The group of Prof. Carell is one of the leading nucleic acid research groups in Europe. The group has prepared in the last five years a collection of DNA lesions not available in any other laboratory in the world. With these lesions, incorporated into oligonucleotides, it was possible to solve crystal structures, which report how these lesions are recognized and repaired by repair enzymes. Crystal structures of these oligonucleotides in complex with polymerases allowed to decipher on an atomic level how polymerase produce mutations. These structural studies will be continued together with the labs of Cramer and Hopfner (area C) or with the X-ray laboratory of Skerra. The prime goal is to get structures of RNA Pol-II in complex with lesion containing DNA and of proteins involved in Nucleotide Excision Repair. The group currently develops small DNA dumbell structures with hemimethylated sides and additionally 5-AzadC, 5-FluorodU or Zebularine incorporated into these structures. These units are strong inhibitors for Dnmt1, a key enzyme needed to keep the methylation pattern in cells. These constructs are being inserted into life cells in collaboration with the groups of H. Leonhard and T. Cremer (LMU Biology, area D) in order to manipulate the methylation process during replication. The hope is to modulate the epigenetic programming of cells. Our long term goal is to make these DNA dumbell structure switchable with light, which would allow us to turn the inhibitory function on or off during specific times in the cell devision process. In a second part of the program, large DNA double strands (100-200bp) with specific lesions inside are constructed and immobilized as large loops on quantum dots. These quantum dots will be inserted in the Cremer lab (area D) into cell nuclei. Using single molecule laser fluorescence microscopy in collaboration with the Bräuchle and Leonhardt group (area A and D) the Carell group will study how repair proteins are moving to the side of damage. The DNA duplexes containing damaged DNA will also be modified with light activateable catcher groups that allow us to crosslink DNA repair proteins or other lesion-binding proteins to the damaged DNA (these studies require a strong proteomics facility, which is provided by the groups of Görg and Mann). For the delivery of the DNA duplexes into cells we will use the cell penetrating peptides. The specificity of the peptides can be improved by incorporating novel targeting strategies. The long term goal is to prepare novel chemical tools which allow to analyse protein functions in the fields of repair and DNA methylation in living cells. Together with Prof. Klein from the MPI for Neurobiology the Carell group is developing new voltage sensitive dyes.

 

The group of Dr. Mayer applies small organic molecules as probes to study the function of molecular motor proteins during mitosis in living cells. These studies are aimed at dissecting the function of dynein and the mitotic kinesins MCAK, Kif18A, and CENP-E during assembly of the bipolar mitotic spindle, the congression and subsequent segregation of chromosomes. Since small molecules are able to perturb protein functions on a fast time scale they are versatile tools to study the highly dynamic process of mitosis. However, currently this approach is limited by the fact that Eg5 is the only kinesin for which specific membrane-permeable inhibitors are available. Thus, the initial task of the Mayer group is to identify appropriate bioactive compounds. To this end, they will perform both protein- and phenotype-based small molecule screens to identify inhibitors of dynein and the mitotic kinesins Kif18A, MCAK, and CENP-E. Commercially available small molecule libraries composed of drug-like molecules and natural products will be used for these inhibitor screens. The phenotype-based screen images will be acquired using an automated microscope and they will be analyzed by image analyses software. Once the specificity of the identified inhibitors has been determined these molecules can be used to dissect the function of the targeted motor proteins in living cells. For these studies the Mayer group will combine classical cell biological approaches with live-cell fluorescence microscopy in collaboration with the Bräuchle group (area A) and the Cremer/Leonhardt groups (area D). Given that small molecules typically act on a fast time scale, these studies will provide novel insights into the function of dynein, MCAK, Kif18A, and CENP-E during mitosis. The long term goal is to create a set of small molecules able to interfere with any step of spindle assembly in living cells.

