RESEARCH REPORT 2009/2010 Leibniz-Institut Für Molekulare Pharmakologie Im Forschungsverbund Berlin E.V

RESEARCH REPORT 2009/2010 Leibniz-Institut für Molekulare Pharmakologie im Forschungsverbund Berlin e.V. im Forschungsverbund Berlin e.V. Leibniz-Institut für Molekulare Pharmakologie 2009/2010 REPORT RESEARCH SCIENTIFIC CONTENT ADVISORY BOARD

PREFACE What’s New at the FMP? Interview with Acting Director Hartmut Oschkinat...... 4

Highlights Propellers, Fibers, and a Magic Angle...... 9 Hiding in the Membrane...... 15 Reporters in the Labyrinth...... 21 Managing the Channels of Perception and the Chemical Workplaces of the Cell...... 27

Prof. Dr. Annette G. Beck-Sickinger Leibniz Graduate School of Molecular Biophysics, Berlin...... 32 Institut für Biochemie Interview with Bernd Reif, Coordinator Universität Leipzig ResearcH groups Prof. Dr. Bernd Bukau Structural Biology Zentrum für Molekulare Biologie Protein Structure H. Oschkinat...... 34 Ruprecht-Karls-Universität Heidelberg* Protein Engineering C. Freund...... 38 Structural Bioinformatics and Protein Design G. Krause...... 42 Prof. Dr. Michael Freissmuth Computational Chemistry/Drug Design R. Kühne...... 46 Institut für Pharmakologie Solid-State NMR Spectroscopy B. Reif...... 50 Medizinische Universität Wien Solution NMR P. Schmieder...... 54 Molecular Imaging – erc Project Biosensorimaging L. Schröder...... 58 Prof. Dr. Christian Griesinger In-Cell nmr P. Selenko...... 62 MPI für Biophysikalische Chemie, Göttingen (Vorsitzender des Beirats) Signal Transduction / Molecular Genetics Physiology and Pathology of Ion Transport T.J. Jentsch...... 66 Prof. Dr. Hans-Georg Joost Molecular Cell Physiology I. Blasig...... 70 Deutsches Institut für Ernährungsforschung Anchored Signalling E. Klußmann...... 74 Potsdam-Rehbrücke Molecular Neuroscience and Biophysics A. Plested...... 78 Protein Trafficking R. Schülein...... 82 Prof. Dr. Gerhard Klebe Biochemical Neurobiology W.E. Siems...... 86 Institut für Pharmazeutische Chemie Cellular Imaging B. Wiesner...... 90 Universität Marburg Chemical Biology Chemical Systems Biology R. Frank...... 94 Prof. Dr. Frauke Melchior Peptide Synthesis M. Beyermann...... 98 Zentrum für Molekulare Biologie Peptide-Lipid-Interactions/Peptide Transport M. Dathe/J. Oehlke...... 102 Ruprecht-Karls-Universität Heidelberg Synthetic Organic Biochemistry V. Hagen...... 106 Biophysics of Membrane Proteins S. Keller...... 110 Prof. Dr. Eckhard Ottow Mass Spectrometry E. Krause...... 114 Bayer HealthCare Pharmaceuticals Screening Unit J.P. von Kries...... 118 Berlin Medicinal Chemistry J. Rademann...... 122 Protein Chemistry D. Schwarzer...... 126 Prof. Dr. Herbert Waldmann MPI für Molekulare Physiologie Administrative and Technical Services...... 130 Dortmund Index...... 131 Map of the Campus...... 134 * Chairperson since 10/2010 Imprint...... 136

2 SCIENTIFIC ADVISORY BOARD CONTENT 3 WHAT‘S NEW AT THE FMP?

Prof. Dr. Hartmut Oschkinat

Prof. Dr. Hartmut Oschkinat has been the acting What do you consider to be the major accomplishments of the external ones. An example is the recent, very nice collaboration new enzyme targets is too restricted right now. Furthermore, the FMP over the past two years? with Volker Haucke, which produced compounds that inhibit the catalytic centers of enzymes are often highly conserved with quite scientific director of the FMP since January 2009. There has been some very nice work from the group of Thomas formation of “clathrin-coated pits” in the cell membrane. These pits specific structures, making the development of highly specific He is also head of the structural biology department. Jentsch, who has established new disease models. Several years are important for the entry of viruses, other pathogens and disease- inhibitors a difficult task. On the other hand, inhibiting or modu- ago, he had shown that mutations in two isoforms of intracellu- related signals into cells. The discovery has already led to very im- lating highly specific protein-protein interactions within the same In this interview, he outlines major projects and lar CLC chloride/proton exchangers underlie renal endocytosis portant tools for research in cell biology. If more specificity in inhib- protein network might offer a way of developing substances that recent developments at the institute. defects and osteopetrosis, respectively. He has now shown that iting these processes can be achieved, such substances may also modulate specific biological functions. their replacement by a pure Cl- conductance in mice leads to similar point the way to the development of new types of treatments for pathologies, a very unexpected finding that is highly relevant for cell diseases in which endocytosis plays a role. Isn’t there still a problem that occurs when you have an interest- biology. Moreover, by studying the intracellular trafficking of these ing discovery and want to hand it off to industry to take it on for transporters, his team could bring ClC-7 to the cell surface, where it Additionally, the group of Ronald Kühne found a surprising solution further development? How are you coping with this? is now amenable to high throughput screens for ClC-7 inhibitors. As to an old problem. In collaboration with Hans-Günther Schmalz in We have two plans. First, we need to consider the big gap the loss of ClC-7 function causes osteopetrosis, i.e. hypercalcified Cologne, he developed molecules that replace prolines as bind- between scientific results obtained in an academic environment and bones, a pharmacological inhibition of ClC-7 may be useful for ers of important protein interaction domains. These domains bind the proof-of-principle stage at which industry is willing to enter into treating a much more common disease – osteoporosis, undercalci- to proline-rich peptide segments in their interaction partners, re- a project. We will continue to maintain a small translation support fied bones. This research gives us an approach for the development presenting a new class of potential drug targets. The scientists group, which looks at in-house projects and gives a realistic esti- of small molecules to treat a common disease – which is an impor- showed that these compounds are able to block the SH3 domain mate concerning the effort that is necessary to close this gap. This tant aim of the FMP’s activities. of a kinase called Fyn. We hope that the new compounds will help group, called the PEAG, plays an important role in deciding at which us to understand the functions of interactions mediated by proline- point a project should be promoted further either by in-house ef- Another area that has been very fruitful is the development of solid- rich motifs and might open new ways to address so-called “undrug- forts, or be the starting point for a spin-off company, or a pre-GoBio- state NMR methodologies, especially by the group of Bernd Reif. gable” targets. type project, or benefit from the introduction of a biotech or phar- One accomplishment of my own lab, with Barth van Rossum, has maceutical company. been to determine the structure of the alphaB-crystallin oligomer. This field of protein-protein interactions is a major focus of the It’s the largest structure determined so far de novo by solid-state FMP, yet isn’t it true that pharmacologically speaking, it’s in its Secondly, we want to convert the screening unit into a chemical NMR and contributes to an understanding of hereditary diseases infancy? biology unit, where follow-up research that involves expertise in such as cataracts in the eye lens and cardiomyopathies. Most im- Yes, research on this topic is relatively new, but in my opinion, this is chemistry and cell biology can take place. In that kind of environ- portantly, the chemical biology projects are thriving. We have exactly what we should be doing in our institute: these interactions ment, promising results can be taken to the next level, especially initial screening results that are very promising for further experi- are still thought to be too difficult to be tackled by small molecules in cycles of compound refinement by in-house chemists coupled to ments, and some compounds have already been developed suf- within industrial development projects. I hope that our work will help cell-biological assay results that can be obtained rapidly in the same ficiently to yield biologically interesting tools. These projects are lower the barriers with respect to considering such targets. We also unit. Our enhancement of this area is primarily aimed at producing partly concerned with in-house targets, but we have also taken on believe that this theme is an obvious choice, since the number of substances that can be used as tools for biological research – of

4 WHAT‘S NEW AT THE FMP? WHAT‘S NEW AT THE FMP? 5 HIGHLIGHTS

course we’d also welcome diagnostic tools for medical imaging! In application. And I am especially pleased that the EU-OPENSCREEN any case, these efforts will certainly help to push interesting com- project has officially started and is in its preparatory phase. pounds further towards the development stage. This project is supported by the European Strategy Forum on Research Infrastructures (ESFRI). It is coordinated by Ronald Frank Another good example of what is possible at the FMP has come of the FMP and links together a wide range of chemical biology from the group of Michael Beyermann in cooperation with Peter platforms throughout Europe. The aim is to combine all the expertise Schmieder and Karola Rück-Braun from the TU in Berlin. Over the required for the development of small molecules that can be used past two years Michael has generated cyclic peptides that bind to as specific modulators or inhibitors of biological functions, making PDZ domains and can be triggered by light to either bind or not high-throughput screening platforms and libraries, resources for hit bind. This means that he can switch biological processes on and off discovery and optimisation, bioinformatics support and databases using laser light. One application that they have demonstrated is to available to groups that may not have access to these tools for car- initiate muscle contraction by stimulating NO release through the rying their chemical biology projects further. As a result, our screen- laser pulse. They went all the way from designing a compound with ing facility and other platforms are offering services to groups across a desired activity to a biological assay in which an organ’s behaviour Europe – and we are becoming linked to a range of interesting sci- is modulated in a specific way. entific projects.

Our efforts in chemical biology highlight the fact that the FMP has a We are also taking part in a second ESFRI project called INSTRUCT, diverse atmosphere and it needs to integrate many disciplines. We whose focus is structural biology. Our particular role is to help develop try to maintain coherence by encouraging groups to work around solid-state NMR as a more widely-used tool in the determination common themes, common molecules, etc. Crucial in this effort will of difficult structures. For example, we have had some important be to make sure that our upcoming new hirings are integrative. The successes in studying polydisperse systems such as alphaB-crystallin candidates that we have identified as our potential next director and oligomers, protofibrils such as the deposits observed in Alzheimer’s head of the chemical biology activities are both very well-known for disease, membrane proteins, and tubulin-associated motors of integrating chemical and biological skills and a deep knowledge of kinases, all of which are difficult to analyze using crystallography and scientific problems. They will also help to bridge the gap between most other techniques. biophysics, molecular genetics, and pharmacology. These are examples of the ways in which the FMP is strengthening Have you established important new collaborations with the its integration into the wider environment of research in Berlin, Ger- wider academic or industrial community? many, and the international community. We are experiencing a process of linking the FMP to excellence initiatives in Berlin, two of which have been invited to make an

6 WHAT‘S NEW AT THE FMP? 7 PROPELLERS,

FIBERS,

AND A MAGIC ANGLE

- - -

Proteins are born when cells use the recipe in a messenger RNA molecule to select amino acids and assemble them into a long string. They don‘t keep this shape for long. Just as a component of a machine needs to have the right size and shape to function, a protein needs to be folded into a precise, three-dimensional structure to carry out its cellular tasks. That shape is partly determined by internal chemistry – specific amino acids attract or repel each other – and also by the watery environment of the cell. The string twists and turns to hide some parts of the sequence inside the molecule and to leave others exposed on the outside. This creates a surface shape and chemistry that allows the protein to bind to other molecules. The chemical contacts inside the molecule often serve as the nuts and bolts that preserve its overall shape, and they may rearrange themselves to help toggle it between active and inactive states.

HIGHLIGHT 9 3

1. Nicotic acethylcholin receptor

2. Ümit Akbey, Sascha Lange, Barth-Jan von Rossum

3. NMR spectrum

4. Liselotte Handel, Anja Voreck

5. aB-crystallin

6. 3-D model of an aB-crystallin

2 7. Cross-section of a 3-D model of a ribosome 4 5

Sometimes molecules called chaperones are needed to help pro- Both types of molecules have been difficult to analyze from a struc- of phosphates, a hallmark of intracellular signaling. Researchers still shapes of the pairs, they exposed them to X-rays in a technique teins achieve their final forms. Chaperones dock onto proteins that tural point of view. Membrane proteins don’t dissolve in water – a don’t have a full explanation for why this happens, or how it con- called small-angle X-ray scattering (SAXS) and computer models are at least partially unfolded, guide them into the proper confor- prerequisite to turning molecules into crystals so that they can be tributes to the development of disease. A structural analysis of the of possible structures. mation, and then release them. If a mutation or another problem analyzed by X-rays. And fibers – or huge complexes containing many αB-crystallin complexes might provide some answers. - - - has changed the amino acid spelling of a protein, the chaperone copies of a protein – usually refuse to form orderly arrays, which they might not recognize it, and the folding process will go awry. If the must do to form crystals. “Here, however, structural biologists have faced a problem: the mol- “Our calculations showed that the pairs bind in chaperone itself is defective, the result may also be misfolded pro- ecule assembles in complexes that usually contain between 24 and teins. Usually they are destroyed by the cell, but if they survive NMR offers an important alternative to working with crystals, and 32 copies,” Barth says. “Groups of different sizes link up in different a curved shape that repeats over and over again in they will probably be unable to carry out some of their functions Hartmut’s lab has applied the method to both problems. One pro- ways, and it has been virtually impossible to obtain samples that larger assemblies. The angle of the curve seems to be because they don’t have the proper shape. And the cell may die or ject undertaken by Stefan Jehle, Barth van Rossum and other mem- contain complexes of only one size – a prerequisite for most struc- undergo changes that cause a disease. bers of Hartmut’s lab, has centered on a structural analysis of small tural studies.” Changes in pH are known to alter its behavior, and dependent on pH. In a highly acidic environment, the heat shock proteins (sHSPs). These molecules play an essential role mutations in the three amino acids that compose the phosphoryla- - - - pairs form a sharp curve, but at high pH values in protecting the cell against misfolded proteins. AlphaB-crystallin, tion site can do so as well. for example, links to a variable number of copies of itself to form they flatten out.” “αB-crystallin was originally discovered in large assemblies that have been difficult to analyze from a structural Recently studies using the electron microscope – which can reveal - - - the eye as a protein that was essential for the point of view. the overall shape of molecules but not the positions of individual atoms – have provided some important information about the Researchers already knew that single αB-crystallin proteins ar- transparency of the lens.” “αB-crystallin was originally discovered in the eye as a protein that shape assumed by 24 copies of αB-crystallin. They form a hollow, ranged themselves in an accordion-like shape consisting of six flat - - - was essential for the transparency of the lens,” Hartmut says. “In the sphere-like particle with a 12-fold symmetry. beta sheets sandwiched together, and a seventh sheet that was meantime we’ve discovered that it has a huge range of functions, likely used to connect it to a second copy. Additional sequences at Two kinds of molecules that have always posed a challenge for including helping to mark defective proteins for destruction and Hartmut and his colleagues reasoned that various complexes of the ends of the molecules also play a role in how two copies link to structural biologists are membrane proteins and large, complex triggering cell death. Patients with multiple sclerosis have too little αB-crystallin were likely built on interactions between single pairs each other. An important aspect of the study was the fact that the assemblies of proteins such as the fibers that accumulate between of the protein in their cerebrospinal fluid, and some experimental of molecules. Changes in the shapes of the pairs might be respon- scientists analyzed these interactions at different pH values. cells. Both are of high interest to pharmacologists. Because therapies with mice have shown that the symptoms of the disease sible for assemblies of different sizes. Getting a look at the pairs they are accessible on the cell surface, membrane proteins are can be reversed by administering the protein to animals. Defects in required a combination of methods. First, the scientists turned to “Our calculations showed that the pairs bind in a curved shape that usually easily accessible to drugs that can modify their behavior. the molecule have been linked to Alzheimer’s disease and Alexan- solid-state NMR, which can be used effectively to study molecules repeats over and over again in larger assemblies,” Hartmut says. And fibers are the hallmark of many diseases – in Alzheimer’s, for der’s disease – another neurodegenerative condition in which fibers that are rather inflexibly bound in large complexes. The samples “The angle of the curve seems to be dependent on pH. In a highly example, they form thick clumps between cells that the body can’t composed of misfolded proteins accumulate between nerve cells.” were handled with magic angle spinning (MAS), a technique that acidic environment, the pairs form a sharp curve, but at high pH dissolve. There they likely interfere with communication between rotates them very rapidly while exposing them at a specific angle values they flatten out.” As this happens, the chemical properties nerve cells, eventually leading to cell death and the symptoms of An analysis of these fibers has shown that they contain copies ofα B- to a strong magnetic field. This technique produces very clean of the surfaces change, which probably accounts for the different the disease. crystallin that have been phosphorylated – tagged with small groups readouts of the relative positions of atoms. To study the overall sizes and shapes of the complexes that it forms.

10 PROPELLERS, FIBERS, AND A MAGIC ANGLE PROPELLERS, FIBERS, AND A MAGIC ANGLE 11 5 6 7

The study also revealed a groove that likely permits αB-crystallin molecules, cells were unable to receive signals that guide crucial in this structural study: the helix comes into contact with a larger, to bind to other molecules. Sometimes the groove is occupied developmental processes in the early embryo. flat surface called a beta sheet for a certain fraction of time, and by sequences from another αB-crystallin protein, which blocks it. at other moments the helix is whirling around, without contacts But if the pair spreads to its more open form, the sequences are Understanding misfolding requires comparing it to the normal to the main core. In the mutant proteins, the contacts between released and other molecules can gain access. situation in healthy cells. In the case of LDLRs, this means ob- helix and core domain do not take place. It is now assumed that taining a clear view of how MESD recognizes target sequences the open form with the helix not in contact with the protein core is The discovery of different conformations for the pairs under dif- and interacts with them. This is the goal of a project undertaken the active one. As the release of the helix from the main body of ferent levels of pH likely explains αB-crystallin’s chaperone func- by Christian Köhler and other members of Hartmut’s lab. After the protein happens, the molecule assumes a shape that can dock tions and some of its other behavior. “The protein is known to several years of work on MESD, the group has now developed onto LDLRs – most likely the region created by the juncture of the bind to cardiac muscle proteins if blood flow is restricted to a model of the way the protein switches between two confor- “propellors” with the EGF motif. However, since for the mutants the heart, for example during cardiac ischemia,” Hartmut says. mations. In one state it recognizes unfolded LDLRs and docks never the closed form is produced, the release of the chaperone “When that happens, there is a drop in pH, and the heat shock onto them. As folding progresses, MESD undergoes a structural from the receptor most likely does not take place, and the receptor proteins are probably needed to protect damage to the proteins. change that probably helps it release well-formed molecules. is not free to serve its native functions. The structure we have discovered shows how αB-crystallin undergoes an overall change of shape that allows it to bind to new Using NMR, Christian and his colleagues pinpointed an area The study provides a clearer view of MESD’s structure that helps partners and assume new functions.” within MESD that undergoes structural change. They began by explain how it identifies and processes LDLRs. Since NMR can studying mutations of MESD that interfered with LDLR functions. discover the dynamic behavior of the helix, Hartmut’s lab has come Hartmut’s group has been also studying ‘private’ chaperones In some cases these mutations had naturally occurred in lines up with an explanation for how MESD might bind to unfolded that are designed to help folding specific and ‘difficult’ proteins of laboratory animals, and researchers had traced the defects to LDLRs and yet release them when the molecules had achieved – such that face particular challenges during the folding process: MESD. Because existing studies didn’t provide enough detailed their proper form. The group studied how the structure chang- the protein ‘mesoderm development’ (MESD) is a chaperone for information, the lab introduced more single “spelling changes” es under different pH values – when levels of acid rise and fall. the family of molecules called the low-density lipoprotein receptors in the MESD amino acid recipe. This gave them two basic sets of As LDLRs fold, they leave a cellular compartment called the (LDLRs). These molecules play a crucial role in transport of hor- MESDs to compare using NMR: those that successfully carried endoplasmic reticulum and enter another structure called the mones into cells and in the development of Alzheimer’s disease, out their chaperone functions, and those that did not. This led to Golgi complex. In doing so, they encounter an environment with a osteoporosis and hereditary eye disorders. LDLRs are notoriously the identification of a region that plays a crucial role in binding lower pH – higher amounts of acid. This likely changes the charges hard to fold – think of intricate origami – because they need to to LDLRs. of some of the amino acids that are critical for binding, and causes be woven into a shape like a propeller with six blades, and the MESD to release the folded protein. Chaperones for other LDLR blades have to be linked to a nearby hub called an EGF domain. The studies homed in on a small spiral structure called an alpha molecules are known to let go for the same reason. In the cells of mammals, folding the propeller and its hub re- helix at the beginning of the amino acid sequence that is con- quires the help of a chaperone called the mesoderm develop- nected with the core of MESD by a loose, hinge-like region. For ment protein (MESD). In mice, due to improper folding of LDLR the normal ‘wildtype’ protein, two different states are observed

12 PROPELLERS, FIBERS, AND A MAGIC ANGLE PROPELLERS, FIBERS, AND A MAGIC ANGLE 13 HIDING IN THE

MEMBRANE

- - -

In most diagrams of the cell – you know, those drawings from your high school textbook – the plasma membrane is represented by a simple line that separates the cell from the surrounding environment. This hides the fact that it is a tremendously complex place. To get a better image, think of two soap bubbles, one tucked tightly inside the other. The two surfaces are fluids made mostly of fat molecules called lipids. Floating in them are proteins that often extend brushy protrusions toward the exterior and have tails hanging into the major cell compartment, the cytoplasm. These proteins have crucial functions. To name just a few: external molecules dock onto them and send signals into the cell; they recognize foreign molecules and trigger immune responses; or shuttle ions and other substances through the membrane.

What happens between the two lipid layers is also crucial to all of these functions. Many membrane proteins weave back and forth in this inner space, creating complex folds – as if you were trying to sew the two layers together with a thread, weaving in and out. Sequences and structures within the membrane allow the proteins to bind to each other and create pores through which substances pass. Or the encounters draw their tails together and trigger chemical reactions that spark a signal destined for the genes. Or they create new surfaces for molecules to bind outside the cell.

While the region within the membrane is tremendously important, it is notoriously hard to investigate using the traditional techniques of structural biology. Membrane proteins rarely, if ever, form crystals, which would permit their study using X-ray crystallography, one of the main methods of structural biology. But biochemical work and computer modeling, along- side the other methods used at the FMP, are steadily revealing details of the inner workings of membrane proteins.

HIGHLIGHT 15 1 Complex model of the TSH Receptor: Hormone bound on extracellular LRR domain (green). Allosteric binding pocket (yellow) between the transmembrane 3 3 helices (red) and intracellular bound G-Protein

2 Antje Schmidt

3 Anita Kinne, Inna Hoyer, Ann-Karin Haas

4 Ann-Karin Haas

5 TSHR model: Amino acid side chains (lilac) surrounding the transmembrane binding pocket (yellow)

6 TSHR model, extracelluar view: Small molecule agonist (yellow) bound into the allosteric binding pocket (green)

2 7 Jonas Protze 4

One of the best places to go to catch a glimpse of this hidden regions can prevent the receptor to binding to an important signal- the thyroid gland to a racing heartbeat, hand tremors, weight loss (LRRD) – whose name derives from the fact that it contains repeated world is Gerd Krause’s lab at the FMP. For years, Gerd’s group has ing molecule. Changes in the tail and other intracellular portions and serious problems with the digestive system. About a quarter sequences with many copies of the amino acid leucine. The second been systematically investigating a family of membrane proteins may prevent the protein from binding to G proteins and passing of the patients also suffer from inflammations of the eye muscles is a hinge-like region that connects the LRRD to the cell and the called G-protein-coupled receptors, or GPCRs. When activated by along a signal. And a defect in regions that span the membrane can that cause their eyes to protrude. seven helices that weave back and forth through the interior of the external signals, these molecules set off a cascade of reactions block the interplay between different molecules that is necessary cell membrane. Other labs have developed a picture of most of the - - - within cells that are amplified by G proteins lying just under the cell for their functions. LRRD in protein crystallography experiments. But the hinge region surface. Working with Ralf Schülein’s lab, the group has exposed has remained largely unexplored. “Ideally, we would find a way to directly inhibit important structural features of the molecules that they have GPCRs have similar forms – they all have seven helical structures assembled into a database that is open to the scientific commu- that weave back and forth between the two membrane layers the pathological activation of TSHR.” The scientists knew that both thyrotropin – the thyroid hormone nity. Recently this work has led the lab to new discoveries about in a serpentine-like pattern – and are relatively easy to detect – and autoimmune antibodies bind mostly to the LRRD, but also - - - the structure of a GPCR called the thyrotropin receptor, or TSHR, from the sequences of genes or proteins. Analyses of the human to the hinge region, to influence the receptor’s activity. A struc- with a particular focus on the structures that lie between the two genome reveal that our cells make at least 800 types, an amazing tural picture of a very similar receptor, which binds to the follicle- layers of the plasma membrane. What happens there, Gerd says, number that accounts for about four percent of all human genes. Treatments usually involve administering drugs which reduce the stimulating hormone (FSH), revealed that the hormone binds to reveals general principles of GPCRs that will be crucial for an Yet by 2010, scientists had obtained detailed structures of only four production of the thyroid hormone, or removing at least part of specific regions called beta-strands on the concave inner surface understanding of diseases and the designing of new drugs to of these molecules and three more from other species. Gerd be- the thyroid gland, but they do not address the underlying cause. of the LRRD. But this structure did not include parts of the mol- combat them. lieves that many of the others can be understood by comparison Nor do they address symptoms involving the eyes. “Ideally,” ecule closest to the hinge region, which might also influence the to these seven examples, and his group has compiled an exten- Gerd says, “we would find a way to directly inhibit the patholo- way the receptor binds to partners. And it said nothing about the - - - sive database to help groups start. The work of his own lab shows gical activation of TSHR. But while substances are known that involvement of the hinge region. how promising this approach might be. increase receptor signaling, finding methods of reducing it has “GPCRs play such a fundamental role in cell been much harder.” The recent project sought to clarify aspects of the hinge struc- signaling that they are the target of a large One major project of the group over the last several years has in- ture – particularly its location relative to the serpentine helices volved a GPCR called the thyrotropin receptor (TSHR). This mole- Here insights into the structure of TSHR might be invaluable, and that cross the membrane – and to determine which amino acids proportion of today‘s drugs.” cule is produced by cells in the thyroid gland and fibroblasts in the Gerd’s group has been systematically investigating the entire were crucial to TSHR signaling. Gunnar, Paul Grzesik and their - - - muscles of the eye. TSHR has received special attention because molecule in hopes of identifying areas that might be susceptible colleagues systematically introduced mutations in single amino in the autoimmune condition called Grave’s disease, which affects to manipulation. Recently Gunnar Kleinau, who was a postdoc acids, or deleted them entirely, hoping to measure an effect on “GPCRs play such a fundamental role in cell signaling,” Gerd says, about two percent of females and a smaller number of males, it in Gerd’s group for several years and now works at the Institute the receptor’s activity. “that they are the target of a large proportion of today’s drugs.” is continually activated by antibodies produced by the body. This for Experimental Pediatric Endocrinology of the Charité, took Malfunctions of the signaling pathways triggered by GPCRs have provokes an overproduction of thyroid hormone that results in a a close look at regions of the receptor that lie outside the cell. Most of the changes led to an increase in signaling. But sev- been linked to diabetes, allergies, some forms of cancer, and disruption of the body’s metabolism. It speeds up a number of eral mutations in the hinge region led to decreased signaling. a number of other serious diseases. Disrupting nearly any part processes, leading to a wide range of symptoms including dis- Gerd says that this extracellular module consists of two distinct Interestingly, Gerd says, combining several of these mutations of a GPCR can lead to serious problems. Mutations in external turbances of the nervous system – from a general enlargement of parts. First comes a module called the leucine-rich repeat domain did not amplify the effect. Some of the changes prevented the

16 HIDING IN THE MEMBRANE HIDING IN THE MEMBRANE 17 5 6

receptor from reaching the membrane at all – probably because the group of Ralf Schülein to mutate all the amino acids that line Using a computational approach, the scientists created a model of If the pocket is so crucial to that activation, could it also be the they caused it to be trapped inside the cell, or caused the cell this pocket between the helices. They identified two functional the pocket which shows that it closely resembles a similar structure key to blocking receptor signaling? Gerd thinks so, and the lab to recognize that it was defective and subsequently destroy it. types of mutations in this pocket region: one that can deactivate in other GPCRs. In these cases, natural substances slip into the has continued to refine its view of the pocket’s structure. Combin- and another that can activate the TSHR. pocket. That contrasts with TSHR, which is normally activated by a ing computer models – which draw on structures that have been - - - molecule that binds to the extracellular region. But TSHR and the better defined in the otherG PCRS – with mutational studies has “When a substance binds to some of the amino acids that line the other molecules seem to have evolved from a common ancestor, revealed seven mutants that lead to an overall decrease in signal- This work has led Gerd‘s group to a structure pocket, receptor activity increases,” Gerd says. “Others seem to and TSHR has retained the pocket. It doesn’t seem to have a natu- ing. “The mutants reveal sites in the pocket that a drug will likely hidden within the membrane itself: a pocket that lock it into an inactive conformation. What we’d like to do is map ral function in human cells, but it might be susceptible to drugs. have to interact with in order to weaken the receptor,” Gerd says. their positions; that could help us find or design a new substance “Our ever-clearer view of their functions and spatial positions may forms between the serpentine helices. that will specifically target them.” - - - help us design a new molecule that specifically does so.” It’s a task - - - he plans to take on with the help of his American collaborators and The team has already seen some preliminary successes, such as in a “We think their function is to help stabilize the FMP Screening Unit. Changing subtle aspects of the way the hinge folds altogether 2008 collaboration with Marvin Gershengorn’s lab at the National the membrane structure of the receptor. seems to lead to an overactivation of the receptor, but the study Institute of Diabetes and Digestive and Kidney Diseases of the Success, he says, might finally provide a long-sought solution to suggests that it will be difficult to use this information to cause National Institutes of Health (U.S). The scientists took a substance These positions are potential points where small some of the dangerous symptoms of Grave’s disease. In addition, its inactivation. And that, Gerd says, is the key to finding a way called Org41841 – which they had shown to enhance GPCR signaling it could open the door to similar approaches that could be taken molecules can trigger its activation.” to prevent the excess of signaling that occurs in Grave’s disease. in an earlier study – and began modifying it in the hope of achiev- with many other GPCRs that are found to be defective in a wide The molecule seems to be able to overcome most attempts to ing the opposite effect. They introduced chemical changes in the - - - range of diseases, and are already common targets of powerful disable it. molecule based on the structure of another molecule called NIDDK/ drugs. CEB-52 that partially inhibited TSHR signaling, in combination with “The mutation studies showed that changing many of the amino If that’s the case, researchers will need to look to other regions a careful look at particular features of TSHR structure. The study acids that line the cavity leads to an increased activity of the recep- of the receptor to find a way to shut it down. And that has led showed that the modified molecule might serve as the basis for a tor,” Gerd says. “We don’t know much about this structure in the Gerd’s group to a structure hidden within the membrane itself: more powerful inhibitor. related molecules, but it suggests that the corresponding amino a pocket that forms between the serpentine helices. Gerd and acids may also influence signaling.” his colleagues have found substances that enter the membrane, The groups have continued to work together to determine snap into this gap and enhance TSHR signaling. It means that the which amino acids in the pocket influence signaling, and how. In The study revealed that six amino acids near the pocket do just that. molecule has a second binding site that influences its activity, and 2010 Gunnar, Ann-Karin, and their colleagues in Ralf and Gerd’s Gerd says, “We think their function is to help stabilize the membrane it may serve as a keyhole to block the receptor’s activity. groups, as well as the American scientists, began mutating structure of the receptor. These positions are potential points where single amino acids in the pocket to determine their effects. At first small molecules can trigger its activation. And further work has shown Recently Ann-Karin Haas, Inna Hoyer, Jens Furkert and Claudia the efforts were directed at finding positions that had a positive that the substance we developed acts on them to introduce slight Rutz promoted a collaborative project between Gerd’s lab and effect on signaling. changes in the TSHR structure that push it toward its active state.”

18 HIDING IN THE MEMBRANE HIDING IN THE MEMBRANE 19 REPORTERS

IN THE

LABYRINTH

- - -

Imagine trying to make a map of a labyrinth by having someone stand at two or three intersections along the way and checking on the progress of someone walking through. You might figure out where he entered, and where he ended up, but you‘d miss most of the twists and turns, false trails, and dead ends. Even if you did this time and time again, your map wouldn‘t be very complete. It would be better to have a reporter sta- tioned at every turn, tracking the progress and path taken by visitors. Cells contain chemical labyrinths whose pathways researchers would like to map because they often hold the key to states of health and disease. Most important events in a cell‘s life – when it divides, what type it be- comes, and even whether it lives or dies – start with a signal from its surroundings. These signals often move from the surface to genes in the nucleus along a labyrinthine path full of starts and stops, sudden moves forward, and sometimes they take odd turns.

