via short peptide spacers with synthetic tripalmitoyl-S-glyceryl-cysteine. which represents the strong membrane anchoring N-terminus of bacterial lipoprotein. Alamethicin-lipopeptide conjugates exhibit high channel forming activities, whereby they self-assemble and adopt preferred pore states with extremely long lifetimes. The novel membrane modifying peptaibol constructs are valuable lead compounds for developments in sensorics related to transmembrane ion conductance.     

Facile functionalization of lipid bilayer vesicles by titania: the use of cerasome-forming lipids for surface and core modification

Two kinds of functionalization of a lipid bilayer vesicle by titania were achieved by utilizing a cerasome-forming lipid, which is the starting material to prepare a cerasome, a morphologically stable lipid bilayer vesicle having an atomic layer of siloxane networks on its surface. One system is the preparation of the titania-coated cerasomes by immobilizing nanaometer-sizes of titania onto the surface siloxane network of cerasomes. The other is the creation of an asymmetric lipid bilayer structure on the surface of the colloidal titania particles. The characteristics of these surface- and core-functionalized vesicles were investigated, and it was found that these conjugates showed photocatalytic activity as evaluated by photolysis experiments of the cationic dye methylene blue.     

Superquenching as a detector for microsphere-based flow cytometric assays.

BACKGROUND: Fluorescent conjugated polymers display high fluorescence quantum yields and enhanced sensitivity to quenching (superquenching) by oppositely charged quenchers through energy or electron transfer. Fluorescent polymers and their quenchers are used in bead-based biosensor applications where the polymers are coated on particles. In this work, we investigate a detection method that utilizes superquenching on microspheres, which can be used for flow cytometric assays. METHODS: Microspheres were coated with the fluorescent cationic polyelectrolyte poly(p-phenylene-ethynylene) (PPE), and its superquenching by 9,10-anthraquinone-2,6-disulfonic acid (AQS) was examined by fluorometric methods in presence and in absence of a barrier to superquenching in the form of an anionic lipid bilayer. RESULTS: Flow cytometry detected superquenching of PPE on microspheres (MS-PPE) by AQS where high levels of reduction in fluorescence were observed. Adding different concentrations of AQS to MS-PPE yielded a Stern-Volmer quenching constant of 0.8x10(6) M-1. While forming an anionic lipid bilayer around the MS-PPE acted as a barrier to superquenching by AQS, disrupting the lipid bilayer allowed superquenching to take place. CONCLUSIONS: The sensitivity of flow cytometry in detecting fluorescence of microspheres and the amplified quenching sensitivity of fluorescent conjugated polymers both offer advantages over other fluorometric methods and conventional quenching detection. This study used superquenching of fluorescent polymers as a new tool in flow cytometry, thus combining the advantages offered by both method and detector. In addition, we employed the formation and the disruption of a supported lipid bilayer in mediating superquenching to offer new biosensing applications.     

Anchoring of a monotopic membrane protein: the binding of prostaglandin H2 synthase-1 to the surface of a phospholipid bilayer

Prostaglandin H₂ synthases (PGHS-1 and -2) are monotopic peripheral membrane proteins that catalyse the synthesis of prostaglandins in the arachidonate cascade. Picot et al. (1994) proposed that the enzyme is anchored to one leaflet of the bilayer by a membrane anchoring domain consisting of a right-handed spiral of amphipathic helices (residues 73–116) forming a planar motif. Two different computational approaches are used to examine the association of the PGHS-1 membrane anchoring domain with a membrane via the proposed mechanism. The electrostatic contribution to the free energy of solvation is obtained by solving numerically the finite-difference Poisson equation for the protein attached to a membrane represented as a planar slab of low dielectric. The nonpolar cavity formation and van der Waals contributions to the solvation free energy are assumed to be proportional to the water accessible surface area. Based on the optimum position determined from the continuum solvent model, two atomic models of the PGHS-1 anchoring domain associated with an explicit dimyristoylphosphatidylcholine (DMPC) bilayer differing by the thickness of the membrane bilayer were constructed. A total of 2 ns molecular dynamics simulation were performed to study the details of lipid- protein interactions at the microscopic level. In the simulations the lipid hydrocarbon chains interacting with the anchoring domain assume various shapes, suggesting that the plasticity of the membrane is significant. The hydrophobic residues in the membrane side of the helices interact with the hydrophobic membrane core, while the positively charged residues interact with the lipid polar headgroups to stabilize the anchoring of the membrane domain to the upper half of the bilayer. The phosphate headgroup of one DMPC molecule disposed at the center of the spiral formed by helices A, B, C and D interacts strongly with Arg120, a residue on helix D that has previously been identified as being important in the activity of PGHS-1. In the full enzyme structure, this position corresponds to the entrance of a long hydrophobic channel leading to the cyclooxygenase active site. These observations provide insights into the association of the arachidonic acid substrate to the cyclooxygenase active site of PGHS-1. Received: 20 December 1999 / Revised version: 26 March 2000 / Accepted: 26 March 2000     

