RESUMEN
We describe a simple method for patterning biomolecular films on surfaces with high resolution. A conventional polymeric resist is structured by electron-beam lithography. The exposed and developed patterns are then used for the directed self-assembly (SA) of a first molecule from solution. Removal of the remaining resist allows the SA of a second species. We illustrate the potential of the approach by assembling on gold (Au) substrates two alkanethiols of contrasting terminal functionality. The patterns have dimensions from the micrometer range down to 40 nm and an edge resolution of 3.5 nm.
RESUMEN
Protein-protein interactions at the cell surface are important in the activity of bacterial toxins such as colicins. We have developed methods to study these events using tethered lipid bilayers, which can be probed by impedance spectroscopy and surface plasmon resonance. Recently we have attached the receptor proteins directly to gold electrodes and this offers new possibilities for measuring protein-protein interactions on solid supports.
Asunto(s)
Porinas/biosíntesis , Sitios de Unión , Colicinas/química , Colicinas/metabolismo , Electrodos , Oro , Membrana Dobles de Lípidos , Porinas/química , Porinas/metabolismoRESUMEN
Shear force mapping on thiolipid Langmuir-Blodgett monolayers probed with Al-coated tips leads to a contrast highly dependent on the monolayer molecular organization, which does not correspond to the topographical relief of the sample. The use of functionalized surface probes offers the possibility to better control the probe-to-sample interaction. In addition, hydrophilic surface probes are totally insensitive to alkyl chain arrangements in the monolayer. Hydrophobic probes, instead, can be chosen to map shear force on soft samples in liquid environment, since their mechanical properties are not influenced by the surrounding liquid.
Asunto(s)
Fosfolípidos/química , Compuestos de Sulfhidrilo/química , Microscopía Fluorescente/métodos , Estrés MecánicoRESUMEN
Monolayers of gramicidin A, pure and in mixtures with dimyristoylphosphatidylcholine (DMPC), were studied in situ at the air/H2O and air/D2O interfaces by polarization-modulated infrared reflection absorption spectroscopy (PM-IRRAS). Simulations of the entire set of amide I absorption modes were also performed, using complete parameter sets for different conformations based on published normal mode calculations. The structure of gramicidin A in the DMPC monolayer could clearly be assigned to a beta6.3 helix. Quantitative analysis of the amide I bands revealed that film pressures of up to 25-30 mN/m the helix tilt angle from the vertical in the pure gramicidin A layer exceeded 60 degrees. A marked dependence of the peptide orientation on the applied surface pressure was observed for the mixed lipid-peptide monolayers. At low pressure the helix lay flat on the surface, whereas at high pressures the helix was oriented almost parallel to the surface normal.
Asunto(s)
Gramicidina/química , Lípidos de la Membrana/química , Aire , Fenómenos Biofísicos , Biofisica , Óxido de Deuterio , Dimiristoilfosfatidilcolina/química , Polarización de Fluorescencia , Modelos Químicos , Estructura Secundaria de Proteína , Espectroscopía Infrarroja por Transformada de Fourier , AguaRESUMEN
The present paper scrutinizes the application of impedance spectroscopy and quartz-crystal microbalance (QCM) measurements in the analysis of composite layers of receptor containing lipid bilayers, and their interaction with external ligands or pore-forming peptides. The formation of supramolecular structures and their analysis will be discussed. Impedance measurement allows one to follow the adsorption of proteins on artificial membranes. This method is even more suitable for quantifying changes in membrane conductivity induced by channel peptides incorporated into the lipid membrane. The QCM is another sophisticated method for analyzing ganglioside-lectin and ganglioside-toxin interactions. A critical comparison between both methods will be given. Moreover, we will demonstrate that the QCM method, especially in combination with impedance analysis, is a completely new approach for determining electrical and viscoelastic properties of epithelial and endothelial cell monolayers that form controlled barriers in vivo.