HIGHLIGHT 21 3

1. Low resolution microscopy image of a thin section of a Xenopus laevis oocyte

2. Alexander Dose

3. Beata Bekei

4. Signaling pathway, scemats presentation

5. Dynamic signaling responds curves

6. Diana Lang

2 7. NMR theory 5 6

“This metaphor captures part of the complexity of signaling path- and organisms. They have identified key molecules in the major something unexpected – for example, if it failed to reach its next For many biophysical methods, such as X-ray crystallography and ways,” says Philipp Selenko. “The diagrams that researchers put in pathways and combined bits and pieces of data into models. But target, or if it were a lot stronger than usual – scientists would have electron microscopy, such artificial setups are a necessary means to their papers often resemble a labyrinthine route connecting protein they haven’t been able to watch a signal in real time as it moves a hint that something was interfering with it. Such a method would an end. Both methods require pure samples and isolated experi- to protein. But there’s another dimension: The arrival of a signal is through the cell. And as a result, important information – such as also give them a better idea of the problems that arise during mental conditions in the test tube. They also only present static like a flash of light that spreads throughout the cell at various inten- the fact that a signal needs to have a particular strength to pro- diseases and thus permit the development of drugs that cor- images of the molecules under investigation – locked in particular sities, affecting a huge range of molecules,” Phil says. voke the proper effect – often gets lost. rect defects in one pathway without disturbing other healthy states – whereas ideally researchers would like to see how their processes. structures change under the varying biological conditions that Such signals usually begin when molecules in the cell’s environ- “A real-time view of signaling would be great to have,” Philipp says, occur in a cell. ment, such as those of its neighbors, dock onto receptor proteins “because each step in a pathway provides an opportunity for the So far the project remains an ambitious plan, at least in its full- on the cell surface. This sets off cascades of biochemical reactions cell to shut off a signal or amplify it. Or to integrate it with other est form, but Philipp’s lab has already had impressive successes “NMR doesn’t have any of these problems,” Philipp says. “It’s a within the cell that usually changes the pattern of active and silent signals to produce a different outcome. Pathways are dynamic. when it comes to visualizing biological processes inside liv- method that gives you structural information about biomolecules genes. As a result, the cell produces new messenger RNAs and They are often disturbed during disease, and they’re often the ing cells. In a number of recent experiments, the lab has been in virtually any environment, including intact cells, and it detects proteins that influence its structure and behavior. targets of drugs – in both cases, you’d like the full picture of how putting single pieces of the “tracking system” into place. dynamic changes in a time-resolved and non-invasive manner. they behave and respond to changes. And you’d like to watch this in You can pick individual molecules or processes out of the crowd - - - intact cells.”Philipp has been thinking about possible solutions to - - - because most atoms in biological molecules are invisible in the this problem for several years. His group is now piecing together an NMR spectrometer, and fade into the background. However, “A real-time view of signaling would be great approach by which molecules will be turned into reporters that “Yet most experiments that aim at investigating the individual proteins can be made visible by labeling them with to have, because each step in a pathway provides will permit the scientists to track signals within cells using NMR. isotopes – ‘heavy’ versions of some of the atoms. This provides structure or function of molecules are typically carried “The idea,” he says, “is to strategically place reporters that can a means of analyzing them in their native, unlabeled environ- an opportunity for the cell to shut off a signal be interrogated using NMR along different signaling pathways. out in artificial and highly simplified environments that ments, whether you’re working with extracts from cells or even or amplify it.” The reporters are engineered versions of peptide molecules that an intact cell. This makes a single protein, for example, stand out we can easily comprehend and manipulate.” ’light up’ when signals pass through. Each reporter relays informa- against the background of all the other molecules present in - - - tion about one step in the pathway, telling us not only whether - - - the cell.” Cells are continuously stimulated by different signals and they a signal travels along a certain route, but also the strength of its have to relay and interpret them correctly in order to respond transmission.” “The cell interior represents a highly crowded, dynamic solution. Recently Philipp and his colleagues have investigated two of the in a healthy way. Radios use different frequencies to solve this It harbors an intricate network of biological activities that are major chemical changes that many proteins undergo during problem, and cells use signaling pathways that pass information The resulting picture would be like moving through a labyrinth carried out by a vast number of molecules,” Philipp says. “Yet cellular signaling events. Proteins are often activated through the along well-established chemical routes, often involving hundreds equipped with motion detectors, where lights come on with dif- most experiments that aim at investigating the structure or func- addition of chemical tags. In phosphorylation, for example, phosphate of different molecules. To get the full picture of how information ferent intensities depending on whether a single person or several tion of molecules are typically carried out in artificial and highly groups are added to specific sites in proteins. The phosphorylation moves from a receptor to genes, researchers have painstakingly have taken a particular route. The signals could then be observed simplified environments that we can easily comprehend and of a protein often changes the other molecules it interacts with, and carried out experiments in the test tube and a variety of cell types in real time to reveal entire signaling pathways. If a signal did manipulate.” thereby changes its functions.

22 REPORTERS IN THE LABYRINTH REPORTERS IN THE LABYRINTH 23 Phosphorylation is one type of post-translational modification a histone called H3, known to undergo both types of tagging. In (PTM) that proteins undergo; another is acetylation. The latter the test tube the scientists exposed H3 to Msk1, the protein that is often used to activate or silence genes. Once again, the pro- phosphorylates it, and Gen5, which acetylates it. They discovered cess involves a transfer of chemical tags (this time acetyl groups) that the tags are applied in parallel to three sites in H3, but at dif- to proteins. In this case, the recipients are often proteins called ferent rates. Acetylation occurred the fastest, and one of the two histones, which form small, spool-like structures. The strands of phosphorylation sites was tagged more quickly than the other. DNA in the nuclei of our cells are wrapped around these spools at regular intervals. They often have to be moved so that other When the experiments were repeated in extracts from the nuclei molecules can get access to particular locations in the strands of human cells, which contained a full set of signaling molecules, and activate genes. Histones play a critical role in this process: the scientists confirmed that H3 was being acetylated, but at a acetylation of the proteins influences the locations of the spools lower rate than in the test tube. This was true for one of the phos- and, ultimately, whether a gene is active or silenced. Other pro- phorylation sites as well. The second site wasn’t being tagged at teins can also be influenced by the addition of acetyl. all. “This may be due to the type of cells that were used,” Philipp says. “They may not produce the- - factors- that normally phosphory- “In most cases it’s a combination of tags applied to a single mole- late this site. Or these proteins may be kept in check by other mol- cule – it may be both phosphorylated and acetylated, for example ecules; we’ll need to do more experiments to determine which is – that determines the outcome of a signal in the cell,” Philipp says. the case.” “This process is highly dynamic and depends on the overall state - - - of the cell. That poses a problem because most research tools fail to detect different modifications in a single experiment. Nor do they observe dynamic states in complex environments such as In some ways H3 was easy to work with, Philipp says, because the cell extracts or full cells.” “tail” region of the molecule that receives tags has a diverse chemical composition. “Because of the way NMR works, this means that - - - each amino acid in the tail has a unique chemical environment that makes it easy to detect changes. But in other contexts, lysines may “We hoped to see not only whether a molecule be clustered together, surrounded by very similar sequences, and had been tagged, but also the exact position in the NMR readout is blurred. You may not be able to see which residue is receiving the tag.” To use another metaphor: if you’re the molecule where this happened.” looking for a book with a red cover, it’s easiest to find on a book- - - - shelf with books of different colors. If all the red books have been placed on the same shelf, you’ll need to find another way to make In 2010 Philipp’s group worked with the lab of Dirk Schwarzer it stand out. 7 to develop a method for detecting phosphorylation and acetylation events in parallel. The work was funded by the Another project carried out by the labs of Philipp and Dirk aimed FMP as an integrated project, which provides additional sup- to find a solution. They wanted to watch the on-again/off-again port to encourage multiple labs to take on a common project. process of chemical tagging in another histone called H4. This “The idea was to use NMR to observe these modifications in real protein is another component of the molecular spools that DNA time,” Philipp says. “And we wanted a second piece of informa- is wrapped around, and it also plays an important role in the tion as well. Often a single molecule can receive the same type of activation of genes. from others. One of these deacetylases, called HDAC, is being another chemical tag. This can be done whether they are located tag at different locations in its structure, with different outcomes. investigated as a pharmaceutical target because of its powerful in the cell nucleus, like the histones, or at any other position along The scientists took a careful look at the sequence of H4 and fig- effects on human genes. In creating potential drugs, pharma- the pathway between the receptor that originally receives a signal We hoped to see not only whether a molecule had been tagged, ured out a way to attach NMR-active isotopes to different parts of cologists must have a precise understanding of their effects – a on the cell surface and DNA in the nucleus. Philipp and his col- but also the exact position in the molecule where this happened.” the molecule so as to discriminate between the different “blurred” substance should help HDACs remove some tags while leaving leagues have begun to modify other molecules so that the nature When the labs of Philipp and Dirk applied NMR to this problem, residues. Each position would now give off a different kind of others undisturbed. This will require a precise analysis of the and strength of particular signals can be detected using NMR. they discovered that both processes yielded distinctively different NMR signal, which made the problem of identifying which sites deacetylase’s behavior under different conditions. The study by Philipp is modest about the accomplishments. NMR signatures that unambiguously indicated whether a protein had become tagged a straightforward one. Philipp’s and Dirk’s labs provides an important starting point. had been phosphorylated or acetylated – as well as exact posi- “In itself, each step is just a proof of principle,” he says. “It’s when tional information. For example, an enzyme called CBP/p300 was The result was a precise view of the sequence in which tags are - - - all the steps come together that we hope we’ll have a full picture. known to acetylate the protein c-myc, which is known to cause added to the histone and then removed again. When the scientists The fact that we can detect several signaling events in a single “In itself, each step is just a proof of principle. cancer if it suffers defects. Previous experiments had shown that studied the behavior of an acetylase called HAT, they discovered experiment is a shortcut to unraveling the very complex com- the tag was applied to a lysine residue at a specific position in the that lysines were tagged with acetyl in a particular sequence – one It‘s when all the steps come together bination of codes that ultimately determines how histones and protein. Now the scientists observed that another nearby lysine site was acetylated first, until all the copies of the histone scat- other proteins behave in the intricate environment of the cell.” that we hope we‘ll have a full picture.” was also being acetylated. Computer analyses had predicted that tered along the DNA molecule had been tagged, and only then If a signal gets sidetracked as it moves through the labyrinth this site might also be acetylated, but its modification had never was the second site acetylated. CBP/p300 was less discriminate: - - - of cell signaling, if it activates the wrong molecules until it pro- been observed in experiments. it tagged four lysines on H4 simultaneously, although at different vokes a disease, the thing to do is to guide it along the right rates. Overall, the projects help set the stage for the larger project path. Achieving that will require a map of the steps by which it Another set of experiments showed that acetylation and phos- Philipp has in mind – to track a signal from its origins all the way to should achieve its proper effects. That plan is still incomplete, phorylation could be detected simultaneously, with this preci- A study of two deacetylases also revealed that the molecules pre- genes. The same basic procedure can be applied to all signaling Philipp admits, but step by step, his group is putting in place sion, in a single molecule. The recipient the scientists chose was ferred to remove tags from some sites in the histones rather than molecules that are phosphorylated or acetylated, or that receive the missing pieces.

24 REPORTERS IN THE LABYRINTH REPORTERS IN THE LABYRINTH 25 MANAGING THE

CHANNELS OF PERCEPTION

AND THE CHEMICAL

WORKPLACES OF THE CELL

- - -

Most people don‘t associate the cells in their bodies with the cell phones in their pockets, but the functions of both depend on electrical charges. A cell phone has a battery whose power drains over time and has to be recharged. A living cell such as a neuron depends on a balance of charged particles called ions that are drawn in and out. This process is essential to balancing the cell‘s internal environment with that of its surroundings and to the transmission of nerve impulses – every time you move your muscles, hear a sound, run your fingers over a surface to feel its texture, or are struck by pain. A balance (or imbalance) is achieved through the work of proteins in cell membranes that provide channels through which ions are shuttled in and out, or which pump charged particles “upstream” against an unfavorable current. Charge is also an issue for compartments inside a cell, which provide specialized environments in which enzymes can carry out their tasks. Here, too, proteins such as channels and transporters play an essential role.

For many years Thomas Jentsch‘s lab at the FMP and MDC has been systematically studying the membrane molecules that perform this electrochemical balancing act. The last two reports from the FMP have followed the lab‘s work on proteins that serve as channels or transporters for ions such as chloride and potassium. The lab continually breaks new ground in explaining the biological functions of such molecules, often demonstrating how defects in their behavior lead to serious diseases. Over the past two years, Thomas and his colleagues have achieved major insights into the way channels and other proteins participate in sense perception and the maintenance of cellular compartments.

HIGHLIGHT 27 3 1 The olfactory mucosa in mice is arranged on the winding structures of the olfactory turbinates. The calcium-activated chloride channel Ano2 (yellow) resides in the olfactory cilia that are located on the very surface of the olfactory mucosa and are in direct contact to the odorant-carrying air. The dendrites and axons of olfactory sensory neurons are shown in red. Blue structures represent nuclei.

2 – 4 Matthias Heidenreich und Gwendolyn Billig

5 In a section of a mouse nose the calcium-activated chloride channel Ano2 has been made visible by labeling with an antibody (green). The Ano2 protein is found in the thin layer of sensory cilia that cover the surface of the main olfactory epithelium (top) and the pheromone-sensing vomeronasal organ 2 4 (bottom). Blue structures represent nuclei. 5

In 2010 the lab published back-to-back papers in the journal Science protein family called ClC-7 which plays a very important role: its loss for chloride diffusion. Instead, it strictly couples chloride transport acidic compartments than in the rest of the cell, to drive the import regarding the way membrane proteins handle chloride ions in estab- in mice or in humans led to defects in lysosomes, neurodegenera- to a countertransport of protons, making ClC-5 an exchanger rather of chloride. If ClC-5 and ClC-7 don‘t function properly, there isn‘t lishing two types of cellular compartments. One of these, called the tion, and an excessive mineralization of bone called osteopetrosis. than a channel. Because it exchanges negative for positive charges, enough chloride in the compartment. And that is clearly important, lysosome, plays a crucial role in breaking down cellular debris and the process efficiently generates electrical current which can com- because it leads to disease.” foreign matter taken up in a process called endocytosis. This is the “The traditional view has been that ClC-7 helps manage levels of acid pensate for the current from the proton pump. The lab showed that - - - job of powerful enzymes inside the compartment that require high in the lysosome,” Thomas says. “But we‘ve found evidence that it is endosomes don‘t acquire their normal levels of acid when ClC-5 is levels of acid to function. actually doing something different. This is crucial to our understand- lost. However, why should nature have chosen such a complicated “This has important implications for our ing of lysosomal storage diseases, in which cells cannot efficiently exchange process, if a simple chloride channel – as the classical understanding of the operation of these cellular Establishing this environment requires snatching protons from out- degrade waste material. This material then accumulates within cells, model suggested – could also do the trick? side the compartment and pumping them inside. Because protons which may eventually lead to their death, as in the case of neurode- compartments and likely many others.” have a positive electrical charge, this would result in a positive volt- generation.” If one could convert these exchangers into pure chloride channels, age inside the compartment, which would very soon inhibit further the scientists believed, they ought to still carry out their functions in - - - pumping. Acidification wouldn‘t be possible without a compensation If ClC-7 were regulating the amount of acid in the compartments, compensating for the rise in charge. If mice with channels – but not for this charge. Over the past several decades, it has been thought then removing the protein ought to cause a change in the acidity exchangers – still developed disease, it wouldn‘t be due to changes Demonstrating that this was the case required the development and that the compensation was achieved by a passive uptake of negative- of the lysosome. But an experiment carried out by Thomas‘ lab in levels of acid; instead, the problem would be that the acquisition improvement of methods to measure chloride concentrations and ly charged chloride ions. In this “classical textbook” model, chloride a few years ago revealed that this wasn‘t the case. The scientists of protons had become uncoupled from the import of chloride. In levels of acid in lysosomes and endosomes. They also needed new was thought to flow through chloride “channels” – proteins in the developed a strain of mouse without ClC-7. Although the loss of the two new studies, headed by postdocs Stefanie Weinert and mathematical models to interpret changes in the concentration of membrane with minute holes through which chloride can pass. the molecule didn‘t change levels of acid in the lysosomes, the Gaia Novarino, the scientists converted the lysosomal ClC-7 and the ions and the behavior of the membranes. animals still developed the symptoms of lysosomal storage diseas- endosomal ClC-5 into “classic” channels. This was possible with only - - - es. Thomas and his colleagues speculated that ClC-7 might have a minute change in the protein: altering only one of the roughly 800 “This has important implications for our understanding of the opera- another important function. amino acids that made up each of the molecules. tion of these cellular compartments and likely many others,“ Thomas “The traditional view has been that ClC-7 helps says. “It suggests that instead of focusing only on changes in acidi- manage levels of acid in the lysosome, but we‘ve found They had a hint about what it might be from previous work on an- “The results were very surprising,” Thomas says. “The alteration in fication as the cause of disease, we need to understand the role of other member of the chloride channel family called ClC-5, found ClC-5 led to mice which developed a phenotype that is the same the accumulation of chloride ions in those compartments. That‘s a evidence that it is actually doing something different.” in compartments called endosomes in kidney cells. Endosomes as you find if they are totally lacking the protein. Converting ClC-7 fundamental change in perspective.” - - - transport proteins that have been taken up at the cell surface to ly- to a ‘channel’ produced the same severe neurodegeneration as in - - - sosomes, which serve as cellular trash bins. Defects in ClC-5 cause the knock-out mouse, with somewhat less severe osteopetrosis. This Members of the family of molecules called CLC anion transporters, the malfunction of the endosomal compartment and lead to Dent‘s demonstrates that the cause of the diseases is not a change in acid Open a biology textbook to the chapter on the senses and you‘ll discovered by Thomas‘ lab, were believed to act as such chloride disease, in which cells don‘t take up wastes properly; instead, pro- levels in the compartments, as most people have thought. We think find a story about the way nerve cells transmit information about channels in lysosomes and other cellular compartments. Thomas’ teins are lost into the urine and patients develop kidney stones. it‘s due to something else: it means that these proteins normally odors to the brain. It usually goes like this: “smelly” molecules lab had shown that lysosome membranes contain a member of this Thomas‘ lab had shown that ClC-5 doesn‘t serve as a passive channel use the downflow of protons, which are more concentrated in these enter the nose and dock onto proteins on the surfaces of neurons.

28 MANAGING THE CHANNELS OF PERCEPTION AND THE CHEMICAL WORKPLACES OF THE CELL MANAGING THE CHANNELS OF PERCEPTION AND THE CHEMICAL WORKPLACES OF THE CELL 29 As a result, ion channels in cell membranes open and allow a pas- Several pieces of evidence showed that the researchers had found the sage of ions, changing the voltage over the outer cell membrane. elusive chloride channel. Björn Schroeder, now a junior group leader This change generates electrical impulses that travel to the next at the MDC, had already proven that Ano2 transported chloride in cell and on to the brain. The ion channel that opens in response response to calcium – so it was the right type of molecule. Now the to “smelly” molecules lets positively charged sodium and calci- scientists labeled it with an antibody that made it visible under the um ions flow into the cell. The entry of calcium has been thought microscope. It appeared to be the only calcium-activated chloride to trigger the opening of another channel by which negatively channel in neurons of the main olfactory epithelium, which is the point charged chloride ions exit the cell, but researchers haven‘t been of arrival for odor molecules. Finally, Gwendolyn and her colleagues able to identify the channel protein. In 2011 Thomas‘ lab found it, demonstrated that in the mouse line which lacked Ano2, these and in the process they encountered a surprise: animals can smell sensory cells no longer generated chloride currents in response to just fine without the channel. high levels of calcium.

“Most researchers have seen the exit of chloride ions as impor- They now had a way to test the importance of chloride in odor detec- tant, probably even crucial, in mammals‘ perception of smells,” tion. They carried out precise measurements of electrochemical stimu- Thomas says. “Presumably its function has been to amplify odor lation in tissue from the main olfactory epithelium of animals without signals. Measurements based on cells from rodents have sug- Ano2. They discovered that the strength of signals was reduced by gested that the release of chloride leads to a five-to-ten-time 40 percent at most – much lower than previous estimates. And the increase in the strength of electrochemical signals.” animals could still detect odors and differentiate between them. In fact, no difference was found between the smell sensitivity of normal - - - mice and those lacking the channel protein. “In understanding the molecular As a result of the study, Thomas says, researchers will have to Section of the mouse hippocampus and KCNQ5 immunostaining. Higher levels of KCNQ5 in mechanics of smell, scientists can now stop rethink the role of chloride in odor reception. “These ions do amplify a hippocampal CA3 pyramidal cells in contrast to lower signal, but much more modestly than people have believed. That levels in CA1 pyramidal cells (dark staining). chasing a Fata Morgana.” boost doesn‘t seem to be necessary for animals to achieve near- - - - normal sensitivity to smells, at least under normal conditions. Interestingly, a few humans who suffer from a condition called von One reason for the focus on chloride channels has surely been Willebrand disease also lack the Ano2 gene. There haven‘t been any the fact that in freshwater animals, this ion is the major player reports about deficits in their ability to smell.” in exciting neurons that transmit information about odors. The cells of mammals accumulate unusually high amounts of it during It‘s possible, he admits, that the channel plays a role in the response “quiet” phases and apparently release it when they are activated. to some odors – that remains to be seen. But it may also simply be a Scientists have identified the molecule that draws the ions into vestige of evolution, predating the rise of mammals. It may have been cells: a co-transporter called Nkcc1. But until now, the channel preserved because its signal-amplifying functions give animals a slight that permits chloride to leave has remained a mystery. evolutionary edge. In any case, Thomas says, it‘s satisfying to have Several different potassium channels are involved in re-establishing called pyramidal neurons, which are among the largest neurons in found the elusive chloride channel. In understanding the molecular the voltage across neuronal membranes after an action potential. the brain, in a region of the hippocampus called CA3. Surprisingly, And questions remained about the impact of chloride on smell, mechanics of smell, scientists can now stop chasing a Fata Morgana. A particular current, leading to what is called medium afterhyper- these neurons also showed a loss of sAHP. It‘s intriguing, Thomas particularly after 2008, when another lab developed a line of polarization (mAHP), is crucial to both to dampening a charge and says, that the same molecule seems to participate in altering two - - - mice that lacked Nkcc1. Without the protein, animal neurons to returning cells to a resting state so that they can fire again. It is types of current that help the cells recover after firing. accumulated much less chloride and thus had little that could accompanied by a somewhat slower response called slow afterhy- “Another project of the group has focused on - - - be released. The researchers confirmed that this caused a drop perpolarization (sAHP). in the strength of electrochemical signaling in the neurons respon- a channel protein called KCNQ5, which permits a “KCNQ5 is the last molecule of the family sible for the reception of smell. However, the animals responded The lab of Roger Nicoll at the University of California in San Fran- passage of potassium ions in nerve cells.” normally to odors. The results might have cast doubts on the cisco (U.S.) and other groups have demonstrated that deleting without a known biological function. Now we have “amplifier” function attributed to chloride, but most researchers - - - other members of the KCNQ family from mice impairs mAHP determined that it has one.” interpreted it differently: in parallel to Nkcc1, cells might have in certain types of cells. In 2010 Matthias Heidenreich and his another mechanism to accumulate chloride. In that case, the Another project of the group has focused on a channel protein called colleagues in Thomas‘ lab mutated KCNQ5 in mice and collabo- - - - ions could still be released to augment the animals‘ sense of KCNQ5, which permits a passage of potassium ions in nerve cells. rated with the Nicoll group to determine whether this protein had smell. While this molecule appears widely in cells throughout the brain, its similar functions. KCNQ5 is found in many other neurons throughout the brain, functions have remained unknown. Its biophysical properties are including other regions of the hippocampus, but the study was Thomas‘ lab has been systematically investigating ion channels, similar to other potassium channels – the KCNQ family of proteins – They discovered that it did – but apparently only in a specific type unable to show any effect on mAHP or sAHP in those cells. “This with a particular focus on chloride channels. Such molecules have which suggested that it might have comparable functions. of cell in the mouse hippocampus. This small structure is known may be because other KCNQ molecules step up to fill in,” he says. been linked to a range of serious diseases; inheriting a defective to be crucial to memory and learning in humans and animals. It is “It‘s known that the different forms can bind to each other to carry version of one of them or losing its functions often causes a dis- “Other members of this family are known to help stop the firing of also the area where neurons produce the most KCNQ5. Injuries to out their functions. KCNQ5 is the last molecule of the family with- ruption of the nervous system. As a part of the group‘s efforts, neurons that have been excited,” Thomas says. “When the action the hippocampus have been linked to amnesia and an inability to out a known biological function. Now we have determined that it PhD student Gwendolyn Billig produced a line of mice lacking a potential of the cells rise – a condition in which they can excite other form long-term memories, as well as defects in spatial navigation. has one, and we are continuing to look for other functions. There protein called Ano2. This molecule belongs to a family of chloride cells – they open an excessive number of channels that permit an is more to come.” channels that open in response to rising concentrations of another influx of sodium. That needs to be counterbalanced, and this happens Electrophysiological recordings from the animals carrying the Billig et al. (2011) Nat. Neurosci. 14:763-769. ion – calcium. through an outward flow of potassium ions.” mutated form of KCNQ5 revealed a significant loss of mAHP in cells Tzingounis et al. (2010) Proc Natl Acad Sci. 22:10232-7.

30 MANAGING THE CHANNELS OF PERCEPTION AND THE CHEMICAL WORKPLACES OF THE CELL MANAGING THE CHANNELS OF PERCEPTION AND THE CHEMICAL WORKPLACES OF THE CELL 31 LEIBNIZ GRADUATE SCHOOL OF MOLECULAR BIOPHYSICS BERLIN

Interview with Bernd Reif, Coordinator RESEARCH

GROUPS

What was the motivation for creating the graduate school? Will there be a “core course” at some point? What “added value” does it bring to the FMP and Leibniz? Every student is expected to attend two 2-week block cours- One of the key aims has been to bring together biophysicists es in a group/field that is not in the primary technique of the in Berlin, who are often using diverse approaches that can be group that the student has joined. This measure should ensure combined effectively to produce stronger science. For me per- that the students acquire a broader background in biophysics sonally, I wanted to help build a critical mass of researchers and are not limited to one technique only. And there is a weekly in this field. Obviously, biophysics is not the main focus of the lecture given by one of the 20 group leaders that all students at FMP. However, it is an essential tool as we try to better under- the FMP have to attend. This structure is helpful in getting every- stand the interactions between proteins and drugs. For the one “on the same page” and finding potential areas of interaction. Leibniz-Gemeinschaft, the program serves to improve communications between the FMP and universities. The FMP (like all Will all graduate students at the FMP eventually be integrated the other Leibniz Institutes) is not able to grant its own PhD. There- into the school, or will some continue to receive their degrees fore, a close collaboration with one or more universities continues from universities or other institutes? Will joint degrees be to remain essential so that students can acquire these awarded? qualifications. Admittedly, this is currently a weak point of the graduate school. For the FMP, it would be better if there were a general, internal gradu- How many students are currently affected, and how do you ate school. However, the research areas that are pursued at the FMP imagine the program will develop in the future in terms of cover a very broad area that includes structural biology, chemistry, numbers of students? biochemistry, molecular biology and cell biology, including animal The program currently involves 20 research groups and, according- experiments. This means that chemists, physicists, pharmacolo- ly, 20 PhD stipends. Currently, funding is available only for 4 years gists and biologists who come from totally different backgrounds (i.e. one group of 20 students every 4 years). But ideally, the pro- are working closely together. While their research interests are gram should start every year and ultimately integrate at least 80 similar (understanding how a small molecule interacts with a pro- students. This, however, would require significant money from other tein), the language and the tools that they employ are really very sources, like scholarships from a DFG program, etc. We are thinking different. The research focus in the new graduate school takes on a of making another attempt at writing a proposal for DFG funding. portion of this spectrum, focussing on biophysics. But by creating a graduate school in the environment of the FMP, students will be ex- How does it compare to graduate schools at the university or posed to other areas that could well become essential to their work. other places? What special features does the FMP have to offer its students? How will the new graduate school affect the process of select- In contrast to most of the other graduate school programs that ing students? I know, the program is not localized at a single, particular insti- In the first year, every PI looks over the applications and chooses tute, but tries to bring scientists working in the field of biophysics one student to invite for interview. I’m pleased to say that we have together from a variety of locations. Without such a program, the had some very good applications, to the extent that there were students would probably never meet, and the potential benefits even some “fights” among the PIs regarding who would get a par- would never be realized. ticular student.

32 LEIBNIZ GRADUATE SCHOOL OF MOLECULAR BIOPHYSICS, BERLIN RESEARCH GROUPS 33 STRUCTURAL BIOLOGY

PROTEIN STRUCTURE

GROUP LEADER Figure 1. Simulated model of Neurotoxin II (PDB Hartmut Oschkinat 1NOR, light blue) bound to nACh-receptors (gray) in native membranes (dark blue). The biradical, required for DNP-NMR, is inactive (non-luminous orange rods) at the surfaces of the analyte, but still active (luminous orange rods) in the surrounding solvent. This radical- Figure 2. Solid-state NMR spectra of 1H-15N correlations in ArtMP. The left spectrum, measured with cross-polariza- distribution leads to high signal enhancements of tion, shows the signals of the ArtMP complex, whilst the right spectrum, recorded with INEPT, shows selectively the well-resolved signals. signals of the ATP binding cassette protein.

Magic-angle-spinning (MAS) solid state NMR delivers high-resolution Dynamic Nuclear Polarization the nicotinic acetylcholine-receptor (nAChR) suitable for medium-sized membrane pro- Methods enabling structural studies of mem- in native membranes (Fig. 1). We saw that teins, we investigate the structure of OmpG in structural information on complex, heterogeneous samples, brane-integrated receptor systems without TOTAPOL, a biradical required for DNP, lo- lipid bilayers. A number of studies concerning independent of molecular weight and without the necessity of the necessity of protein purification or the de- calizes at the membrane and protein surfaces. labeling strategies, flexibility of the loop form- termination of structures of proteins bound to The concentration of active, membrane-at- ing the lid of the porin, and of suitable pulse crystallization. It is an attractive method for structural investigations the cytoskeleton offer attractive prospects in tached biradical decreases with time, prob- sequences, are ongoing. of ‘difficult’ systems, such as proteins embedded in lipid bilayers structural biology. Dynamic nuclear polariza- ably due to reactive components of the mem- tion (DNP) magic angle spinning NMR allows brane preparation. The presence of inactive Interfering with protein-protein interactions or attached to the cytoskeleton. We aim, in the long run, at the investigation of such systems, delivering TOTAPOL in membrane-proximal situations, In a third line of projects, we search for small- structural investigations within the ‘real space’ of a cell, capitalizing the required sensitivity. With this method, the but with active biradical in the surrounding molecule inhibitors of protein-protein interac- very strong polarization of electron spins is water/glycerol ‘glass’, leads to well-resolved tions, using PDZ (PSD-95, Dlg, ZO-1) domains also on a 20–100 fold increase in signal-to-noise through dynamic transferred to nuclear spins, which can then spectra concomitantly benefitting from a con- as an example. They play important roles in nuclear polarization (DNP). Our short-term aims are a structural be detected at much higher signal-to-noise. siderable enhancement (e=12) is observed. cellular signaling pathways and are structur- We have a number of ongoing studies using The resulting spectra are, to our knowledge, ally characterised by a hydrophobic pocket description of the transport cycle of an ABC-transporter embedded DNP and involving crystalline preparations the first to show resolved signals of a protein surrounded by a conserved sequence motif, in lipid bilayers, and the determination of hormones bound to of soluble proteins (SH3 domain), membrane ligand bound to receptors in native mem- G-L-G-F. This pocket binds the C-termini of proteins (neurotoxin II bound to the nico- brane patches while applying DNP conditions. target proteins, in most cases receptors and receptors without purification of the respective membrane protein. tinic acetyl choline receptor, OmpG, mistic), ion channels. Their functional diversity and amyloid fibrils (Stefin B) and even selectively Structures of membrane proteins in native characteristic, relatively small binding pocket, labeled ribosomes. Initial results from these lipids by solid-state NMR make them attractive targets for the design studies include the enhancement of sensitiv- Four membrane protein systems are investi- of small-molecule inhibitors which may, in the HARTMUT OSCHKINAT ity through additional application of protein gated in our lab. We want to determine the long run, allow for the treatment of several

deuteration, studies of the effects of low structure of the hormone endothelin bound PDZ-related human disorders such as neuro- 1975 – 1976 and 1978 – 1983 Chemistry 1986 – 1987 Postdoctoral work with Since 1998 Head of the department temperatures on protein structures, and first to its receptor and of toxins bound to the pathic pain, congenital diseases, psychiatric degree at the University of Frankfurt; Prof. Dr. Bodenhausen at the University “NMR-supported Structural Biology“ at the assignments of residues in the nascent pro- acetylcholine receptor. Furthermore, we pre- disorders, and cancer. A large number of high- Diploma of Lausanne, Switzerland Leibniz-Institut für Molekulare Pharmakolo- tein chain emanating from the ribosome. In a pared the ABC-transporter ArtMP from geo- resolution structures of PDZ-ligand complexes gie, Professor of Structural Chemistry at the 1983 – 1984 Visit to the laboratory of 1987 – 1991 Position as NMR-spectrosco- nutshell, the enhancements achieved by DNP bacillus stearothermophilus to investigate provide an excellent basis for rational design. Free University in Berlin Prof. Dr. Ray Freeman, Oxford, England pist at the Max-Planck-Institute for Bio- make possible experiments previously out of in detail the structural changes upon ATP Inhibitors with low to medium affinity for sev-

chemistry (Martinsried, Germany), first in the Since 2009 Acting Director of the Leibniz- reach; however the situation is complicated by hydrolysis during the transport cycle in a na- eral PDZ domains were identified and mem- 1983 – 1985 Completion of dissertation in Clore/Gronenborn group, later independent- Institut für Molekulare Pharmakologie a multiplication of resonance lines in the spec- tive lipid environment. Deuterated samples bers of the respective substance classes were Prof. Kessler’s Laboratory at the University ly in the department of Prof. Huber (FMP), Berlin tra known as ‘inhomogeneous broadening’. of ArtMP have been prepared, and CP- and collected in a ‘PDZ-library’. Improvements of of Frankfurt This observation calls for an improvement of J-coupling-based HN-correlations were show- the AF6 PDZ domain inhibitors by modeling- 1992 Habilitation in Biophysical Chemistry 1986 Graduate thesis: “Analysis of the sample preparation conditions – e.g. applying either the signals of the whole transporter chemistry cycles resulted in compounds with at the Technical University of Munich conformation of Cyclosporin in solution ing shock freezing techniques – and spec- or selectively the ATP binding cassette pro- ten to twenty micromolar dissociation con- using NMR-spectroscopy: development 1992 – 1998 Group leader at the EMBL, troscopic means of handling this situation. tein, respectively (Fig.2). This offers tremen- stants, disrupting the AF6-Bcr interaction in and use of new methods” Heidelberg In one of these pilot studies we were able to dous potential for functional studies by al- cell lysates. We intend to exploit our results

solve some of these problems. We observed lowing for the editing of subunits on a simple further by targeting three PDZ domains (AF6, well-resolved solid-state NMR spectra of ex- spectroscopic level. As a pilot project for de- DVL, Shank3), as we seek an understanding of tensively 13C labeled neurotoxin II bound to veloping a structure determination concept the biology of the respective proteins.