 

The group of Dr. Sieber utilizes a chemical proteomic strategy referred to as activity-based protein profiling (ABPP) to develop active site-directed probes for the specific readout of enzyme activity and function directly in whole cells, tissues, and fluid proteomes. Probe-labelled proteins will be detected and visualized by standard gel-electrophoresis procedures as well as several advanced analytical platform technologies including antibody microarrays and multidimensional gel electrophoresis and mass spectrometry (together with the Görg group, TUM-WZW and the Mann group at the MPI) which provide the advantages of high sensitivity and direct target identification. With these methodologies in hand, we propose to generate and utilize potent small molecule probes for the selective labelling and detection of multiple enzyme families including several proteases and phosphatases to identify differentially expressed markers in several different human diseases such as cancer and bacterial infections. The Sieber group anticipates that many of these enzyme activities arise from uncharacterized proteins, which may represent new targets for the diagnosis and treatment of cancer and other diseases. Since many cellular processes are dynamic, the Sieber group would like to complement our profiling studies with real time in vivo imaging to evaluate function, movement and localization of disease associated proteins in living cells. For this application they propose to design and synthesize specific fluorescent, cell-permeable probes. The ultimate goal of this strategy is to discover potent and selective chemical inhibitors of novel disease-associated enzymes and then test the effects of specific inhibitors on their in vivo behaviour such as cancer cell proliferation/invasion (chemical knockout). In order to understand also the function of enzymes lacking a specific chemical inhibitor, they will apply RNAi inactivation in cooperation with the group of Dr. Meister.

 

The group of Dr. Meister is currently developing novel strategies that allow for the design of highly specific and efficient siRNAs. This lab will contribute these reagents to all the groups of the proposed cluster. Their expertise in RNA synthesis and siRNA design will be provided to the individual groups (e.g. Sieber) of the proposed cluster of excellence. The group will further develop RNA interference (RNAi) as a highly specific tool to inactivate protein functions in living cells. RNAi describes a double-stranded RNA (dsRNA)-triggered gene silencing mechanism, which is guided by short interfering RNA (siRNA). Characteristic features of ds siRNAs are 5’ phosphate groups as well as 3’ overhangs of two nucleotides. The functional siRNA, however, is single-stranded and it is still unclear how one strand is selected. One project in our lab focuses on chemical modifications of the 5’ end of the individual siRNA strands. The Meister group found that methylation of the 5' end strongly inhibits strand selection. They are currently investigating different chemical modifications and developing general tools that allow for the design of siRNA with high specificity and low off-target effects. The long term goal is to make RNAi a reliable and well understood tool in cell biology. To this end the group will link up with the Carell group to make synthetically modified RNA strands with improved properties.

 

The group of Dr. Berg focuses on the identification of small molecule inhibitors of the protein-protein interactions required for the activity of dimeric transcription factors. The novel inhibitors to be identified in this project will improve our understanding for the chemical entities suitable for the inhibition of transcription factors, and will be useful tools to study the functions of transcription factors with high temporal control. In one part of the project, they propose to apply their expertise to the identification of selective, cell permeable compounds which bind to the SH2 domains of the transcription factors STAT1 and STAT6. In addition, they aim to identify compounds which inhibit dimerization and DNA-binding of dimeric transcription factors which belong to the basic leucine zipper (bZip) and basic helix-loop-helix motif (bHLH) protein families. These include Jun/Jun homodimers (bZip), Jun/Fos heterodimers (bZip), as well as E47/MyoD heterodimers (bHLH). Binding between the monomeric STAT SH2 domains and fluorescein-labelled peptides comprising known SH2 domain binding motifs (for STAT1 and STAT6), and protein dimers and their fluorescein-labelled DNA binding sites (for Jun/Jun, Jun/Fos, and MyoD/E47), respectively, will be analyzed by fluorescence polarization (in collaboration with the biophysics people in area A). Chemical libraries will be screened for inhibitors of the respective protein-protein interactions. The selectivity profiles of the active compounds will be analyzed in the primary screening assay and in independent in vitro assays. The most active and specific compounds will be tested in cellular assays. The long term goal is to create rules that allow a more systematic manipulation of protein-protein interactions with small organic molecules.

 

