New EPR Method for Cellular Surface Characterization

An electron paramagnetic resonance (EPR)-based membrane surface characterization method is presented to detect the properties of the carbohydrate-rich part of membrane surfaces as well as carbohydrate interaction with other membrane constituents and water-soluble molecules. The proposed method relies on the spin-labeling and spectral decomposition based on spectral simulation and optimization with EPRSIM software. In order to increase the sensitivity of characterization to the carbohydrate-rich part of the membrane surface, the sucrose-contrasting approach is introduced. With this method, which was established on model membranes with glycolipids and tested on erythrocyte membrane, we were able to characterize the surface and lipid bilayer lateral heterogeneity. Additionally, some properties of the interaction between glycocalyx and lipid bilayer as well as between glycocalyx and sucrose molecules were determined. The experiments also provided some information about the anchoring and aggregation of the glycosylated molecules. According to the results, some functions of the glycosylated surface are discussed.     

Immobilization of native membrane-bound rhodopsin on biosensor surfaces

In this paper, we evaluated the grafting of G-protein-coupled receptors (GPCRs) onto functionalized surfaces, which is a primary requirement to elaborate receptor-based biosensors, or to develop novel GPCR assays. Bovine rhodopsin, a prototypical GPCR, was used in the form of receptor-enriched membrane fraction. Quantitative immobilization of the membrane-bound rhodopsin either non-specifically on a carboxylated dextran surface grafted with long alkyl groups, or specifically on a surface coated with anti-rhodopsin antibody was demonstrated by surface plasmon resonance. In addition, a new substrate based on mixed self-assembled multilayer that anchors specific anti-receptor antibodies was developed. Electrochemical impedance spectroscopy performed upon deposition of membrane-bound rhodopsin of increasing concentration exhibited a significant change, until a saturation level was reached, indicating optimum receptor immobilization on the substrate. The structures obtained with this new immobilization procedure of the rhodopsin in its native membrane environment are stable, with a controlled density of specific anchoring sites. Therefore, such receptor immobilization method is attractive for a range of applications, especially in the field of GPCR biosensors.     

Lipid Raft-Mediated Regulation of G-Protein Coupled Receptor Signaling by Ligands which Influence Receptor Dimerization: A Computational Study

G-protein coupled receptors (GPCRs) are the largest family of cell surface receptors; they activate heterotrimeric G-proteins in response to ligand stimulation. Although many GPCRs have been shown to form homo- and/or heterodimers on the cell membrane, the purpose of this dimerization is not known. Recent research has shown that receptor dimerization may have a role in organization of receptors on the cell surface. In addition, microdomains on the cell membrane termed lipid rafts have been shown to play a role in GPCR localization. Using a combination of stochastic (Monte Carlo) and deterministic modeling, we propose a novel mechanism for lipid raft partitioning of GPCRs based on reversible dimerization of receptors and then demonstrate that such localization can affect GPCR signaling. Modeling results are consistent with a variety of experimental data indicating that lipid rafts have a role in amplification or attenuation of G-protein signaling. Thus our work suggests a new mechanism by which dimerization-inducing or inhibiting characteristics of ligands can influence GPCR signaling by controlling receptor organization on the cell membrane.     

Lipid nanoparticle technologies for the study of G protein-coupled receptors in lipid environments

G protein-coupled receptors (GPCRs) are a large family of integral membrane proteins which conduct a wide range of biological roles and represent significant drug targets. Most biophysical and structural studies of GPCRs have been conducted on detergent-solubilised receptors, and it is clear that detergents can have detrimental effects on GPCR function. Simultaneously, there is increasing appreciation of roles for specific lipids in modulation of GPCR function. Lipid nanoparticles such as nanodiscs and styrene maleic acid lipid particles (SMALPs) offer opportunities to study integral membrane proteins in lipid environments, in a form that is soluble and amenable to structural and biophysical experiments. Here, we review the application of lipid nanoparticle technologies to the study of GPCRs, assessing the relative merits and limitations of each system. We highlight how these technologies can provide superior platforms to detergents for structural and biophysical studies of GPCRs and inform on roles for protein-lipid interactions in GPCR function.     