34 RESEARCH GROUPS // STRUCTURAL BIOLOGY RESEARCH GROUPS // STRUCTURAL BIOLOGY 35 Fom left to right: Barth-Jan van Rossum, Sascha Lange, Trent Franks

Gregorio de Palma, Florian Seiter

Liselotte Handel, Britta Kunert, Anja Voreck

MEMBERS OF THE GROUP Arne Linden (doctoral student), EF, PT, * SELECTED PUBLICATIONS COLLABORATIONS EXTERNAL FUNDING Europäische Kommission

“European Drug Initiative on Channels Stefan Markovic (doctoral student), EF, PT, * Jehle S, Rajagopal P, Bardiaux B, Markovic International Deutsche Forschungsgemeinschaft and Transporters” Dr. Ümit Akbey Gregorio de Palma (doct. student), EF, PT, * S, Kühne R, Stout JR, Higman VA, Klevit RE, “Bestimmung der Raumstrukturen von EDICT/Health-F4-2007-201924 A. Arseniev Dr. Linda Ball, EF, * van Rossum BJ, Oschkinat H (2010) Rezeptoren sowie gebundener Agonisten 02.2008 – 01.2012, 179.325 Euro Arndt Pechstein (doctoral student), EF, PT, * Shemyakin Institute Moskau, Russland Solid-state NMR and SAXS studies provide und Antagonisten mittels Festkörper- “Structural Biology of Membrane Proteins” Dr. Benjamin Bardiaux Joren Sebastian Retel (doct. student), EF, PT, * a structural basis for the activation of alphaB- I. Bertini, Florence University, Italy NMR-Spektroskopie” SBMPs/PITN-GA-2008-211800

Dr. Victoria Ann Davies, EF, * crystallin oligomers. SFB 449, TP B1, with B. v. Rossum 09.2008 – 08.2012, 475.244 Euro Florian Seiter (doctoral student), PT, * L. Emsley, ENS Lyon, France Nat Struct Mol Biol 17: 1037-1042 01.2002 – 12.2010, 526.746 Euro “Exploiting the Potential of Structural Dr. Anne Diehl (laboratory head) Ahmad Shahid Shakeel (doct. student), EF, PT, * A. Fersht, MRC-LMB, Cambridge, UK “Multivalenz als chemisches Organisations- Biology through NMR and Associated Tech- Akbey U, Franks WT, Linden A, Lange S, Dr. Trent Franks und Wirkprinzip: Neue Architekturen, nologies. Opportunities for the Economic Anja Voreck (doctoral student), PT, * Griffin RG, van Rossum BJ, Oschkinat H R. G. Griffin, MIT, Cambridge, USA Funktionen und Anwendungen“ Development of the Biotechnology and Dr. Matthias Hiller, EF, PT (2010) Dynamic nuclear polarization of Anne Wartenberg (doct. student), EF, PT,* H. de Groot SFB 765, TP C4, with C. Freund Pharmaceutical Industries in Tuscany, Berlin- deuterated proteins. Dr. Stefan Jehle, * Leiden University, The Netherlands 01.2007 – 12.2011, 291.200 Euro Brandenburg and Beyond.” Janet Zapke (doctoral student), EF, PT, * Angew Chem Int Ed Engl, 49: 7803-7806 “Modulation of PDZ-domain-mediated EPISODE/FP7-REGIONS-2008-229761 Dr. Jana Körner, EF, PT, * R. Klevit Nils Cremer (technical assistant), EF, * Lange V, Becker-Baldus J, Kunert B, van protein-protein interactions” 01.2009 – 12.2011, 107.505 Euro University of Washington, Seattle, USA Dr. Vivien Lange, EF, PT, * Rossum BJ, Casagrande F, Engel A, Roske Y, FOR 806, TP 05 (OS 106/7-1,7-2) “Focusing NMR on the machinery of life” Natalja Erdmann (technical assistant), EF Scheffel FM, Schneider E, Oschkinat H C. Luchinat, Florence University, Italy with G. Krause, J. Rademann LSHG-CT-2005-018758 Dr. Nikolaus Loening, EF, * Lieselotte Handel (technical assistant), PT (2010) A MAS NMR study of the bacterial 03.2010 – 02.2013, 682.000 Euro 12.2005 – 02.2009, 34.185 Euro M. Macias, University of Barcelona, Spain Dr. Silke Radetzki, PT ABC transporter ArtMP. “Analyse von Wechselwirkungen in Martina Leidert (technical assistant), PT Land Berlin Chembiochem 11: 547-555 R. W. Martin β-Faltblattstrukturen ausgesuchter WW- Dr. Barth-Jan van Rossum “Anwendung eines Hochzelldichte-Fermen- Thao Thi Bich Nguyen (techn. assistant), PT * University of California, Irvine, USA Domänen durch den Einbau synthetischer Akbey U, Oschkinat H, van Rossum BJ tationssystems zur effektiven Produktion Dr. Monica Santos de Freitas, EF, * Dipeptidisostere” Kristina Rehbein (technical assistant), PT (2009) Double-nucleus enhanced A. Milon, Universtity Toulouse, France von Proteinen in Insektenzellkultur” OS 106/5-5 Dr. Tobias Werther, EF; * recoupling for efficient 13C MAS NMR IBB 10138845, A. Diehl Sarah Wistuba M. Waterman 09.2007 – 09.2009, 2.300 Euro correlation spectroscopy of perdeuterated 01.2008 – 02.2010, 133.170 Euro Francesca Camponeschi (technical assistant apprentice), * Vanderbilt University, Nashville, USA “Eine erste MAS-NMR-Struktur eines proteins. J Am Chem Soc 131: 17054-17055 “HIF-1alpha Stabilisatoren” (doctoral student), EF, PT, * Membranproteins: Die Struktur von OmpG Katja Riemann (student), PT, * National IBB 10138782 Krabben L, van Rossum BJ, Jehle S, und die Dynamik der Porenöffnung in Anup Choudhury (doctoral student), EF, PT, * 12.2007 – 01.2009, 65.149 Euro Kristian Händler (student), PT, * Bocharov E, Lyukmanova EN, Schulga AA, W. Baumeister nativer Lipidumgebung”

Feng Ge (doctoral student), EF, PT, * Arseniev A, Hucho F, Oschkinat H (2009) MPI für Biochemie, Martinsried OS 106/9-1 Alexander v. Humboldt-Stiftung Philip Mauch (student) PT, * Loop 3 of short neurotoxin II is an additional 11.2007 – 10.2009, 147.520 Euro Forschungskostenzuschuss an Gastgeber Jan Hendrik Holtmann (doct. student), EF, PT, * B. Bukau, Universtiät Heidelberg interaction site with membrane-bound “Interfering with intracellular protein- von Stipendiatin M. Santos de Freitas Nestor Kamdem (doctoral student), EF, PT, * nicotinic acetylcholine receptor as detected T. Carlomagno, EMBL, Heidelberg protein interactions – probing protein 11.2008 – 10.2010, 19.200 Euro

* Part of period reported by solid-state NMR spectroscopy. functions with small molecules” Christian Köhler (doctoral student), EF, PT, * W. Kühlbrandt, MPI für Biophysik, Frankfurt Novo Nordisk PT part time J Mol Biol 390: 662-671 FOR 806, TP Z2 (OS 106/11-2) “Development of new methods for Britta Kunert (doctoral student), PT; * EF position funded externally T. Müller, Universität Würzburg 03.2010 – 02.2013, 145.250 Euro characterization of membrane proteins” (3rd-party funds) for at least part Daniela Lalli (doctoral student), EF, PT, * G. Radziwill, Universität Freiburg 07.2008 – 06.2010, 54.000 Euro of the reporting period FMP authors Sascha Lange (doctoral student), EF, PT, * Current group members (31.12.2010) Group members H.-G. Schmalz, Universität zu Köln

36 RESEARCH GROUPS // STRUCTURAL BIOLOGY RESEARCH GROUPS // STRUCTURAL BIOLOGY 37 STRUCTURAL BIOLOGY

PROTEIN ENGINEERING

GROUP LEADER Christian Freund Figure 1. Comparison of the Smy2 (A) and CD2BP2- GYF (B) domains. Aspartate 22 in Smy2 is held away from the binding pocket and allows for the formation of an extended cleft accommodating a hydrophobic amino acid of the ligand. In CD2BP2 (B), the same cleft is closed by the bulky tryptophane 22.

Our goals relate to the assembly process of multiprotein complex- Proline-rich sequence recognition. was identified as a direct interactor and cor- We recently determined the three-dimen- roborates a direct link between the ADAP- es. On the one hand this involves the transient interactions mediated sional structure of the Smy2-GYF domain SKAP55 module and the actin cytoskeleton. by proline-rich sequences in the spliceosome. Adaptor domains in complex with a peptide from the yeast branch-point binding protein. This is the first MHC-peptide interactions such as GYF and WW are critical for the formation of the early complex structure of the larger Smy2-GYF We recently obtained the first NMR back- spliceosome, and the structural prerequisites of individual domain- domain family and it highlights the subtle bone assignment of an MHC class II mol- differences between Smy2- and CD2BP2- ecule and are now in the position to monitor peptide interactions have been elucidated in our own lab and many type GYF domains. We have, furthermore, peptide binding and exchange at atomic other labs. However, the importance of multivalent interactions for used pulldown experiments in combina- level in solution. We are not only interested tion with stable isotope labelling of amino in the exchange dynamics, but also want to defining specificity and assembly dynamics is poorly understood acids in cell culture (SILAC) to delineate understand the mechanism of how the natu- and is one focus area of our research. Dynamic rearrangements are the putative proline-rich interaction part- ral exchange catalyst HLA-DM (an MHC-like ners of mono- and bi-functional GYF, WW molecule) catalyses the process at acidic pH CHRISTIAN FREUND also center-stage in another project where we want to understand and UEV domains. For CD2BP2-GYF and values (Figure 2). Another interesting obser-

the influence of post-translational modifications on membrane Tsg101-UEV, we have deconvoluted the rela- vation emerges from the fact that the place- 1983 – 89 Studied chemistry tive importance of the proline-rich binding holder peptide CLIP (processed from the so- in Düsseldorf and München recruitment of an integrin-regulating intracellular complex in T cells. site relative to a second protein interaction called invariant chain chaperoning the MHC 1990 – 1994 Ph.D. at the Max-Planck- SH2 domain and phosphoinositol-mediated networks are investi- site. Futhermore, we could show that Smy2 on its way to the late endosomes) can bind in Institute of Biochemistry, Martinsried and its human counterpart GIGYF2 are as- two perpendicular orientations to the MHC gated by SILAC-MS, biophysical methods and functional interfer- (Dr. Tad Holak, Dr. Wolfram Bode) sociated with mRNA surveillance factors. The class II molecule DR1. The question arises Figure 2. (A) Superimposed regions of 1H-15N HSQC spectra showing HLA-DR1(14Na/15Nb)/CLIP when ence. A third topic in the group is related to antigen-presentation localization of the latter protein to cytoplas- of whether this is a more common phenom- 102-120 1994 – 1997 Post-Doc at the University freshly co-refolded (blue) or at indicated time points mic granula upon arsenite treatment of HeLa enon; if so, this will be of importance for epi- of Zürich (Prof. Plückthun), Zürich by MHC class II molecules. Loading and exchange of self- and (red). Representative resonances indicate an increas- cells suggests that it plays a role in the stress tope prediction and for the potential breaking population of inverted CLIP102-120 over time. Rapid 1997 – 2000 Post-Doc at Harvard antigenic peptides is probed by NMR spectroscopy in the presence response of mammalian cells. age of self-tolerance in the periphery, in inter-conversion of the same resonances is seen when equimolar amounts of HLA-DM were added Medical School (Prof. Wagner and cases where peptide processing and display of natural and non-natural exchange catalysts. prior to spectral acquisition (right). Prof. Reinherz), Boston ADAP-SKAP55 complex differs between thymus and peripheral sites. (B) Superimposed 1H-15N-HSQC spectra showing We delineated the individual tyrosine phos- In addition, we have started to use Xe- HLA-DR1(14Na/15Nb)/CLIP representing canoni- Since 2000 Group Leader at the FMP 102-120 phorylation sites of the T cell scaffolding NMR to monitor the binding of antigenic cal CLIP (blue), HLA-DR1/CLIP106-120 representing inverted CLIP (red), and spectra of these two protein ADAP by mass spectrometry (with peptide to MHC molecules. Eventually samples after HLA-DM catalyzed exchange against E. Krause, FMP). We experimentally tested this will allow for the magnetic resonance HA306-318 (light and dark grey, respectively). The latter the role of individual tyrosine motifs by imaging of MHC molecules in cell cultures two spectra superimpose well indicating the formation of HLA-DR1/HA from both HLA-DR1/CLIP overexpression of the corresponding mu- and in mouse models of immune diseases. 306-318 complexes. tants in Jurkat T cells, and we subsequently (C) Model for flipping and exchange of peptide, measured adhesion and migration (with S. Finally, we have used phage display in vari- as it is catalyzed by HLA-DM. The N-terminus of CLIP Kliche, Univ. Magdeburg). The phosphory- ous projects to delineate peptide binders is indicated in green and binding can occur in two lation of certain tyrosines is critical for ad- of adaptor domains, or conversely we have orientations. Both MHC-CLIP-complexes can exchange against the viral hemagglutinin antigen hesive strengthening, and these motifs bind evolved adaptor domains as alternative scaf- HA, that binds to HLA-DR1 in a single canonical to SH2 domain-containing proteins. Nck folds able to interact with a variety of targets. orientation.

38 RESEARCH GROUPS // STRUCTURAL BIOLOGY RESEARCH GROUPS // STRUCTURAL BIOLOGY 39 From left to right: Sebastian Günther, Daniela Kosslick

Fabian Gerth

Gesa Albert

Andreas Schlundt

MEMBERS OF THE GROUP SELECTED PUBLICATIONS COLLABORATIONS EXTERNAL FUNDING

Dr. Kirill Piotukh Günther S, Schlundt A, Sticht J, Roske Y, Kofler M, Schümann M, Merz C, Kosslick International BMBF-Innovationspreis Medizintechnik Deutsche Forschungsgemeinschaft

Heinemann U, Wiesmüller KH, Jung G, D, Schlundt A, Tannert A, Schaefer M, “Biosensor-basierte 129Xe-Magnetreso- „Multivalente Protein-Protein-Interak- Dr. Jana Sticht, PT Christoph Wülfing Falk K, Rötzschke O, Freund C (2010) Lührmann R, Krause E, Freund C (2009) nanz-Tomographie in Zellen und Maus- tionen zwischen WW-Domänen und UT Southwestern Medical Center Gesa Albert (doctoral student), PT Bidirectional binding of CLIP peptides to Proline-rich sequence recognition I: Marking modellen der Autoimmunität“ Prolin-reichen Segmenten“

an MHC class II molecule. GYF and WW domain assembly sites in early Ellis Reinherz with Physikalisch-Technische Bundesanstalt, SFB 765, TP C4 Sebastian Günther (doctoral student), PT Proc Natl Acad Sci (USA) 107: 22219-24 spliceosomal complexes. Harvard Medical School Berliner Institut, AG MR-Messtechnik with H. Oschkinat

Daniela Kosslick (doctoral student), PT Mol Cell Proteom 8: 2461-2473 05.2010 – 05.2013 01.07 – 12.2010 Ash MR, Fälber K, Kosslick D, Albert GI, John Gross 534.976 Euro 291.200 Euro Roland Lehmann (doctoral student), PT Roske Y, Kofler M, Schümann M, Krause E, Schlundt A, Kilian W, Beyermann M, University of California, San Francisco

Freund, C (2010) Conserved β-hairpin Sticht J, Günther S, Höpner S, Falk K, Bundesministerium für Bildung und Deutsche Forschungsgemeinschaft Bernhard Meineke (doctoral student), PT, * Balaji Prakash recognition by the GYF domains of SMY2 Rötzschke O, Mitschang L, Freund C (2009) Forschung “Regulatory phosphorylation of Indian Institute of Technology, Kanpur Andreas Schlundt (doctoral student), PT and GIGYF2 in mRNA surveillance and A Xenon-129 biosensor for monitoring “Structural and biophysical investigations scaffolding proteins of the inside-out-

vesicular transport complexes. MHC-peptide interactions. Olaf Rötzschke of MHC-ligand interactions” signalling complex” Marc Sylvester (doctoral student), PT, * Structure 18: 944-954 Angew Chem Int Ed 48: 4142-5 Singapur Immunology Network 01GU0514-FR SFB 740, TP D4 (FR 1325/4-1)

Marek Wieczorek (doctoral student), PT, * 01.2006 – 05.2009 with T. Höfer (DKFZ) Sylvester M, Kliche S, Lange S, Geithner S, National 174.526 Euro 01.2007 – 12.2010 Fabian Gerth (student), * Klemm C, Schlosser A, Großmann A, Stelzl U, FMP authors 82.500 Euro Schraven B, Krause E, Freund C (2010) Group members Burkhart Schraven Deutsche Forschungsgemeinschaft Stefan Klippel (student), * Adhesion and degranulation promoting Immunology, Universität Magdeburg “Analysis and inhibition of GYF domain

Kathrin Motzny (technical assistant), PT adapter protein (ADAP) is a central hub for mediated protein interactions” Guido Stoll phosphotyrosine-mediated interactions in FOR 806, TP 04 (FR 1325/3-1) Sindy Blank (technical assistant), *, PT Neurology, Universität Würzburg T cells. PlosOne 5: e11708 01.2007 – 01.2010

Christian Spahn 249.000 Euro

* Part of period reported Charité – Universitätsmedizin Berlin Deutsche Forschungsgemeinschaft PT part time Udo Heinemann “Assemblierung und Membran-Rekrutie-

MDC Berlin rung des ADAP/SKAP55-Komplexes” EF position funded externally SFB 854, TP12 (3rd-party funds) for at least part with B. Schraven, S. Kliche of the reporting period 01.2010 – 12.2013 Current group members (31.12.2010) 177.600 Euro

40 RESEARCH GROUPS // STRUCTURAL BIOLOGY RESEARCH GROUPS // STRUCTURAL BIOLOGY 41 STRUCTURAL BIOLOGY

STRUCTURAL BIOINFORMATICS AND PROTEIN DESIGN

GROUP LEADER Gerd Krause Figure 1. Flow chart showing our web-accessible interactive resource system for the sequence-structure- function analysis of glycoprotein hormone receptors (GPHR), which is fed by data from more than 1100 mutations, by sequences/sequence alignments, and by protein structures using homologous structural models.

The group focuses on the sequence-structure analysis of proteins On the one hand we are developing bioin- fects. The strength of this database and together with NIH cooperation partners. We formatic tools (Worth et al. 2009; Schillinger these web-tools lies with the comprehensive have now proceeded to mutate all 30 resi- using structural bioinformatics combined with experimental et al. 2009) for investigating such structure- GPHR dataset of unified scaled mutation dues surrounding this allosteric small ligand functional studies of changed sequence(s) to reveal structure- function relationships; on the other hand we data and the link between structural infor- binding site and delineated distinguishing are applying them to particular molecular mation and functional data that permits a features for small molecule agonist and an- function relationships of proteins and potential interaction partners. biological projects of protein/ligand, pro- semi-quantitative structure function analysis tagonist binding modes. For verification of structure-function hypotheses we perform tein/substrate or protein/protein interac- (Kleinau & Kreuchwig et al. 2010). tions and to verify the biological function With our combined strategy we deduced de- As part of an integrated FMP project, and experimentally site-directed mutagenesis of selected particular by site-directed mutagenesis. Bioinformatic terminants of i) protein interaction between in cooperation with the group of R. Schül- residues and are analyzing available mutation data to deduce tool/database development and molecular claudins and C. Perfringens enterotoxin ein, we yielded two types of mutations. (A) biology applications mutually support one to modulate tight junctions (Winkler et al. Constitutive active mutants (CAMs) indicate structure-function relationships. another. 2009, Veshnyakova et al. 2010, cooperation residues of signalling sensitive positions Amino acid side-chain substitutions that with J. Piontek), ii) inter- and intramolecular (Kleinau & Haas 2010) and mark potential modify the receptor phenotypes are of activation mechanisms of GPCRs such as for trigger points for receptor activation by GERD KRAUSE The main aims of the group are: great importance for the elucidation of endothelin receptor subtypes ET and ET LMW agonists (Neumann et al. 2009). Since A B structural-functional properties of the wild- (Lättig et al. 2009), and extensively for the TSHR is characterized by a high level of basal 1970 – 1975 Studied chemistry at the • A detailed understanding of intramolecular mechanisms type receptor. Utilizing the huge amount of thyroptropin receptor (TSHR). activity, we found second type (B) mutants University in Leipzig of membrane proteins functional data available from both naturally with impaired basal cAMP signalling activity, 1982 Ph.D. in biochemistry at the occurring and designed mutations (> 1100) For the extracellular region of TSHR (hor- which constrain the TSHR in an inactive con- • The rational discovery of molecular mechanisms and sites Martin-Luther-Universität Halle-Wittenberg in the Glycoprotein hormone receptors mone TSH binding) we not only identified a formation (Haas et al. 2010). They indicate

for protein-protein interactions and protein-ligand or (GPHRs), we developed an online resource second binding site in the hinge region for preferred contact points, where potential 1982 – 1986 Worked in a research unit system for a sequence-structure-function bovine TSH (Müller & Kleinau et al. 2009), inactivating molecules can block receptor in pharmaceutical industry protein-substrate interactions analysis of glycoprotein hormone receptors but also summarized our proposed extra- activation. These comprehensive 3D phar- 1986 – 1991 Research Position at the • Narrowing down locations for potential pharmacological (www.ssfa-GPHR.de). In this recently up- cellular activation mechanisms, and others, macophore patterns covering the allosteric Institute of Drug Design, Berlin dated web resource we describe new opin a highly ranked review journal (Kleinau & ligand binding site will serve in the future interventions to the amino acid and atomic level. tions for interactive data analyses within Krause 2009). as a basis for rational antagonist develop- 1991 – 1992 Visiting Scientist at the three-dimensional structures. The system ment by distinguishing between essential Washington University (Prof. Marshall) allows for the comparison and classification At the intracellular region, we provided the recognition patterns for small agonistic and in St. Louis, MO, USA of data from different GPHR subtypes and first complete map of contact points for a antagonists. 1992 – 1997 Project Leader at the from different experimental approaches. GPCR regarding preferences to the G-pro- Forschungsinstitut für Molekulare Furthermore, we provide new tools for data tein subtypes Gs and Gq, and revealed that Pharmakologie, Berlin visualization and interpretation, such as re- the interface for binding Gs is a subset of

ceptor morphing and interactive selection of that for Gq. (Kleinau et al. 2010) Since 1998 Group Leader of Structural particular amino acid positions within a 3D Bioinformatics and protein design at structure or homology model, which simu- At the transmembrane region, and in con- the FMP late the intramolecular activation process trast to the extracellular orthosteric bind- at the 3D protein structure and provides ing site of the endogenous hormone, an the user with information about side-chain allosteric binding site for small molecules variations and their particular functional ef- at the TSHR was previously identified by us,

42 RESEARCH GROUPS // STRUCTURAL BIOLOGY RESEARCH GROUPS // STRUCTURAL BIOLOGY 43 From left to right: Gerd Krause, Jonas Protze, Franziska Kreuchwig, Annika Kreuchwig

Anita Kinne, Inna Hoyer, Ann-Karin Haas

Inna Hoyer

MEMBERS OF THE GROUP SELECTED PUBLICATIONS COLLABORATIONS EXTERNAL FUNDING

Dr. Sebastian Müller, EF, * Kleinau G, Haas A-K, Neumann S, Worth CL, Neumann S, Huang W, Titus S, Krause G, International Deutsche Forschungsgemeinschaft Deutsche Forschungsgemeinschaft

Hoyer I, Furkert J, Rutz C, Gershengorn M, Kleinau G, Zeng W, Southall NT, Inglese J, “Modulation of PDZ-domain-mediated “Differenzierung molekularer Determi- Dr. Gunnar Kleinau, EF Marvin Gershengorn Schuelein R, Krause G (2010) Signaling- Austin CP, Gavrilova O, Thomas CJ, Raaka BM, protein-protein interactions” nanten der G Protein Selektivität des Susanne Neumann Dr. Catherine Sargent, EF, * sensitive amino acids surround the allosteric Gershengorn MC (2009) Small molecule with H. Oschkinat, G. Krause, J. Rademann TSH-Rezeptors” NIH Bethesda, MD, USA ligand binding site of the thyrotropin agonists for the thyrotropin receptor 01.2007 – 01.2010 KR 1273/2-1 V Dr. Anna Veshnyakova, EF, * receptor. FASEB J 24 (7): 2347-54 stimulate thyroid function in human 342.000 Euro 11.2008 – 11.2009 National Ann-Karin Haas (doctoral student), PT, * thyrocytes and mice. 60.000 Euro Kleinau G, Kreuchwig A, Worth CL, Deutsche Forschungsgemeinschaft Proc Natl Acad Sci 106 (30): 12471-12476 Michael Fromm Jonas Protze (doctoral student), PT, * Krause G (2010) An interactive web-tool for “Modulatoren für den Thyrotropin- Deutsche Forschungsgemeinschaft Heike Biebermann molecular analyses links naturally occurring Schillinger C, Boisguerin P, Krause G (2009) Rezeptor: Molekulare Mechanismen al- “Molekulare und strukturelle Muster para- Christian Schillinger Josef Köhrle mutation data with three-dimensional Domain Interaction Footprint: a multi- losterischer Bindung und Wirkungsweise zellulärer Poren durch subtypabhängige (doctoral student), EF, PT, * Ulrich Schweizer structures of the rhodopsin-like Glycoprotein classification approach to predict domain- kleiner Moleküle” Wechselwirkungen in tight junctions” Charité – Universitätsmedizin Berlin Paul Grzesik (doctoral student), EF, PT, * hormone receptor. peptide interactions. KR 1273/4-1 FOR 721 TP 6 (KR 1273/3-1)

Human Mutation 31 (6): E1519-25 Bioinformatics 25 (13): 1632-9 Ralf Paschke 01.2010 – 12.2012 with P. Schmieder Annika Kreuchwig Leipzig 224.250 Euro 11.2006 – 11.2009 (doctoral student), EF, PT, * Kleinau G, Jaeschke H, Worth CL, Mueller S, 263.000 Euro Gonzalez J, Paschke R, Krause G (2010) FMP authors Deutsche Forschungsgemeinschaft Inna Hoyer (doctoral student), EF, PT, * Principles and determinants of G-protein Group members “Funktion und Struktur von rezeptorin- Deutsche Forschungsgemeinschaft Anita Kinne (doctoral student), EF, PT, * coupling by the rhodopsin-like thyrotropin ternem Transmitter und multipler intra- “Molekuare und strukturelle Muster para-

receptor. PLoS One 5 (3): e9745 molkularer Aktivierungsmrechanismus zellulärer Poren durch subtypabhängige Franziska Winkler (student), PT, * des TSH Rezeptors” Claudin-Claudin-Wechselwirkungen in FOR 721 TP 6 (KR 1273/3-1) tight junction” * Part of period reported 09.2008 – 08.2010 FOR 721 (KR 1273/3-2)

71.500 Euro with J. Piontek PT part time 01.2010 – 12.2012 Deutsche Forschungsgemeinschaft EF position funded externally 315.840 Euro “Differenzierung molekularer Determi- (3rd-party funds) for at least part nanten der G Protein Selektivität des of the reporting period TSH-Rezeptors” Current group members (31.12.2010) FOR 721 TP 6 (KR 1273/2-1) 05.2007 – 05.2009 93.318 Euro

44 RESEARCH GROUPS // STRUCTURAL BIOLOGY RESEARCH GROUPS // STRUCTURAL BIOLOGY 45 STRUCTURAL BIOLOGY Figure 1. Effect of KU-1 with com- pletely replaced PPII core motif on the actin filament synthesis of MDA-MB-231 COMPUTATIONAL cells. (A) + PBS (B) + KU-1 (cyan: core; yellow: actin CHEMISTRY/ fibres; red: VASP). Untreated cells show that DRUG DESIGN VASP is concentrated at the focal adhesion points (yellow arrow) and the tips of the filopodia (green GROUP LEADER arrow). KU-1 causes a Ronald Kühne delocalization of the VASP. The compound containing the poly-proline core motif did not change either the actin filaments or VASP localization. (A) (B)

The rational design of compounds which bind to biologically Development of ligands interfering with MHC class II loading enhancers Library design and automated manage- Major histocompatibility complex (MHC) ment of screening data important target proteins integrates a variety of scientific poly-proline-mediated protein-protein interactions class II molecules are peptide receptors dis- Success in small-molecule screening relies disciplines – from computational chemistry, to bioinformatics, In the course of our research program aiming playing protein fragments derived from ex- heavily on the pre-selection of a library of po- ogenous proteins for surveillance by T cells. tentially bioactive compounds from virtually to molecular modeling – and needs to be closely linked to an at the development of small molecules specifically interfering with intracellular protein- In order to prevent ‘accidental’ loading of unlimited chemical diversity integrating the experimental examination of predicted chemical entities. The protein interactions, we have been searching these molecules with free peptides, empty collected knowledge of Medicinal Chemistry. MHC II molecules that have lost their ligand Within this task, we developed in-house soft- most effective way to organize this relationship is to combine for conformationally defined di-proline mimics in a PPII helix conformation that could be rapidly ‘inactivate’ by acquiring a ‘non-recep- ware for the design of screening libraries us- both experimental and theoretical work in one research unit, incorporated into peptide chains or related tive’ conformation. This non-receptive state ing an innovative fragment-based fingerprint is characterized by the inability to bind any that allows for both the selection of small as is done in our research group. Within our group the full modular constructs. Our modeling studies suggested a list of different scaffolds that free peptides and is presumably based on a sub-libraries with high chemical diversity, repertoire of in-silico methods for homology modeling, virtual should mimic a di-proline motif in PPII orien- closure of the binding cleft. In simulations of and for the combinatorial analysis of struc- tation. The stereoselective synthesis of these the HLA-DR1 without bound peptide, and tural fragments within libraries of annotated, RONALD KÜHNE screening, and ligand optimization is enhanced through novel compounds was successfully devel- in comparison to the antigen complexed biologically active compounds. Furthermore,

experimental binding studies in combination with different oped by our project partner (Prof. Schmalz, HLA-DR1, we found a very rapid collapse of we refined our in-house ADMET- and reac- 1969 – 1973 Studied biochemistry at Köln). For the EVH1 domain, our binding the P1-pocket. Further molecular dynamics tivity filters, which are an important basis the Martin-Luther-Universität Halle biochemical and cell-based methods. studies indicated that complete replacement studies with the αG86Y allele show a strongly for the judgment of screening compounds. Witttenberg of the PPII core by our new building blocks comparable behavior of the MHC dynamic In order to facilitate the design of hit-based 1973 Diploma in Biochemistry enhances ligand binding to EVH1. SPOT- with the bound antigen. This means that focused libraries, we decided to develop

arrays using non-natural amino acids were blocking the P1 collapse inhibits the conver- and constantly update a database of com- 1973 – 1976 Research associate at synthesized to find organic scaffolds address- sion of the receptive into the ‘non-receptive’ mercially available compounds. Currently, the Zentralinstitut für Molekularbiologie ing affinity relevant flanking epitopes. Pep- state, resulting in a reloadable HLA-structure. this database contains approximately 10 und Medizin der Akademie der tides containing the most promising unnatu- Additionally, we found that during the mo- million unique compounds, equipped with Wissenschaften ral amino acids in combination with our PPII lecular dynamics of dipeptides known as purchase order numbers, sources of supply, 1976 – 1992 Research associate at mimetics showed remarkable high affinities MHC-loading enhancers (MLE), these com- and diverse structure-related descriptors. the Institut für Wirkstofforschung der against EVH1 domains of VASP family pro- pounds hinder the collapse of the P1 pocket The basis for the automated analysis of Akademie der Wissenschaften teins (K < 500 nM). These new compounds in a way similar to that shown in the αG86Y screening data is a set of standard operating d are used to study the biological functions of allele simulation. Hence, it follows that the procedures (SOPs) defined for each experi- 1980 Ph. D. at the Martin-Luther- EVH1 domain-containing proteins in the pro- MHC-enhancing activity of the studied com- mental setup. We developed a program to Universität Halle Witttenberg cess of cell migration and cytoskeleton syn- pounds is related to the interaction with the analyze screening data, allowing for the doc- Since 1993 Group leader at the FMP thesis (Figure 1). P1 pocket of HLA-DR. Experimental studies umentation of the raw data, statistical evalua- done by our project partners (Ch. Freund, O. tion after data normalization, identification of Rötzschke) confirmed the theoretical results. hits, and automated reporting for customers. The binding models and their mechanistic This automated reporting system creates an implications can now be used to facilitate the exhaustive pdf-report containing all impor- rational design of highly selective and potent tant quality parameters and screening results. MLE, which promise very interesting therapeutic applications.

