Biomedical subjects

Dietmar Pum

Publications and source records attributed to Dietmar Pum.

11 recordsLinked to original sources

PubMed2006

S-Layers as a basic building block in a molecular construction kit.

Crystalline arrays of protein or glycoprotein subunits forming surface layers (S-layers) are the most common outermost envelope components of prokaryotic organisms (archaea and bacteria). The wealth of information on the structure, chemistry, genetics, morphogenesis, and function of S-layers has revealed a broad application potential. As S-layers are periodic structures, they exhibit identical physicochemical properties for each molecular unit down to the subnanometer level and possess pores of identical size and morphology. Many applications of S-layers in nanobiotechnology depend on the ability of isolated subunits to recrystallize into monomolecular lattices in suspension or on suitable surfaces and interfaces. S-Layer lattices can be exploited as scaffolding and patterning elements for generating more complex supramolecular assemblies and structures, as required for life and nonlife science applications.

Animals↗

PubMed2006

Fluorescence emission properties of S-Layer enhanced green fluorescent fusion protein as a function of temperature, pH conditions, and guanidine hydrochloride concentration.

The fluorescent properties of the S-layer enhanced green fluorescent fusion protein (rSbpA31-1068/EGFP) were investigated as a function of temperature, pH conditions, and guanidine hydrochloride concentration. These results were compared to the fluorescent properties of the recombinant enhanced green fluorescent protein (EGFP) and an equimolar mixture of the S-layer protein rSbpA and EGFP. The intensity of the fluorescence emission of the EGFP at 510 nm, after excitation at 490 nm, is not affected by the presence of rSbpA, either as a fusion partner or as a free protein in solution. In each of the three protein systems, the emission intensity at 510 nm reaches its maximum value between pH 7 and 9 at 20 degrees C and at 0 M guanidine hydrochloride. No fluorescence could be measured at pH 4 and 6 M guanidine hydrochloride. These results show that the S-layer fusion protein (rSbpA31-1068/EGFP) is a suitable candidate for future applications in nanobiotechonology at a wide range of pH, temperature, and guanidine hydrochloride concentrations.

Green Fluorescent Proteins↗

PubMed2005

Structure, surface interactions, and compressibility of bacterial S-layers through scanning force microscopy and the surface force apparatus.

Two-dimensional crystalline bacterial surface layers (S-layers) are found in a broad range of bacteria and archaea as the outermost cell envelope component. The self-assembling properties of the S-layers permit them to recrystallize on solid substrates. Beyond their biological interest as S-layers, they are currently used in nanotechnology to build supramolecular structures. Here, the structure of S-layers and the interactions between them are studied through surface force techniques. Scanning force microscopy has been used to study the structure of recrystallized S-layers from Bacillus sphaericus on mica at different 1:1 electrolyte concentrations. They give evidence of the two-dimensional organization of the proteins and reveal small corrugations of the S-layers formed on mica. The lattice parameters of the S-layers were a=b=14 nm, gamma=90 degrees and did not depend on the electrolyte concentration. The interaction forces between recrystallized S-layers on mica were studied with the surface force apparatus as a function of electrolyte concentration. Force measurements show that electrostatic and steric interactions are dominant at long distances. When the S-layers are compressed they exhibit elastic behavior. No adhesion between recrystallized layers takes place. We report for the first time, to our knowledge, the value of the compressibility modulus of the S-layer (0.6 MPa). The compressibility modulus is independent on the electrolyte concentration, although loads of 20 mN m-1 damage the layer locally. Control experiments with denatured S-proteins show similar elastic properties under compression but they exhibit adhesion forces between proteins, which were not observed in recrystallized S-layers.

Bacterial Proteins↗

PubMed2005

Recrystallization of bacterial S-layers on flat polyelectrolyte surfaces and hollow polyelectrolyte capsules.

Polyelectrolyte multilayer (PE) deposition and S-layer technology have been combined to make novel robust biomimetic surfaces and membranes. Isolated subunits of the bacterial cell surface layer from Bacillus sphaericus CCM2177 SbpA was self-assembled on PE multilayer supports, with the composition of the multilayer playing a crucial role in determining the structure of the resulting supported protein layers. Flat substrates were studied using atomic force microscopy and neutron reflectometry; protein on suitable PE combinations showed a crystalline structure with lattice constants equal to those found in vivo on bacterial surfaces. The mechanical stability of the S-layer is higher when recrystallized on PEs than directly on silicon supports. The recrystallization process was subsequently used to coat colloidal particles, permitting the determination of zeta potentials before and after coating. Hollow capsules could also be coated in the same way, as proven by various techniques. Our results suggest that electrostatic interactions via divalent cations are important for the assembly process. The results also demonstrate that the versatility of the PE multilayer membranes can be successfully combined with the well-defined surface chemistry and structure of 2D protein crystals.