New insights into the nature of the wool fibre surface

Wool fibres have been treated to remove the covalently bound lipid and characterised using lipid analysis, wettability and scanning probe microscopy. A model substrate consisting of alternating stripes of hydrophobic (predominantly CH₃ terminated molecules) and hydrophilic (COOH terminated molecules) surfaces, micro-printed onto a gold-coated mica surface was assessed using the SPM techniques of adhesion, friction and phase imaging and showed that SPM can easily distinguish these surfaces. When KOH/methanol treated wool fibres were examined, SPM showed an increase in coefficient of friction and a decrease in adhesion as the lipid is removed. The increased friction is consistent with studies on the model surface and confirms the hypothesis that the lipid layer decreases the surface friction of fibres. The decreased adhesion is consistent with results in the literature on hair, but is at odds with the results on the model surface. The strong contrast shown between the methyl and carboxylic acid surfaces in the friction image of the micro-patterned surface, and the complete absence of any such contrast developing with time of treatment of the wool fibres strongly suggests that the surface lipid is not present as a discrete outer layer on the fibre. A new model is proposed in which the lipid is intimately associated with the surface proteins and allows for changes in lipid concentration at the surface in response to changes in environmental conditions.     

Fullerene modified supported lipid membrane as sensitive element of sensor for odorants

A supported lipid bilayer membrane (s-BLMs) formed on a freshly cleaved metallic surface by the Tien method was applied for the design of an electrochemical sensor for detection of neutral odorant molecules. The lipid bilayer was modified by saturation with fullerene C₆₀, which possesses electron mediator properties and facilitates a redox reaction occurring at the border of the lipid membrane and metal surface. I₂/I⁻ and ferrocenyl trimethyl bromide were used as electroactive marker ions. The smell compounds adsorb on the surface of the lipid layer and change its structure. As a consequence the ratio of marker ion penetration to the lipid membrane is altered. The magnitude of these changes depends on the amount and chemical structure of adsorbed molecules. The research presented was carried out by cyclic voltammetry. The magnitude of the electrochemical signal generated by smell compounds was correlated with other parameters describing their molecular properties such as: octanol/water partition coefficients and dipole moments.     

Molecular dynamics of a vasopressin V2 receptor in a phospholipid bilayer membrane

Molecular dynamics simulations were carried out for a V2 receptor (V2R) model embedded in a dimyristoylphosphatidylcholine (DMPC) bilayer. Both free and ligand-bound states of V2R were modeled. Our initial V2R model was obtained using a rule-based automated method for GPCR modeling and refined using constrained simulated annealing in vacuo. The docking site of the native vasopressin ligand was selected and justified upon consideration of ligand-receptor interactions and structure-activity data. The primary purpose of this work was to investigate the usefulness of MD simulation of an integral membrane protein like a GPCR receptor, upon inclusion of a carefully parameterized surrounding lipid membrane and water. Physical properties of the system were evaluated and compared with the fully hydrated pure DMPC bilayer membrane. The solvation interactions, individual lipid-protein interaction and fluctuations of the protein, the lipid, and water were analyzed in detail. As expected, the membrane-spanning helices of the protein fluctuate less than the peripheral loops do. The protein appears to disturb the local lipid structure. Simulations were carried out using AMBER 4.1 package upon constant number-pressure-temperature (NPT) conditions on massively parallel computers Cray T3E and IBM SP2.     

Interfacial Binding Sites for Cholesterol on G Protein-Coupled Receptors

A docking procedure is described that allows the transmembrane surface of a G protein-coupled receptor (GPCR) to be swept rapidly for potential binding sites for cholesterol at the bilayer interfaces on the two sides of the membrane. The procedure matches 89% of the cholesterols resolved in published GPCR crystal structures, when cholesterols likely to be crystal packing artifacts are excluded. Docking poses are shown to form distinct clusters on the protein surface, the clusters corresponding to “greasy hollows” between protein ridges. Docking poses depend on the angle of tilt of the GPCR in the surrounding lipid bilayer. It is suggested that thermal motion could alter the optimal binding pose for a cholesterol molecule, with the range of binding poses within a cluster providing a guide to the range of thermal motions likely for a cholesterol within a binding site.     

Surface potential of mycoplasma membranes

Abstract The outer membrane surfaces of several mycoplasma species carry a dense layer of anionic charges, i.e., lipid phosphate groups. They induce a negative surface potential ψ at the membrane-aqueous phase interface. This surface potential strongly affects the distribution of ions including protons. Accordingly, the pH at the interface differs from the bulk pH. By using the fluorescent lipoid pH indicator 4-heptadecyl-7-hydroxycoumarin the pH at the membrane surface was determined. From the difference of the bulk and the interfacial pH the membrane surface potential of Mycoplasma mycoides subsp. capri was calculated to be ψ = −68 mV.     