46 RESEARCH GROUPS // STRUCTURAL BIOLOGY RESEARCH GROUPS // STRUCTURAL BIOLOGY 47 From left to right: Martyna Pawletta, Lara Kuhnke

Michael Lisurek

Matthias Müller, Matthias Barone

MEMBERS OF THE GROUP SELECTED PUBLICATIONS COLLABORATIONS EXTERNAL FUNDING

Dr. Michael Lisurek Jehle S, Rajagopal P, Bardiaux B, Markovic Ahsanullah, Schmieder P, Kühne R, International Deutsche Forschungsgemeinschaft Bayer Schering Pharma AG

S, Kühne R, Stout JR, Higman VA, Klevit Rademann J (2009) Metal-free, regioselec- “Design and synthesis of low molecular “Ligandendesign von G-Protein- Dr. Bernd Rupp, EF Kjetil Tasken RE, van Rossum BJ, H Oschkinat (2010) tive triazole ligations that deliver locked cis weight prolin-rich motif (PRM) mimetics gekoppelten Rezeptoren auf der University of Oslo Dr. Federica Morandi, EF, * Solid-state NMR and SAXS studies provide peptide mimetics. recognized by PRM binding domains” Basis von Rezeptormodellen” Institute of Molecular genetics, Prague a structural basis for the activation of aB- Angew Chem Int Ed Engl 48: 5042-5045 FOR 806 TP 03 (KU 845/2-1) 9108 Dr. Anna Schrey, EF, * crystallin oligomers. Antonio Monge 01.2007 – 01.2010 01.2009 – 12.2009

Dr. Jörg Wichard, EF, * Nat Struct Mol Biol 17: 1037-1042 University of Navarra 122.106 Euro 30.000 Euro FMP authors Robert Opitz (doctoral student), EF, PT, * Zaminer S, Brockmann C, Huy P, Opitz R, Group members Deutsche Forschungsgemeinschaft caprotec bioanalytics GmbH National Reuter C, Beyermann M, Freund C, Müller “Design, synthesis and functional “Modellierung von capture- Matthias Müller (doctoral student), EF, PT, * M, Oschkinat H, Kühne R, Schmalz H-G Hans-Günther Schmalz characteristics of low molecular weight compounds” Lara Kuhnke (student), * (2010) Addressing protein-protein inter- University of Köln proline-rich motif (PRM) mimetics 07.2009 – 06.2010

actions by small molecules: a designed recognized by PRM binding domains” 85.000 Euro Martyna Pawletta (student), * Günther Jung pro-pro dipeptide mimic with a PPII helix FOR 806 TP 03 (KU 845/2-2) University of Tübingen Charité – Universitätsmedizin Berlin Dr. Frank Eisenmenger (technical assistant) conformation as a module for the synthesis 03.2010 – 02.2013 “n-silico Optimierung von GPCR- of PRD-binding ligands. Angew Chem Int Olaf Rötzschke, Kirsten Falk 123.000 Euro Liganden” Ed Engl 49: 7111-7115 Singapore Immunology Network (SIgN), * Part of period reported Bundesministerium für Bildung und 04.2008 – 03.2010 Agency for Science, Technology and Lisurek M, Rupp B, Wichard J, Forschung, Forschungsprämie 50.000 Euro PT part time Research (A*STAR), Singapore Neuenschwander M, von Kries J, “Modellierung von von poly-prolin- MDC, Berlin NOXXON AG EF position funded externally Frank R, Rademann J, Kühne R (2010) Motiv-vermittelten Protein-Protein- “Homologiemodellierung von (3rd-party funds) for at least part Design of chemical libraries with potentially Wechselwirkungen” Proteinfragmenten” of the reporting period bioactive molecules applying a maximum 03FPD00036 02.2010 – 04.2010 common substructure concept. 01.2010 – 08.2010 Current group members (31.12.2010) 15.000 Euro Mol Divers 14: 401-408 7.500 Euro

Cirauqui N, Schrey AK, Galiano S, Ceras J, Bundesministerium für Bildung und Pérez-Silanes S, Aldana I, Monge A, Kühne Forschung, Forschungsprämie R (2010) Building a MCHR1 homology model “Moleküldynamik-basierte Methode provides insight into the receptor-antagonist zur Aufklärung von Bindungsmodi contacts that are important for the develop- von Protein-Liganden” ment of new anti-obesity agents. 03FPD00023 Bioorg & Med Chem 18: 7365-7379 05.2009 – 12.2009

7.500 Euro

48 RESEARCH GROUPS // STRUCTURAL BIOLOGY RESEARCH GROUPS // STRUCTURAL BIOLOGY 49 STRUCTURAL BIOLOGY

SOLID-STATE NMR SPECTROSCOPY

GROUP LEADER Bernd Reif Figure 1. Electronmicrographs of Ab40 fibrils (left). 1H, 15N MAS solid-state NMR correlation spectrum of a Ab40 fibril preparation containing u-2H, 13C, 15N isotopically-enriched peptide (middle). Strips extracted from a HNCACB experiment recorded for u-2H, 13C, 15N Ab40 (right).

We use Nuclear Magnetic Resonance (NMR) in order to character- We started to apply MAS solid-state NMR Furthermore, we worked on the develop- In addition, we could show that MAS solid- techniques to structurally characterize fibrils ment of methods for MAS solid-state NMR state NMR methods are applicable to pro- ize biomolecular systems, which are at the interface between solu- of the Alzheimer’s disease (AD) b-amyloid spectroscopy. Perdeuteration of proteins teins in solution, in case their tumbling cor- tion and solid. In this context, we focus on membrane proteins and peptide (Ab). The disease is characterized in the solid-state, and application of inter- relation time is smaller than the MAS rotor by deposition of plaques of this peptide in mediate up to fast magic-angle-spinning, period (Mainz et al., 2009). This approach amyloidogenic peptides and proteins. So far, about 20 proteins are the brains of AD patients. Solid-state NMR enables a resolution in the proton dimen- applies to protein complexes with a mo- known for which a correlation between aggregation and disease is is therefore the method of choice to pro- sion which is comparable to the resolution lecular weight of greater than 300 kDa. In vide structural information at atomic resolu- achievable in solution-state NMR spectros- the future, this method will allow to probe established. The most prominent examples are Alzheimer’s disease tion. It is the goal of these studies to obtain copy of medium size proteins (Chevelkov protein-ligand interactions without the (AD), the prion diseases (BSE, CfJ) and Huntington’s disease. How- a better understanding of the mechanisms, et al., 2006). The spectral quality allows the need of co-crystallization which is of impor- which result in fibril formation and aggre- inclusion of a proton dimension in reso- tance for weakly interacting molecules, e.g. ever, little is know about the mechanism, which leads to aggrega- gation. We prepare Ab peptides, tag-free, nance assignment experiments, which in chaperone-amyloid interactions. tion, as well as about the structure of the amyloid fibrils. We would which are overexpressed in inclusion bod- turn increases the reliability of the assign- ies. Figure 1 shows an electronmicrograph- ment process (Linser et al., 2007). Using BERND REIF like to gain further insight into the structure of oligomeric inter- ic picture of the obtained b-amyloid fibrils. dipole, CSA cross-correlated relaxation

mediate states, which are associated with protofibril formation. Using perdeuterated samples of fibrillar experiments (Chevelkov et al., 2007; Chev- 1987 – 1994 Study of physics and Ab40, we could show that the spectral reso- elkov et al. 2009; Linser et al., 2010), we biochemistry at the University of Bayreuth In addition, we are interested in characterizing dynamic chemical lution in comparison to previous studies were able to characterize the time scale 1994 – 1998 Ph.D. in chemistry at the exchange processes between the soluble and aggregated state of (Tycko, 2006) can be increased by a factor and amplitude of slow motional processes University of Frankfurt of ca. 100. The 1H, 15N spectrum of a fibril in the solid-state. We find that motional the respective proteins. preparation containing u-2H, 13C, 15N isoto- correlation times can be as high as 500 ns. 1998 – 1999 Post-Doc at the pically labeled peptide is displayed in the Solid-state NMR is ideally suited to charac- Massachusetts Institute of Technology middle. The amide resonances are readily terize motional processes. In solution-state (MIT), Cambridge, USA assigned using HNCACB type experiments NMR relaxation is mostly caused by overall 2000 – 2002 Emmy Noether Research (Figure 1, right). Scalar coupling-based ex- tumbling. Rotational diffusion is, however, Group Leader at the TU München periments like HNCO, HNCA, HNCACB, absent in the solid-state. Conformational HNCACO and HNCOCA were tested first fluctuations can therefore be sampled in Since 2003 Research Group Leader using a micro-crystalline sample of the the solid-state with high accuracy (Agar- at the FMP α-spectrin SH3 domain (Linser et al., 2008; wal et al., 2008). For a similar reason, high- Linser et al., 2010). resolution deuterium NMR spectra can be obtained in the solid-state.

50 RESEARCH GROUPS // STRUCTURAL BIOLOGY RESEARCH GROUPS // STRUCTURAL BIOLOGY 51 From left to right: Andi Mainz, Juan Miguel Lopez del Amo

Andi Mainz, Tomas Jacso, Juan Miguel Lopez del Amo

MEMBERS OF THE GROUP SELECTED PUBLICATIONS COLLABORATIONS EXTERNAL FUNDING

Dr. Veniamin Chevelkov, EF, * Asami S, Schmieder P, Reif B (2010) High Jacso T, Grote M, Daus ML, Schmieder P, International Deutsche Forschungsgemeinschaft Deutsche Forschungsgemeinschaft

resolution 1H-detected solid-state NMR Keller S, Schneider E, Reif B (2009) The “Physiologische Bedeutung membran- “Integrated approach to protein Dr. Katja Faelber, EF, *, Alexej Krushelnitzky spectroscopy of protein aliphatic resonanc- periplasmic loop P2 of the MalF subunit of ständiger Formen des Amyloid-Vor- dynamics: Bringing together solid- University Kazan, Russia Dr. Tomas Jacso, PT, EF es: Access to tertiary structure information. the maltose ATP binding cassette transport- läuferproteins (APP) und strukturbasierte and solution-state NMR data”

J Am Chem Soc 132: 15133-15135 er is sufficient to bind the maltose binding Nikolai Skrynnikov Vorhersage für die Bindung von Substrat Re1435/6 Dr. Mangesh Joshi, EF, * protein MalE. Biochemistry 48: 2216-2225 Purdue University, West Lafayette, USA und Inhibitoren an Sekretasen“ with N. Skrynnikov, Purdue University Richter L, Munter LM, Ness J, Hildebrand Dr. Juan Miguel Lopez del Amo, EF SFB 449 TP B15 11.2007 – 11.2010 PW, Dasari M, Unterreitmeier S, Bulic B, Mainz A, Jehle S, van Rossum BJ, Salvador Ventura with G. Multhaup, FU Berlin 255.000 Euro Vipin Agarwal (doctoral student), PT, * Beyermann M, Gust R, Reif B, Weggen Oschkinat H, Reif B (2009) Large protein Universitat Autonoma Barcelona, Spain 01.2005 – 12.2010 S, Langosch D, Multhaup G (2010) Ab42- complexes with extreme rotational cor- Deutsche Forschungsgemeinschaft Sam Asami (doctoral student), PT, EF 81.600 Euro lowering compounds directly bind to Aß relation times investigated in solution by National “Strukturelle und funktionelle

Muralidhar Dasari (doctoral student), PT, EF and interfere with APP transmembrane magic-angle-spinning NMR spectroscopy. Deutsche Forschungsgemeinschaft Charakterisierung von b-Amyloid- Jan Bieschke dimerization. JACS 131: 15968-15969 “Structural characterization of chaperone Aggregation“ Rasmus Linser (doctoral student), PT, EF MDC Berlin Proc Natl Acad Sci USA 107: 14597-14602 modulated protein aggregation in Re1435/10

Andi Mainz (doctoral student), PT, EF Teresa Carlomagno S. cerevisiae” with J. Bieschke, MDC Berlin Linser R, Fink U, Reif B (2010) Detection FMP authors EMBL Heidelberg SFB 740 TP B02 03.2010 – 03.2013 Uwe Fink (technical assistant) of dynamic regions in biological solids ena- Group members 01.2007 – 12.2010 351.600 Euro bled by spin-state selective NMR experi- Marcus Fändrich Kerstin Steinhagen (technical assistant) 301.386 Euro ments. J Am Chem Soc 132: 8891-8893 MPI Halle LGS-Springmeeting

Deutsche Forschungsgemeinschaft 03.2010 – 03.2010 Gerd Multhaup * Part of period reported “Charaterisation of dynamic processes 3000 Euro FU Berlin in proteins by combining solid- and PT part time Erich Wanker solution-state NMR spectroscopy” EF position funded externally MDC Berlin RE 1435/7-1 (3rd-party funds) for at least part 01.2008 – 12.2010 of the reporting period 227.550 Euro

Current group members (31.12.2010)

52 RESEARCH GROUPS // STRUCTURAL BIOLOGY RESEARCH GROUPS // STRUCTURAL BIOLOGY 53 STRUCTURAL BIOLOGY

SOLUTION NMR

Figure 1. The cyclic peptide Cyc15AMPB (a) was designed to be photoswitchable due to its azo-benzene GROUP LEADER moiety and to exhibit binding to the Syntrophin-PDZ- Peter Schmieder domain in one conformation and no binding in the other, depending on the state of the photo-switch. To control the photostate during the NMR-measurements a device was constructed to allow for continuous irradiation in the magnet (b). The desired binding properties were confirmed using NMR (c) and isothermal titration calorimetry (ITC) (not shown).

The group focuses on the investigation of the structure and For many organisms on earth light is not only (harboring a flavin chromophore) and aSTAS protein interaction domains (PIDs). Interfer- an essential source of energy but also an ex- (Sulphate Transporters AntiSigma-factor ing with these interactions can be interest- dynamics of biomolecules at atomic resolution using solution ternal stimulus that regulates developmen- antagonist) domain. We use biophysical ing for two reasons. One is the design of state NMR-spectroscopy as the major technique. The full tal processes. It can, however, also be po- methods to obtain information on the three- small molecules that can inhibit a particular tentially dangerous at shorter wavelengths. dimensional structure and in particular the interaction, influence the biological process repertoire of multidimensional techniques is used in combination Consequently, organisms have developed domain interactions of the full-length pro- and thus act as a drug molecule. The other with appropriate labelling schemes and other biophysical mechanisms to evaluate light intensity, di- tein and to study changes occurring after the is the design of molecular probes that can rection, duration and color. This is accom- illumination with blue light. Using analytical be used to disentangle the complex signal techniques (analytical ultracentrifugation (AUC), isothermal plished by several types of photoreceptors ultracentrifugation (AUC) and small-angle transduction pathways of a cell by selec- titration calorimetry (ITC) and small-angle x-ray scattering (SAXS)). that usually consist of a protein component x-ray scattering (SAXS) we have shown that tively inhibiting individual proteins in the and an organic chromophore cofactor, of- YtvA is a dimer and has a dumbbell-shaped signalling chain. One PID of great interest Current aims are the elucidation of the structural basis of ten covalently attached. The properties of domain orientation in the dark and after ac- is the PDZ domain, a 100 amino-acid-resi- activation mechanism of the blue-light receptor YtvA and the the organic molecule bound to the protein tivation by blue light. Most importantly we due domain. Most PDZ domains recognize are tuned by the protein environment such find that no significant change takes place short, C-terminal peptide motives. In the PETER SCHMIEDER determination of the role of dynamics in the presentation of that the light stimulus can be perceived. We upon activation. SAXS profiles of YtvA were heterodimer of the nNOS-PDZ domain and

peptides by MHC class one molecules and their recognition by have investigated the influence of photo- obtained in the dark and after illumination the a-syntrophin PDZ domain, however, the 1982 – 84/85-87 Studied chemistry chromism on the structure and dynamics of and used to calculate an ab initio shape of former exhibits a b-finger that binds to the at the University of Frankfurt T-cell receptors. prokaryotic phytochromes, of the blue-light the molecule in solution. The obtained mo- latter. In collaboration with the groups of K. 1988 Diploma thesis in Prof. Kessler’s receptor YtvA from Bacillus subtilis and of lecular envelope of YtvA corresponds to a Rück-Braun (TU Berlin) and M. Beyermann group a photoswitchable peptide derived from li- dumbbell with two bulges on either side of we have designed a cyclic, photoswitch-

gands binding to a PDZ domain. the molecule. The attempt to obtain a more able peptide (Figure 1a) from the b-finger 1989 – 1992 Ph.D. at the TU Munich detailed picture by rigid body modelling of of nNOS and it has been shown that bind- (Prof. Kessler) Structure determination and domain the structure of the protein using a recently ing of the peptide to the PDZ domain of a- 1992 – 1995 Post-Doc at Harvard interaction of the photochromic protein published high-resolution model of YtvA syntrophin can be switched with light, even Medical School (Prof. Wagner) YtvA from Bacilus subtilis failed to provide a unique answer. Two struc- in vivo. NMR spectroscopic investigations

Phototropins are blue-light photoreceptors, tural models both consistent with the SAXS showed that the binding pocket is con- Since 1995 Group Leader at the FMP for which bacterial analogues exist as well. profiles but clearly different were obtained. served, that the binding is reversible (Figure

We study the blue light absorbing and fla- We are currently using solution NMR tech- 1c) and ITC investigations showed that the vin binding photoreceptor YtvA from Bacil- niques to obtain more detailed structural binding constants are in the same range as lus subtilis. It has been shown that YtvA is information. those of the parent peptides. Interestingly, part of the stress response in Bacillus sub- the peptide exhibits a high flexibility in both tilis but the detailed mechanism is not yet Structure and PDZ binding of the photos- forms of the photoswitch. understood. It is located in the stressosome, witchable peptide Cyc15AMPB a 25 S macromolecular complex responsible The majority of biological processes are not for the activation of the transcription regula- performed by a single protein but rather by tor σB via a phosphorylation cascade and a assemblies of proteins that can consist of partner switching mechanism. This photo- ten or more proteins. The individual binding receptor is a two-domain protein, consist- partners are assembled via protein-protein ing of a LOV (light, oxygen, voltage) domain interactions that are mediated via special

54 RESEARCH GROUPS // STRUCTURAL BIOLOGY RESEARCH GROUPS // STRUCTURAL BIOLOGY 55 From left to right: Matthias Dorn, Marcel Jurk, Marco Röben

Philipp Schramm, Martin Ballaschk

Monika Beerbaum, Brigitte Schlegel

MEMBERS OF THE GROUP SELECTED PUBLICATIONS COLLABORATIONS EXTERNAL FUNDING

Martin Ballaschk (doctoral student), EF, PT, * Jurk M, Dorn M, Kikhney A, Svergun D, Hahn J, Strauss HM, Schmieder P (2009) National Deutsche Forschungsgemeinschaft Hans-Fischer-Gesellschaft

Gärtner W, Schmieder P (2010) Heteronuclear NMR investigation on the “NMR-spektroskopische Untersuchungen “NMR-Spektroskopische Untersuchungen Matthias Dorn (doctoral student), EF, PT The switch that does not flip – the blue- structure and dynamics of the chromophore Wolfgang Gärtner von lichtreduzierten Strukturveränderun- an linearen Tetrapyrollen“ Tolga Helmbrecht (doctoral student), EF, PT light receptor YtvA from Bacillus subtilis binding pocket of the cyanobacterial MPI für Bioanorganische Chemie, gen in Protein-Chromophorkomplexen” HFG Schmieder

adopts an elongated dimer conformation phytochrome Cph1. JACS 130: 11170-11178 Mülheim an der Ruhr SFB 498 TP B6 01.2009 – 12.2009 Marcel Jurk (doctoral student), EF, PT independent of the activation state as 01.2006 – 12.2009 23.000 Euro Peter Hildebrand Marco Röben (doctoral student), EF, PT revealed by a combined AUC and SAXS Hoppmann C, Seedorff S, Richter A, 306.759 Euro TU Berlin EMP Biotech GmbH study. J Mol Biol 403: 78-87 Fabian H, Schmieder P, Rück-Braun K, Sabine Seedorff (doctoral student), EF, PT Deutsche Forschungsgemeinschaft Aufnahme von Routine NMR-Spektren Beyermann M (2009) Light-directed protein Jon Hughes “Structure determination and domain 01.2009 – 12.2010 Aaron Hahn (student), PT, * Seedorff S, Appelt C, Beyermann M, binding of a biologically relevant beta- University of Giessen interaction of the photochromic 1.880 Euro Schmieder P (2010) Design, synthesis, sheet. Angew Chem Int Edit 48: 6636-6639 Philipp Schramm (student), PT, * Tilman Lamparter protein YtvA from Bacillus subtilis” structure and binding properties of PDZ University of Karlsruhe SCHM 880/8-1 Monika Beerbaum binding, cyclic beta-finger peptides. FMP authors 01.2009 – 12.2011 (technical assistant), EF, PT Biochem Biophysic Res Comm 395: 535-539 Richard Lucius Group members 203.400 Euro HU Berlin Brigitte Schlegel (technical assistant) Röben M, Hahn J, Klein E, Lamparter T, Volkswagenstiftung Karola Rück-Braun Psakis G, Hughes J, Schmieder P (2010) “Regulation of phytochrome biological TU Berlin * Part of period reported NMR spectroscopic investigation of function through light-induced

mobility and hydrogen bonding of the Roderich Süssmuth conformational changes: A study using PT part time chromophore in the binding pocket of TU Berlin solid-state NMR spectroscopy”

EF position funded externally phytochrome proteins. VW I/82 630 Andreas Ziegler und (3rd-party funds) for at least part ChemPhysChem 11: 1248-1257 10.2007 – 10.2009 Barbara Uchanska-Ziegler of the reporting period 67.700 Euro Charité – Universitätsmedizin Berlin Current group members (31.12.2010)

56 RESEARCH GROUPS // STRUCTURAL BIOLOGY RESEARCH GROUPS // STRUCTURAL BIOLOGY 57 STRUCTURAL BIOLOGY

MOLECULAR IMAGING – ERC PROJECT BIOSENSORIMAGING

GROUP LEADER Leif Schröder

Figure 1. Schematic illustration for SEOP-based hy- perpolarization of Xe-129. The polarization of photons is first used to spin-polarize electrons of rubidium, which eventually will increase the spin polarization of xenon nuclei.

Pharmacological research, i.e. the understanding of drug action, The LEIPNIX Polarizer Multinuclear CEST Imaging Initial studies revealed that differences in the chemical shift of xenon for discrimina- would benefit substantially from imaging modalities that directly For the flexible production of hyperpolar- Installation of the multinuclear NMR micro- ized xenon we developed a new design for imaging system (completed in March 2011) tion of its chemical environment are in- visualize interactions occurring between a living organism and a mobile setup that can be used at different was followed by characterizing various se- creased when trapping the noble gas in the sensor than in the absence of a sensor. It chemicals. The Molecular Imaging group aims to establish a novel NMR scanners. The polarization process lective saturation pulse patterns for CEST is based on spin exchange optical pump- applications. Modifications of existing MRI could also be demonstrated to selectively approach to magnetic resonance imaging (MRI) for improving drug ing (SEOP) and is performed with a new pulse programs are first tested for exchange- address the sub-pools by Hyper-CEST detection and use both micro-environments development and therapy monitoring. Most of today’s protocols type of line-narrowed laser diode emit- able proton systems like barbituric acid for ting at high power levels (>100 W) in cw 1H saturation transfer imaging. This allowed separately to generate image contrast. rely on the detection of abundant water molecules and are serious- mode. This ensures a photon density for for making flexibleCEST techniques acces- pumping the Rb D transition that is much sible with the current software implemented Novel Xenon Biosensors ly limited in illustrating the spatial distribution of biomarkers 1 higher than in conventional systems. The on the 400 MHz NMR spectrometer. These As a new type for an enzyme-specific sen- at low concentrations. Xenon biosensors have an outstanding relatively compact design of the LEIPNIX protocols are then modified for 129Xe imag- sor, a construct with a cleavage motif for setup (Laser Enabled Increase of Polar- ing to realize sensitive biosensor detection of MMP-11 is currently being developed. LEIF SCHRÖDER potential to improve MRI with the high sensitivity of hyperpolarized ization for Nuclei of Imprisoned Xenon) caged xenon. Various NMR inserts, ranging MMP-11 is a marker for pancreatic cancer,

xenon and the specificity of functionalized contrast agents. is optimized for continuous flow applica- from 5 – 30 mm inner diameter, have been and the enzymatic amplification allows 1995 – 1997 Studies of Physics and tions that allow for permanent production characterized to ensure a flexible use of the for converting substrate-labeled crypto- Chemistry, Georg-August Universität Research focuses on high-sensitivity in vitro and in vivo diagnostics of solutions saturated with hyperpolarized new MR micro-imaging lab, including suit- phane cages into a modified construct that Göttingen for localization of, for example, tumor cells and their response xenon to be detected in molecular cages. able quality assurance protocols for future shows a shift in the Xe NMR signal of ca. 1997 – 2001 Studies of Physics and 2 ppm. The design of enzyme-responsive to drug delivery. Such xenon biosensors will enable detection of The improved setup is based on a single experiments. Astronomy, Ruprecht-Karls Universität beam path with optimized polarization MR contrast agents has been the focus of Heidelberg, Diploma in Physics tumors at high sensitivity without any background signal. This optics, incorporating a Glan-Laser beam Interaction of Cryptophanes with considerable recent research, driven by

splitter cube with an extinction ratio of Lipid Membranes the goal of collecting biochemical infor- 2001 – 2003 Ph.D. student, Deutsches approach has the potential to close the sensitivity gap between 1:100’000. Compared to standard compo- While there is great potential in using xe- mation from optically dense tissues, such Krebsforschungszentrum and Ruprecht- modalities of nuclear medicine like PET/SPECT and MRI without nents with extinction ratios of only 1:2’500, non biosensors for targeted molecular im- as tumors. Monitoring the catalytic activity Karls Universität Heidelberg of an enzyme, as opposed to stoichiomet- using ionizing radiation or making compromises in penetration this ensures much more accurate manipu- aging in vivo, e.g., in the determination 2003 – 2005 Research Assistant, Deutsches lation of the laser light polarization and of distributions of cell surface receptors, ric protein binding, dramatically improves Krebsforschungszentrum, Heidelberg depth that are linked to optical imaging. allows us to work with only one compact their non-specific interactions with many MR detection sensitivity. This is now, for the

beam path instead of two paths that have biological materials are poorly understood. first time, paired with Hyper-CEST detec- 2005 – 2007 Emmy Noether Fellow of to be collimated in the optical cell. Further- Some interaction between the hydrophobic tion to further improve NMR sensitivity for the DFG, University of California, Berkeley more, an active cooling system controls xenon-binding moiety (a cryptophane mol- disease-specific marker recognition. Matrix 2007 – 2009 Research Fellow, Lawrence the distribution of Rb vapour in the optical ecule) that has been targeted to a cell sur- metalloproteinases (MMPs), which degrade Berkeley National Laboratory pumping cell. Controlling the temperature face receptor and the hydrophobic interior all components of the extracellular ma-

conditions using this device yields a sig- of the cell membrane is likely. Interactions trix and thereby influence a wide range of Since 2009 Junior research group leader nificant increase in spin polarization when between cryptophane and lipid vesicles physiological and pathological processes, at FMP compared to commercial setups. that are comparable in size and composi- are particularly attractive candidates. tion to cell membranes are studied using xenon NMR and fluorescent microscopy.

58 RESEARCH GROUPS // STRUCTURAL BIOLOGY RESEARCH GROUPS // STRUCTURAL BIOLOGY 59 From left to right: Jörg Döpfert, Martin Kunth

Leif Schröder, Chris Witte

Federica Rossella

MEMBERS OF THE GROUP COLLABORATIONS EXTERNAL FUNDING

International Dr. Christopher Witte, EF, * Deutsche Forschungsgemeinschaft

Alexander Pines “Molekulare Bildgebung mittels Jörg Döpfert (doctoral student), EF, PT, * Materials Sciences Division hyperpolarisierter Biosensoren” Martin Kunth Lawrence Berkeley National Laboratory SCHR 995/2-1 (doctoral student), EF, PT, * and qb3 Institute 07.2009 – 11.2009

University of California at Berkeley (approved budget: 1.151 370 Euro) Federica Rossella 26.577 Euro (doctoral student), EF, PT, * David Wemmer Physical Biosciences Division Europäische Kommission Lawrence Berkeley National Laboratory (7. Rahmenprogramm) * Part of period reported and qb3 Institute “BiosensorImaging”: Hyperpolarized

University of California at Berkeley Biosensors in Molecular Imaging PT part time (ERC-Grant) Alexander Ziegler EF position funded externally 12.2009 – 11.2014 Museum of Comparative Zoology (3rd-party funds) for at least part 1.851.000 Euro Harvard University, Cambridge, USA of the reporting period SAW-Verfahren der WGL

Current group members (31.12.2010) “Development of Novel NMR Probes: National Impoving Cell Profiling for Early Franz Schilling Diagnosis” SELECTED PUBLICATIONS Department Chemie with C. Freund Technische Universität München 01.2011 – 12.2013

Meldrum T, Schröder L, Denger P, Wemmer 761.613 Euro Andreas Hennig, Ute Resch-Genger DE, Pines A (2010) Xenon-based molecular Bundesanstalt für Materialforschung sensors in lipid suspensions. und –prüfung, Berlin J Magn Reson 205: 242-246 (cover article)

Figure 2. Schematic illustration for CEST imaging with exchangeable nuclei. Figure 3. A schematic of the various pools of xenon in intralipid emulsion. Xe in Christian Hackenberger Selective saturation pulses allow for amplifying the signal from low-concentration lipids (green) and xenon in water (red) correspond to unbound xenon. Xe@cage Schilling F, Schröder L, Palaniappan KK, Institut für Chemie und Biochemie analytes by transferring it into a pool of high abundance nuclei that can be detected in lipids (dark blue) and Xe@cage in water (yellow) appear ca. 130 ppm upfield of Zapf S, Wemmer DE, Pines A (2010) MRI at much better signal-to-noise conditions. the red peak. The cage is represented by the light blue hexagon. The cryptophane Freie Universität Berlin

cage used is shown in the inset. thermometry based on encapsulated hyperpolarized xenon. Carmen Birchmaier-Kohler ChemPhysChem 11(16): 3529–3533 Developmental Biology/Signal Transduction, Max-Delbrück-Centrum für Molekulare

Medizin, Berlin FMP authors Group members

60 RESEARCH GROUPS // STRUCTURAL BIOLOGY RESEARCH GROUPS // STRUCTURAL BIOLOGY 61 STRUCTURAL BIOLOGY

IN-CELL NMR

Figure 1. Artistic representation of the intracellular GROUP LEADER environment. Despite the fact that the eukaryotic cytoplasm is a highly complex and very crowded Philipp Selenko mixture, proteins and other biomolecules have evolved to exert their individual functions under exactly those kind of conditions. Analyzing protein structures and functions in native cellular settings allows us to uncover the hidden world of Cellular Structural Biology.