Supported double membranes

Planar model membranes, like supported lipid bilayers and surface-tethered vesicles, have been proven to be useful tools for the investigation of complex biological functions in a significantly less complex membrane environment. In this study, we introduce a supported double membrane system that should be useful for studies that target biological processes in the proximity of two lipid bilayers such as the periplasm of bacteria and mitochondria or the small cleft between pre- and postsynaptic neuronal membranes. Large unilamellar vesicles (LUV) were tethered to a preformed supported bilayer by a biotin–streptavidin tether. We show from single particle tracking (SPT) experiments that these vesicle are mobile above the plane of the supported membrane. At higher concentrations, the tethered vesicles fuse to form a second continuous bilayer on top of the supported bilayer. The distance between the two bilayers was determined by fluorescence interference contrast (FLIC) microscopy to be between 16 and 24 nm. The lateral diffusion of labeled lipids in the second bilayer was very similar to that in supported membranes. SPT experiments with reconstituted syntaxin-1A show that the mobility of transmembrane proteins was not improved when compared with solid supported membranes.     

Lateral diffusion and fluorescence microscope studies on a monoclonal antibody specifically bound to supported phospholipid bilayers

Supported phospholipid bilayers prepared by Langmuir-Blodgett techniques were introduced recently as a new model membrane system [Tamm, L.K., & McConnell, H.M. (1985) Biophys. J. 47, 105-113]. Here, supported bilayers are applied to study the lateral diffusion and lateral distribution of membrane-bound monoclonal antibodies. A monoclonal anti-trinitrophenol antibody was found to bind strongly and with high specificity to supported phospholipid bilayers containing the lipid hapten (trinitrophenyl)phosphatidylethanolamine at various mole fractions. The lateral distribution of the membrane-bound antibodies was studied by epifluorescence microscopy. The bound antibodies aggregated into patches on a host lipid bilayer of dimyristoylphosphatidylcholine below the lipid chain melting phase transition and redistributed uniformly on fluid-phase supported bilayers. Lateral diffusion coefficients and mobile fractions of fluorescent phospholipid analogues and fluorescein-labeled antibodies were measured by fluorescence recovery after pattern photobleaching. The lateral diffusion coefficients of the membrane-bound antibodies resembled those of the phospholipids but were reduced by a factor of 2 in the fluid phase. The lipid chain melting phase transition was also reflected in the lateral diffusion coefficient of the bound antibody but occurred at a temperature about 3 deg higher than the phase transition in supported bilayers of pure phospholipids. The antibody lateral diffusion coefficients decreased in titration experiments monotonically with increasing antibody surface concentrations by a factor of 2-3. Correspondingly, a relatively small decrease of the antibody lateral diffusion coefficient was observed with increasing mole fractions of lipid haptens in the supported bilayer.     

The Nanomechanical Properties of Lipid Membranes are Significantly Influenced by the Presence of Ethanol

Ethanol has a profound impact on biological systems and is moreover used in various medical and nonmedical applications. Its interaction with the lipid part of biological membranes has been the subject of intensive studies, but surprisingly, to our knowledge, no study has examined the influence of ethanol on lipid bilayer nanomechanics. We performed atomic force microscopy-based measurements to assess the influence of ethanol on the nanomechanical properties of fluid supported lipid bilayers. Ethanol significantly reduces membrane stability, bilayer thickness, Young’s modulus, area stretch modulus, and bending stiffness. Altogether, our data suggest that ethanol addition to supported lipid bilayers supports both the hydrophobic and the hydrophilic permeation pathways by a decrease of bilayer thickness and reduced stability, respectively.     