Differential targeting of glucosylceramide and galactosylceramide analogues after synthesis but not during transcytosis in Madin-Darby canine kidney cells

A short-chain analogue of galactosylceramide (6-NBD-amino-hexanoyl- galactosylceramide, C6-NBD-GalCer) was inserted into the apical or the basolateral surface of MDCK cells and transcytosis was monitored by depleting the opposite cell surface of the analogue with serum albumin. In MDCK I cells 32% of the analogue from the apical surface and 9% of the analogue from the basolateral surface transcytosed to the opposite surface per hour. These numbers were very similar to the flow of membrane as calculated from published data on the rate of fluid-phase transcytosis in these cells, demonstrating that C6-NBD-GalCer acted as a marker of bulk membrane flow. It was calculated that in MDCK I cells 155 microns membrane transcytosed per cell per hour in each direction. The fourfold higher percentage transported from the apical surface is explained by the apical to basolateral surface area ratio of 1:4. In MDCK II cells, with an apical to basolateral surface ratio of 1:1, transcytosis of C6-NBD-GalCer was 25% per hour in both directions. Similar numbers were obtained from measuring the fraction of endocytosed C6-NBD-GalCer that subsequently transcytosed. Under these conditions lipid leakage across the tight junction could be excluded, and the vesicular nature of lipid transcytosis was confirmed by the observation that the process was blocked at 17 degrees C. After insertion into one surface of MDCK II cells, the glucosylceramide analogue C6-NBD-GlcCer randomly equilibrated over the two surfaces in 8 h. C6-NBD-GalCer and -GlcCer transcytosed with identical kinetics. Thus no lipid selectivity in transcytosis was observed. Whereas the mechanism by which MDCK cells maintain the different lipid compositions of the two surface domains in the absence of lipid sorting along the transcytotic pathway is unclear, newly synthesized C6-NBD-GlcCer was preferentially delivered to the apical surface of MDCK II cells as compared with C6-NBD-GalCer.     

Characterization of the surfaces generated by liposome binding to the modified dextran matrix of a surface plasmon resonance sensor chip

The dextran matrix of a surface plasmon resonance (SPR) sensor chip modified with hydrophobic residues (BIAcore sensor chip L1) provides an ideal substrate for liposome adsorption. Liposomes of different lipid compositions are captured on the sensor chips by inserting these residues into the liposome membrane, thereby generating stable lipid surfaces. To gain a more detailed understanding of these surfaces, and to prove whether the liposomes stay on the matrix as single particles or form a continuous lipid layer by liposome fusion, we have investigated these materials, using atomic force microscopy (AFM) and fluorescence microscopy. Force measurements with AFM probes functionalized with bovine serum albumin (BSA) were employed to recognize liposome adsorption. Analysis of the maximal adhesive force and adhesion energy reveals a stronger interaction between BSA and the dextran matrix compared to the lipid-covered surfaces. Images generated using BSA-coated AFM tips indicated a complete and homogeneous coverage of the surface by phospholipid. Single liposomes could not be detected even at lower lipid concentrations, indicating that the liposomes fuse and form a lipid bilayer on the dextran matrix. Experiments with fluorescently labeled liposomes concurred with the AFM studies. Surfaces incubated with liposomes loaded with TRITC-labeled dextran showed no fluorescence, indicating a complete release of the encapsulated dye. In contrast, surfaces incubated with liposomes containing a fluorescently labeled lipid showed fluorescence.     

Lipid-induced conformation and lipid-binding properties of cytolytic and antimicrobial peptides: determination and biological specificity

While antimicrobial and cytolytic peptides exert their effects on cells largely by interacting with the lipid bilayers of their membranes, the influence of the cell membrane lipid composition on the specificity of these peptides towards a given organism is not yet understood. The lack of experimental model systems that mimic the complexity of natural cell membranes has hampered efforts to establish a direct correlation between the induced conformation of these peptides upon binding to cell membranes and their biological specificities. Nevertheless, studies using model membranes reconstituted from lipids and a few membrane-associated proteins, combined with spectroscopic techniques (i.e. circular dichroism, fluorescence spectroscopy, Fourier transform infra red spectroscopy, etc.), have provided information on specific structure-function relationships of peptide-membrane interactions at the molecular level. Reversed phase-high performance chromatography (RP-HPLC) and surface plasmon resonance (SPR) are emerging techniques for the study of the dynamics of the interactions between cytolytic and antimicrobial peptides and lipid surfaces. Thus, the immobilization of lipid moieties onto RP-HPLC sorbent now allows the investigation of peptide conformational transition upon interaction with membrane surfaces, while SPR allows the observation of the time course of peptide binding to membrane surfaces. Such studies have clearly demonstrated the complexity of peptide-membrane interactions in terms of the mutual changes in peptide binding, conformation, orientation, and lipid organization, and have, to a certain extent, allowed correlations to be drawn between peptide conformational properties and lytic activity.     