(Image, courtesy of Adrain Elcock, Univ. of Iowa, USA)

So what do proteins actually look like inside live cells? Naïve as this Our research is roughly divided between atomic resolution. Here, in-cell NMR spec- The answer to this question, we believe, purely structural questions – such as how troscopy provides the unique opportunity lies in the specific cellular environment that question may seem, we do not yet have an answer to it. After all, neurodegenerative disease proteins con- to be able to ‘look into live cells’ in a non-in- these cells ‘impose’ on alpha-synuclein. most of the ‘classical’ Structural Biology techniques require protein vert to form amyloid fibrils in human brain vasive, non-disruptive manner and to follow Something in the macromolecular compo- cells – and then questions that are more directly the establishment of different PTMs sition of dopaminergic neurons must ren- samples in very artificial conditions – consider a crystallized protein, closely related to how cellular processes in a time-resolved manner. Comparable to der synuclein more prone to aggregation. for example, and how it compares to the physiological environ- like post-translational protein modifications a time-lapse video microscope with atom- The obvious question is, what exactly is this (PTMs) change the shape (and consequent- level resolution, in-cell NMR spectroscopy component? On the way to finding an an- ment of a live cell. Just to be clear, ‘classical’ Structural Biology has ly the biological properties) of functional is the only method that enables such high- swer to this question, we study the struc- provided a wealth of accurate structural information about numer- protein modules. As a matter of fact, both resolution in vivo studies. tural properties of IDPs like alpha-synuclein of these processes are inherently connect- in different mammalian cell lines, including ous folded proteins, or protein domains, and we can safely assume ed to intrinsically disordered proteins (IDPs), We focus this ‘microscope’ of ours on pro- large sets of neuronal cells. Uncovering the that their cellular in vivo conformations deviate little from their or intrinsically disordered protein regions teins that play important roles in key biolog- conformational differences that certain cel- (IDRs). ical processes, such as human oncoproteins, lular environments induce in these proteins PHILIPP SELENKO structural in vitro properties. But what about intrinsically disordered transcriptional regulators, or apoptotic may provide us with important clues about

proteins (IDPs)? What about these proteins that do not exhibit Conformational Plasticity of messengers. The key questions we wish to the molecular origins of these devastating 2002 Ph.D. at the European Molecular Functional Protein Modules answer are how post-translational protein diseases. Biology Laboratory (EMBL), Heidelberg structural properties in the ‘classical’ sense? How do IDPs look like All eukaryotic biology is governed by post- modifications lead to functional reprogram- 2003 – 2008 Post-Doc at Harvard inside live cells? Does the crowded intracellular environment induce translational protein modifications. Every ming of these proteins, and to what extent Medical School decision making process in intact cells, such processes depend on protein plasticity conformational properties that are not populated under isolated whether it is cell-cell communication, cell and structural in vivo conversions. 2003 – 2004 EMBO fellowship in vitro conditions? Do post-translational protein modifications or cycle progression, cell differentiation, or 2004 – 2006 Human Frontiers programmed cell death, is triggered by Cell/Structure Anomalies intracellular protein-protein interactions establish structural features in Science (HFSP) fellowship cascades of reprogramming signals that oc- Another focus of our work is on structural

that are not sampled by in vitro methods? These are the kinds of cur mostly in the form of covalent chemical protein anomalies that are only displayed 2006 – 2007 Max Kade fellowship amendments that are established on indi- in highly specialized cell types. One such by the Austrian Academy of Science questions that our research group is trying to answer. In the course vidual protein residues by different sets of example is the human neurodegenerative Since 2007 Group Leader in the of our work we explore that hidden world of Cellular Structural modifying enzymes. These chemical modifi- disease protein alpha-synuclein. It occurs Structural Biology Section of the FMP, cations can alter the functional properties of throughout the brain at comparably high Biology, and seek to uncover some of the secrets that it holds. Emmy Noether fellowship by the proteins in many ways, sometimes resulting levels, but only converts and deposits in the Deutsche Forschungsgemeinschaft in opposing biological activities (like chang- form of amyloid fibrils in dopaminergic neu- (DFG) ing a transcriptional activator into a repres- rons of the substantia nigra (a very tiny area sor, for example). in the brain). We ask what makes this cellular environment so special that it promotes While we appreciate the complexity of eu- aggregation of this intrinsically disordered karyotic signaling networks as an integral protein, especially given the fact that neigh- part of modern biology, we collectively lack boring neurons – carrying equally heavy the appropriate tools to study the structural loads of the same protein – are spared from consequences of cellular PTM reactions at amyloid formation?

62 RESEARCH GROUPS // STRUCTURAL BIOLOGY RESEARCH GROUPS // STRUCTURAL BIOLOGY 63 From left to right: Jonas Kosten, Francois-Xavier Theillet

Rossukon (Tim) Thongwichian, Andres Binolfi

Honor May Rose, Beata Bekei

MEMBERS OF THE GROUP SELECTED PUBLICATIONS COLLABORATIONS EXTERNAL FUNDING

Dr. Andres Binolfi, * Liokatis S, Dose A, Schwarzer D, International National Deutsche Forschungsgemeinschaft Selenko P (2010) Simultaneous detection “The protein disorder paradox: Dr. Stamatios Liokatis, * Axel Behrens Wolfgang Fischle of protein phosphorylation and acetylation What do natively unfolded proteins look Cancer Research UK, London Max Planck Institute of Dr. Honor May Rose, EF* by high-resolution NMR spectroscopy. like inside living cells?” Biophysical Chemistry, Göttingen JACS 132 (42): 14704-5 Donal O’Carroll SE 1794/1-1 Dr. Francois-Xavier Theillet, EF* European Molecular Biology Laura Hartmann 11.2007 – 03.2012 Ito Y, Selenko P (2010) Cellular structural Dr. Silvain Tourel, EF* Laboratory (EMBL), Rome Max Planck Institute of 665.389 Euro biology. Curr Opin Struct Biol 5: 640-8 Colloids and Interfaces, Berlin Beata Bekei Gary Daughdrill Deutsche Forschungsgemeinschaft

(doctoral student), EF, PT* University of South Florida Remco Sprangers “The protein disorder paradox: FMP authors Max Planck Institute for What do natively unfolded proteins look Jonas Kosten Group members Stephan Grieszek Developmental Biology, Tübingen like inside living cells?” (doctoral student), EF, PT* Biozentrum Basel SE 1794/1-1, II. Ulrich Stelzl Rossukon Thongwichian Kyou-Hoon Han 11.2010 – 10.2012 Max Planck Institute for (doctoral student), EF, PT* Korea Reserach Institute of Bioscience 230.020 Euro Molecular Genetics, Berlin and Biotechnology (KRIBB) Silvia Verzini SAW-Verfahren der Leibniz-Gemeinschaft Erich Wanker, Jan Bieschke (doctoral student), EF, PT* Guy Lippens (‚Pakt für Forschung und Innovation‘ I) MDC Berlin University of Lille “In vivo NMR-Spektroskopie in humanen Sandy Goyette Zellen: Ein neues Instrument in der (technical assistant), EF * Roberta Pieratelli, Dr. Isabella Felli Systembiologie“ Center for Magnetic Resonance (CERM), Marleen van Rossum 01.2010 – 12.2012 Florence (technical assistant), PT 761.000 Euro Peter Tompa

Humgarian Academy of Science * Part of period reported and University of Brussels PT part time Valdimir Uversky EF position funded externally University of South Florida (3rd-party funds) for at least part of the reporting period

Current group members (31.12.2010)

64 RESEARCH GROUPS // STRUCTURAL BIOLOGY RESEARCH GROUPS // STRUCTURAL BIOLOGY 65 SIGNAL TRANSDUCTION/ MOLECULAR GENETICS

PHYSIOLOGY AND PATHOLOGY OF ION TRANSPORT

GROUP LEADER Thomas J. Jentsch Figure 1. Back to classics. In the classical picture of ve- converted selected CLC exchangers into channels using the luminal charge accumulation blocks acidification (d). sicular acidification, electrical currents of the +H -ATPase single point mutations (c). These mice should display nor- We suggest that there is an important, previously unrec- are neutralized by a Cl- channel (a). However, we have mal acidification of endosomes (for ClC-5) or lysosomes ognized role of luminal chloride concentration. These shown that endosomal CLC proteins, instead of being (for ClC-7). Surprisingly, both mouse models (Clcn5unc and exciting results, which have profound implications for cell Cl- channels, are rather Cl-/H+ exchangers (b), raising Clcn7unc, unc for uncoupled from protons) display pheno- biology of endolysosomal trafficking and function, have the question what this exchange is good for. In our most types (impaired endocytosis or neurodegeneration) that just been published in Science. See Weinert et al. and recent work, we generated knock-in mice in which we largely overlap with those of the respective KOs, in which Novarino et al.

We aim to understand ion transport processes from the molecular CLC chloride channels and transporters Cl-/H+-exchanger, and have investigated the dominant negative KCNQ4 mutation found Proteins of the CLC gene family, discovered selectivity of vesicular CLCs in a structure- in humans), and a KCNQ5 KI mouse carrying (structure-function analysis) to the subcellular and cellular level by us in 1990, reside in the plasma membrane function study. a similar dominant mutation. Using the lat- (e.g. role in endosomes) up to the level of the organism. The latter and intracellular vesicles. We have generated ter mice, we have shown in a collaboration KO mouse models for most CLCs and have Anoctamin (TMEM16) Ca2+-activated with Roger Nicoll (UCSF) that KCNQ5 me- aspects are addressed by investigating the phenotypes of knock- identified corresponding human diseases, Cl- channels diates a component of the afterhyperpolar- out (KO) and knock-in (KI) mice, and by analyzing human diseases. yielding insights into their diverse physi- We have started new projects to define ization current in the hippocampus. We are ological functions. We have also identified functions of members of the newly identi- currently investigating the role of KCNQ4 in We focus on CLC chloride channels and transporters, KCNQ two ancillary β-subunits (barttin and Ostm1). fied Anoctamin family of Ca2+-activated Cl- dorsal root ganglia and in mechanosensa- potassium channels, KCC and NKCC cation-chloride cotransport- Mutations in their genes also cause human channels. As a first result, we have shown tion. About ten years ago, we had identi- disease. Vesicular CLCs were believed to be that Ano2 is the long-sought Ca2+-activated fied KCNE3 as a -subunit of KCNQ1 which ers, and Anoctamin Ca2+-activated chloride channels. An important β Cl- channels that facilitate vesicular acidifica- Cl- channel of olfactory sensory neurons, renders this channel constitutively open. We research area concerns the role of vesicular pH and chloride in the tion by shunting proton pump currents, but which is supposed to play an important role have now generated and analyzed a KCNE3 surprisingly, most (or all) vesicular CLCs are in amplifying olfactory responses. We have knock-out mouse and have shown that endosomal/lysosomal system. We study many organs, including the Cl-/H+-exchangers. To determine whether generated Ano2 knock-out mice and found KCNQ1/KCNE3 channels are important as a brain, inner ear, eye, olfactory epithelium, kidney, intestine, Cl-/H+-exchange can be replaced by a Cl- that Ca2+-activated Cl- currents are abolished recycling pathway for intestinal and tracheal conductance as in the classical model of ve- in olfactory receptor neurons of the main ol- Cl- secretion. pancreas, and bone. sicular acidification, we have now generated factory epithelium. Surprisingly, the electrical ClC-5 and ClC-7 KI mice in which we con- response of the olfactory epithelium to odors Cation-Cl cotransporters verted these exchangers with single point as measured in electroolfactograms was We have previously reported on the roles of THOMAS J. JENTSCH mutations into pure Cl- conductors. Surpris- reduced by only up to 40%, and mice were KCC cotransporters that were gleaned from

ingly, the phenotypes of these mice largely indistinguishable to wild-type mice in sev- our respective KO mice. We are currently in- 1972 – 1978 Studied medicine at the 1986 – 88 Postdoctoral fellow at the 1995 – 1998 and 2001 – 2003 Director recapitulated those seen in the respective eral tests for olfaction. Hence the textbook vestigating the role of the neuronal KCC2 in FU Berlin Whitehead Institute (Harvey F. Lodish, of the Centre for Molecular Neurobiology KOs, pointing to a previously unrecognized model that stipulates that Ca2+-activated defined neuron populations in which we dis- MIT), Cambridge MA Hamburg (ZMNH) 1974 – 1980 Studied physics at the role of vesicular Cl- accumulation. In other Cl- currents powerfully amplifies the olfac- rupted the cotransporter using our ‘floxed’ FU Berlin 1988 – 93 Research group leader Since 2006 Head of department at FMP projects, we demonstrated that ClC-7 is im- tory response has to be revised. This work has Kcc2 mice. Whereas KCC2 lowers the intra-

at the Centre for Molecular Neurobiology and MDC, Berlin (joint appointment), portant for lysosomal protein degradation, been published in 2011 (Billig, Pál, Fidzinski, neuronal Cl- concentration, a process that is 1981 – 1985 Staff scientist at the Institut Hamburg (ZMNH), Hamburg University Full Professor (W3) at Charité – Universitäts- but that the enlargement of lamp-1-positive Jentsch, Nature Neurosci. 14: 763-769). important for the inhibitory response to the für Klinische Physiologie (Prof. Wiederholt), medizin Berlin compartments is not a consequence of pro- neurotransmitters GABA and glycine, the FU Berlin 1991 “Habilitation” in Cell Biochem- tein accumulation; that lysosomal acidifica- KCNQ potassium channels NaK2Cl-cotransporter NKCC1 raises intra- istry at the Medical School of Hamburg Since 2007 Member of Neurocure 1982 Ph.D. in physics at the Fritz-Haber- tion is prominently supported by a lysosomal We cloned and characterized the K+ chan- neuronal Cl- above its electrochemical equi- University Cluster of Excellence Institute (Prof. Block), Berlin cation conductance; that ubiquitylation is nels KCNQ2-5, have shown that mutations librium, potentially leading to an excitatory

Since 1993 Full professor (C4) of not important for the role of ClC-5 in renal in KCNQ2 and 3 cause neonatal epilepsy, response to these neurotransmitters. Using 1984 M.D. at the Institut für Klinische Molecular Neuropathology at the ZMNH, endocytosis, nor is there any compensation and mutations in KCNQ4 a form of domi- Nkcc1-/- mice, we have now shown that spon- Physiologie (Prof. Wiederholt), FU Berlin Hamburg University; Director of the Institut by ClC-3 or ClC-4. We have also investigat- nant deafness (DFNA2). KCNQ2-5 mediate taneous neuronal activity is reduced in brain für Molekulare Neuropathobiologie ed sorting signals for all vesicular CLCs, have highly regulated‚ M-type currents that are slice preparations of these mice and that the

compared in detail the pathology caused by important for the regulation of neuronal ex- neuronal excitability that is dependent on disruption of ClC-7/Ostm1 and ClC-6, have citability. We have recently generated mouse Nkcc1 drives synaptic network maturation shown for the first time thatClC-6 is also a models for KCNQ4 (KO, and a KI carrying a early in development.

66 RESEARCH GROUPS // SIGNAL TRANSDUCTION/MOLECULAR GENETICS RESEARCH GROUPS // SIGNAL TRANSDUCTION/MOLECULAR GENETICS 67 Figure 2. Smelling without amplifier. Calcium- activated chloride currents are believed to powerfully amplify the olfactory signal during the transduction process. In our recent publication in Nature Neuro- science we identify Ano2 as the calcium-activated chloride channel of olfactory sensory neurons. Antibody labeling of an anterior part of the mouse nose shows expression of Ano2 (green) exclusively in the ciliary layer of the main olfactory epithelium (top) and in sensory protrusions of the pheromone sensing vomeronasal organ (bottom). Surprisingly, disruption Left: of Ano2 in mice did not affect olfactory performance. Anyess von Bock In contrast to textbook knowledge, normal olfaction does not need signal amplification by calcium-activat- Right: ed chloride currents. Sebastian Schütze

MEMBERS OF THE GROUP SELECTED PUBLICATIONS COLLABORATIONS EXTERNAL FUNDING

Dr. Luiza Bengtsson, EF Patricia Preston (doctoral student), PT, * Novarino G, Weinert S, Rickheit G, International Deutsche Forschungsgemeinschaft Deutsche Forschungsgemeinschaft

Jentsch TJ (2010) Endosomal chloride- Seth Alper, Harvard, USA “Ionentransporter und GABAerge “Dissektion der Rolle von CIC-5 bei Dr. Pawel Fidzinski Sebastian Schütze (doct. student), EF, PT, * proton exchange rather than chloride Andrea Ballabio, Transmission“ der renalen Endozytose und tubulären Dr. Chandresh Gajera, EF, * Patricia Seja (doctoral student), EF, PT, * conductance is crucial for renal endocytosis. Telethon, Napoli, Italy, and Houston, USA JE 164/8-1 Transportprozessen“

Science 328: 1398-1401 Jacques Barhanin, Valbonne, France with C. Hübner FOR 667 (JE 164/6-1) Dr. Hermann-Josef Kaiser, * Felizia Voss (doctoral student), EF, PT, * Christian Chabbert, Montpellier, France 07.2008 – 06.2011 07.2006 – 08.2008 (verlängert bis 06.2010) Preston P, Wartosch L, Günzel D, Fromm M, Dr. Ioana Neagoe, EF, * Niclas Gimber (diploma student), PT, * Jon Cooper, King’s College, London, UK 491.895 Euro 211.583 Euro Kongsuphol P, Ousingsawat J, Kunzelmann Cor Cremers, Nijmegen, Holland Dr. Gaia Novarino, * Verena Perneczky (diploma student), PT, * K, Barhanin J, Warth R, Jentsch TJ (2010) Deutsche Forschungsgemeinschaft Europäische Kommission Dominique Eladari, Paris, France Disruption of the K+ channel β-subunit “Protein modules involved in vesicular (6. Forschungsrahmenprogramm) Dr. Carsten Pfeffer, EF Cathleen Rohleder (diploma student), PT, * Raúl Estévez, Universitat de Barcelona, Spain KCNE3 reveals an important role in acidification and trafficking: focus of Teilprojekt des Integrated Project Euro- Miguel Ángel Moreno-Pelayo, Dr. Balazs Pál, EF, * Florian Wagner (diploma student), PT, * intestinal and tracheal Cl- transport. CIC-6” Hear: “Advances in hearing science: Hospital Ramón y Cajal, Madrid, Spain J Biol Chem 285: 7165-7175 SFB 740, TP C05 from functional genomics to therapies” Dr. Vanessa Plans, EF Anyess von Bock (technical assistant) Kyoshi Mori, Kyoto University, Japan 01.2007 – 12.2010 LSHG-CT-2004-512063 Stauber T, Jentsch TJ (2010) Sorting motifs Roger Nicoll, UCSF, USA Dr. Guillermo Spitzmaul, EF Alexander Fast (technical assistant), EF, * 313.200 Euro 07.2006 – 11.2009 of the endosomal/lysosomal CLC chloride Michael Pusch, CNR, Genova, Italy 310.104 Euro Dr. Tobias Stauber, EF Inga Freyert (technical assistant), EF, * transporters. J Biol Chem 285 (45): 34537-48 Francisco Sepúlveda, CECS, Valdivia, Chile Deutsche Forschungsgemeinschaft

Richard Smith, Iowa, USA “Der CIC-7/Ostm1 Chloridtransporter in “Leibniz-Vorhaben im Rahmen des Dr. Lena Wartosch, EF, PT, * Petra Göritz (animal care taker) Weinert S, Jabs S, Supanchart C, Schweizer Marie Trudel, Montréal, Canada Lysosomen und Osteoklasten“ ‘Paktes für Forschung und Innovation‘“ M, Gimber N, Richter M, Rademann J, Dr. Stefanie Weinert, EF Nicole Krönke (technical assistant) Chris de Zeeuw, Rotterdam, Netherlands JE 164/7-1 01.2009 – 12.2011 Stauber T, Kornak U, Jentsch TJ (2010) 01.2007 – 31.07.2010 128.000 Euro p.a., davon 1x IIa, 1x Va Dr. Dietmar Zimmer Ina Lauterbach (technical assistant), EF, * Lysosomal pathology and osteopetrosis National 533.844 Euro (davon 1xIIa, 2xIIa/2, 1xIVb) (Research Coordinator), PT upon loss of H+-driven lysosomal Cl- Bernd Fakler, Freiburg Exzellenzinitiative an der Humboldt- Rainer Leben (technical assistant), EF accumulation. Science 328: 1401-1403 Michael Fromm Deutsche Forschungsgemeinschaft Universität zu Berlin, Projekt NeuroCure: Eun-Yeong Bergsdorf (doct. student), EF, PT, * Janet Liebold (technical assistant) Charité – Universitätsmedizin Berlin “Strukturelle Grundlagen und physiolo- “Towards a better outcome of central Pfeffer CK, Stein V, Keating DJ, Maier H, Gwendolyn Billig (doctoral student), EF, PT Maik Gollasch gische Funktion des Cl-/H+-Gegenaus- nervous system disorders” Ruth Pareja (technical assistant), EF Rinke I, Rudhard Y, Hentschke M, Rune G, Charité – Universitätsmedizin Berlin tausches bestimmter CLC-Chloridtrans- 01.2008 – 12.2010 Gregor Däubler (MD doctoral student), PT, * Jentsch TJ, Hübner CA (2009) NKCC1- Patrick Seidler (technical assistant) Christian Hübner, Jena portproteine“ 30.000 Euro Sachm./J. dependent GABAergic excitation drives Kathrin Gödde (doctoral student), PT, * Uwe Kornak ZD 58/1-1 Stephanie Wernick (technical assistant), * synaptic network maturation during early Alexander v. Humboldt-Stiftung Charité – Universitätsmedizin Berlin with A. Zdebik Matthias Heidenreich (doctoral student), PT hippocampal development. Guillermo Spitzmaul Silke Zillmann (technical assistant), EF und MPI Mol. Genet., Berlin 07.2006 – 10.2010 J Neurosci 29: 3419-3430 06.2008 – 01.2010 Sabrina Jabs (doctoral student), EF, PT Karl Kunzelmann, Regensburg 110.950 Euro 14.400 Euro Gary Lewin, MDC Berlin continued as: Lilia Leisle (doctoral student), EF, PT, * * Part of period reported FMP authors Hannes Maier, Hamburg JE 164/9-2 Prix Louis Jeantet Carmen Ludwig (doctoral student), EF, PT,* PT part time Group members Tobias Moser, Göttingen 01.2011 – 12.2013 04.2006 – 12.2010

EF position funded externally Dietmar Schmitz 169.200 Euro 308.008 Euro Kristin Natho (doctoral student), EF, PT (3rd-party funds) for at least part Charité – Universitätsmedizin Berlin Oberheide, Karina (doctoral student), PT, * of the reporting period Michaela Schweizer, Hamburg Current group members (31.12.2010) Richard Warth, Regensburg

68 RESEARCH GROUPS // SIGNAL TRANSDUCTION/MOLECULAR GENETICS RESEARCH GROUPS // SIGNAL TRANSDUCTION/MOLECULAR GENETICS 69 SIGNAL TRANSDUCTION/ MOLECULAR GENETICS

MOLECULAR CELL PHYSIOLOGY

Figure 1. Model of formation and molecular organiza- GROUP LEADER tion of heteropolymeric claudin strands. Cld1, -2, -3, -5, Ingolf E. Blasig and -12, assumed to be expressed at cerebral barriers, are shown. Black double arrows, cis-interaction; green spheres, cis-oligomers; n, number of molecules; red arrows, homo- and heterophilic trans-interactions; black/yellow arrows, Cld3/Cld5 trans-interactions; CldX, claudin that incorporates Cld12 by heterophilic trans- or cis-interaction.

We focus on the structure, function, and modulation of cell Elucidation of the tightening New modulators of TJ the CPE peptide was used as a new method mechanism of TJ Claudins form the backbone of the TJ strands, to enrich claudins and to identify new clau- contacts to explore tight junctions (TJ) in tissue barriers, in order Our innovative approach of quantifying the and their combination determines the barrier din-binding proteins in a mass spectrometric to disclose pathological mechanisms that might lead to improved interactions between TJ proteins (Haseloff et function in a tissue. Modulation of such bar- approach (Lohrberg et al., 2009). The FRET al., 2010) revealed molecular principles that riers is therefore of potential pharmaceutical assays were adapted to single cells for FACS therapies. New strategies specifically manipulating selected determine how claudins may close the para- interest. As an example modulator, the bind- analysis, to discriminated between trans- barriers are proposed for improving drug delivery and to prevent cellar cleft (Piehl et al., 2010, Zhang et al., ing of Clostridium perfringens enterotoxin to and cis-interaction and to increase the cell 2010). For the first time, a complete picture of claudins was analyzed. We identified the ami- number investigated, and to cell extracts for barrier dysfunction. the homologous and heterologous interac- no acid motif and the interaction mechanism fluorimetric measurements in the screening The paracellular tightness is determined by membrane proteins: tion possibilities between the members of a resulting in a molecular model of the bind- of modulators. For a better interpretation of highly conserved claudin group, called classic ing patch, the second extracellular loop of the FRET data, especially of heterologous claudins, TAMPs and associated scaffolding proteins. Structure, claudins, was established (Fig. 1). We showed claudin-3 (Krause et al., 2009; Winkler et al., claudin-claudin, claudin-TAMP, and TAMP- regulation, and protein interactions within TJ are unclear concern- that conserved sequences in the second ex- 2009; Veshnyakova et al., 2010). This model is TAMP interactions, fluorescence recovery tracellular loop of these claudins tighten the relevant to the highly homologous group of after photobleaching (FRAP) was performed. INGOLF E. BLASIG ing opening or tightening of TJ. Our work elucidates the oligo- gap between the cell surfaces of two oppos- classic claudins. In addition to the use of toxin To compare the local tightness of a claudin or

merization of key proteins of cerebral TJ and mechanisms to open ing cells through an aromatic core. Claudin-5 ligands of the extracellular loop 2 of claudins TAMP barrier, a novel test was designed by 1970 – 1974 Studied biology and is essential for the BBB, which we established as TJ-modulator, we found claudin peptides measuring the paracellular diffusibility. Based biochemistry in Leipzig, diploma thesis and/or to reconstitute the TJ after opening. as a new pharmacological and diagnostic tar- specifically and transiently opening neuro- on the interaction studies, a new enrichment on cancer research at the Robert- To disseminate news of progress in the field, we organize an get. Investigation of the interactions within logical barriers. These peptides allowed the method for claudins has been worked out Rössle-Hospital in Berlin-Buch another group of the transmembrane TJ utilization of drugs that are otherwise ineffec- which also allowed the identification of new annual international symposium; the results presented at these 1984 Dissertation on the pharmacology proteins, the TJ-associated marvel proteins tive: drug uptake enhancers. Patents are cur- binding partners of TJ proteins (Lohrberg et of myocardial infarction at the Academy have stimulated additional publications and have attracted (TAMP) (Westphahl et al., 2010), as well as rently in preparation. al., 2009). of Sciences, Berlin between claudins and TAMP, exhibited re- increased funding. duced binding possibilities and a reduced Methodological goals Translation 1992 Habilitation for investigations on binding intensity. Powerful protein-protein binding assays We recommended clinical studies both to reactive species in myocardial dysfunction were modified or evolved. Size-exclusion confirm our findings that oxidative stress in- and venia legendi for biochemical New regulatory role of occludin chromatography, native polyacrylamide gel jures the blood-brain barrier, and to optimize pharmacology, University of Halle- The exact function of the TAMP is not known. electrophoresis, dynamic light scattering, therapy. In mood disorders, an opening of the Wittenberg For occludin, a unique TJ marker, we now and mass spectrometry were applied for the blood-brain barrier was found. Both are re- Since 1992 Head of the independent know that its oligomerisation is redox-sen- first time to TJ proteins. They demonstrated duced by selected antipsychotics (Schroeter research group for Molecular Cell Physiology sitive (Walter et al., 2009b), and that the TJ a dimerisation potential of extracellular loops et al., 2009 and 2010). at the FMP, teaching of pharmacology, function is regulated via occludin by the cel- of claudins and occludin’s coiled coil-domain functional biochemistry, neurochemistry at lular redox potential (Walter et al., 2009a). (Winkler et al., 2009; Walter et al., 2009a), the universities in Potsdam and Berlin This alteration is of relevance for pathological and the TAMP family. We developed quanti-

conditions with oxidative stress, such as hy- fication assays for the association between TJ 1993 – 1995 Awarded project leader at poxia or blood flow disturbances (Leblanc et proteins in living cells: FRET assays to analyze the NIH, Washington, DC/USA. al., 2009). Moreover, the oligomerization sta- the cis-interaction, and a cell scan method for tus of occludin influenced its association with the trans-interaction (Fig. 2). The interaction other TJ-proteins, e.g. claudin-5 and ZO-1. measurements with ligands of claudins, e.g.,

70 RESEARCH GROUPS // SIGNAL TRANSDUCTION/MOLECULAR GENETICS RESEARCH GROUPS // SIGNAL TRANSDUCTION/MOLECULAR GENETICS 71 From left to right: Tarek Saleh, Benjamin Lau

Olga Breitkreuz-Korff, Nora Gehne,

Christian Staat, Nadja Käding

MEMBERS OF THE GROUP SELECTED PUBLICATIONS COLLABORATIONS EXTERNAL FUNDING

Dr. Reiner Haseloff Dr. Jörg Piontek Piehl C, Piontek J, Cording JD, Wolburg H, International Deutsche Forschungsgemeinschaft (7. Forschungsrahmenprogramm)

(independent funding), EF Blasig IE (2010) Participation of the second “Alterationen von Occludin bei Project: JUSTBRAIN Dr. Uwe Schlomann, EF, PT, * EU project consortium ‘JUSTBRAIN’ extracellular loop of claudin-5 in paracellular oxidativem Stress und Aufklärung der “Blood-brain barrier junctions as Jan Rossa (doctoral student), PT, *, # Dr. Giovanna Del Vecchio, EF, * tightening against ions, small and large European consortium ‘Brains4brain’ Funktion von Claudin-12” targets for paracellular drug delivery

Jonas Protze (doctoral student), PT, EF, *, # molecules. Cell Mol Life Sci 67: 2131-2140 FOR 721/2, TP5 (BL 308/9-1) to the brain” Dr. Denise Zwanziger, EF, * Danica Stanimirovic 01.2010 – 12.2012 11.2009 – 10.2013 Ivonne Heise (student), PT, *, # Zhang J, Piontek J, Wolburg H, Piehl C, IBS, NRC, Ottawa, Canada Christian Bellmann (doctoral student), PT, * 126.000 Euro 606.925 Euro Liss M, Otten C, Christ A, Willnow TE, Fränze Vorreiter (student), PT, *, # Elga de Vries, Amsterdam Victor Manuel Castro Villela Blasig IE, Abdeliah-Seyfried S (2010) Deutsche Forschungsgemeinschaft B4B Foundation (doctoral student), EF, PT, * Negar Waziri (student), PT, *, # Establishment of a neuroepithelial barrier Jerry Turner, Chicago, USA “Modulation der Claudinoligomeri- “Modifying arylsulphatase A with

by Claudin5a is essential for zebrafish sierung zur Beeinflussung der peptides to bypass the blood-brain Jimmi Cording (doctoral student), PT, * Christian Kern (student), PT, *, # Walter Hunziker, Singapur brain ventricular lumen expansion. Blut-Hirnschranke” barrier” Sebastian Dabrowski Proc Natl Acad Sci USA 107: 1425-1430 Tetsuja Terasaki, Sendai, Japan BL 308/7-4 07.2010 – 03.2012

(doctoral student), EF, PT, * * Part of period reported 01.2009 – 12.2011 80.000 Euro Walter JK, Castro V, Voss M, Gast K, Jean-Michel Heard, Paris, France 116.000 Euro Anja Kublik (doctoral student), PT, * # funding and supervision: Jörg Piontek Rueckert C, Piontek J, Blasig IE (2009) Land Berlin

Redox-sensitivity of the dimerization of Deutsche Forschungsgemeinschaft “Blut-Hirnschrankenmodulatoren” Inga Newie (doctoral student), EF, PT, * PT part time National occludin. Cell Mol Life Sci 66: 3655-3662 “Molekuare Organisation von hetero- (Programm zur

Christian Piehl (doctoral student), EF, PT, * EF position funded externally DFG Forschergrupe ‘TJ’ polymeren Tight-Junction-Strängen” Förderung von Forschung, Innovationen Lohrberg D, Krause E, Schümann M, (3rd-party funds) for at least part PI 837 / 2-1 und Technologien – ProFIT ) Christian Staat (doctoral student) EF, PT, * Piontek J, Winkler L, Blasig IE, Haseloff RF Salim Abdelliah-Seyfried, MDC Berlin of the reporting period J. Piontek IBB 10142936 (2009) A strategy for enrichment of claudins Christian Tscheik (doctoral student), EF, PT, * Dominik Müller 07.2009 – 06.2012 07.2009 – 12.2011 Current group members (31.12.2010) based on claudin affinity to Clostridium Charité – Universitätsmedizin Berlin 228.000 Euro 275.200 EUR Juliane Walter (doctoral student), EF, PT; * perfringens enterotoxin.