The laboratory of Prof. Skerra (Biological Chemistry) is specialized and internationally leading in the field of protein engineering and design. The research goals within CIPSM are (I) to fundamentally understand the phenomenon of molecular recognition of ligand by proteins in a constructive approach and (II) to apply the principles of rational as well as combinatorial protein design to the generation of biomolecules with novel properties to be used not only as research tools but also for medical therapy. The group applies a broad spectrum of experimental research methods in molecular biology, ranging from gene synthesis, library selection techniques, bacterial protein production, protein purification, spectroscopic characterisation (UV/VIS, fluorescence, CD, Biacore, DLS, ITC, stopped flow) to protein crystallization and X-ray structural analysis. Currently, the group works on five major topics: (I) engineering of antibodies, (II) development of the Strep-tag method for the standardized purification of recombinant proteins, (III) structural characterization of various proteins with relevance, for example, in neurobiology and developmental biology, (IV) structure-function analysis of lipocalin proteins, and (V) development and application of the ‘Anticalin’ technology. In the field of antibody engineering emphasis is on the de novo design of antibodies as well as on the so-called humanization and functional optimization of antibody fragments for the treatment of cancer (e.g. Hodgkin's lymphoma) or for stimulation of neurological regeneration (e.g. by addressing the target receptors Nogo or L1). A recent example for the research on lipocalins is the successful crystallization and X-ray structural analysis of human apolipoprotein D, a notoriously hydrophobic protein with medical relevance in lipid metabolism and, notably, for neurological and psychiatric disorders, due to its binding activity for arachidonic acid. During the past years all ten human lipocalins – as well as some additional members from other organisms – were produced in E. coli and successfully subjected to a first comparative ligand-binding analysis, which is going to shed new light on this functionally diverse protein family. It is now planned to systematically crystallize at least four more human lipocalins whose three-dimensional structure is not known to date. Lipocalins also provide the scaffold for so-called Anticalins, a novel class of engineered binding proteins with prescribed specificities not only for low molecular weight ligands but also for protein 'antigens', which were first developed in this laboratory. Anticalins are generated in a combination of rational and combinatorial protein design – involving targeted random mutagenesis and phage display selection as well as colony screening – and provide valuable reagents both for basic research and for medical applications, especially in the areas of cancer, cardiovascular, and autoimmune diseases. The anticalin technology is also currently exploited in cooperation with PIERIS Proteolab AG (http://www.pieris.biz), a biotech startup company located at the campus Weihenstephan. The possibility to generate Anticalins with modulating effects and binding activities for spectroscopic probes or for functionally interesting small molecules and to use them, for example, for in vivo delivery offers interesting options for collaboration between laboratories with different expertise at the interface between organic chemistry and protein science. Within CIPSM, the Skerra group plans the engineering of anticalins – and also of other protein scaffolds – in order to generate novel enzymatic activities. Furthermore, efforts have been launched to develop methods for the site-specific incorporation of non-natural amino acids into recombinant proteins in order to create biosynthetic proteins with totally new functionalities. These projects will provide further interesting possibilities for interdisciplinary research in the future. Most of these highly topical approaches rely on powerful selection and screening techniques. Whereas phage display was the method of choice to select proteins with novel activities from large combinatorial libraries during the past 15 years, cell-based selection, especially from libraries of transformed yeast and E. coli cells, emerges as a powerful new technique. To this end a high throughput FACS cell sorter will be needed and is planned to be part of the advanced protein analytics platform of CIPSM. In order to make such state of the art cellular selection and analysis methodology available to all CIPSM members, matching funds from the central project will be required.

 

The research of the group of Prof. Langosch (Biopolymer Chemistry) revolves around the structure/function relationships of integral membrane proteins. Sequence-specific interactions and conformational dynamics of various transmembrane domains are related to functions of the respective proteins. Previously regarded as mere membrane anchors, transmembrane domains are now recognized as independent domains whose homo- and heterotypic interactions contribute to membrane protein folding and oligomerization. Further, recent evidence points to functionally relevant conformational dynamics of transmembrane domains. In project 1, we will focus on the functional role of protein transmembrane domains in biological membrane fusion processes, which is at the heart of intracellular vesicle transport, postmitotic organelle fusion, neurotransmitter release etc.. Transmembrane domains from various fusogenic proteins, such as SNAREs from different species or viral fusion proteins, were shown to homo-dimerize in a sequence-specific way. Moreover, they exhibit substantial conformational flexibility, as probed by CD spectroscopy and hydrogen/deuterium exchange reactions. In project 2, we will investigate transmembrane domain interactions in a general and systematic way. To this end, we continue our development of the ToxR/POSSYCCAT system that reports protein/protein interactions in natural membranes and allows for selection of combinatorial libraries in vivo. By combining in-depth analysis of existing membrane protein structures, rational design of a prototypical membrane-spanning leucine zipper interaction motif, and data base mining, we have previously predicted homotypic interactions of E-cadherin and erythropoietin receptor transmembrane domains. These predictions were verified and their functional relevance could be demonstrated in vivo (as part of external collaborations). Screening of combinatorial libraries with partially randomized transmembrane domains yielded novel self-interacting sequences. Interestingly, tryptophan was found to be enriched in a position-specific way in self-interacting sequences and thus recognized as a critical determinant of interaction in membranes. In project 3 we will investigate the use of ESI mass spectrometry for detection of protein/protein and protein/ligand interactions. Here, we will use different model systems including both transmembrane domains and soluble enzymes. Further, we are involved in a number of collaborative projects in the Munich region by contributing our technical expertise.