Magnetic cell separation using nano-sized bacterial magnetic particles with reconstructed magnetosome membrane

Magnetic nanoparticles produced by magnetotactic bacterium, bacterial magnetic particles (BacMPs), covered with a lipid bilayer membrane (magnetosome membrane) can be used to separate specific target cells from heterogeneous mixtures because they are easily manipulated by magnets and it is easy to display functional proteins on their surface via genetic engineering. Despite possessing unique and valuable characteristics, the potential toxicity of BacMPs to the separated cells has not been characterized in detail. Here, a novel technique was developed for the reconstruction of magnetosome membrane of BacMPs expressing protein A (protein A-BacMPs) to reduce cytotoxicity and the newly developed nanomaterial was then used for magnetic cell separation. The development of the magnetosome membrane-reconstructed protein A-BacMP was based on the characteristics of the Mms13 anchor protein, which strongly binds to the magnetite surface of BacMPs. Treatment of protein A-BacMPs with detergents removed contaminating proteins but did not affect retention of Mms13-protein A fusion proteins. The particle surfaces were then reconstructed with phosphatidylcholine. The protein A-BacMPs containing reconstructed magnetosome membranes remained dispersible and retained the ability to immobilize antibody. In addition, they contained few membrane surface proteins and endotoxins, which were observed on non-treated protein A-BacMPs. Magnetic separation of monocytes and B-lymphocytes from the peripheral blood was achieved with high purity using magnetosome membrane-reconstructed protein A-BacMPs.     

Phosphatidylethanolamine distribution and fluidity in outer and inner membranes of the gram-negative bacterium Erwinia carotovora.

1. The distribution of phosphatidylethanolamine, the major lipid of Erwinia carotovora, was investigated in intact bacteria, spheroplasts and outer- and inner-membrane preparations, with the amino-group reagent 2,4,6-trinitrobenzenesulphonic acid. Only 4% was found on the external surface of the outer membrane with 30% on the internal surface, whereas the inner membrane had 27 and 38% on its external and internal surfaces respectively. Some comparative studies were made with three other bacteria. 2. The fluidity of the membranes of E. carotovora was studied by using the fluorescent probe 1,6-diphenylhexa-1,3,5-triene. Results were consistent with the hydrocarbon region of the outer membrane bilayer being less fluid than that of the inner one. 3. On the basis of these and other results a model for the outer- and inner-membrane structures of E. carotovora is proposed.     

Stable self-assembly of a protein engineering scaffold on gold surfaces

The outer membrane protein OmpF from Escherichia coli is a member of a large family of beta-barrel membrane proteins. Some, like OmpF, are pore-forming proteins whilse others are active transporters or enzymes. We have previously shown that the receptor-binding domain (R-domain) of the toxin colicin N binds with high affinity to OmpF reconstituted into tethered lipid bilayers on gold electrodes. The binding can be measured by surface plasmon resonance (SPR) and ion channel blockage (impedance spectroscopy, IS). In this paper we report the use of a mutant OmpF-E183C in which a single cysteine had been introduced on a short periplasmic turn. OmpF-E183C binds directly to gold surfaces and creates high-density protein layers by self-assembly from detergent solution. When the gold surface is pretreated with beta-mercaptoethanol and thiolipids are added after the protein immobilisation step, the protein is shown, by Fourier transform infrared spectroscopy (FTIR), to retain its beta-rich structure. Furthermore, we could also measure R-domain binding by SPR and IS, confirming the functional reconstitution of a self-assembled membrane protein monolayer at the gold surface. Because these beta-barrel proteins are recognized protein engineering scaffolds, the method provides a generic method for the simple self-assembly of protein interfaces from aqueous solution.     