BMC Mol Biol 10:61: 1-8 Hartwig Wolburg, Universität Tübingen Deutsche Forschungsgemeinschaft Johanna Berg (student), PT,* “Molekulare und strukturelle Muster Winkler L, Gehring C, Wenzel A, Mueller SL, Bernd Nürnberg, Universität München Corinna Gagell (student), PT, * parazellulärer Dichtheit durch Piehl C, Krause G, Blasig IE, Piontek J (2009) Heike Rittner, Universität Würzburg subtypabhängige Claudin-Claudin- Inga Eichhorn (student), PT, * Molecular determinants of the interaction Wechselwirkungen in Tight Junctions” between Clostridium perfringens enterotoxin Klaus Gast, Universität Potsdam Sophie Jankowski (student), PT,* FOR 721, (KR 1273/3-2) and claudins. J Biol Chem 284: 18863-18872 Otmar Huber, Universität Jena J.Piontek with G.Krause Benjamin Lau (student), PT, * 1.2010 – 12.2012 Matthias Schroeter, MPI Leipzig Kristin Trapp (student), PT, * FMP authors 315.840 Euro

Group members Stefan Liebner, University Frankfurt/M. Maria Walter (student), PT, *

Barbara Eilemann (technical assistant)

72 RESEARCH GROUPS // SIGNAL TRANSDUCTION/MOLECULAR GENETICS RESEARCH GROUPS // SIGNAL TRANSDUCTION/MOLECULAR GENETICS 73 SIGNAL TRANSDUCTION/ MOLECULAR GENETICS

Figure 1. A strategy for the identification of novel ANCHORED AKAPs. A. An AKAP pattern, derived from the RII- binding domains of known AKAPs, was used to screen the human proteome (Swissprot database) for potential SIGNALLING novel AKAPs. Out of 4,519 candidate peptides, 2,572 were spot-synthesized and assayed for an interaction with RII subunits of PKA by RII overlay (incubation with radioactively labeled RII). 829 peptides bound RII. B. 27 GROUP LEADER putative novel AKAPs were identified (rows C-E). When Enno Klußmann/Walter Rosenthal the RII probe used for RII overlays was preincubated with an AKAP peptide (AKAP18δ-L314E) the binding to the peptide spots was reduced whereas an inactive control peptide (AKAP18δ-PP) had no effect, demonstrating that the peptide spots bind to the same site on RII as established AKAPs. One of the identified AKAP candidates was GSKIP (spot E03, box).

Protein kinase A (PKA) is a ubiquitous serine/threonine kinase that Identification of a newAKAP , Glycogen in response to cAMP elevation. We have re- in the framework of our GoBio project, we Synthase Kinase 3 Interaction Protein cently shown that the elevation of cAMP in- have established a technology platform for controls a large variety of cellular functions. Specificity ofPKA β The interaction between AKAPs and PKA creases AQP2 protein levels within 30 min- the identification of small molecule disrup- action is achieved by controlling its cellular localization through is conserved within the AKAP family. It is utes in primary cultured rat inner medullary tors of such interactions. In cooperation direct and mediated by the dimerization collecting duct (IMCD) cells, in human em- with the screening unit, we carry out prima- A-kinase anchoring proteins (AKAPs). AKAPs bind PKA through an and docking domain of regulatory subunits bryonic kidney (HEK) 293 cells ectopically ry high throughput small molecule library amphipathic α-helical structure (RII-binding domain), which inter- of PKA and the PKA-binding domain of expressing AQP2, and, most importantly, screenings using cell-based assays and in AKAPs. Through detailed analysis of the in the mouse kidney. The increased abun- vitro assays such as Alpha screen and ELISA. acts directly with the dimerization and docking (D/D) domain of interactions, we generated an AKAP signa- dance of AQP2 could not be explained by Hits are validated in vitro, in cell culture regulatory subunit dimers. Besides PKA, AKAPs directly bind other ture motif. A combination of bioinformatics increased transcription or translation. We and in animal experiments. We have estab- and peptide array screening based on this observed that cAMP inhibited p38-mito- lished a medicinal chemistry laboratory for signalling proteins such as other protein kinases, protein phospha- signature motif identifiedG SKIP (glycogen gen-activated protein kinase (p38-MAPK) the optimization of hits with regard to affin- tases, phosphodiesterases (PDEs), GTPases, adaptor proteins, and synthase kinase 3β interaction protein) as via activation of PKA, and that this inhibition ity and specificity. The identified molecules an AKAP. In vertebrates, GSKIP binds PKA was associated with decreased phosphory- are not only valuable tools for studying the ENNO KLUSSMANN substrate proteins of PKA through unique interacting domains. and GSK3β (glycogen synthase kinase 3β) lation (serine 261) and polyubiquitination of role of AKAP-PKA interactions in cellular

Thus AKAPs coordinate multi-protein signalling complexes estab- directly, and facilitates the phosphorylation AQP2. Consequently, proteasomal degra- processes, but may also pave the way to 1985 – 1988 Studied genetics at – and thus inactivation – of GSK3β by PKA. dation of AQP2 was decreased, explaining new concepts for the treatment of chronic the University of London, Queen lishing compartmentalized signalling. Our aim is to elucidate the GSKIP is expressed in renal principal cells the increase in its abundance. Thus AVP el- heart failure. Mary College, UK role and the molecular mechanisms of compartmentalized cAMP/ and cardiac myocytes. A potential role in evates AQP2 protein abundance through a 1988 – 1992 Studied biology at the control of AVP-mediated water reab- PKA- and p38-MAPK–dependent pathway PKA signalling in controlling arginine-vasopressin (AVP)-mediated the Philipps-University, Marburg sorption and cardiac myocyte contractility that controls the proteasomal degradation

water reabsorption in renal principal cells and cardiac myocyte is currently being investigated. In inverte- of AQP2. (J Am Soc Nephrol, 2010) 1992 – 1996 Ph.D. at the Department brates, GSKIP only interacts with GSK3β, so of Gastroenterology (Prof. Hanski), contractility. the AKAP function was apparently gained Identification of small molecules for the Charité – Universitätsmedizin Berlin with the evolution of vertebrates. (J Biol inhibition of AKAP-PKA interactions 2005 Habilitation in pharmacology Chem 2010) A focus of the work in the group has been and toxicology at the Charité – the identification of small molecule disrup- Universitätsmedizin Berlin Crosstalk between PKA and p38-MAP tors of AKAP-PKA interactions. AKAP-PKA

Kinase regulates aquaporin-2 abundance interactions play key roles in the control of 1996 – 1997 Post-Doc at the Through control of the water channel aqua- AVP-mediated water reabsorption in renal Department of Gastroenterology and the porin-2 (AQP2) in the renal collecting duct, principal cells and contractility of cardiac Institute of Pharmacology at the Charité – AVP maintains homeostasis of body water. myocytes (Skroblin et al., 2010). Both cell Universitätsmedizin Berlin AVP activates vasopressin V2 receptors types are involved in the development of 1997 – 2003 Post-Doc at the FMP, (V2R), which leads to an increase in cAMP, chronic heart failure. Preliminary data ob- Group of Prof. Rosenthal activation of PKA, and PKA-catalyzed tained by us and others in cell, organ, and

phosphorylation of AQP2. This, in turn, animal experiments indicated that disrup- 2003 – 2010 Head of Anchored promotes the translocation of AQP2 from tion of AKAP-PKA interactions in the two Signalling Group at the FMP intracellular vesicles into the plasma mem- cell types positively influences the devel- brane. Transcription of AQP2 also increases opment of chronic heart failure. Therefore,

74 RESEARCH GROUPS // SIGNAL TRANSDUCTION/MOLECULAR GENETICS RESEARCH GROUPS // SIGNAL TRANSDUCTION/MOLECULAR GENETICS 75 From left to right: Dörte Faust

Marie Moutty, Jessica Tröger, Frank Götz, Kerstin Zühlke, Philipp Skroblin

Jana Bogum

MEMBERS OF THE GROUP SELECTED PUBLICATIONS COLLABORATIONS EXTERNAL FUNDING

Dr. Andrey Christian da Costa Goncalves, Manja Lukesch (student), PT, * Nedvetsky PI, Tabor V, Tamma G, International Deutsche Forschungsgemeinschaft Bundesministerium für Bildung EF, * Beulshausen S, Skroblin P, Kirschner A, “The function of AKAP-dependent und Forschung Christoph Schult (student), PT, * M. Houslay Mutig K, Boltzen M, Petrucci O, protein-protein interactions in the “GO-Bio 2: Entwicklung neuartiger Wirk- Dr. Adeeb El-Dashan, EF, * University of Glasgow, Scotland Beate Eisermann (technical assistant), EF Vossenkämper A, Wiesner B, Bachmann S, regulation of vaso-pressin-mediated stoffe zur Behandlung von Herzkreislauf- Dr. Sabine Friedl, EF, * Rosenthal W, Klussmann E (2010) Reciprocal D. M. Cooper water reabsorption and cardiac erkrankungen durch Modulation Andrea Geelhaar (technical assistant) regulation of Aquaporin-2 abundance and University of Cambridge, England myocyte contractility” von Protein-Protein-Wechselwirkungen“ Dr. Frank Götz, EF, PT, Jürgen Malkewitz, (technical assistant), EF, * degradation by protein kinase A and FOR 806, TP 01 (KL 1415/4-2) 315097 K. Tasken Dr. Evelina Grantcharova, EF p38-MAP kinase. with W. Rosenthal 01.2008 – 10.2011 Anita Neumann (technical assistant) University of Oslo, Norway J Am Soc Nephrol 21: 1645-1656 06.2010 – 05.2013 2.586.862 Euro Dr. Solveig Großmann, EF, * Sylvia Niquet (technical assistant), EF, M. Zaccolo 225.750 Euro Hundsrucker C, Skroblin P, Christian F, Europäische Kommission Dr. Nico Kotzur, EF, * University of Glasgow, Scotland Benjamin Leibrandt (apprentice), * Zenn HM, Popara V, Joshi M, Eichhorst J, Deutsche Forschungsgemeinschaft (6. Forschungsrahmenprogramm) Dr. Jelena Milic, EF, * Wiesner B, Herberg FW, Reif B, G. Valenti “Defining cellular functions of the STREP thera-cAMP: Vivian Schulz (apprentice), EF, * Rosenthal W, Klussmann E (2010) Glycogen University of Bari, Italien A kinase-anchoring protein AKAP18 “Identification of therapeutic molecules Dr. Marie Moutty, EF, * Aline Kirschner (apprentice), EF, * synthase kinase 3beta interaction protein by pharmacological ablation of its to target compartmentalized cAMP G. Tamma Dr. Elvira Rohde, * functions as an A-kinase anchoring protein. interaction sites” signaling networks in human disease” Cindy Büssow (apprentice), * University of Bari, Italy J Biol Chem 285: 5507-21 FOR 806, TP 01 (KL 1415/4-1) LSH-2005-1.2.5-3 Dr. Vedrana Tabor, EF, M. Schmidt with W. Rosenthal, R. Volkmer 09.2006 – 09.2009 Skroblin P, Grossmann S, Schäfer G, Dr. Kerstin Zühlke, EF, * * Part of period reported University of Groningen, The Netherlands (Charité – Universitätsmedizin Berlin) 798.149 Euro Rosenthal W, Klussmann E (2010) 01.2007 – 08.2010 Sven Beulshausen (doctoral student), EF, PT, * PT part time Mechanisms of protein kinase A anchoring. National 364.500 Euro Int Rev Mol Cell Biol 283: 235-330 Jana Bogum (doctoral student), EF, PT EF position funded externally (invited review) F.W. Herberg Deutsche Forschungsgemeinschaft (3rd-party funds) for at least part Frank Christian (doctoral student), EF, PT, * Universität Kassel “Identifizierung und Charakterisierung of the reporting period Meng D, Lynch MJ, Huston E, Beyermann M, von Proteinen, die den Transport von Verena Ezerski (doctoral student), PT, EF Eichhorst J, Adams DR, Klussmann E, M. Bergmann Current group members (31.12.2010) Aquaporin-2 in renalen Hauptzellen Houslay MD, Baillie GS (2009) MEK1 binds Asklepius Klinik St. Georg, Hamburg Dörte Faust (doctoral student), EF, PT, * kontrollieren“ directly to betaarrestin1, influencing both and MDC, Berlin FOR 667, TP 03 (KL 1415/3-2) Claudia Noack (doctoral student), EF, PT, * its phosphorylation by ERK and the timing O. Daumke with W. Rosenthal of its isoprenaline-stimulated internalization. Gesa Schäfer (doctoral student); EF, PT, MDC, Berlin 01.2009 – 12.2011 J Biol Chem 284: 11425-35 252.150 Euro Philipp Skroblin (doctoral student), EF, PT S. Bachmann Nedvetsky PI, Tamma G, Beulshausen S, Charité – Universitätsmedizin Berlin Jessica Tröger (doctoral student), EF, PT Valenti G, Rosenthal W, Klussmann E (2009) Regulation of aquaporin-2 trafficking. Tordis Borowski (student), EF, PT, * Handb Exp Pharmacol 190: 133-57 Christian Courault (student), PT, *

Hendrikje Göttert (student), PT, * FMP authors Group members

76 RESEARCH GROUPS // SIGNAL TRANSDUCTION/MOLECULAR GENETICS RESEARCH GROUPS // SIGNAL TRANSDUCTION/MOLECULAR GENETICS 77 SIGNAL TRANSDUCTION/ MOLECULAR GENETICS

MOLECULAR NEUROSCIENCE AND BIOPHYSICS

Figure 1. Disulfide bridges trap GluK2 ligand binding GROUP LEADER cores at two sites. Andrew J. R. Plested Left panel: The tetramer arrangement of the ligand binding cores and the Cysteines introduced to crosslink subunits. Centre panel Western blots reveal dimers in oxidizing, but not reducing, conditions. Right panel: Brief pulses of glutamate activate fast desensitizing currents that are differently inhibited by oxidizing conditions (red fitted traces).

Our aim is to understand the molecular basis of neurotransmitter Domain organization and function in The desensitized state of AMPA and Mapping gating and interactions of AMPA receptor activation. Our main interest is the glutamate receptors AMPA and Kainate receptors (A. Plested) kainate receptors (Anna Carbone) receptors using orthogonal chemistry The AMPA-type glutamate receptor has an Despite their structural similarities, AMPA (Viktoria Klippenstein) that mediate excitatory transmission at ~60% of synapses in the brain. unexpected arrangement of the extracellular and kainate receptors have divergent We hypothesize that structural changes that We study these channels because they are essential for learning domains (Sobolevsky et al. Nature 2009). In properties, including the 100-fold longer underlie glutamate receptor gating occur collaboration with the laboratory of Mark May- lifetime of the desensitized state in kain- within the plasma membrane. To address and memory, and are important for many diseases of the nervous er, we used a combination of electrophysiol- ate receptors. This difference has a physi- these interactions, we will express iGluRs system. To achieve this goal, we use patch clamp electrophysiology, ogy, X-Ray crystallography, and biochemistry ological role in the processing of infor- harboring genetically-encoded photoactive to show that the same arrangement is found mation in the retina and hippocampus, crosslinkers in mammalian cells. Our tools specializing in rapid agonist application and single-channel record- in the kainate-selective glutamate receptor. but its molecular mechanism is unknown. are the unnatural amino acids (UAAs) Benzo- ing. We also employ biochemistry, chemical biology and structural Using crystallographic data we identified We constructed functional chimeras of AMPA ylphenylalanine (BzoF) and Azidophenylala- potential interaction sites in the extracellu- and kainate receptors, swapping the ligand nine (AziF). Upon soft UV irradiation (365 nm), studies to investigate the assembly and dynamics of glutamate lar amino terminal domains (ATDs) of GluK2 binding domains, and were able to entirely these derivatives form free radicals that cova- ANDREW J. R. PLESTED receptor complexes. Our long-term aim is to develop tools for kainate receptors. Western Blotting showed exchange the lifetime of the desensitized lently crosslink to neighbouring protein back- that Cysteine residues introduced at these states (see Figure 2). This exchange shows bone. We will use these crosslinks to map manipulating these complexes in vivo, and, in so doing, understand 1994 – 1998 M.Sci. 1st Class Hons, Physics, sites were able to induce selective disulfide that the ligand binding domains are the sole helix movements during gating, and con- Imperial College, University of London, UK their role in the brain in health and disease. crosslinking in full-length receptors. Thus, the determinants of the recovery rate in AMPA tacts between iGluRs and partner proteins.

dimer interactions in the ATD layer, and the and kainate receptors. In contrast to previ- We incorporate UAAs via suppression of the 1998 – 2002 Ph.D. Imperial College small footprint interdimer interface, identi- ously published work, we were able to show amber stop codon (TAG), by inclusion of a (Prof. Franks and Prof. Lieb), fied from crystal structures of GluK2 ATDs, that this exchange does not involve spurious coevolved tRNA-synthetase pair (a gift from University of London, UK are both present in the full-length recep- changes in receptor function, such as large T. Sakmar, Rockefeller University). We screen 2003 – 2005 Post-Doc, Dept. of tor. Furthermore, we showed for the first shifts in binding affinity or ligand efficacy. for rescue of TAG-mutants by incorporation Pharmacology (Prof. Colquhoun), time that restraint of the ATD could alter Kinetic modelling suggests that our results of BzoF and AziF, using iGluR-GFP fusions. University College London, UK receptor function in non-NMDA receptors. are most economically explained by a de- We have confirmed membrane expression

We extended the investigation to the ligand sensitized state from which ligands are very of ~20 iGluR-GFPs harboring Azi and Bzo 2005 – 2008 Visiting Research Fellow, binding cores, and paid particular attention slow to unbind. Ongoing mutagenesis stud- at specified sites using confocal micoscopy. Laboratory of Cellular and Molecular to interdimer interactions, which have not ies have identified some of the residues that We also subject Azi-iGluRs and Bzo-iGluRs Neurophysiology (Dr. Mayer), NICHD, previously been reported. We were able to underlie this difference, and also a correlation to fast-solution exchange electrophysiology, NIH, Bethesda, USA crosslink subunits at two sites (see Figure 1). between desensitization and gating. and flash photolysis. So far, we have shown Since 2008 Junior Group Leader, FMP, These results suggest that Kainate receptors that 365 nm light has little effect on wild-type Berlin share a common architecture with AMPA receptors, or on the GluA2 mutants F541AziF

and NMDA receptors, including the subunit and Y768AziF. We confirmed the selective in- Since 2008 Assistant Professor, Molecular crossover between extracellular domains. corporation of the AziF side chain using the Neuropharmacology (W1) NeuroCure, Further, our data provide the first evidence L483AziF mutant, which had the same prop- Charité – Universitätsmedizin Berlin that chemically identical subunits have dis- erties to the L483F mutant, distinct from wild- tinct roles in receptor gating, dependent on type channels. AziF and BzoF mutants will be their positions in the complex. This work was used for biochemical studies, and AziF mu- published in PNAS (Das et al. 2010). tants can be used for orthogonal labelling by Click chemistry.

78 RESEARCH GROUPS // SIGNAL TRANSDUCTION/MOLECULAR GENETICS RESEARCH GROUPS // SIGNAL TRANSDUCTION/MOLECULAR GENETICS 79 Left: Valentina Ghisi From left to right: Jean-Baptiste Esmenjaud (intern), Right: Marcus Wietstruk Héctor Salazar

MEMBERS OF THE GROUP SELECTED PUBLICATIONS COLLABORATIONS

Figure 2. Ligand binding domains control recovery Dr. Anna Carbone, EF, * Das U, Kumar J, Mayer ML, Plested AJR International

from desensitization. AMPA receptors (GluA2) recover (2010) Domain organization and function in very rapidly from desensitization after a long pulse of Dr. Héctor Salazar, EF, * Chris Ahern GluK2 subtype kainate receptors. glutamate. Kainate receptors (GluK2) recover much University of British Columbia, Canada more slowly. A chimera in which the ligand binding Viktoria Klippenstein PNAS 107: 8463-8

domain (LBD) of GluA2 is swapped into the GluK2 (doctoral student), PT, EF, * Baron Chanda backbone (B2P6) recovers as fast as wild-type GluA2 Vijayan R, Plested AJR, Biggin PC and University Wisconsin Madison, USA (red curve). The inverse chimera (B6P2) recovers at the Marcus Wietstruk (technical assistant) Mayer ML (2009) Selectivity and Cooperativ- same rate as GluK2 (blue curve). Dashed lines show ity of modulatory ions in a neurotransmitter Mark Mayer the time course of wild-type receptor recovery. receptor. Biophysical Journal 96: 1751-1760 NIH, USA * Part of period reported

Plested AJR, Mayer ML (2009) Engineer- PT part time National ing a high-affinity allosteric binding site for EF position funded externally divalent cations in kainate receptors. Oliver Daumke (3rd-party funds) for at least part Neuropharmacology 56: 114-20 MDC, Berlin

of the reporting period Plested AJR, Mayer ML (2009) AMPA Current group members (31.12.2010) receptor ligand binding domain mobility EXTERNAL FUNDING revealed by functional cross linking. J Neurosci 29: 11912-23 Deutsche Forschungsgemeinschaft Chaudhry C, Plested AJR, Schuck P, “Trapping the activation mechanism Mayer ML (2009) Energetics of glutamate of AMPA-type glutamate receptors” receptor ligand binding domain dimer PL 619/1-1 assembly are modulated by allosteric ions. 09.2010 – 09.2013 PNAS 106: 12329-34 216.000 Euro

NeuroCure – Innovation Grant FMP authors 01.2010 – 12.2010 Group members 65.000 Euro

80 RESEARCH GROUPS // SIGNAL TRANSDUCTION/MOLECULAR GENETICS RESEARCH GROUPS // SIGNAL TRANSDUCTION/MOLECULAR GENETICS 81 SIGNAL TRANSDUCTION/ MOLECULAR GENETICS 600 SP2-

500 CRF 1R

400 CRF R PROTEIN 2(a) 300 Figure 1. Adenylyl cyclase activity assay using transiently transfected HEK 293 cells expressing CRF2(a)R (black; uncleaved pseudo signal peptide), CRF R TRAFFICKING 1 200 (grey; conventional cleaved signal peptide) and the

signal peptide exchange mutants SP2-CRF1R, and CRF1R SP1-CRF2(a)R. Intact cells were stimulated with increas- GROUP LEADER 100 cAMP formed [pmoles/well] ing concentrations of the agonist sauvagine and a Ralf Schülein cAMP RIA was performed. When the pseudo signal SP1- is present, monophasic concentration response 0 CRF2(a)R curves are recorded (Gs stimulation only). When the conventional signal peptide is present, bell-shaped 10 -12 10 -11 10-10 10 -9 10 -8 10-7 10 -6 10 -5 concentration response curves are detected (Gs coupling at low ligand occupancy, Gi coupling at high Sauvagine [M] ligand occupancy).

G protein-coupled receptors (GPCRs) and other integral membrane Functional significance of cleavable signal Development of a novel method to study Quality control of mutant V2Rs in post-ER peptides of GPCRs receptor trafficking using fusions with the compartments and identification of novel proteins must reach their correct subcellular location to function The ER insertion of GPCRs and other integral photoconvertible Kaede protein substances improving folding and/or traf-

properly. Transport is enabled by the secretory pathway and starts membrane proteins is mediated by signal se- To visualize GPCR trafficking between dif- ficking of misfolded 2V Rs by high through- quences and the protein-conducting Sec61 ferent subcellular compartments, we have put screening and automated microscopy with the signal sequence-mediated insertion of the proteins into channel of the translocon complex. Membrane developed a novel methodology using the Mutations in the genes of membrane pro- the membrane of the endoplasmic reticulum (ER). In the ER, fold- proteins have two types of signal sequences. photoconvertible Kaede protein from the teins frequently lead to misfolded proteins, The majority of the proteins possess signals stony coral Trachyphyllia geoffroyi. The fluo- which are recognized by the QCS and may ing of the proteins is checked by a quality control system (QCS) and forming part of the mature protein (“signal rescence of Kaede can be switched from cause inherited diseases such as X-linked only correctly folded forms enter the later vesicular transport steps anchor sequence”; usually TM1). A smaller green (gKaede) to red (rKaede) using UV nephrogenic diabetes insipidus (NDI; mu- group, however, possesses additional N-ter- irradiation, in contrast to that of the widely tations in the V R). Folding and transport to the plasma membrane. Mutations in the genes of membrane pro- 2 minal signal peptides that are cleaved off dur- used green fluorescenct protein G( FP) and of the mutant receptors can be rescued teins frequently lead to misfolded proteins, which are recognized ing the insertion process. We could show that its derivatives. We have shown that Kaede by “pharmacological chaperones”. These a third type of signal sequence occurs in the fusions do not change the pharmacologi- substances (hydrophobic agonists or an- RALF SCHÜLEIN by the QCS and retained intracellularly. These mutations may cause case of corticotropin-releasing factor receptor cal properties of GPCRs and we have used tagonists) bind to misfolded receptors in the

inherited diseases. The aim of the Protein Trafficking group is to type 2a (CRF R). Here, an N-terminal “pseu- these fusions to study the recycling behav- early secretory pathway and facilitate correct 1982 – 1989 Biology studies at the 2(a) do signal peptide” is present, which is un- iour of the CRF R (3): cell surface receptors folding of the ligand binding pocket, and in University of Würzburg understand the trafficking mechanisms of GPCRs in the early secre- 1 cleaved and forms an additional hydrophobic were internalized by agonist treatment, their turn correct folding of the full length recep- 1989 Diploma in Biology tory pathway and to find novel substances influencing GPCR folding domain at the N tail of the receptor. Recently, gKaede signals were specifically photocon- tor. We recently started a screening project

we have assessed the functional significance verted in the endosomes to rKaede, and the to find new pharmacological chaperones 1990 – 1993 Ph.D. thesis on toxin transport and transport. of this novel GPCR domain and could show fate of the rKaede signals was analyzed mi- for the V R or substances influencing quality in E. coli at the Department of Microbiology 2 that its presence prevents Gi-mediated inhi- croscopically. Using this technique, we could control. Screening algorithms were devel- (Prof. Goebel), University of Würzburg bition of adenylyl cyclase activity, a property, show that the CRF1R belongs to the group of oped to analyze trafficking of GFP-tagged 1995 – 1997 Post-Doc in the Department which could be transferred in signal peptide GPCRs, which are able to recycle to the mutant receptors in HEK 293 cells by auto- of Pharmacology (vasopressin V2 receptor, exchange experiments to the CRF1R, which plasma membrane (Figure 2; Schmidt et al., mated microscopy. We then screened the Prof. Rosenthal), University of Gießen normally possesses a conventional cleaved 2009). ChemBioNet library (17.951 compounds) of

signal peptide (Figure 1; Schulz et al., 2010). the FMP and found one substance, which Since 1997 Group Leader at the FMP The pseudo signal peptide of the CRF2(a)R rep- rescued the transport of a mutant in a con- 2002 “Habilitation” in Pharmacology resents the first example of a signal sequence centration-dependent manner (Ridelis et al., and Toxicology at the Charité – Univer- influencing signal transduction processes. In manuscript submitted). sitätsmedizin Berlin addition to the ER targeting/insertion process, signal peptides also mediate gating of the protein-conducting Sec61 channel. Re- cently, we have shown that the signal peptide

of the ETBR alone is unable to open the Sec61 channel and that the 26 amino acids following the signal peptide are required for this process (gating domain; Alken et al., 2009).

82 RESEARCH GROUPS // SIGNAL TRANSDUCTION/MOLECULAR GENETICS RESEARCH GROUPS // SIGNAL TRANSDUCTION/MOLECULAR GENETICS 83 From left to right: Jens Furkert, Carolin Westendorf

Ingrid Ridelis Rivas

Carolin Westendorf

MEMBERS OF THE GROUP SELECTED PUBLICATIONS COLLABORATIONS

Dr. Martina Alken, * Schulz K, Rutz C, Westendorf C, Ridelis I, International

Vogelbein S, Furkert J, Schmidt A, Dr. Jens Furkert, PT Ramanujan Hegde Wiesner B, Schülein R (2010) The pseudo NIH, Bethesda, USA Dr. Antje Schmidt, EF signal peptide of the corticotropin-releasing Figure 2. Recycling assay using the Kaede-tagged CRF R in stably transfected HEK 293 cells. The gKaede factor receptor type 2a decreases receptor Nikos Tsopanoglou 1 Dr. Susanne Vogelbein signals (upper panels, green) and rKaede signals expression and prevents Gi-mediated University of Patras, Greece (doctoral student), PT, * (lower panels, red) of the receptor were recorded by inhibition of adenylyl cyclase activity. confocal laser scanning microscopy (LSM) prior to Ingrid Ridelis Rivas (doctoral student), PT, * J Biol Chem 285: 32878-32887 National agonist stimulation (0). Cells were stimulated for 30 min with sauvagine and another scan was made Katharina Schulz (doctoral student), EF, PT, * Alken M, Schmidt A, Rutz C, Furkert J, Ulrike Alexiev (internalization). The agonist was removed and photoconversion was induced specifically in the endosomal Kleinau G, Rosenthal W, Schülein R (2009) FU Berlin Carolin Westendorf compartments after ROI setting by UV irradiation The sequence after the signal peptide of (white circles). The fate of the rKaede signals was (doctoral student), EF, PT, * Gunnar Kleinau the G protein-coupled endothelin B receptor analyzed in a time series in the same cells (one picture Charité – Universitätsmedizin Berlin every 5 min; only the final picture after the 45 min Dimitrios Zampatis is required for efficient translocon gating at recycling period is shown). (doctoral student), EF, PT, * the endoplasmic reticulum membrane. Richard Kroczek

Mol Pharmacol 75: 801-811 Robert-Koch-Institut, Berlin Dr. Gisela Papsdorf (technical assistant), * Schmidt A, Wiesner B, Weißhart K, Schulz Walter Rosenthal

K, Furkert J, Lamprecht B, Rosenthal W, MDC Berlin Dr. Claudia Rutz Schülein R (2009) Use of Kaede fusions to (technical assistant), PT Klaus Weißhart visualize recycling of G protein-coupled Carl Zeiss MicroImaging GmbH, Jena Maria Kotte (student), PT, * receptors. Traffic 10: 2-15

Jan Wolkenhauer (student) PT, * FMP authors EXTERNAL FUNDING

Group members * Part of period reported Deutsche Forschungsgemeinschaft PT part time “Struktur und Funktion von Transportsig-

nalen des Vassopressin-V -Rezeptors“ EF position funded externally 2 SFB 449, TP A3 (3rd-party funds) for at least part with W. Rosenthal of the reporting period 01.2009 – 12.2010 Current group members (31.12.2010) 213.900 Euro

84 RESEARCH GROUPS // SIGNAL TRANSDUCTION/MOLECULAR GENETICS RESEARCH GROUPS // SIGNAL TRANSDUCTION/MOLECULAR GENETICS 85 SIGNAL TRANSDUCTION/ MOLECULAR GENETICS

BIOCHEMICAL NEUROBIOLOGY

GROUP LEADER Wolf Eberhard Siems

The Biochemical Neurobiology Group deals/dealed with the bio- Our group investigated biochemical, phar- the control switch for appetite control, Finally, we analyzed possibilities for an maceutical, and molecular aspects of mem- since candoxatril cannot cross the blood- up-regulation of NEP activity. In 2006 M.F. chemical and molecular aspects of peptidases. Our current research brane-bound peptidases. The research was brain barrier. Thus, lack of NEP activity, Melzig et al. described that components is focussed on angiotensin-converting enzyme (ACE), neutral en- focused on angiotensin-converting enzyme mediated genetically or pharmacologically, of green tea extracts activate NEP under (ACE), the neutral endopeptidase (nepri- leads to a gain in body weight and in sig- in-vitro conditions (using SK-N-SH hybrid- dopeptidases (NEP) and some related enzymes. We evaluate their lysin/NEP), and some related peptidases. nificant fat-accumulation. Consequently, oma cells). This effect was of particular biochemical and functional relations to various disorders in man, ACE, and especially neprilysin, cleave a the described obesity model should be interest because green tea and some of very broad spectrum of physiologically an ideal tool for research on the develop- its components are repeatedly reported like seeking for alcohol, obesity, problems in male fertility, neuronal important peptide hormones, and conse- ment, molecular mechanisms, diagno- to reduce body weight and abdominal disorders and heart diseases. quently play essential roles in several bodi- sis, and therapy of the obesity pandemic. fat amounts (Klaus, et al. 2005, Shimotoy- ly functions. In the period under review we odome, et al. 2005). Our own studies also finished former experiments on neuronal confirm that under in-vivo conditions the disorders and heart diseases and focused NEP activity is slightly up-regulated after on a DFG-supported project and on a close oral consumption of green tea extracts. WOLF EBERHARD SIEMS cooperation with groups of the FMP, the Moreover, in cooperation with the Screen-

Charité – Universitätsmedizin Berlin, the EC- ing Unit of the FMP (Dr. J. von Kries), we 1963 – 1968 Studied chemistry at the CPS in Gießen (Germany), and the HYMS searched for small molecules up-regulat- University Rostock in Hull (UK), on the biochemical and func- ing the activity, as well as the expression, 1968 – 1972 Scientific Assistant at the tional relations of the NEP to obesity. of NEP. Using membrane-bound murine University Rostock Our own former studies, along with work NEP, or NEP-expressing cells (SK-N-SH),

incorporated from other groups, has lead some compounds with slightly neprilysin- 1972 Ph.D. at the University Rostock us to postulate that NEP activity and the activating properties were, in fact, found. 1972 – 1979 Scientific Co-worker at the onset of obesity are closely related. We The specificity and selectivity of these Institute of Artificial Indemination (IfKB), observed, for example, that NEP-knockout compounds are still under investigation. Schönow mice develop a late-onset, mature obesity. As originally scheduled, the work on NEP

