Publications de Horst Arthur Vogel

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The surface attachment and detection of DNA probes are essential in the design of nucleic acid-based biosensors. A new strategy for the covalent immobilization of single-stranded oligonucleotides on gold-covered planar supports is presented. Optimization of the surface d. in the resulting DNA arrays permits a high hybridization efficiency to be achieved. Surface plasmon resonance and, for the first time, ATR-FTIR spectroscopy are used to follow in situ the oligonucleotide layer formation and the subsequent

A bilayer lipid membrane sensor comprises (1) a gold recording surface; (2) a 1st lipid layer which is an imperfect layer of a thiolipid comprising the 2 phospholipid mols. linked to each end of a disulfide group, each through an oxyethylene chain short enough to allow the thiolipid to become anchored to the gold surface by self-assembly, but long enough to trap an aq. layer between the gold surface and the bottom of the thiolipid layer, the thiolipid being attached to the gold surface and the imperfect lay

The effect of melittin on lipid order in dimyristoylphosphatidylcholine bilayers was investigated by Raman spectroscopy and fluorescence anisotropy with diphenylhexatriene as fluorescent probe. In the fluid lipid phase, the Raman results indicated a slight increase in the conformational order of the lipid chains, and the fluorescence anisotropy results indicated a considerable increase in the rigid-body orientational order of the lipid chains. These results were contrasted with the reported decrease in the

The glucagon-like peptide-1 receptor (GLP-1R), which belongs to the class B1 G protein-coupled receptor (GPCR) family, is an important target for treatment of metabolic disorders, including type 2 diabetes and obesity. The growing interest in GLP-1R-based ...

We demonstrate that the combined application of Langmuir-Blodgett and self-assembly techniques allows the fabrication of patterns with contrasting surface properties on gold substrates. The process is monitored using fluoroscence microscopy and surface plasmon spectroscopy and microscopy. These structures are suitable for the investigation of biochem. processes at surfaces and in ultrathin films. Two examples of such processes are shown. In the first example, the structures are addressed through the binding

Time-resolved fluorescence anisotropy (FA) measurements are reported for 5 helical bilayer-spanning henicosapeptides, each contg. 1 tryptophan at sequence position 1, 6, 11, 16, or 21. The FA decay reflects 2 mol. processes in all cases: local internal fluctuations of the tryptophan side chain with a relaxation time of 200-500 ps, and motions of the whole polypeptide mol. with a relaxation time of 9-10 ns. The amplitudes of the fast fluctuation are largest at the helix ends and decrease toward the center of

Characterizing the movement, interactions, and chem. microenvironment of a protein inside the living cell is crucial to a detailed understanding of its function. Most strategies aimed at realizing this objective are based on genetically fusing the protein of interest to a reporter protein that monitors changes in the environment of the coupled protein. Examples include fusions with fluorescent proteins, the yeast two-hybrid system, and split ubiquitin. However, these techniques have various limitations, and

Regioselectivity addressable functionalised templates (RAFT) represent topological cyclic peptides containing orthogonally protected attachment sites pointing to opposite faces of the template backbone. Functionalisation of the template with coordinating groups and alkane chains result in metal-binding and self-association properties CI solution without major disruption of the preferred template conformation as inferred by NMR, CD and surface plasmon resonance, (SPR) spectroscopy studies. This suggested tha

Using transient expression, high amounts (>20 mg/mL) of secreted anti-human Rhesus-D IgG1 were produced in a suspension-adapted HEK293 EBNA cell line (Meissner et al., Biotechnol Bioeng 75: 197-203, 2001). Time of harvest was 3 days after transfection. For the estimation of transfection efficiencies, we routinely co-transfected EGFP reporter DNA. At higher reporter plasmid concentrations, >2% of total transfecting plasmid DNA, a substantial reduction of recombinant antibody synthesis, was observed. This phe

A deriv. of the integral membrane protein, cytochrome b5, was prepd. in which the native heme group was replaced by the structurally similar Rh(III)-protoporphyrin IX. This metalloporphyrin had a finite triplet yield with a single exponential decay time of 22 ms in water. After insertion of the metalloporphyrin into the protein, its triplet-state decay became strongly nonexponential with >=3 equal amplitude components with time consts. varying over a range of 100. The derivatized protein was incorporated in

Raman spectroscopy demonstrated that (1) in pure lipid membranes (dihexadecylphosphatidic acid or phosphatidylcholines), the lipid chains are mainly in an all-trans conformation in the ordered membrane phase, and in the fluid phase, lipid chains contain all-trans segments of 11 or 8 C atoms, resp.; (2) in charged membranes (dimyristoylmethylphosphatidic acid at pH 9), in addn. to a phase transition shift from 46 to 32 Deg, the lipid chain conformation is disordered, and the disordering effect is concd. at t

The amino-amido-thiol AT (HS-(CH2)10-CO-NH-CH2-CH2-NH2)(HS-(CH2)10-CO-NH-CH2-CH2-NH2), adsorbed on Au(111), was studied by sum frequency generation (SFG) and IR reflection absorption spectroscopy in the CH2 stretching vibration region. The SFG and IR spectra are qual. similar; both show the sym. stretching band and an intense and unusually broad antisym. stretching band. The IR spectra show a reversible difference between a sample dried after rinsing in electrolyte and dried after rinsing in ethanol, thus e