 

In project 1 (membrane fusion), we relate self-interaction, conformational dynamics, and protein/lipid interactions of transmembrane domains to various stages of the fusion reaction in vitro and in vivo (external collaborations) and thus aim at a detailed understanding of the interdependence of fusion protein structure and lipid reorganisation. Further, we use the insights gained from basic research to design novel fusogenic transmembrane domains for potential use in liposome-mediated drug delivery.

 

In project 2 (combinatorial library screening), we will investigate the precise role of tryptophan in transmembrane domain interactions by biophysical methods. Further, we expand the technical features of the POSSYCCAT system to be able to detect heterotypic interactions and to identify small organic molecules that may induce interactions of transmembrane domains. Here, the long-term goal is to develop molecular switches to be used in biosensor devices etc.. In project 3 (mass spectrometry), we will investigate the stoichiometry of protein/protein and protein/ligand complexes, which is difficult to access by other methods.

 

The group of Dr. Braun is active in the engineering and design of cofactor-binding membrane proteins. De-novo and model proteins are engineered to study the folding and assembly of membrane proteins, with a focus on proteins in which bound cofactors can be used as probes for the assembly. The interplay of proteins, the cofactors and lipids is investigated experimentally and theoretically. Computational and statistical analyses are applied to identify cofactor-binding motifs, specifically (bacterio)chlorophyll, carotenoid and heme binding motifs in -helical membrane proteins. Identified key interaction motifs are tested by ‘rescue mutagenesis’ in the context of simplified model BChl-proteins. For example, assembly of inactivated model proteins can be rescued by a single amino acid capable of forming a H-bond at the BChl-protein interface. The established experimental system also helps to shed light on membrane biogenesis in purple bacteria, where assembly of BChl-proteins is accompanied by accumulation of non-bilayer lipids, and generation of vesicular membrane structures. These prior achievements are initial milestones on the way to reach our principal goals: (I) to help understand the underlying principles of cofactor recognition and docking, (II) to build simple model proteins for studies of protein assembly, structure, and dynamics in the cell membrane, (III) to help understanding how membrane proteins steer cellular differentiation e.g. biogenesis of membranes which are differentiated in shape and composition.

 

The group of Prof. Görg will perform systematic identification and characterization of proteins and protein patterns in order to analyse cellular protein organisation in health and disease and thus to improve the knowledge of fundamental biological processes. This will also be performed in collaboration with the groups working on the synthesis of chemical proteomics tools such as Carell and Sieber. The information can be used as diagnostic and prognostic markers and may yield targets for pharmaceutical drugs. The global analysis of differentially expressed proteins will enable to elucidate (I) protein signatures of specific cellular states in response to internal and external stimuli, and (II) the organisation and dynamics of the metabolic, signalling, and regulatory networks through which the life of the cell is transacted. The Görg group will use two-dimensional electrophoresis (2DE) technology with immobilized pH Gradients (IPG / IPG-Dalt), thus enabling the separation of complex protein mixtures such as whole cell lysates. Moreover, they will continue to refine 2DE/MS proteomics technology for a wide range of medical and biological questions. This highly sophisticated 2DE technology has become – in combination with mass spectrometry (MS) and bioinformatics tools – the basis for the majority of the ongoing proteome projects (clinical, pharmaceutical, microbial and plant proteomics) worldwide. However, due to the enormous number, the high dynamic range, and diversity of expressed proteins, particularly in eukaryotic tissues, proteome analysis is technically challenging. Hence, the development of technologies that facilitate separation, detection, und quantification of all proteins expressed in the cell routinely and systematically would be a significant achievement. Major shortcomings persist with respect to the analysis of (I) proteins that are expressed in rather low copy numbers (low abundance proteins), (II) extremely hydrophobic proteins (i.e., membrane proteins), and (III) very alkaline proteins which exhibit isoelectric points > pH 10. The objectives of our future research projects are, first, to develop advanced proteomic technologies, with the special emphasis on the separation and identification of these ‚critical’ protein species and, second, to apply these novel technologies for the quantitative analysis of differentially expressed proteins obtained from cells representing different states, thus providing detailed diagnostic patterns for classifying cellular or pathological states and yielding new insights into the function and control of biological processes. These research plans will strongly benefit from the synthesis of specific small molecule markers by the groups of Carell and Sieber. Their small molecules can be used to specifically label relevant subsets of proteins, which allows analysis of their precise role and interaction. The activities in the field of proteomics at the campus Weihenstephan will soon be boosted by the erection of the new TUM chair of Bioanalytics. The appointment of an internationally renowned candidate is expected in the course of 2006. Hence, this campus will be able to make a significant contribution to the advanced protein analytics platform as part of the core facilities of CIPSM. To complement existing and planned equipment a MALDI-MS instrument is needed. Since powerful proteomice methods are of central importance for CIPSM, matching funds from the central project will be required.