Bacillus↗

PubMed2005

S-layers as patterning elements for application in nanobiotechnology.

Two-dimensional bacterial cell surface layer protein crystals (S-layers) are the most commonly observed cell surface structure in bacteria and archaea. Isolated S-layer proteins have the intrinsic tendency to self-assemble into crystalline arrays in suspension and on various interfaces. Basic research on the structure, genetics, chemistry, morphogenesis and function of S-layers has led to a broad spectrum of applications in nanotechnology and biomimetics. The possibility to change the properties of S-layer proteins by genetic engineering opens new ways for tuning their functional and structural features. Functionalized S-layer proteins that maintain their ability to self-assemble have led to new affinity matrices, diagnostic tools, vaccines or biocompatible surfaces, as well as to biological templating or specific biomineralisation strategies at surfaces.

Bacterial Proteins↗

PubMed2005

Nanotechnology with S-layer proteins.

The cross-fertilization of biology, chemistry, material sciences, and solid-state physics is opening up a great variety of new opportunities for innovation in nanosciences. One of the key challenges is the technological utilization of self-assembly systems wherein molecules spontaneously associate under equilibrium conditions into reproducible supramolecular aggregates. The attractiveness of such processes lies in their capability to build uniform, ultrasmall functional units and the possibility of exploiting such structures at meso- and macroscopic scale for life and nonlife science applications. The use of crystalline bacterial cell-surface proteins (S-layer proteins) provided innovative approaches for the assembly of supramolecular structures and devices with dimensions of a few to tens of nanometers. S-layers have proven to be particularly suited as building blocks in a molecular construction kit involving all major classes of biological molecules. The immobilization of biomolecules in an ordered fashion on solid substrates and their controlled confinement in definite areas of nanometer dimensions are key requirements for many applications including the development of bioanalytical sensors, biochips, molecular electronics, biocompatible surfaces, and signal processing among functional membranes, cells, and integrated circuits.

Animals↗

PubMed2004

Highly robust lipid membranes on crystalline S-layer supports investigated by electrochemical impedance spectroscopy.

In the present work, S-layer supported lipid membranes formed by a modified Langmuir-Blodgett technique were investigated by electrochemical impedance spectroscopy (EIS). Basically two intermediate hydrophilic supports for phospholipid- (DPhyPC) and bipolar tetraetherlipid- (MPL from Thermoplasma acidophilum) membranes have been applied: first, the S-layer protein SbpA isolated from Bacillus sphaericus CCM 2177 recrystallized onto a gold electrode; and second, as a reference support, an S-layer ultrafiltration membrane (SUM), which consists of a microfiltration membrane (MFM) with deposited S-layer carrying cell wall fragments. The electrochemical properties and the stability of DPhyPC and MPL membranes were found to depend on the used support. The specific capacitances were 0.53 and 0.69 microF/cm(2) for DPhyPC bilayers and 0.75 and 0.77 microF/cm(2) for MPL monolayers resting on SbpA and SUM, respectively. Membrane resistances of up to 80 mega Ohm cm(2) were observed for DPhyPC bilayers on SbpA. In addition, membranes supported by SbpA exhibited a remarkable long-term robustness of up to 2 days. The membrane functionality could be demonstrated by reconstitution of membrane-active peptides such as valinomycin and alamethicin. The present results recommend S-layer-supported lipid membranes as promising structures for membrane protein-based biosensor technology.

Alamethicin↗

PubMed2004

Chemical and thermal denaturation of crystalline bacterial S-layer proteins: an atomic force microscopy study.