NMR-spectroscopy studies showed that and its relation to obesity and fat accumula- 1980 – 1992 Scientist at the Institute of the higher body weight in NEP-/- mice is tion at the FMP was concluded in February Drug Research (IWF, Berlin-Friedrichsfelde) exclusively due to an accumulation of fat. 2011 (coinciding with the retirement of the 1985 – 1988 Postgraduate studies in As often observed in polygenetic human group leader). The work will be continued biochemistry, Academy of Advanced obesity, neprilysin-knockout mice were by the team of Thomas Walther in Hull, and Medical Training, Berlin characterized by higher blood glucose in Gießen.

levels and a significantly impaired glucose 1992 – January 2011 Head of the Bio- tolerance. The key role of NEP in determin- chemical Neurobiology Group at the FMP ing body mass was confirmed by pharmacological approaches. In wild-type mice, the neprilysin inhibitor candoxatril (Pfizer) increased body weight due to a stimulation of food intake and accumulation of fat. More peripheral than central, NEP is

86 RESEARCH GROUPS // SIGNAL TRANSDUCTION/MOLECULAR GENETICS RESEARCH GROUPS // SIGNAL TRANSDUCTION/MOLECULAR GENETICS 87 Bettina Kahlich

MEMBERS OF THE GROUP SELECTED PUBLICATIONS COLLABORATIONS EXTERNAL FUNDING

Sandra Schröder (student), PT, * Walther T, Albrecht D, Becker M, Sun X, Rentzsch B, Gong M, Eichhorst J, International Deutsche Forschungsgemeinschaft

Schubert M, Kouznetsova E, Wiesner B, Pankow K, Papsdorf G, Maul B, Bader “Die Neutrale Endopeptidase – ein Anja Schwiebs (student), EF, PT, * Thomas Walther Maul B, Schliebs R, Grecksch G, Furkert J, M, Siems WE (2010) Signal transduction in Schlüsselenzym bei der Entstehung und Nils Tappenbeck Stephanie Fürl (technical assistant), EF, PT, * Sterner-Kock A, Schultheiss HP, Becker A, CHO cells stably transfected with domain- Therapie der Adipositas“ Anja Schwiebs Siems WE (2009) Improved learning and selective forms of murine ACE. SI 483/8-2 Bettina Kahlich (technical assistant), * University of Hull, UK memory in aged mice deficient in amyloid Biol Chem 391: 235-44 02.2010 – 01.2011

Matthias Münzer (technical assistant), EF, * beta-degrading neutral endopeptidase. R.A. Skidgel 51.450 Euro Becker M, Siems WE, Kluge R, Gembardt F, PLoS One 4(2): e4590 S. Danilov Robert Rülke (technical assistant), * Schultheiss HP, Schirner M, Walther T (2010) Deutsche Forschungsgemeinschaft University of Chicago, USA Pankow K, Schwiebs A, Becker M, New function for an old enzyme: NEP “Die Neutrale Endopeptidase – ein

Siems WE, Krause G, Walther T (2009) deficient mice develop late-onset obesity. Schlüsselenzym bei der Entstehung und * Part of period reported National Structural substrate conditions required for PLoS One 5(9): e12793 Therapie der Adipositas“ PT part time neutral endopeptidase-mediated natriuretic Thomas Walther SI 483/8-1

Peptide degradation. Florian Gembardt with T. Walther, EF position funded externally FMP authors J Mol Biol 393: 496-503 Y. Wang Justus-Liebig-Universität Gießen (3rd-party funds) for at least part Group members Justus Liebig Universität Gießen 02.2008 – 01.2010 of the reporting period 91.843 Euro Cornelia C. Metges Current group members (31.12.2010) FBN-Dummerstorf, Rostock

Reinhard Kluge DIfE-Potsdam

M. Bader MDC-Berlin

88 RESEARCH GROUPS // SIGNAL TRANSDUCTION/MOLECULAR GENETICS RESEARCH GROUPS // SIGNAL TRANSDUCTION/MOLECULAR GENETICS 89 SIGNAL TRANSDUCTION/ MOLECULAR GENETICS

CELLULAR IMAGING

GROUP LEADER Burkhard Wiesner

Metastasizing MDA-MB-231 breast cancer cells A nuclei B actin filaments C vasodilator stimulated phosphoprotein (VASP) (Collaboration with the group of Ronald Kühne)

The group cooperates with all research groups of the FMP. We work Since 2006, this group has existed as a core The current equipment of the group: Sample preparation: facility. For better technical support and or- • LSM510 – META – UV equipped with • resin embedding of biological probes on different sub-projects, which we resolve independently, and ganization, we have our group split into two exaction wavelenghts of 364nm, 458nm, for morphological studies which therefore justifies our claim to joint publications. For detailed sub-units, the “unit of light microscopy” and 488nm, 514nm, 543nm, and 633nm, three • room temperature or freeze substitution the “unit of electron microscopy”. detector channels (one of these is a technique information on thematic priorities, see the reports of the research META-detector) • Tokuyasu cryo-embedding and cryo-sec- groups of the FMP. Introduction of the functions of the • LSM510 – META – NLO equipped with tioning technique light microscopy core facility exaction wavelenghts of 458nm, 488nm, • RT- and cryo- sectioning The light microscopy facility supports all re- 514nm, 543nm, 633nm, and 720nm-930nm • Pre- and post-embedding immunogold search groups of the FMP with technology (tunable for two-photon microscopy), labelling and expertise for studying their biological three detector channels (one of these is a • negative staining of protein particles, samples (living cells, fixed cells, tissue, so- META-detector two-dimensional protein crystals, or fibril lutions). The central purpose of our group • LSM510 – ConfoCor2 (FCS) equipped with structures is the establishment of different single cell exaction wavelenghts of 458nm, 488nm, • preparation of formvar- and carbon-coat- BURKHARD WIESNER techniques and the application of the most 514nm, 543nm, and 633nm, three detector ed EM grids diverse microscopic methods for the de- channels and additional two Avelange scription of cellular signal transduction. diodes for FC Current equipment of the group: Microscopic methods such as FRET (Fluo- • LSM710 – ConfoCor3 (FCS) equipped with • Transmission electron microscope Zeiss rescence Resonance Energy Transfer), FRAP exaction wavelenghts of 405nm, 458nm, 902a, 80 kV, (1994), equipped with Mega- (Fluorescence Recovery After Photobleach- 488nm, 514nm, 561nm, and 633nm, view III camera (2003) ing) or FCS (Fluorescence Correlation Spec- three detector channels (one of these • Transmission electron microscope troscopy) are of central importance while, in is a META-detector) and additional two FEI Tecnai G2, FEG, 200 kV, (05/2008) before bleach after bleach addition, we examine the use of differently Avelange diodes for FCS equipped with Gatan Cryo-holder, tomog- 4.4.55 caged compounds in connection with intra- • Imaging system “AxioVision”, UV flash raphy holder, Gatan CCD-camera (2kx2k) 44 before bleach cellular Ca2+ measurements using UV- and/ low dose imaging 3.3.55 after bleach CFP or IR-irradiation for the process of uncaging. Introduction of the functions of the • Vitrobot (FEI, 11/2010) for vitrification 33 Furthermore, we develop opportunities for EM core facility • glow discharge and carbon coating device 2.5 2.5 data analysis and ort he use of algorithms for The EM facility of the FMP assists interested • RT-ultramicrotome and Cryo-ultramicro-

intensity 22 YFP biophysical issues (e.g. we create macros ort groups of the institute in visualising the cel- tome 1.1.55 he analyses). When using new dyes (e.g. use lular architecture and in localising individual 11 of the photoconversion of Kaede), we always proteins at a high resolution. In addition, high 0.0.55 test and create protocols for general use. resolution imaging of 2D protein crystals, as 00 overlay well as fibril structures, is supported. The lab 400 450 500 550 600 650 700 40 04 50 5005 50 6006 50 700 also provides standard and advanced speci- wavelength (nm) men preparation techniques. Recently at the FMP, a FEI Tecnai G2 FEG 200kV electron mi- Principle of fluorescence resonance energy transfer croscope with cryo-equipment has become Left Fluorescence spectra of CFP fused to YFP before and after bleaching of YFP Bleaching of YFP results in an increase of CFP intensity if molecules are in close proximity available. Currently we are establishing cryo- Right Corresponding picture to photobleaching-FRET process methods for protein and cell imaging.

90 RESEARCH GROUPS // SIGNAL TRANSDUCTION/MOLECULAR GENETICS RESEARCH GROUPS // SIGNAL TRANSDUCTION/MOLECULAR GENETICS 91 From left to right: Martina Ringling

Jenny Eichhorst

Anke Teichmann

Thorothea Lorenz

Martina Ringling

MEMBERS OF THE GROUP SELECTED PUBLICATIONS COLLABORATIONS

Dr. Dorothea Lorenz Hundsrucker C, Skroblin P, Christian F, Noack C, Bergmann M, Kass R, Hampel K, Walther T, Albrecht D, Becker M, International National

Zenn HM, Popara V, Joshi M, Eichhorst J, Kashin D, Genieser HG, Herberg FW, Schubert M, Kouznetsova E, Wiesner B, Anke Teichmann (doctoral student), PT Uwe Vinkemeier Anje Sporbert Wiesner B, Herberg FW, Reif B, Rosenthal Willoughby D, Cooper DM, Baillie GS, Maul B, Schliebs R, Grecksch G, Furkert J, School of Biomedical Science, Core Facility Light Microscopy, Martina Ringling (technical assistant), PT W, Klussmann E (2010) Glycogen synthase Houslay MD, von Kries JP, Zimmermann B, Sterner-Kock A, Schultheiss HP, Becker A, University of Nottingham, UK MDC Berlin kinase 3 beta interaction protein functions Rosenthal W, Klussmann E, (2010) Siems WE (2009) Improved learning and Jenny Eichhorst (technical assistant) as an A-kinase anchoring protein. Small molecule AKAP-protein kinase A (PKA) memory in aged mice dieficient in amyloid Roger A. Johnson Bettina Erdmann Svea Hohensee (technical assistant), PT J Biol Chem, 285: 5507-5521 interaction disruptors that activate PKA beta-degrading neutral endopeptidase. Physiology and Biophysics, Cora Facility Electron Microscopy:

interfere with compartmentalized cAMP PloS ONE, 4: e4590 Stony Brook University New York; USA MDC Berlin Nedvetsky PI, Tabor V, Tamma G, signaling in cardiac myocytes. * Part of period reported Beulshausen S, Skroblin P, Kirschner A, Mollajew R, Zocher F, Horner A, Wiesner B, Thomas Walter Andreas Hermann J Biol Chem. Epub 2010 Dec 22. Mutig K, Boltzen M, Petrucci O, Klussmann E, Pohl P (2010) Routes of Cardiovascular Physiology Molecular Biophysics and Cell Biology, PT part time Vossenkämper A, Wiesner B, Bachmann S, Hagen V, Dekowski B, Kotzur N, Lechler R, epithelial water flow: Aquaporins versus University of Hull, UK Humboldt University of Berlin

EF position funded externally Rosenthal W, Klussmann E (2010) Recipro- Wiesner B, Briand B, Beyermann M (2009) cotransporters. Biophys J, 99: 3647-3656 Peter Pohl U.B. Kaupp (3rd-party funds) for at least part cal regulation of aquaporin-2 abundance {7-[Bis(carboxymethyl)amino]coumarin-4-yl} (2010) Institute of Biophysics, Molecular Sensory Systems, of the reporting period and degradation by protein kinase A and methoxycarbonyl derivatives for photore- Schulz K, Rutz C, Westendorf C, Ridelis I, University of Linz, Austria Caesar Research Center Bonn p38-MAP kinase. lease of carboxylic acids, alcohols/phenols, Current group members (31.12.2010) Vogelbein S, Furkert J, Schmidt A, J Am Soc Nephrol, 1645-1656 (2010) thioalcohols/thiophenols, and amines. Elena Pohl Oliver Daumke Wiesner B, Schuelein R (2010) The pseudo Chemistry, 14: 1621-1627 Institute of Medical Physics and Structure and Membrane Interaction Orthmann A, Zeisig R, Koklic T, Sentjurc M, signal peptide of the corticotropin-re- Biostatistics, University of Veterinary of G-Proteins, MDC Berlin Wiesner B, Lemm M, Fichtner I (2010) Hava D, Forster U, Matsuda M, Cui S, leasing factor receptor type 2a decreases Medicine Vienna, Austria Impact of membrane properties on uptake Link BA, Eichhorst J, Wiesner B, Chitnis A, receptor expression and prevents Gi-medi- Dorothea Eisenhardt and transcytosis of colloidal nanocarriers Abdelilah-Seyfried S (2009) Apical mem- ated inhibition of adenyly cyclase activity. Systemic Neurobiology, across an epithelial cell barrier model. brane maturation and cellular rosette forma- J Biol Chem, 285: 32878-32887 (2010) Department of Neurobiology J Pharm Sci, 99: 2423-2433 tion during morphogenesis of the zebrafisch Schwefel D, Fröhlich C, Eichhorst J, FU Berlin lateral line. J Cell Sci, 122: 687-695 Turner Y, Wallukat G, Saalik P, Wiesner B, Wiesner B, Behlke J, Daumke O (2010) Pritz S, Oehlke J (2010) Cellular uptake Kilic F, Kashikar ND, Schmidt R, Alvarez L, Structural basis of oligomerization in and biological activity of peptide nucleic Dai L, Weyand I, Wiesner B, Goodwin N, septin-like GIMAP2. acids conjugated with peptides with and Hagen V, Kaupp UB (2009) Caged progester- PNAS, 107: 20399-20234 (2010) without cell-penetrating ability. one: A new tool for studying rapid non-

J Pept Sci, 16: 71-80 genomic actions of progesterone. FMP authors JACS, 131: 4027-4030 Christian F, Szaszák M, Friedl S, Group members Drewianka S, Lorenz D, Goncalves A, Schmidt A, Wiesner B, Weisshart K, Schulz Furkert J, Vargas C, Schmieder P, K, Furkert J, Lamprecht B, Rosenthal W, Götz F, Zühlke K, Moutty M, Göttert H, Schülein R (2009) Use of kaede fusions to Joshi M, Reif B, Haase H, Morano I, visualize recycling of G protein-coupled Grossmann S, Klukovits A, Verli J, Gáspár R, receptors.Traffic, 10: 2-15

92 RESEARCH GROUPS // SIGNAL TRANSDUCTION/MOLECULAR GENETICS RESEARCH GROUPS // SIGNAL TRANSDUCTION/MOLECULAR GENETICS 93 CHEMICAL BIOLOGY

CHEMICAL SYSTEMS BIOLOGY

GROUP LEADER Figure 1. Technical steps in the preparation of chemical Ronald Frank microarrays following the SC2 process: Parallel SPOT synthesis of a combinatorial library of compounds; punching out spots and dissolving the cellulose matrix in acid, followed by precipitation, washing and dissolu- tion in DMSO; printing the microarrays by transfer of nL aliquots onto a prepared glass slide; probing with fluorescently labelled target.

The Chemical Systems Biology group was newly established in Biology focus Chemistry focus The focus of the department is the develop- Upon loading the four calcium-binding A part of the work of our group is to sup- September 2010 and is still in its build-up phase. The group works ment of approaches for the directed modu- sites with calcium ions, a conformational port the drug research efforts of the insti- on two research projects: advancement of chemical microarrays and lation of complex regulatory networks as change in CaM is rapidly induced resulting tute with new technologies and resources. a basis to discover novel pharmacological in the exposition of hydrophobic patches We have implemented all technical steps investigation of the calmodulin regulated cellular protein network. principles. Stimulated by earlier work of on its surface, which enable CaM to bind for the production of chemical libraries in the PI, we have chosen the large group of to a variety of different target proteins the format of microarrays. A new library of Chemical Microarray Technology will be established, advanced and proteins regulated by calcium/Calmodulin with extraordinary high affinity. A number almost 2500 diketopiperazines was synthe- (CaM). CaM is a small acidic ubiquitous proof organic hydrophobic molecules such sized and will be tested for specific CaM a) used as a tool for the discovery of small molecule ligands of tein and conserved extraordinarily through- as derivatives of naphthalene and natural binders. This generic binder library is now protein targets in the projects of the group, and out the evolution of eukaryotes. Essentially peptides such as peptide hormones, neu- available to be probed with many other tar- all vertebrates contain identical CaMs, even rotransmitters and venoms also bind CaM get proteins. b) offered, under the auspices of the Chemical Biology Unit, to though encoded by different genes. CaM is with high affinity. Libraries of soluble compounds will be pre- other internal and external cooperation partners. an integral modulator of many calcium-de- For a first part of our Calmodulinome proj- pared to complement the chemical collec- pendent processes in virtually every eukary- ect we have scanned all protein compo- tion of the FMP in regions of low populated The Calmodulinome project will investigate the growing family of otic cell type. It functions as a cytosolic cal- nents of the human ribosome in the format chemical space. A synthetic route to tetra- Calmodulin (CaM) target proteins, their complex Calcium-mediated cium receptor and binds up to four calcium of 21mer overlapping peptide fragments and penta-substituted dihydropyrroles (Zhu ions with very high affinity. In this way it re- (5000 peptides altogether, including ref- et al., J Comb Chem 2009) has been estab- regulation, and their exploration for a systems pharmacological sponds to a variety of different extra cellu- erence peptides and proteins) for CaM lished. intervention. lar signals which increase the cytosolic cal- binding. We have confirmed many sites cium level. Thereby CaM decodes the Ca2+ reported in the literature and have identi- signal that is brought about by the influx of fied several new ones. Functional analysis RONALD FRANK Ca2+ through respective Ca-channels in the of these regulatory sites is underway. plasma membrane and activates or deacti- 1967 – 1973 Studied chemistry, biochemis- 1980 – 1984 Post-Doc at the Gesellschaft Since 2008 Coordinator of ChemBioNet vates both kinases and phosphatases. Fur- try and microbiology at the University für Biotechnologische Forschung mbH national infrastructure initiative for academic thermore, calmodulin regulates enzymes of Hamburg, Germany (GBF, Dr. Blöcker) in Braunschweig, Germany research in chemical biology at the FMP involved in the signal transduction such as

in Berlin-Buch cyclic nucleotide phospodiesterases, ade- 1974 Diploma in chemistry at the University Since 1985 Staff scientist at the GBF nylate cyclases, nitric oxide synthetase and of Hamburg, Germany (since 2005 Helmholtz Centre for Infection Since 2009 Coordinator of the European plasma membrane calcium ATPases. There Research, HZI) Research Infrastructure Initiative EU-OPEN- 1975 – 1979 Ph.D. at the University of are, however, protein families including SCREEN at FMP Hamburg (Prof. Köster) 1995 – 2003 Head of research group neuromodulin, myosin, ionchannels that

“Molecular Recognition” at the GBF Since 2010 Head of Department Chemical bind CaM at remarkably low concentrations 1979 – 1980 Post-Doc at the Institute Systems Biology at FMP (acting coordinator or even in the absence of calcium. of Organic Chemistry and Biochemistry 2003 – 2010 Head of department of of Chemical Biology Section) (Prof. Köster) at the University of Hamburg Chemical Biology at the HZI

94 RESEARCH GROUPS // CHEMICAL BIOLOGY RESEARCH GROUPS // CHEMICAL BIOLOGY 95 From left to right: Khalid Abu Ajaj, Lioudmila Perepelittchenko

Edelgard Schmeisser

Edelgard Schmeisser, Irina Nickeleit

MEMBERS OF THE GROUP SELECTED PUBLICATIONS COLLABORATIONS EXTERNAL FUNDING

Dr. Khalid Abu Ajaj Weiß SM, Ladwein M, Schmidt D, Ehinger Andreetto E, Yan LM, Matarek-Nossol M, International EU-OPENSCREEN

J, Lommel S, Städing K, Beutling U, Disanza Frank R, Kapurniotu A (2010) Identification EU-funded Preparatory Phase Project of Dr. Irina Nickeleit EU-OPENSCREEN A, Frank R, Jänsch L, Scita G, Gunzer F, of hot regions of the Aß-IAPP interaction an ESFRI (European Strategy Forum on institutes from 12 member states Dr. Bahne Stechmann Rottner K, Stradal TEB (2009) IRSp53 links interface as high affinity binding sites in Research Infrastructures) Roadmap Research (Scientific Manager EU-OPENSCREEN), EF the enterohemorrhagic E.coli effectors Tir both cross- and self-association. Annie Andrieux, Didier Job, Infrastructure

and EspFU for actin pedestal formation. Angew Chem Int Ed Engl 49: 3081-3085 Christophe Bosc 11.2010 – 10.2013 Dr. Anne Höner Cell Host & Microbe 5: 244-258 Grenoble-Institute des Neurosciences, 3.700.000 Euro (Project Manager EU-OPENSCREEN), PT Maenz B, Goetz V, Wunderlich K, Eisel J, Grenoble Lisurek M, Rupp B, Wichard J, von Kries Kirchmair J, Stech J, Stech O, Chase G, Martin McLean, MA The group actively supports the building J P, Frank R, Rademann J, Kühne R (2009) Frank R, Schwemmle M (2011) Disruption Andreas Meyerhans (Assistant Project Manager of a European research infrastructure of Design of chemical libraries with potentially of the viral polymerase complex assembly University Pompeu Fabra, Barcelona EU-OPENSCREEN), EF screening platforms for Chemical Biology bioactive molecules applying a maximum as a novel approach to attenuate influenza in the frame of the ESFRI roadmap Dr. Lioudmila Perepelittchenko common substructure concept. a virus. J Biol Chem 286: 8414-8424 National (see page 4 – 6). (technical assistant) Mol Divers 14: 401-408 Hans-Joachim Fritz Edelgard Schmeißer (technical assistant) Bocquet A, Berges R, Frank R, Robert P, FMP authors Varignost, Göttingen

Peterson AC, Eyer J (2009) Neurofilaments Group members Marianne Dreißigacker (Secretary) Intavis Bioanalytical Instruments bind tubulin and modulate its polymeriza- Cologne Torsten Meiners (guest EU-Openscreen) tion. J Neurosci 29: 11043-11054

Martin Schwemmle

Institute of Virology, University of Freiburg * Part of period reported

PT part time

EF position funded externally (3rd-party funds) for at least part of the reporting period

Current group members (31.12.2010)

96 RESEARCH GROUPS // CHEMICAL BIOLOGY RESEARCH GROUPS // CHEMICAL BIOLOGY 97 CHEMICAL BIOLOGY

PEPTIDE SYNTHESIS

GROUP LEADER Michael Beyermann

Figure 2. “Like a fisherman who angles for fish, we will Figure 1. Light-directed inhibition of the interaction between a-1-syntrophin and neuronal NO synthase in living use the soluble receptor model in ongoing studies to skeletal muscle with a cell permeable photo-switchable nNOS-derived peptide ligand. After illumination at 365 nm catch new ligands, hopefully with improved behaviours, the voltage-stimulated concentration is inhibited. in peptide libraries.”

Vital signalling functions in organisms are frequently controlled by Ligand-directed signalling of GPCR fact that led us to assume that the activat- in the CRF ligands is important for their Corticotropin-Releasing Factor (CRF) re- ing domain for Gs is not identical to that biological potency at the CRF receptor, we peptide-protein interactions. Thus, there is considerable interest in ceptors belong to the biologically impor- for Gi, but may be located in the C-terminal incorporated appropriate units into Urocor- knowing those structural features, which are important for recogni- tant class B of GPCRs and are promiscu- receptor binding site of urocortin-1. This tin-1. As expected, dependent on the pho- ous, which means that they can couple to hypothesis contradicts the widely accepted to-isomerization state of the incorporated tion of proteins by their peptide ligands. It is the main topic of the multiple G proteins and, thereby, activate receptor binding/activation model and is, azobenzene system, the helical conforma- group to develop and apply chemical and enzymatic methods for different intracellular signalling events. CRF therefore, the subject of ongoing studies. tion of the linker unit is affected and the receptors are involved in mediating anxiety two isomers showed different potencies in the synthesis of specifically modified peptides, and even proteins, and depressive disorders and other stress- Light-directed peptide ligands activating the receptor. In ongoing studies to discover such structural features, aiming at the development of associated pathologies. These receptors An interesting approach for the develop- we will investigate which position in the li- are activated by polypeptides (Urocortins, ment of new protein ligands comes from gand is most effective in this regard. new peptide ligands showing either a desired specific activity (e.g. Sauvagine, Urotensin-I, CRF) that activate a joint project with Peter Schmieder (FMP) receptor agonists which activate only one signalling pathway and both Gs and Gi proteins. We have demon- and Karola Rück-Braun (TU Berlin) based on Ligand fishing with artificial receptor strated that an agonist-directed signalling the use of photo-switch elements for light- Another searching approach for new pro- MICHAEL BEYERMANN inhibit, at the same time, another one), or a specific behaviour such of the CRF receptor (CRFR1) takes place directed control of peptide conformation. tein ligands is based on the chemosynthe- as being controlled by light-activation. when Urocortin-1 is modified at specific By embedding a photo-switchable unit into sis of protein models. After achieving the 1971 – 1975 Studied chemistry at the positions of its N-terminal site, which is a cyclic peptide that mimics the beta-finger first synthesis of a soluble GPCR receptor Humboldt University, Berlin believed to be the activating domain. We motif in nNOS, which is crucial for binding model consisting of the four extracellular 1975 – 1978 Ph.D. (protein semisynthesis) have shown that certain benzoyl-phenylal- of nNOS to alpha-1-syntrophin, the binding ligand binding domains of CRFR1 on a lin- at the Humboldt University, Berlin anine-substituted analogues of urocortin between the two can be controlled by light. ear peptide template, we have improved

1 exhibit both full Gs activation and com- In skeletal muscle, nNOS interacts with syn- the synthesis protocol in a way that we have 1978 – 1991 Research Associate at the plete inhibition of Gi coupling. We named trophin to recruit nNOS to the dystrophin- now obtained not only a small (~300 µg) Institute of Drug Research of the Academy this behaviour (Gs)ago-(Gi)antagonism and associated protein complex at the plasma but a large quantity (15 mg) of a new re- of Sciences of the GDR have assumed that by segregating func- membrane, coupling the production of ceptor model which, moreover, is based on 1988 – 1989 Research Associate at the tional domains, peptides offer a means for the second messenger nitric oxide (NO) to a cyclic peptide template. Like a fisherman Department of Chemistry of the University the rational design of signalling-selective muscle contraction. In the presence of the who angles for fish, we will use the soluble of Massachusetts receptor ligands. Following systematic trans-form of our photo-switchable pep- receptor model in ongoing studies to catch

single amino acid substitutions, replacing tide mimic, the voltage-stimulated muscle new ligands, hopefully with improved be- Since 1992 Head of the Peptide Synthesis all positions of the activating domain by contraction is not affected, but illumination haviours, in peptide libraries (Fig. 2). group at the FMP amino acids with side-chains of increasing resulted in formation of the corresponding size, we obtained exclusively either non-se- cis-form that inhibits the contraction com- lective agonists or (Gs)ago-(Gi)antagonists. pletely (Figure 1), showing that peptide Interestingly, the size of the side-chain is tools can be used for light-directed control important for activating Gi proteins, in of intracellular function in living systems. that very bulky side-chains revealed Gi an- The promising results prompted further tagonists. On the contrary, the potency for studies of applying photo-switchable ami- Gs activation is only decreased by bulkier no acids to the control of biological activ- side-chains. Not a single substitution in the ity in other systems like the CRF ligands. activating domain led to Gs antagonism, a Knowing that the helical linker structure

98 RESEARCH GROUPS // CHEMICAL BIOLOGY RESEARCH GROUPS // CHEMICAL BIOLOGY 99 From left to right: Dagmar Michl, Nadja Heinrich

Bernhard Schmikale

Sabine Abel

MEMBERS OF THE GROUP SELECTED PUBLICATIONS COLLABORATIONS EXTERNAL FUNDING

Dr. Nadja Heinrich Carpino LA, Nasr K, Abdel-Maksoud AA, El- International Deutsche Forschungsgemeinschaft Deutsche Forschungsgemeinschaft

Faham A, Ionescu D, Henklein P, Wenschuh “Synthesis, optimisation, and screening Forschergruppe FG 806 (Teilprojekt 3) Sabine Abel (doctoral student), PT, * Louis A. Carpino H, Beyermann M, Krause E, Bienert M of small molecule libraries targeting KU 845/2-2 Department of Chemistry Benoit Briand (doctoral student), EF, PT, * (2009) Dicyclopropylmethyl peptide back- protein-protein interactions” with R. Kühne, H.-G. Schmalz University of Massachusetts, Amherst bone protectant. Org Lett 11: 3718-3721 FOR 806, TP Z1 (RA 895/5-1) 03.2010 – 02.2013 Christian Hoppmann with J. Rademann, J.P. von Kries 123.000 Euro (doctoral student), EF, PT, * Coin I, Beerbaum M, Schmieder P, Bi- National 01.2007 – 01.2010 enert M, Beyermann M (2009) Solid-phase Deutsche Forschungsgemeinschaft Angelika Ehrlich (technical assistant) Prof. Gerd Multhaup 81.000 Euro synthesis of a cyclodepsipeptide: Cotransin. Forschergruppe FG 806 (Teilprojekt 3) Institut for Chemistry and Biochemistry Annerose Klose (technical assistant) Org Lett 10: 3857-3860 Deutsche Forschungsgemeinschaft BE 1434/6-2 FU Berlin “Strukturelle Untersuchungen früher with H.-G. Schmalz, R. Kühne Dagmar Krause (technical assistant) Hagen V, Dekowski B, Kotzur N, Lechler Prof. Karola Rück-Braun Phasen der Fehlfaltung des Proteins 01.2011 – 12.2013 R, Wiesner B, Briand B, Beyermann Dagmar Michl (technical assistant), PT TU Berlin beta2-Mikroglobulin – ein Beitrag 15.000 Euro M (2009) {7-[Bis(carboxymethyl)amino] zum Verständnis der Enstehung von Bernhard Schmikale (technical assistant) coumarin-4-yl}methoxycarbonyl derivatives Dr. Heinz Fabian Amyloidosen“ for photorelease of carboxylic acids, alco- RKI Berlin NA 226/12-2 hols/phenols, thioalcohols/thiophenols, and * Part of period reported Dr. Frank Bernhard with B. Uchanska-Ziegler amines. Chemistry 14: 1621-1627 Centre for Biomolecular Magnetic (Charité – Universitätsmedizin Berlin), PT part time Hoppmann C, Seedorff S, Richter A, Resonance, Institute for Biophysical D. Naumann (Robert-Koch-Institut) EF position funded externally Fabian H, Schmieder P, Rück-Braun K, Chemistry, University Frankfurt/Main 08.2005 – 07.2011 (3rd-party funds) for at least part Beyermann M (2009) Light-directed protein 126.333 Euro

of the reporting period binding of a biologically relevant beta- Volkswagenstiftung sheet. Angew Chem Int Edit 48: 6636-6639 Current group members (31.12.2010) “Control of protein-protein-interactions through conformational changes induced FMP authors by light: photoswitchable ligand mol- Group members ecules for PDZ domains” VW I/80 771 06.2005 – 07.2009 93.500 Euro

Hoffmann La Roche GmbH Chemierelevante Forschung und Lehre F. Hoffmann-La Roche Ltd. 04.2008 – 12.2011 7000 Euro

100 RESEARCH GROUPS // CHEMICAL BIOLOGY RESEARCH GROUPS // CHEMICAL BIOLOGY 101 CHEMICAL BIOLOGY

PEPTIDE-LIPID- INTERACTIONS/ PEPTIDE TRANSPORT Figure 1. CLSM spectroscopic studies of the cellular uptake of fluorescence-labeled potential micellar drug carriers (D): Micelles of fluorescence-labeled P2A2 GROUP LEADER are not internalised into endothelial cells of large Margitta Dathe, Johannes Oehlke vessels (A) and peptide-lipid-mixed micelles show little uptake into macrophages (red lipid fluorescence in B) but, micelle uptake in brain capillary endothelial cells (C) is highly efficient (green peptide fluorescence). Red (A and C) and blue (B) trypan blue labeling of the cell membranes reflect the viability of the cells.