Anchoring mechanisms of membrane-associated M13 major coat protein

Bacteriophage M13 major coat protein is extensively used as a biophysical, biochemical, and molecular biology reference system for studying membrane proteins. The protein has several elements that control its position and orientation in a lipid bilayer. The N-terminus is dominated by the presence of negatively charged amino acid residues (Glu2, Asp4, and Asp5), which will always try to extend into the aqueous phase and therefore act as a hydrophilic anchor. The amphipathic and the hydrophobic transmembrane part contain the most important hydrophobic anchoring elements. In addition there are specific aromatic and charged amino acid residues in these domains (Phe 11, Tyr21, Tyr24, Trp26, Phe42, Phe45, Lys40, Lys43, and Lys44) that fine-tune the association of the protein to the lipid bilayer. The interfacial Tyr residues are important recognition elements for precise protein positioning, a function that cannot be performed optimally by residues with an aliphatic character. The Trp26 anchor is not very strong: depending on the context, the tryptophan residue may move in or out of the membrane. On the other hand, Lys residues and Phe residues at the C-terminus of the protein act in a unique concerted action to strongly anchor the protein in the lipid bilayer.     

A CNBR peptide located in the middle region of diphtheria toxin fragment B induces conductance change in lipid bilayers: Possible role of an amphipathic helical segment

Conductance measurements on planar lipid bilayers demonstrate that CB1, a CNBr peptide of diphtheria toxin fragment B located in its middle region, possesses the unique property to destabilize the lipid bilayer organization. It is suggested that a segment of 25 amino acids in the N-terminal sequence of CB1 could be responsible for this effect. Its very low polarity, its predicted amphipathic helical structure and a helix length corresponding to the thickness of the hydrocarbon region of the lipid bilayer should specifically favor its insertion in the membrane. The existence of such a transverse lipid-associating domain could confer upon the molecule the properties leading to the anchoring of diphtheria toxin in the cytoplasmic membrane.     

固体支撑的自组装的双层类脂膜

具有通常BLM_s某些相似特性的固体支撑的双层类脂膜(S-BLM)能够通过两步自组装到新劈开的金属丝上面。如:(1)包有聚四氟乙烯的铂丝头部浸在类脂溶液里,用解剖刀把顶部切开;(2)包有类脂溶液的新铂丝末端转移到0.1mol/L KCl溶液里,电测定证实,数分钟后,在金属丝末端自动地形成了稳定的类脂双层。本文报道了这种固体支撑的BLM(S-BLM)在检测Pb²⁺离子中的应用。S-BLM为液晶结构,它可用于基础研究、生物传感器和分子电子器件等技术上的应用。     

DNA-tethered membranes formed by giant vesicle rupture

We have developed a strategy for preparing tethered lipid bilayer membrane patches on solid surfaces by DNA hybridization. In this way, the tethered membrane patch is held at a controllable distance from the surface by varying the length of the DNA used. Two basic strategies are described. In the first, single-stranded DNA strands are immobilized by click chemistry to a silica surface, whose remaining surface is passivated to prevent direct assembly of a solid supported bilayer. Then giant unilamellar vesicles (GUVs) displaying the antisense strand, using a DNA–lipid conjugate developed in earlier work [Chan, Y.-H.M., van Lengerich, B., et al., 2008. Lipid-anchored DNA mediates vesicle fusion as observed by lipid and content mixing. Biointerphases 3 (2), FA17–FA21], are allowed to tether, spread and rupture to form tethered bilayer patches. In the second, a supported lipid bilayer displaying DNA using the DNA–lipid conjugate is first assembled on the surface. Then GUVs displaying the antisense strand are allowed to tether, spread and rupture to form tethered bilayer patches. The essential difference between these methods is that the tethering hybrid DNA is immobile in the first, while it is mobile in the second. Both strategies are successful; however, with mobile DNA hybrids as tethers, the patches are unstable, while in the first strategy stable patches can be formed. In the case of mobile tethers, if different length DNA hybrids are present, lateral segregation by length occurs and can be visualized by fluorescence interference contrast microscopy making this an interesting model for interactions that occur in cell junctions. In both cases, lipid mobility is high and there is a negligible immobile fraction. Thus, these architectures offer a flexible platform for the assembly of lipid bilayers at a well-defined distance from a solid support.     

Modification of supported lipid membranes by atomic force microscopy

The atomic force microscope (AFM) was used to structurally modify supported lipid bilayers in a controlled quantitative manner. By increasing the force applied by the AFM tip, lipid was removed from the scanned area, leaving a cut through the lipid bilayer. Cuts were repaired with the AFM by scanning the region with a controlled force and driving lipid back into the cut. A slow self-annealing of cuts was also observed.