MD Simulations of a 5-HT2A Receptor Model in DOPC Membranes

Abstract

This work presents MD calculations on a model 5-HT_2A G protein-coupled receptor embedded in DOPC membrane bilayers at different lipid:protein ratios. The primary purpose is to evaluate physical properties of the system to probe membrane dynamics and the solvation interactions. This showed several kinds of apparent cooperativity phenomena in distributions, lipid dynamics, and hydrogen bond interactions. The integral protein appears to cause a disordered condensation of the local lipid structure which does not extend far beyond the surface layer of lipids. This, and changes in the lipid-protein interaction profiles give a start toward understanding membrane-protein selectivity. The presence of a partially immobilized surface layer of lipids may generally slow down kinetics of solute-solute interactions in similar liquid-crystal membranes. A hydration algorithm was also applied to the GPCR model producing a detailed water structure in some of the internal cavities, including in the areas proposed for ligand binding. Other properties examined included the distributions of lipid groups, the membrane electrostatic potential, and some of the short-time protein dynamics.     

A new easy method for determination of surface adhesion of phototrophic biofilms

Terrestrial cyanobacteria grow as phototrophic biofilms and offer a wide spectrum of interesting products. For cultivation of phototrophic biofilms different reactor concepts have been developed in the last years. One of the main influencing factors is the surface material and the adhesion strength of the chosen production strain. In this work a flow chamber was developed, in which, in combination with optical coherence tomography and computational fluid dynamics simulation, an easy analysis of adhesion forces between different biofilms and varied surface materials is possible. Hereby, differences between two cyanobacteria strains and two surface materials were shown. With longer cultivation time of biofilms adhesion increased in all experiments. Additionally, the content of extracellular polymeric substances was analyzed and its role in surface adhesion was evaluated. To test the comparability of obtained results from the flow chamber with other methods, analogous experiments were conducted with a rotational rheometer, which proved to be successful. Thus, with the presented flow chamber an easy to implement method for analysis of biofilm adhesion was developed, which can be used in future research for determination of suitable combinations of microorganisms with cultivation surfaces on lab scale in advance of larger processes.     

Preparation of Mica Supported Lipid Bilayers for High Resolution Optical Microscopy Imaging

Supported lipid bilayers (SLBs) are widely used as a model for studying membrane properties (phase separation, clustering, dynamics) and its interaction with other compounds, such as drugs or peptides. However SLB characteristics differ depending on the support used. Commonly used techniques for SLB imaging and measurements are single molecule fluorescence microscopy, FCS and atomic force microscopy (AFM). Because most optical imaging studies are carried out on a glass support, while AFM requires an extremely flat surface (generally mica), results from these techniques cannot be compared directly, since the charge and smoothness properties of these materials strongly influence diffusion. Unfortunately, the high level of manual dexterity required for the cutting and gluing thin slices of mica to the glass slide presents a hurdle to routine use of mica for SLB preparation. Although this would be the method of choice, such prepared mica surfaces often end up being uneven (wavy) and difficult to image, especially with small working distance, high numerical aperture lenses. Here we present a simple and reproducible method for preparing thin, flat mica surfaces for lipid vesicle deposition and SLB preparation. Additionally, our custom made chamber requires only very small volumes of vesicles for SLB formation. The overall procedure results in the efficient, simple and inexpensive production of high quality lipid bilayer surfaces that are directly comparable to those used in AFM studies.     

Real-time monitoring of binding events on a thermostabilized human A2A receptor embedded in a lipid bilayer by surface plasmon resonance

Membrane proteins (MPs) are prevalent drug discovery targets involved in many cell processes. Despite their high potential as drug targets, the study of MPs has been hindered by limitations in expression, purification and stabilization in order to acquire thermodynamic and kinetic parameters of small molecules binding. These bottlenecks are grounded on the mandatory use of detergents to isolate and extract MPs from the cell plasma membrane and the coexistence of multiple conformations, which reflects biochemical versatility and intrinsic instability of MPs. In this work ,we set out to define a new strategy to enable surface plasmon resonance (SPR) measurements on a thermostabilized and truncated version of the human adenosine (A_2A) G-protein-coupled receptor (GPCR) inserted in a lipid bilayer nanodisc in a label- and detergent-free manner by using a combination of affinity tags and GFP-based fluorescence techniques. We were able to detect and characterize small molecules binding kinetics on a GPCR fully embedded in a lipid environment. By providing a comparison between different binding assays in membranes, nanodiscs and detergent micelles, we show that nanodiscs can be used for small molecule binding studies by SPR to enhance the MP stability and to trigger a more native-like behaviour when compared to kinetics on A_2A receptors isolated in detergent. This work provides thus a new methodology in drug discovery to characterize the binding kinetics of small molecule ligands for MPs targets in a lipid environment.