Asunto(s)
Técnicas Biosensibles , Membrana Dobles de Lípidos , Receptores de Superficie Celular/metabolismo , Animales , Impedancia Eléctrica , Humanos , LigandosRESUMEN
The interaction between tetanus toxin and its fragments with gangliosides and negatively charged phosphatidylglycerols has been studied in phosphatidylcholine host membranes by protein circular dichroism measurement, calorimetry to determine lipid phase transitions, and by fluorescence spectroscopy to follow the toxin-induced pore formation by measuring the release of intravesicular entrapped dye. CD-spectroscopic secondary structure analysis showed conformational change of the toxin only in the presence of GT1b clearly demonstrating the involvement of the ganglioside headgroups for this lipid-protein-interaction. In a dot-blot analysis we showed that fragment C binds to GT1b in reconstituted vesicles and that this fragment is then accessible to a fragment C specific antibody which is only possible if fragment C is exposed at least partially on the surface of the vesicle. Our calorimetric study demonstrates the preferential binding of tetanus toxin to ganglioside GT1b. However, this protein is also able to bind to other gangliosides and also to negatively charged phospholipids causing phase separation due to electrostatic interaction. Since tetanus toxin preferentially binds short chain phosphatidylglycerol, we conclude that the protein adopts lipids with respect to charge, head group structure and chain length from the bulk phase. One consequence of this lipid-protein interaction is the ability of tetanus toxin to permeabilize lipid vesicles. Pore formation is favoured in the presence of GT1b in phosphatidylcholine membranes but only at a sufficiently high enough ganglioside content. Gangliosides others than GT1b are less effective in pore formation. In the presence of negatively charged phosphatidylglycerol tetanus toxin causes a dye release which in contrast to GT1b-containing vesicles is not saturable. We conclude that tetanus toxin acts in combination with a given number of GT1b molecules. Twenty ganglioside molecules are found to be necessary to form the stable pore. Other negatively charged lipids also cause the toxin to intercalate into the membrane but in this case the release velocity is determined by the formation of membrane defects.
Asunto(s)
Gangliósidos/química , Membrana Dobles de Lípidos/química , Toxina Tetánica/química , Dicroismo Circular , Membranas Artificiales , PermeabilidadRESUMEN
One topic of this study is the comparison of different preparation techniques to build up solid supported lipid bilayers onto gold substrates. The deposited lipid bilayers were investigated by a.c. impedance spectroscopy. Three different strategies were applied: (1) The gold surface was initially covered with a chemisorbed monolayer of octadecanethiol or 1,2-dimyristoyl-sn-glycero-3-phosphothioethanol (DMPTE). The second monolayer consisting of phospholipids was then deposited onto this hydrophobic surface by (i) the Langmuir-Schaefer-technique, (ii) from lipid solution in n-decane/isobutanol, (iii) by the lipid/detergent dilution technique or (iv) by fusion of vesicles. (2) Charged molecules carrying thiol-anchors for attachment to the gold surface by chemisorption were used. Negatively charged surfaces of 3-mercaptopropionic acid were found to be excellent substrates that allow the attachment of planar lipid bilayers by applying positively charged dimethyldioctadecylammoniumbromide (DODAB) vesicles or negatively charged 1,2-dipalmitoyl-sn-glycero-3-phosphoglycerol vesicles in the presence of chelating Ca2+-ions. If positively charged first monolayers of mercaptoethylammoniumhydrochloride were used we were able to attach mixed 1,2-dimyristoyl-sn-glycero-3-phosphoglycerol/1,2-dimyristoyl-sn-glycero- 3-phosphoethanolamine vesicles to form planar lipid bilayers via electrostatic interaction. (3) Direct deposition of lipid bilayers is possible from vesicles containing 1,2-dimyristoyl-sn-glycero-3-phosphothioethanol (DMPTE). A critical amount of more than 50 mol% of DMPTE was found to be necessary to form a solid supported lipid bilayer. Bilayers obtained with these different preparation techniques were scrutinized with respect to their capacitances, kinetics of formation and their long-term stabilities by impedance spectroscopy. The second feature of this paper is the application of the supported bilayers to study ion transport through channel-forming peptides. We used a DODAB-bilayer for the reconstitution of gramicidin D channels. By circular dichroism measurements we verified that the peptide is in its channel conformation. The ion transport of Cs+-ions through the channels was recorded by impedance analysis.