 

The junior group of Dr. Budisa is active in the in vitro and in vivo biosynthesis of proteins with an expanded set of amino acids. Fundamental results have been achieved in this respect using the green fluorescent protein as a model system. Replacement of the aromatic side chain of the fluorophoric group in the bacterially expressed GFP by the non-natural amino acid 4-aminotryptophan yielded the so-called golden fluorescent protein, which exhibits unique spectral properties and fluorescence quantum yields. Furthermore, this group studies protein folding and stability after incorporation of synthetic amino acids and it produces recombinant proteins with specific amino acid labels for phase determination in X-ray structural analysis. The mission is to expand the number of amino acids for protein biosynthesis beyond the natural set of 20 (or so) residues. This requires the programming of the cellular protein translation machinery by changing the standard genetic code.

 

The groups of Prof. Wester and Prof. Schwaiger (Molecular Imaging and Nuclear Medicine) focus on the imaging of protein expression in vivo and molecular imaging. The determination of the individual protein expression patterns as a result of gene function and regulation is a fundamental part of current approaches in individualized medicine. The selection of treatment strategies will depend on the individual “molecular signature” for a specific disease. The early or even “pre-treatment” evaluation of a therapeutic response will be based on the assessment of changes of patient-specific molecular key processes or disease-specific protein profiles, respectively. To investigate metabolic processes, protein expression, function, and regulation in a non-invasive manner in vivo, molecular imaging modalities have become powerful tools, both in the experimental set-up and in a clinical environment. Among the imaging modalities used, positron emission tomography (PET) and combined PET/CT-imaging provide significant advantages. Using suitable radiolabeled probes, molecular targets or processes, both outside or inside a cell, can be detected quantitatively with high sensitivity and contrast and co-registered with high resolution CT, thus reflecting the anatomy. For determination of protein function, tracer addressing receptors (e.g. avß3, sst2, CXCR4) enzymes (e.g. hexokinase, thymidine kinase, choline kinase, fatty acid synthase), transporters (e.g. natrium-iodine-symporter, norepinephrine transporter) or antigens (e.g. CD20, tumor-specific mutant E-cadherin, PSMA) are used or under evaluation. In vivo reporter genes expressing the aforementioned targets (e.g. HSV tK-1, NET or NIS) are under evaluation for the isotope-based assessment of gene regulation. Metabolic activity determined by 18F-Fluoro-deoxy-glucose has been shown to reflect tumor viability and has been used to monitor cytotoxic effects before structural changes occur. Early assessment of therapy allows stratification in responders and non-responders which has been useful to guide therapeutic intervention based on PET results obtained 2 weeks after initiation of therapy. In patients with gastrointestinal tumours, metabolic response to neoadjuvant therapy was significantly related to patient survival, allowing improved selection of patients for this form of therapy. Corresponding studies have been initiated for therapy monitoring with the proliferation marker 18F-Fluoro-thymidine during therapy of patients with NHL. As part of our efforts to target key proteins for the characterization of tumour biology, we investigate compounds to address processes such angiogenesis, metastasis, proliferation, apoptosis, amino acid transport, lipid synthesis or the expression of various G-protein coupled receptors.

 

For this purpose Radiopharmaceutical Chemistry (located at the Department of Nuclear Medicine of MRI and at the campus Garching, Institute for Radiochemistry) has been established at the TUM as a linking unit between chemistry/protein science and clinical research. With the aim to make target-specific Anticalins amenable to tumour therapy and monitoring, an ongoing collaboration has been established with Prof. Skerra.