The highly efficient Semliki Forest virus (SFV) system has been applied to express several recombinant 7TM receptors and ligand-gated ion channels. High affinity binding and functional coupling to G proteins were obtained for the receptors. The ligand-gated ion channels showed high specific binding activity and high-amplitude electrophysiol. responses in whole-cell patch clamp recordings. The rapid and simple generation of high titer virus stocks has made the SFV system also useful for site-directed mutagen

Classically, cellular signalling is investigated measuring different optical or elec. properties of either single cells or microliter vols. of suspensions of live cells. Here we show how ligand binding to cell surface receptors and subsequent signalling reactions can be monitored in single submicrometer sized vesicles derived from biol. cells. These vesicles are the smallest autonomous containers capable of performing cellular signaling reactions, thus opening the door to downscale anal. of cellular functio

The formation of supported lipid layers incorporating promastigote surface protease (PSP), a glycosylphosphatidylinositol-anchored protein, is investigated using surface plasmon resonance. Both hydrophilic and hydrophobic substrates are used for the formation of lipid layers, and results are consistent with the formation of lipid bilayers and monolayers, resp. Specific antibody binding to layers contg. PSP is obsd., whereas nonspecific binding of the antibody to the surface is effectively suppressed by the

A Ca2+ sensor based on the admittance change of synthetic membranes supported on derivatized silicon electrodes is reported. The ion-sensitive membranes consist of mixed monolayers of phospholipid and the Ca2+-ligand ETH 1001. The electrodes are characterized electrochem. and their sensor performances are described. Through ion complexation by the neutral ligand, elec. charges are incorporated in the low-dielec.-const. environment of the sensitive layer. The no. of charged complexes and thus the membrane ad

The kinetics of incorporation of proteins into phospholipid liposomes were studied using fluorescence of the protein tryptophan residue; fluorescence was detd. at varying protein/lipid ratio and at varying temp. Cytochrome b5, purified from liver microsomes, was exposed to unilamellar dimyristoyllecithin liposomes in a fluorescence cuvette. Tryptophan fluorescence of cytochrome b5 increased rapidly as the protein was incorporated into the lipid bilayer. Protein incorporation was biphasic at low lipid/protei

A review with 6 refs. of the authors' work in developing a biophys. technique to investigate the structure, function and dynamics of membrane receptors. A fluorescence-based approach has been developed and applied to the prototypic G protein-coupled receptor NK2. A fluorescent unnatural amino acid was introduced at known sites into NK2 by suppression of nonsense codons with the aid of a chem. misacylated synthetic tRNA specifically designed for the incorporation of unnatural amino acids during expression in

The reversible oriented immobilization of proteins on solid surfaces is a prerequisite for the investigation of mol. interactions at interfaces or the construction of supramol. assemblies. We demonstrate a generally applicable method using a synthetic chelator thioalkane which can self-assemble on a gold surface via its thiol group. It exposes its nitrilotriacetic acid group which serves as a chelator for transition metal ions. Reversible binding of a Fab fragment modified with a C-terminal hexahistidine ex

Ligand binding to the nicotinic acetylcholine receptor is studied by surface plasmon resonance. Biotinylated bungarotoxin, immobilized on a streptavidin-coated gold film, binds nicotinic acetylcholine receptor both in detergent-solubilized and in lipid vesicle-reconstituted form with high specificity. In the latter case, nonspecific binding to the sensor surface is significantly reduced by reconstituting the receptor into poly(ethylene glycol)-lipid-contg. sterically stabilized vesicles. By preincubation of

Horst Arthur Vogel

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Design of Oligonucleotide Arrays at Interfaces

Lipid membrane sensors

Unifying description of the effect of membrane proteins on lipid order. Verification for the melittin/dimyristoylphosphatidylcholine system

Exploring Conformational Transitions in Biased and Balanced Ligand Binding of GLP-1R

Biologically addressable monolayer structures formed by templates of sulfur-bearing molecules

Structural fluctuations of a helical polypeptide traversing a lipid bilayer

A general method for the covalent labeling of fusion proteins with small molecules in vivo

Functionalisation of gold surfaces via topological templates

Balancing GFP reporter plasmid quantity in large-scale transient transfections for recombinant anti-human Rhesus-D IgG1 synthesis

The rotational diffusion of cytochrome b5 in lipid bilayer membranes. Influence of the lipid physical state

Determination of the conformational order of lipid membranes from Raman spectroscopy

Conformations of an amino-amido-thiolate self-assembled layer on gold in air and in electrolytes

The Semliki Forest virus system: receptor expression and scale-up

Investigating cellular signalling reactions by micro- and nanotechnology

Incorporation and antibody recognition of a lipid-anchored membrane protein in supported lipid layers

Calcium(2+)-sensitive monolayer electrodes

Kinetics of the incorporation of cytochrome b5, an integral membrane protein, into unilamellar dimyristoyllecithin liposomes

Probing ligand-receptor recognition in G protein-coupled receptors through biosynthetic incorporation of fluorescent amino acids at specific sites and measurement of distances by fluorescence energy transfer

Reversible oriented immobilization of histidine-tagged proteins on gold surfaces using a chelator thioalkane

Ligand binding to nicotinic acetylcholine receptor investigated by surface plasmon resonance