Our research is focused on peptides as targeting and transmem- Peptide-modified liposomal and out cell-penetrating ability, were assessed in the membrane-disturbing helical cationic micellar carriers parallel. The results suggest that the bioactiv- peptide. The membrane translocating ability brane transport-promoting tools for bioactive molecules and lipid- Nanoparticles equipped with cell-targeting ity of PNA-peptide conjugates is not primarily of the cyclic peptides is currently under inves- based drug carrier systems. One major aim is to improve the and uptake-mediating peptides have attract- related to the membrane-penetrating ability tigation. ed much attention as a means of overcoming of the peptide tags. Moreover, transwell ex- transport of molecules from the blood to the brain across the tight cellular barriers. We introduced a sequence periments also revealed an extensive trans- Our cooperation with three other institutions

cell layer called the blood brain barrier (BBB). Furthermore, we (LRKLRKRLLR)2, named A2, as a target-rec- location of unmodified PNA into and across (Biosyntan; IZW and FLN, Schönow) showed ognizing and uptake-mediating compound. various cell types and suggested that the that cyclic R-,W-rich peptides are promising are interested in elucidating the structural basis of the microbial Dipalmitoylation of the peptide (P2A2) allowed low intracellular availability of PNA is mainly antimicrobial alternatives for the conservation selectivity of peptides, and optimising them as antimicrobial the rapid and easy formation of micelles and caused by export mechanisms. Our most re- of semen for in vitro fertilization in animal pro- lipopeptide-tagged liposomes of variable size cent investigations have revealed the ability duction: the bacterial contamination of boar additives for selected applications (semen conservation, treatment and lipid composition. The monomeric pep- of specific components of foetal calf serum semen was low and synergistic activities al- of diseases caused by blood-born parasites). Additionally, we have tide, P2A2 micelles and liposomes translocate to circumvent the cellular export machineries lowed drastic reductions in dosages. Further- into cells (brain capillary endothelial cells, en- and to significantly enhance the intracellular more, several peptides were proven to influ- been interested in peptides for generating bacteriocidal surfaces. dothelial cells of large vessels, macrophages, delivery of PNA, PNA-peptide conjugates, ence the motility and movement of sperm in keratinocytes) with very high efficiency. The and probably other nuclease-stable oligo- a highly favorable way. Current in vivo studies uptake is endocytotic, partially non-endocy- nucelotides. Our efforts in the near future will on fertilisation with peptide-conserved semen MARGITTA DATHE JOHANNES OEHLKE totic and mediated by different membrane focus on the isolation and characterization of preparations are very promising.

constituents. The observations imply that in these serum components. 1974 Diploma thesis in physics at the 1975 Ph.D. in pharmaceutical chemistry the various cell species, different transport Surface immobilisation of CAPs Humboldt-Universität Berlin at the University of Leipzig routes are activated, and different properties Cationic antimicrobial peptides (CAPs) Biofilms present a serious threat to human 1978 Ph.D. at the Academy of Sciences 1975 – 1980 Research Associate on of the particulate carriers such as the size and Small cyclic antimicrobial peptides health. We found that tethering of antimicro- of the GDR glycoside synthesis at the von Ardenne surface density of the vector peptide and the Small (less expensive in production) and bial peptides is suitable for the generation

Institute Dresden peptide conformation influence the process. proteolytically stable (not degradable dur- of antimicrobial surfaces. Using peptide im- 1979 – 1992 Research Associate at Small micellar particles with a high sur- ing application) antimicrobial peptides are of mobilisation at resin beads as model solid the Institute of Drug Research Since 1980 research in the fields of face density and flexibility of the cationic particular interest as lead structures for the de- surfaces, we identified coupling conditions organic chemistry, radioactive labeling, 1992 – 1999 Team Leader of the peptide were found selective for uptake velopment of new antimicrobial compounds. which preserve the advantageous properties cellular uptake of peptides, and Conformational Analysis Group at into capillary endothelial cells (Figure 1). Our studies on the structural and functional of soluble antimicrobial peptides and render oligonucleotide delivery at the Institute the FMP In cooperation with the University Hospital Mün- principles of small R-,W-rich hexapeptides surfaces antimicrobial. We found the length of of Drug Research, since 1992 FMP ster P2A2-modified liposomes have successful- resulted in cyclic sequences with pronounced spacer and the amount of surface-located, tar- Since 1999 Team Leader of the ly been used to deliver recombinant transglu- activity towards Gram negative bacteria. The get-accessible peptide as critical parameters Peptide Lipid Interaction / Peptide taminase-1 (TGase-1) into keratinocytes. O-antigen of the lipopolysaccharides (LPS) of and proved that the chain position of linkage Transport Group at the FMP the outer E. coli membrane is essential for a is less sensitive for the activity. Immobilization Intracellular delivery and biological maximal biological effect (cooperation with reduced the activity but did not influence the activity of naked and peptide-tagged R. Harvey, Kings College, London) and the activity pattern on a biological level and the peptide nucleic acids low permeabilising activity against the inner mode of action. The parameters that have The cellular uptake and the antisense activity membrane points to a non-membrane per- been analyzed are relevant for the establish- of peptide nucleic acids (PNAs), after disulfide meabilising mode of action. Thus, the cyclic ment of a more general approach to obtain bridging with various peptides with and with- R-,W- peptides behave quite differently from efficient biocidal surfaces.

102 RESEARCH GROUPS // CHEMICAL BIOLOGY RESEARCH GROUPS // CHEMICAL BIOLOGY 103 From left to right: Aleksandra Djoric (intern), Margitta Dathe, Kathi Scheinpflug, Heike Nikolenko, Johannes Oehlke Front: Oxsana Krylova, Karl Sydow

Kathi Scheinpflug, Heike Nikolenko

Karl Sydow

MEMBERS OF THE GROUP SELECTED PUBLICATIONS COLLABORATIONS EXTERNAL FUNDING

Mojtaba Bagheri (doctoral student), PT, EF Bagheri M, Beyermann M, Dathe M Turner Y, Wallukat G, Saalik P, Wiesner B, International DAAD Stipendium, Internationales Büro des BMBF im Projekt-

(2009) Surface-bound cationic antimicro- Pritz S, Oehlke J (2010) Cellular uptake Working stay Christof Junkes; träger beim Deut. Zentrum für Luft und Christof Junkes (doctoral student), PT, EF Robert Hancock bial peptides: immobilization reduces the and biological activity of peptide nucleic 08.2009 – 11.2009 Raumfahrt e.V. University of British Columbia Eik Leupold (doctoral student), PT, EF activity with no influence upon the activity acids conjugated with peptides with and King’s College, London, Great Britain “Optimierung kleiner Peptide als poten- Vancouver, Canada spectrum. Antimicrob Agents Chemother without cell-penetrating ability. tielle Wirkstoffe gegen intrazelluläre Kathi Scheinpflug (doctoral student), PT Foundation for Ichthyosis and Related Skin 53(3): 1132-1141 J Pept Sci 16: 71-80 Richard Harvey menschliche Pathogene“ Types Research Grand Program, USA Karl Sydow (doctoral student), PT, EF King´s College London SUA 09/048 Leupold E, Nikolenko H, Dathe M (2009) “In vitro and in vivo models for transglu- with M. Beyermann, FMP and M. Rauten- Heike Nikolenko (technician) Apolipoprotein E peptide-modified FMP authors taminase-1 deficient lamellar Ichthyosis” National bach, Stellenbosch University, South Africa colloidal carriers: the design determines Group members with H. Traupe, University Hospital Münster Gabriela Vogelreiter (technician) EF approval: 28.01.2010 the mechanism of uptake in vascular Monika Schäfer-Korting 2009 – 2010 7.200 Euro und 150 days exchange of stu- endothelial cells. FU Berlin 2.500 Euro for the FMP dents (corresponds to 13.300 Euro ) * Part of period reported Biochim Biophys Acta 1788(2): 442-449 Alfred Blume Deutsche Forschungsgemeinschaft Alexander v. Humboldt Stiftung, PT part time Vucic E, Sanders H, Arenam F, Terrenon E, MLU Halle “Innovative Arzneistoffe und Träger- “Synthesis and study of small antimicro- Aime S, Klaase N, Leupold E, Dathe M, systeme - Integrative Optimierung zur EF position funded externally Heiko Traupe bial peptides containing conformationally Sommerdijk N, Mulder W (2009) Well- Behandlung entzündlicher und hyperpro- (3rd-party funds) for at least part University Hospital Münster constrained arginine analogues” defined, multifunctional nanostructures liferativer Erkrankungen“ of the reporting period 2.3-DEU/1136332 of a paramagnetic lipid and a lipopeptide FG 463, Project 7B, DA 324/5-3 with I. Komarov, Shevchenko University, Current group members (31.12.2010) for macrophage imaging. 2008 – 2009 Kiev, Ukraine, J Am Chem Soc 131(2): 406-407 13.500 Euro approval: 22.04.2010 Arouri A, Dathe M, Blume A (2009) PRO INNO II, 55.000 Euro

Peptide-induced demixing in PG/PE lipid Ministerium für Wirtschaft und Technologie Foundation for Ichthyosis and Related Skin mixtures: a mechanism for the specificity of “Development of antimicrobial peptides Types Research Grand Program, USA antimicrobial peptides towards biological and their application in reproduction “Liposomal Packing of recombinant trans- membranes. biology” glutaminase-1” Biochim Biophys Acta 1788: 650-659 KF 0376001MD6 with H. Traupe, University Hospital, Münster 2006 – 2009 approval: 2010 103.500 Euro 2.500 Euro for the FMP Deutsche Forschungsgemeinschaft “Peptide-modified micellar nanocarriers to mediate transport at the blood brain barrier (BBB)” DA 324/9-1 approval: 13.01.2010 38.800 Euro and 1 Doc-Position for 3 years

104 RESEARCH GROUPS // CHEMICAL BIOLOGY RESEARCH GROUPS // CHEMICAL BIOLOGY 105 CHEMICAL BIOLOGY

SYNTHETIC ORGANIC BIOCHEMISTRY

GROUP LEADER Volker Hagen

Figure 1. Photocleavages of BBHCM-caged biomolecules

Our research group focuses on the development of so-called caged Design and synthesis of novel caging tadpoles. The levels of UV light needed for Furthermore, we synthesized BBHCM-based groups transgene induction were harmless, as no progesterone and bourgeonal phototrig- compounds. Caged compounds are light-sensitive, inactive deriva- We introduced variants of the (6-bromo- signs of toxicity were detected. Thus, a novel gers. The introduction of the methyleneimi- tives of molecules that, on illumination using UV/Vis or IR light, 7-hydroxycoumarin-4-yl)methyl (Bhc) caging system for high-resolution conditional trans- nodiacetic acid group in position 8 of the group and used them for caging carboxylic gene expression in complex organisms could coumarin chromophore has been achieved rapidly release the biomolecule. In this way, almost instantaneous acids, amines, phenols, and carbonyl com- be established. by Mannich reaction, starting with the corre- jumps in the concentration of the biomolecule can be achieved. pounds. In particular, {8-[bis(carboxymethyl) Furthermore, we developed BBHCM-caged sponding Bhc-caged derivatives. The caged aminomethyl]-6-bromo-7-hydroxycoumarin- compounds of the protein synthesis inhibitors compounds (cyclic ketales and acetales) are Caging and uncaging of biomolecules are very useful techniques 4-yl}methyl (BBHCM) and {8-[(piperazin-1-yl) emetine and anisomycin (cooperation with D. important new tools for studying rapid pro- for studies of the mechanisms and the kinetics of rapid cellular methyl]-6-bromo-7-hydroxycoumarin-4-yl} Eisenhardt and B. Wiesner). The caged com- gesterone and bourgeonal effects in human methyl (PBHCM) caged compounds were pounds show high solubilities in aqueous buf- sperm and other cell types (U. B Kaupp). To processes and their precise resolution in time and space. We aim to found to be very useful. They are distin- fers, long-wavelengths absorptions, relatively assist the Kaupp group in studies for the design novel photoremovable protecting groups for the caging of guished by a high solubility in aqueous high quantum yields, high photorelease rates, identification of the putative progesterone buffers and high photoefficiencies at long- and perform excellently in an in vitro transla- receptor in human sperm, we developed VOLKER HAGEN different functional groups and are concerned with the synthesis, wavelengths irradiations. Additionally, BBH- tion assay as well as in Hela-S pE.GFP cells. progesterone crosslinker constructs and a

photochemical characterization, and application of photoactivatable CM- and PBHCM-caged derivatives are suf- Therefore, the compounds should be useful cell membrane impermeable progesterone 1966 Diploma in chemistry from the ficiently resistant to solvolysis, are sensitive tools for studying physiological processes tripeptide conjugate. Humboldt University, Berlin biomolecules. to two-photon excitation (740–770 nm), and associated with translation. Emetine and an- 1970 Ph.D. in chemistry from the the primary step of the photocleavages oc- isomycin are used in neuroscience to interfere In another project (in cooperation with D. Humboldt University, Berlin curs with rate constants of about 109 s-1. The with protein synthesis dependent processes Eisenhardt and B. Wiesner) we introduced

phototriggers can be easily synthesized and like long-term synaptic plasticity and memory caged octopamine derivatives and caged 1970 – 1972 Post-Doc at the HU Berlin permit efficient photorelease at a lower pH consolidation. The Eisenhardt group will use epinastine. Octopamine belongs to the 1972 – 1982 Leader of a research group than the corresponding Bhc derivatives. the caged compounds in studies of memory group of biogenic amines and plays a ma- at the Institute for Pharmacological consolidation in the honeybee brain. jor neuromodulatory role in invertebrates. Research of the Pharmaceutical Industry Caged compounds for the control of In the honeybee, octopamine is involved in

gene or protein expression Caged compounds for studying learning and memory function. Epinastine is 1982 – 1992 Team Leader at the Institute In cooperation with S. Cambridge we devel- signalling pathways a highly specific antagonist of insect octo- for Drug Research of the Academy of the oped 1-(4,5-dimethoxy-2-nitrophenyl)ethyl- Using novel coumarinylmethyl protecting pamine receptors. Caged octopamine and Sciences of the GDR caged cyanodoxycycline for precise spatial groups, we prepared greatly improved caged caged epinastine will help to identify brain 1990 Habilitation in medicinal chemistry and temporal light-controlled activation of versions of cyclic nucleoside monophos- regions in the honeybee that are critical at the Institute for Drug Research of transgenes based on the ‘Tet-on’ system. Af- phates (cNMPs). With {7-(bis(carboxymethyl) for octopamine-dependent behaviour (D. the Academy of the Sciences of the GDR ter incubation with the caged compounds, amino]coumarin-4-yl}methyl-caged 8-(NBD- Eisenhardt).

gene expression could be topically induced aminoethylthio)cNMPs we developed caged Since 1992 Team Leader of the Synthetic by local irradiation with UV light or by 2-pho- cNMPs with fluorescence marker proper- Organic Biochemistry research group at ton uncaging. Cambridge et al. showed that ties, and with 4,5-dimethoxy-2-nitrobenzyl- the FMP photoactivated gene expression can be ac- caged ADP-ribose for the first time a caged complished in diverse biological systems in- derivative of ADP-ribose. The usefulness of cluding mouse organotyping brain cultures, the caged compounds was demonstrated in developing mouse embryos, and Xenopus physiological studies by the Kaupp group.

106 RESEARCH GROUPS // CHEMICAL BIOLOGY RESEARCH GROUPS // CHEMICAL BIOLOGY 107 Janina Schaal Brigitte Dekowski

MEMBERS OF THE GROUP SELECTED PUBLICATIONS COLLABORATIONS EXTERNAL FUNDING

Nico Kotzur (doctoral student), EF, PT, * Hagen V, Kilic F, Schaal J, Dekowski B, Kilic F, Kashikar ND, Schmidt R, Alvarez L, International Deutsche Forschungsgemeinschaft Schmidt R, Kotzur N (2010) Dai L, Weyand I, Wiesner B, Goodwin N, “Orthogonale photolabile SH-Schutzgrup- Funda Kilic (doctoral student), EF, PT, * Roger Johnson [8-[Bis(carboxymethyl)aminomethyl]-6-bro- Hagen V, Kaupp UB (2009) Caged pro- pen in der Proteinsynthese“ University of New York, USA Janina Schaal (doctoral student), EF, PT mo-7-hydroxycoumarin-4-yl]methyl moieties gesterone: a new tool for studying rapid HA 2694/3-2 as photoremovable protecting groups for nongenomic actions of progesterone. Andreas Barth 01.2008 – 06.2009 Brigitte Dekowski (technical assistant) compounds with COOH, NH2, OH, and JACS 131: 4027-4030 The Arrhenius Laboratories for Natural for 2009: 15385 Euro C=O functions. J Org Chem 75: 2790-2797 Sciences, Stockholm University, Sweden Kotzur N, Briand B, Beyermann M, Fonds der Chemischen Industrie * Part of period reported Bönigk W, Loogen A, Seifert R, Kashikar Hagen V (2009) Wavelength-selective Chemierelevante Forschung und Lehre National PT part time ND, Klemm C, Krause E, Hagen V, Krem- photoactivatable protecting groups for 2003 – 2011

mer E, Strünker T, Kaupp UB (2009) An thiols. JACS 131: 16927-16931 Pablo Wessig 2.024 Euro EF position funded externally atypical CNG channel activated by a single Institute of Chemistry, University Potsdam (3rd-party funds) for at least part cGMP molecule controls sperm chemotaxis. of the reporting period FMP authors Reinhard Schmidt Sci Signal 2: 68 Group members Institute of Physical and Theoretical Current group members (31.12.2010) Cambridge SB, Geissler D, Calegari F, Chemistry, J.W. Goethe-University, Anastassiadis K, Hasan M, Steward F, Frankfurt/Main Huttner WB, Hagen V, Bonhoeffer T (2009) U. Benjamin Kaupp Doxycyclin-dependent photoactivated caesar Bonn gene expression in eukaryotic systems. Nat Methods 6: 527-531 Klaus Benndorf F. Schiller-University, Jena

Sidney B. Cambridge MPI of Neurobiology, Munich and University Heidelberg

Stephan Frings University Heidelberg

Dorothea Eisenhardt FU Berlin

108 RESEARCH GROUPS // CHEMICAL BIOLOGY RESEARCH GROUPS // CHEMICAL BIOLOGY 109 CHEMICAL BIOLOGY

BIOPHYSICS OF MEMBRANE PROTEINS

GROUP LEADER Sandro Keller

Our group is interested in deciphering the molecular forces that Membrane binding and permeation Solubilisation and reconstitution of Protein-ligand and protein-protein A molecule’s ability to cross lipid membranes membranes and membrane proteins interactions govern protein-protein and protein-ligand interactions, with special is of the utmost importance for the extracel- Microcalorimetry is an excellent method of Isothermal titration calorimetry is the gold emphasis on integral membrane proteins. To this end, we apply a lular application of intracellularly active com- gaining detailed knowledge of the phase standard for the quantitative characterisa- pounds in cell biology, medicinal chemistry, state of lipid/detergent mixtures, which is a tion of protein-ligand and protein-protein in- broad range of spectroscopic, calorimetric, and chromatographic and drug development. A combination of prerequisite for the controlled solubilisation teractions. However, reliable determination

techniques to study the reconstitution and stability of model mem- microcalorimetric uptake and release as- of biological membranes and the rational of the dissociation constant (KD) is typically says is a fast and universally applicable tool reconstitution of membrane proteins. We limited to the range 100 µM > K > 1 nM. brane proteins. Moreover, we have developed novel assays for D to measure the membrane permeation of a provided the first fully quantitative account Nevertheless, interactions characterised by

quantifying membrane permeation of various compounds, as well wide variety of compounds. By accounting of the thermodynamics of vesicle solubilisa- a higher or lower KD can be assessed indi- for electrostatic effects at the membrane tion and reconstitution by ionic detergents, rectly, provided that a suitable competitive as ultratight drug-target interactions by high-sensitivity isothermal surface with the aid of Gouy-Chapman the- demonstrating that the kinetics of membrane ligand is available whose KD falls within the titration calorimetry. ory, we were able to render this approach translocation of a given detergent determine directly accessible window. Unfortunately, amenable to ionic compounds, including its suitability for thermodynamically-controlled the established competitive assay faces seri- SANDRO KELLER proteins and peptides, experimentally and solubilisation and reconstitution processes. ous problems when applied to studying pro-

clinically relevant detergents, and small mol- We could exploit this assay to guide the de- tein binding of poorly water-soluble small- 2003 – 2006: Dissertation, Department ecules. Thus, we succeeded in demonstrat- velopment of a detergent-like lipopeptide molecule ligands, which comprise a major of Chemical Biology, FMP, Berlin ing that a cell-penetrating peptide, dubbed intended for the targeted delivery of chemi- portion of most compound libraries used for 2006 Dr. rer. nat., Faculty of Natural penetratin, cannot translocate across lipid cal probes and drugs. When combined with screening or drug development. In collabo- Sciences - Chemistry and Physics, membranes in a passive fashion, which had phospholipids, and depending on the mixing ration with Bayer Schering Pharma AG, we Martin-Luther-University Halle-Wittenberg, long remained a controversial issue. Using ratio, the lipopeptide forms defined micellar have recently developed a new calorimetric Halle this assay, we could show that: (i) a doxy- or liposomal supramolecular structures and competition assay that overcomes this limi-

cycline derivative developed for photoacti- thus can be employed for the application of tation, thus allowing for a precise thermody- 2006 – 2010 Junior Research Group vated gene expression can indeed reach its both hydrophobic and hydrophilic cargo mol- namic description of high- and low-affinity Leader, Department of Chemical Biology, nuclear target by transmembrane diffusion ecules. The lipopeptide can accommodate protein-ligand interactions involving poorly FMP, Berlin upon extracellular application; (ii) the cellu- large amounts of phospholipid in mixed mi- water-soluble compounds. Since 2009 Assistant Professor of lar internalisation of cyclic nucleotides and celles, which, in spite of their small size, offer Molecular Biophysics (with tenure track), photolabile capsaicin precursors is not due a membrane-like environment and thus en- Department of Biology, University of to transmembrane diffusion; (iii) phototrig- able recognition of its binding sequence by Kaiserslautern, Kaiserslautern gers for protons can be developed in order membrane-bound receptor proteins. This has to acidify lipid membranes specifically on made possible its use as an excellent carrier either only one or both sides. system for the targeted delivery of contrast agents for the magnetic resonance imaging of atherosclerotic plaques in mouse models. Very recently, we have pioneered the application of high-sensitivity microcalorimetry for monitoring the solubilisation of complex biological membranes and the functional reconstitution of ion channels and other membrane proteins.

110 RESEARCH GROUPS // CHEMICAL BIOLOGY RESEARCH GROUPS // CHEMICAL BIOLOGY 111 From left to right: Oxana Krylova, Wiebke Kleinschmidt (intern)

Wiebke Kleinschmidt (intern), Nadin Jahnke

MEMBERS OF THE GROUP SELECTED PUBLICATIONS COLLABORATIONS EXTERNAL FUNDING

Prof. Dr. Fateh Sing Nandel, EF, * Bordag N, Keller S (2010) Alpha-helical Schmidt MF, El-Dahshan A, Keller S, International National Deutsche Forschungsgemeinschaft

transmembrane peptides: a “divide and Rademann J (2009) Selective identification “Thermodynamics of Mistic protein Dr. Oxana Krylova, PT, * Margarida Bastos Sidney Cambridge conquer” approach to membrane proteins. of cooperatively binding fragments in a folding” Department of Chemistry, Interdisciplinary Center for Neurosciences, Natalie Bordag (doctoral student), PT Chem Phys Lipids 163: 1-26 Review high-throughput ligation assay enables KE 1478/1-1 University of Porto, Portugal University of Heidelberg development of a picomolar Caspase-3 10.2007 – 06.2010 Jana Bröcker (doctoral student), EF, PT Fiedler S, Broecker J, Keller S (2010) inhibitor. Angew Chem Int Ed 48: 6346-6349 Amitabha Chattopadyhay Volker Haucke 128.650 Euro Protein folding in membranes. Nadin Jahnke (doctoral student), PT Centre for Cellular and Molecular Biology, Institute of Chemistry and Biochemistry, Cell Mol Life Sci 67: 1779-1798 Review Deutsche Forschungsgemeinschaft Hyderabad, India FU, Berlin Sebastian Fiedler (doctoral student), EF, PT FMP authors Zusammenarbeit im Rahmen der Kemmer G, Keller S (2010) Nonlinear Group members Heiko Heerklotz Daniel Huster DFG-INSA-Vereinbarung: Förderung des Monika Georgi (technical assistant), * least-squares data fitting in Excel Leslie Dan Faculty of Pharmacy, Institute of Medical Physics and Gastaufenthaltes eines Wissenschaftlers spreadsheets. Nat Protoc 5: 267-281 Gerdi Kemmer (student), EF, PT, * University of Toronto, Canada Biophysics, University of Leipzig aus Indien (Prof. Fateh Singh Nandel,

Krylova OO, Jahnke N, Keller S (2010) Panjab University, Chandigarh) Anja Sieber (student), EF, PT, * Peter Poh Ingo Morano Membrane solubilisation and reconstitution KE 1478/2-1 Institute of Biophysics, MDC, Berlin Elisabeth Fischermeier (student), EF, PT, * by octylglucoside: Comparison of synthetic 04.2009 – 06.2009 Johannes Kepler University, Linz, Austria lipid and natural lipid extract by isothermal Erwin Schneider 6.900 Euro Georg Krainer (student), EF, PT, * titration calorimetry. Holger Strauss Institute of Biology, HU, Berlin

Sandra Gusewski (student), EF, PT, * Biophys Chem 150: 105-111 Novo Nordisk, Copenhagen, Denmark Anne S. Ulrich Alekos Tsamaloukas Karlsruhe Institute of Technology

* Part of period reported Rensselaer Polytechnic Institute, Troy, USA Rudolf Volkmer PT part time Institute of Medicinal Immunology,

Charité – Universitätsmedizin Berlin EF position funded externally (3rd-party funds) for at least part of the reporting period

Current group members (31.12.2010)

112 RESEARCH GROUPS // CHEMICAL BIOLOGY RESEARCH GROUPS // CHEMICAL BIOLOGY 113 CHEMICAL BIOLOGY

MASS SPECTROMETRY

GROUP LEADER Figure 1. Representative mass spectra of the analysis Eberhard Krause of protein-protein interactions by quantitative mass spectrometry/SILAC approach. A-C, isotope pattern of tryptic peptides show heavy/light ratios greater than 10, indicating that these proteins bind specifically to the bait structure. D, typical spectrum of a tryptic peptide derived from a protein, which non-specifically binds to the matrix.

In recent years our group has focussed on the quantitative analysis Detection and, in particular, quantitative vation of a receptor guanylyl cyclase (GC) in (competiting PRS peptide) and with a GST- analysis, of post-translational protein modifi- sea urchin sperm that controls chemotaxisis. tagged U5-15K as bait in the presence and of peptide-protein and protein-protein interactions and the charac- cations by mass spectrometry is often ham- The GC is phosphorylated at rest and be- in the absence of a PRS inhibitor, provided terization of protein post-translational modifications and their func- pered by changes in the ionization/detection comes dephosphorylated after binding to an insight into PRS-dependent complex assem- efficiencies caused by amino acid modifi- interaction partner, resact. Since phosphory- bly of endogenous full-length CD2BP2/52K. tional consequences. Proteomics strategies have been evaluated cations. In order to increase confidence in lation analysis by mass spectrometry relies on Among the proteins identified in all experi- with regard to their suitability for identifying modification-mediated the analysis of protein phosphorylation and accurate sequence information and the pub- ments were SmB/B’ and NpwBP. These pro- methylation, and to enhance the reliability lished sequence lacks the conserved catalytic teins contain several PRS binding motifs protein interactions and protein assemblies using pull-down and of the quantitative assessment of the degree domain, the primary structure of the GC was and are known in vitro interaction partners TAP experiments. We established different stable isotope label- of modification based on ion intensities, we re-examined by cloning of cDNA from a tes- for CD2BP2/52K-GYF. Our study has shown studied the influence of phosphorylation and tis library of A. punctulata. Mass spectrometry that domain-based pull-down experiments in ling techniques in combination with high-accuracy and high-MS/MS methylation on responsiveness of peptides to analysis of flagellar proteins confirmed the combination with site-specific inhibition of in- capacity mass spectrometry and performed methodological studies, MALDI and ESI mass spectrometry. Our re- presence of peptides covering almost entire- teraction and advanced nanoLC-mass spec- sults clearly show that the ion yields are gen- ly the new C-terminal region that includes the trometry/stable isotope labelling (SILAC) al- EBERHARD KRAUSE which include attempts to improve the analysis of post-translation erally lower and the signal intensities are re- canonical motif for the catalytic site. We iden- lows one to deconvolute the contributions

modification of proteins. duced with phosphopeptides than with their tified 6 phosphorylated serine residues in the of individual epitopes to protein complex 1975 Diploma degree in physical nonphosphorylated analogs and that this unstimulated GC. Four of the 6 phosphory- formation (with C. Freund, FMP). chemistry at the Humboldt University has to be taken into account in MALDI and lated residues underwent dephosphorylation of Berlin ESI mass spectrometry. The unpredictable after resact stimulation. The rapid inactivation 1982 Ph.D. at the Humboldt University impact of phosphorylation does not depend of GC by dephosphorylation is one of the of Berlin on the hydrophobicity and net charge of the functional features that allow GC to encode

peptide, indicating that reliable quantifica- rapid changes in ligand concentration over 1984 – 1986 Research Group Leader tion of phosphorylation by mass spectrom- many orders of magnitude (with U.B. Kaupp, “Drug Development” in the etry requires the use of internal standards. Research Center, Jülich and Caesar, Bonn). pharmaceutical industry

1987 – 1991 Research Associate at In addition, we have developed novel (shot- Proline-rich sequences (PRS), which are the Institute of Drug Research, Berlin gun and quantitative) mass spectrometry ap- mostly assigned to unstructured regions of

proaches for studying membrane proteins the corresponding full-length proteins, serve Since 1992 Senior Scientist and and complex protein mixtures resulting from as docking sites for proline-rich sequence head of the Mass Spectrometry group pull-down experiments. These approaches recognition domains (PRDs). One interest- at the FMP included orthogonal multi-dimensional sepa- ing protein that mediates PRS interactions ration methods by reversed-phase capillary is the spliceosomal protein CD2BP2/52K, LC (RP-RP) in combination with ESI-LTQ-Or- which contains a GYF adaptor domain. In bitrap and MALDI-TOF-TOF mass spectrom- order to define PRS-mediated interactions etry. of CD2BP2/52K, pull-down experiments with SILAC labelling were performed. Pull-down Comprehensive phosphorylation analyses experiments using GYF domains with muta- were performed in the context of experi- tions within the PRS site (W8R/Y33A mutant), ments examining the activation and deacti- or a high concentration of a peptide inhibitor

114 RESEARCH GROUPS // CHEMICAL BIOLOGY RESEARCH GROUPS // CHEMICAL BIOLOGY 115 From left to right: Michael Schümann, Benno Kuropka

Heike Stephanowitz, Sabine Anker

MEMBERS OF THE GROUP SELECTED PUBLICATIONS COLLABORATIONS EXTERNAL FUNDING

Benno Kuropka (doctoral student), EF, PT, * Lange S, Sylvester M, Schümann M, Broedel O, Krause E, Stephanowitz H, International Biosyntan GmbH Freund C, Krause E (2010) Identification Schuemann M, Eravci M, Weist S, Brunkau “Massenspektrometrische Charakter- Diana Lang (doctoral student), EF, PT, * Miles Housley of phosphorylation-dependent interaction C, Wittke J, Eravci S, Baumgartner A (2009) isierung von Fluoreszenz-markierten University of Glasgow, Scotland Sabine Lange (doctoral student), PT, * partners of the adapter protein ADAP using In-gel O-18 labeling for improved identifica- Glyko- und Phosphopeptiden“ quantitative mass spectrometry: SILAC vs tion of proteins from 2-DE gel spots in com- Louis A. Carpino Biosyntan – EK1 Inga Eichhorn (student), PT, * 18O-labeling. J Proteome Res 9: 4113-4122 parative proteomic experiments. J Proteome University of Massachusetts, Amherst, USA 01.2009 – 12.2011 Michael Schümann (technical assistant) Res 8: 3771-3777 4.500 Euro

National Heike Stephanowitz (technical assistant) Gropengiesser J, Varadarajan BT, Stepha- Institut für Biochemie der FU nowitz H, Krause E (2009) The relative influ- U. Benjamin Kaupp “Massenspektrometrische Charakter-

ence of phosphorylation and methylation on Center of Advanced European Studies isierung von Lysin-phosphorylierten * Part of period reported responsiveness of peptides to MALDI and and Research, Bonn Peptiden und deren Interaktionspartner“ PT part time ESI mass spectrometry. J Mass Spectrom 44: 04.2011 – 03.2013 Bernard Hoflack 821-831 6.000 Euro EF position funded externally TU Dresden (3rd-party funds) for at least part Kofler M, Schuemann M, Merz C, Kosslick Kurt Engeland of the reporting period D, Schlundt A, Tannert A, Schaefer M, Universität Leipzig Lührmann R, Krause E, Freund C (2009) Current group members (31.12.2010) Proline-rich sequence recognition: I. Marking Iris Bruchhaus GYF and WW domain assembly sites in early Bernhard Nocht Institute for Tropical spliceosomal complexes. Mol Cell Proteom- Medicine, Hamburg ics 8: 2461-2473 Volker Haucke Schlundt A, Sticht J, Piotukh K, Kosslick FU Berlin D, Jahnke N, Keller S, Schuemann M, Uta-Maria Bauer Krause E, