Research Report 2002 > Centre for Biotechnology and Biomedicine (BBZ)

The overall goal of research at the Chair of Bioanalytics (Prof Ralf Hoffmann) is to understand the mechanisms of protein regulation by posttranslational modifications. These enzymatic modifications are typically reversible, but are sometimes irreversible chemical modifications of specific positions within a protein chain. Important, well-investigated modifications include phosphorylations on the hydroxyl groups of serine, threonine and tyrosine residues (phosphoric acid esters), and glycosylation on the side chains of serine, threonine and asparagine residues. Such modified proteins are analysed with the latest techniques including mass spectrometry, HPLC and 2D gel electrophoresis. Methods are optimised and developed to analyse these and other less investigated protein modifications.

The current target of analysis is the tau protein, which is highly phosphorylated in the brains of Alzheimer's disease patients and highly likely to carry disease-specific modifications (PHF tau). Despite worldwide efforts by many research groups for several decades, the exact causes of Alzheimer's disease are still unknown. Light could be shed on the disease by the tau protein, which mainly consists of 441 different amino acids. Research is focused on disease-related alterations of this protein in the brains of Alzheimer's patients. In collaboration with two research groups in the USA, new modifications in PHF tau have been identified. Analyses of the complex phosphorylation patterns of bovine and human tau samples using mass spectrometry and 2D gel electrophoresis indicate that the phosphorylation patterns of bovine tau and PHF tau from Alzheimer's patients are very similar.

The main achievement of the Chair of Molecular Biological-biochemical Processing Technology (Prof Andrea Robitzki) in 2002 was the development and application of planar cell-based chip systems and 3D tissue-based multi-microcapillary arrays for functional biomonitoring and bioanalysis. The second generation of biochips for research in the field of proteomics and metabolics as well as for pharmaceutical and pharmacotoxicological screening was established at the interface of cellular and molecular biology, biotechnology, microsystems and sensor technology. Various cell types (e.g. neuronal, glial cell lines, squamous fibroblast models) and tissue models (e.g. 3D in vitro retina models, heart muscle cell aggregates and tumour models) were generated, established and connected to biosensors. This novel technological approach was patented nationally and internationally. Applied industrial R&D is divided into two areas geared to the commercial market. The Biotechnological Processing Technology and Biochip Modules project group conducts projects in the field of bioelectronic assay development, planar electrophysiological and patch clamping modules (MEA, multielectrode arrays), optoelectronic sensor elements for in vivo diagnosis and single-cell monitoring, biotechnological micro-ultrasound systems, and processing technology for automated, 3D tissue culture. Optimum synergy for the development of biohybrid systems ("living chips" or stent biosensors for cardiovascular monitoring) was achieved by the second project group, Cell and Tissue Modelling. Various cell and tissue models for diagnosis, functional receptor assays and primary cell cultures were generated, while 3D in vitro retina based screening modules, bioelectronic photoreceptor based assays (MEA) as well as squamous fibroblast models for medical regeneration and biocompatibility testing (ISO) were developed and characterised.

Research at the Chair of Structural Analysis of Biopolymers (Prof Norbert Sträter) is concentrated on the relationship between the 3D structure of proteins and their function by using biochemical and biophysical methods, mainly protein crystallography. Enzymes are currently being studied: mostly metallo-enzymes, but also other proteins of medical or biotechnological relevance. Aside from the study of the structure of the native protein, another prime interest is the characterisation of the catalytic mechanism or function of a protein on a molecular basis. Current projects focus on dinuclear metallohydrolases, pharmacologically relevant extracellular receptor proteins, and proteins involved in signal transduction. The group's research is aligned at the interface between chemistry and life sciences. Prominent examples include the analysis of the chemical catalytic mechanisms of enzymes and the molecular mode of action of synthetic drugs on biological targets by analysing the complex structures.

The junior research group Molecular Diagnostics - Microarray Techiques (Dr Peter Ahnert) analyses interindividual diversity in the etiology and pathogenesis of autoimmune diseases, especially rheumatoid arthritis. Autoimmune diseases such as rheumatoid arthritis are complex diseases with various causes. Genetic predisposition is generally accepted to play a role in disease susceptibility and disease progression. Although it is highly probable that more than just the well-known HLA locus is involved, exactly what other genes and variants thereof may be important is not yet clear. Studying candidate genes and their polymorphisms appears to be a promising approach to elucidate this problem. Combinations or patterns of low penetrance gene variants are likely to be important here as well as for complex diseases in general, taking research in the direction of systems biology. The goal of this project is to help identify the genetic component in the etiology and pathogenesis of rheumatoid arthritis and to further the development of methods to investigate the genetics of complex diseases in general.

The junior research group Applied Molecular Evolution (Dr S. Brakmann) employs techniques of directed evolution to the functional optimisation of nucleic acid polymerases and exonucleases. The main result last year was the pinpointing of an exonuclease which hydrolyses DNA with one strand consisting only of fluorescently labelled analogues of the natural nucleobases. Together with previous work on the high-density labelling of DNA, these results create the biochemical basis for enabling single-molecule sequencing.

The junior research group Protein Engineering (Dr Thomas Greiner-Stöffele) deals with the rational design or in vitro evolution of a thermostable exonuclease III. The automation of a method for the homologous in vitro recombination of proteins requires a thermostable exonuclease variant. The genes of five proteins homologous to exonuclease III from E. coli were isolated by using sequence comparisons. The corresponding gene product from Archaeoglobus fulgidus was overproduced in E. coli, purified and biochemically characterised for the first time. This enzyme was found to be an unspecific DNase, in contrast to exonuclease III from E. coli. Structural investigations to analyse the differences between the two proteins concerning thermostability and substrate specificity were begun. Apart from starting in vitro evolution assays to increase the thermostability of exonuclease III, we introduced various mutations into the protein as suggested from computer modelling studies. This increased the stability of the enzyme by 15°C.

The junior research group Solid-state NMR Studies of the Structure of Membrane-associated Proteins (Dr Daniel Huster) focused on the investigation of the membrane structure of the peptides B18 and N-ras. B18 is the fusogenic sequence of the fusion protein binding. The N-ras peptide represents the double lipid-modified C-terminus of the human N-ras protein.
Using solid-state NMR approaches, it was shown that B18 assumes a relatively rigid oligomeric parallel b-sheet structure in lipid membranes. The peptide penetrates the hydrophobic part of the membrane.
By contrast, the N-ras peptide exhibits high dynamics at the membrane surface, and there are no signatures of a defined secondary structure. The peptide is located at the lipid-water interface of the membrane, and hydrophobic amino acids penetrate the membrane core. This topology based on NMR measurements was also confirmed by neutron scattering experiments.
Furthermore, a method was developed that allows the insertion depth of membrane protein segments to be determined by means of simple relaxation time measurements in the presence of paramagnetic EPR probes. This method was demonstrated for the model peptide WALP-16.
Finally, the group studied the complex dynamics of bacteriorhodopsin, the physicochemical characterisation of cholesterol analoga, and the membrane binding of flavaniods.

The junior research group Protein-Ligand Interaction by Ion Cyclotron Resonance Mass Spectrometry (Dr Andrea Sinz) established Nano-HPLC/nano-ESI-FTICR (electrospray ionisation Fourier transformation ion-cyclotron resonance mass spectrometry) for use in proteome analysis. This method allows the identification of low abundant proteins, which is important for comparing protein patterns in various tissues, e.g. healthy versus diseased state. This method was successfully applied for the proteome analysis of thyroid nodule tissue in a cooperation project with the Faculty of Medicine at Universität Leipzig.

Novel methods for the characterisation of protein 3D structures and protein interfaces in protein complexes were established using mass spectrometry combined with chemical cross-linking. After chemical cross-linking, the highly complex reaction mixtures were directly analysed by nano-HPLC/nano ESI-FTICR mass spectrometry without prior separation steps. A method for the 3D structure determination of proteins was developed using the proteins cytochrome C and lysozyme, while the well-characterised calmodulin/melittin complex was used to establish a method to identify protein interfaces.

The interactions of the sea urchin peptide B18 with different divalent metal ions were analysed by ESI-FTICR mass spectrometry. B18 is derived from the protein bindin, which is known to play a key role in membrane fusion during fertilisation. Although interactions of B18 with lipid membranes are known to be modified by divalent metal ions such as Zn 2+, the metal-ion binding of B18 itself is not yet understood. ESI-FTICR mass spectrometry allowed the determination of both stoichiometries and relative binding affinities of B18 and seven mutants to various divalent metal ions.

The junior research group Molecular Medicine of Contagious Diseases (Dr Reinhard Straubinger) deals with molecular mechanisms of persistent infection in Borrelia burgdorferi. Borrelia burgdorferi sensu lato is a group of spiral, active mobile bacteria that belongs to the family of spirochetes. These organisms cause a disease known as Lyme borreliosis or Lyme disease in humans and animals. Borrelia can be found in tissue samples of hosts that have mounted a strong humoral and cellular immune response or have even received antibiotic treatment. Recent research data have provided evidence that Borrelia burgdorferi organisms are not only found in their typical spiral appearance, but can change into spherical entities (cysts) if the environmental conditions are unsuitable. So far, the group's findings have shown that a few encysted organisms can survive at room temperature under hypotonic conditions (distilled water) for up to 24 hours. However, after 4 days' exposure to hypotonic conditions, messenger RNA (mRNA) and surface proteins of these bacteria exhibit changes that can be interpreted as signs of degradation. These changes might be the reason for the reduced survival rates at this time.

Centre for Biotechnology and Biomedicine (BBZ)

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