Anja Blüher

Regular arrangement of nanoparticles from the gas phase on bacterial surface-protein layers

FePt nanoparticles from the gas phase are deposited onto the two-dimensional crystalline surface layer protein from the bacterium Bacillus sphaericus NCTC 9602. The potential of this protein layer to facilitate the ordered spatial arrangement of the otherwise statistically distributed nanoparticles on the substrate is studied. Transmission electron microscopy reveals the particles positions to be directed by the regular protein template.

Formation of Tubes during Self-Assembly of Bacterial Surface Layers

Based on experimental studies on tube formation during self-assembly of bacterial surface (S)-layers, a mechanistic model for describing the underlying basic mechanisms is proposed and the effect of process parameters on growth velocity and tube radius is investigated. The S-layer is modeled as a curved sheet with discrete binding sites for the association of monomers distributed along the S-layer edges. Reported changes of the tube radius owing to genetic protein modifications are explained within the framework of continuum mechanics. S-layer growth velocity and shape development are analyzed by Monte Carlo simulation in their dependence on the attachment and detachment frequencies of monomers at the S-layer. For curved S-layer patches, a criterion for the formation of S-layer tubes is derived. Accordingly, tubes can form only within a certain range of the initial monomer concentration. Furthermore, the effect of calcium ion concentration on tube formation is discussed, including recent experimental findings on the calcium effect.

Real-Time Study of the Modification of the Peptide Bond by Atomic Calcium

Strong chemical interaction between bacterial surface protein layers and calcium atoms deposited in situ on top was revealed by means of photoemission spectroscopy. The interaction appears to mainly happen at the oxygen site of the peptide bonds and involves a large charge transfer from Ca 4s states into the peptide backbone. Chemical kinetics of this reaction was characterized using time-dependent valence band photoemission, and the reaction rate constant was determined.

Insight into bio-metal interface formation in vacuo: interplay of S-layer protein with copper and iron.

The mechanisms of interaction between inorganic matter and biomolecules, as well as properties of resulting hybrids, are receiving growing interest due to the rapidly developing field of bionanotechnology. The majority of potential applications for metal-biohybrid structures require stability of these systems under vacuum conditions, where their chemistry is elusive, and may differ dramatically from the interaction between biomolecules and metal ions in vivo. Here we report for the first time a photoemission and X-ray absorption study of the formation of a hybrid metal-protein system, tracing step-by-step the chemical interactions between the protein and metals (Cu and Fe) in vacuo. Our experiments reveal stabilization of the enol form of peptide bonds as the result of protein-metal interactions for both metals. The resulting complex with copper appears to be rather stable. In contrast, the system with iron decomposes to form inorganic species like oxide, carbide, nitride, and cyanide.

Magnetic field induced improvement of the arrangement of nanomagnets from the gas phase on S layers

FePt nanoparticles from the gas phase are deposited onto regular two-dimensional bacterial surface layer proteins with a four-fold lattice symmetry and a lattice constant of 12.5 nm. Transmission electron microscopy shows that the particle arrangement adopts both, the symmetry and periodicity of the protein template. The influence of the particle density on both the degree of agglomeration and the regularity of the particle arrangement is studied. Depositing the particles in a magnetic field applied parallel to the substrate leads to a significant decrease in agglomeration and thereby to an increase in the fraction of regularly arranged particles at high particle densities.

X-ray Damage in Protein—Metal Hybrid Structures: A Photoemission Electron Microscopy Study

Bacterial surface layer protein sheets (S layer) coated with an ultrathin cobalt or silver film were studied by means of laterally resolved near-edge X-ray absorption fine structure spectroscopy performed by photoemission electron microscopy. Comparison with results obtained on pristine S layers allowed us to characterize both chemical interaction and X-ray damage in these protein-metal hybrid systems. In particular, we found that besides direct damage upon exposure to X-ray radiation the biomolecules experience additional contribution of the deposited metals, by low-energy electron generation in the metal particles.

Regular Arrangement of Gas Phase Prepared In-Flight Annealed FePt Nanoparticles on S Layers

To achieve a regular quadratic particle arrangement, gas phase prepared FePt nanoparticles which were annealed in flight in order to obtain the highly anisotropic tetragonal L10 structure are deposited onto the regular 2-D bacterial surface-protein layer (S layer) of Bacillus sphaericus NCTC 9602 which exhibits a four-fold lattice symmetry with a lattice constant of 12.5 nm. The degree of regularity is studied by the statistical evaluation of the particles positions observed by transmission electron microscopy. Although the obtained regularity for the annealed hard magnetic particles is less than for previously studied disordered fcc FePt nanoparticles, a template-directed transfer of both lattice symmetry and periodicity from the 2-D protein crystal to the particle arrangement is observed. In addition, particle agglomeration which has been shown to have a strong effect on the arrangements regularity, is clearly reduced for the annealed particles because of the mutual magnetic repulsion of adjacent particles.

Extraction and long-term storage of S-layer proteins and flagella from Lysinibacillus sphaericus NCTC 9602: Building blocks for nanotechnology

Self‐assembling surface layer (SL) proteins of bacteria have been widely studied, in particular their use as molecularly defined, 2D coatings of technical surfaces. An important prerequisite is the availability of a sufficient amount of protein. However, a detailed and optimized protocol for the complete SL extraction is so far not available. Here, we describe the complete purification and reassembly procedure of an SL protein of Lysinibacillus sphaericus NCTC 9602, starting from the cultivation of cells, the preparation and purification of SL proteins up to the long‐term storage and in vitro self‐assembly of the proteins. All crucial steps of the procedure are assessed by different microscopic techniques, such as light microscopy, atomic force microscopy, and scanning electron microscopy as well as by SDS‐PAGE as a biochemical method. We demonstrate that storage of the protein in the presence of sodium azide or upon lyophilization allows the preservation of the self‐assembly properties for at least 9 years. Additionally, we describe a method allowing the extraction of intact flagella with lengths in the range up to 4 μm. Flagella may have applications in bio‐nanotechnology, for example as templates for metallic nanowires.

Investigation of in-vitro bacterial surface layer formation by FBARs

We report investigations on the adsorption and in-vitro recrystallization kinetics of the bacterial surface layer protein of Bacillus sphaericus NCTC 9602 on gold surfaces by means of film bulk acoustic resonators. The acoustic resonators were operated in shear mode at about 800 MHz. From the measured changes of frequency and in dissipation, the mass and the viscoelasticity of biomolecular films formed at the top electrode of the device could be derived, respectively. The measured data revealed that protein adsorption is a fast process while the time constant for the recrystallization of the monomers into ordered two-dimensional protein crystals is typically on the order of 1 h.