Crystalline monomolecular cell surface layers, S-layers, are one of the most common outermost cell envelope components of the prokaryotic organisms (bacteria and archaeda) that protects them from competitive habitats. Since isolated S-protein subunits are able to re-assemble into crystalline arrays on lipid films and solid supports making biomimetic surfaces, S-layer technology is currently used in nanobiotechnology. An important aspect of the biomimetic surfaces built with S-layers is their stability under extreme solvent conditions or temperature. Chemical (pH, alcohol) and physical (thermal) denaturant conditions were employed to test the stability of S-layers. Recrystallized bacterial surface layers from Bacillus sphaericus (SbpA) on hydrophilic silicon wafers loses the crystalline structure at 80% ethanol/water mixtures, the change in structure being reversible after treating the surface with buffer solution. SbpA on silicon supports denatures at pH 3 and at 70 degrees C, and the process is irreversible. Cross-linking of SbpA enhances the stability for high ethanol and acidic conditions, but it does not improve thermal stability. Recrystallized SbpA on secondary cell wall polymer (SCWP), a natural environment for the protein layer, is more resistant to ethanol and pH exposure than recrystallized SbpA on hydrophilic silicon supports. Atomic force microscopy (AFM) was used to monitor the loss of stability and the changes in protein layer conformation.

Bacillus↗

PubMed2004

S-layer proteins as supporting scaffoldings for functional lipid membranes.

A great challenge is the coupling of very sensitive microelectronic devices to wet biological systems in the generation of biomimetic sensors. Lipid membranes on solid supports (electrodes or semiconductors) may become the matrix of future bioelectronic devices probing and controlling biomolecular processes for scientific and technical applications. A sufficient electrical isolation between the coupling region and the surrounding electrolyte is mandatory. An attached lipid membrane with integral natural or designed proteins may be the material of choice if the biological components are kept in a proper environment. To retain the fluidity and stability of the lipid membrane and to provide an ion reservoir and space for bulky integral proteins, a separating layer composed of crystalline arrays of monomolecular proteinaceous subunits, termed S-layer, can be self-assembled on metal or semiconductor surfaces. In this way S-layer-supported lipid membranes are biomimetic structures mimicking the supramolecular principle of archeal cell envelopes. These composite architectures may ascend toward exciting new key devices, particularly in fields of membrane protein-based biosensors or lab-on-a-chip technology.

Bacterial Proteins↗

PubMed2002

S-layer-streptavidin fusion proteins as template for nanopatterned molecular arrays.

Biomolecular self-assembly can be used as a powerful tool for nanoscale engineering. In this paper, we describe the development of building blocks for nanobiotechnology, which are based on the fusion of streptavidin to a crystalline bacterial cell surface layer (S-layer) protein with the inherent ability to self-assemble into a monomolecular protein lattice. The fusion proteins and streptavidin were produced independently in Escherichia coli, isolated, and mixed to refold and purify heterotetramers of 1:3 stoichiometry. Self-assembled chimeric S-layers could be formed in suspension, on liposomes, on silicon wafers, and on accessory cell wall polymer containing cell wall fragments. The two-dimensional protein crystals displayed streptavidin in defined repetitive spacing, and they were capable of binding d-biotin and biotinylated proteins. Therefore, the chimeric S-layer can be used as a self-assembling nanopatterned molecular affinity matrix to arrange biotinylated compounds on a surface. In addition, it has application potential as a functional coat of liposomes.

Biotinylation↗

PubMedDate not supplied

Formation of nanoparticle arrays on S-layer protein lattices.

Crystalline bacterial cell surface layers (S-layers) composed of identical protein units have been used as binding templates for well-organized arrangements of nanoparticles. Isolated S-layer proteins were recrystallized into monomolecular arrays on solid substrates (such as silicon wafers and SiO2-coated grids) and in suspension forming so-called self-assembly products. These S-layer assemblies were studied by atomic force microscopy and transmission electron microscopy (TEM). The orientation of the S-layer lattice, exhibiting anisotropic surface properties, on the solid surface and on the self-assembly products, was compared with the orientation on the bacterial cell. On both bacterial cells and SiO2 surfaces the outer face of the S-layer protein was exposed. On the self-assembly products occasionally the inner face was also visible. Metal- and semiconductor nanoparticles 2 to 10 nm in mean diameter were covalently or electrostatically bound to the solid-supported S-layers and self-assembly products. TEM studies reveal that upon activation of carboxyl groups in the S-layer lattice with 1-ethyl-3,3'(dimethylaminopropyl)carbodiimide (EDC), a close-packed monolayer of 4-nm amino-functionalized CdSe nanoparticles could be covalently established on the S-layer lattice. Because of electrostatic interactions, anionic citrate-stabilized Au nanoparticles (5 nm in diameter) formed a superlattice at those sites where the inner face of the S-layer lattice was exposed. In contrast, cationic semiconductor nanoparticles (such as amino-functionalized CdSe particles) formed arrays on the outer face of the solid-supported S-layer lattices.

Adsorption↗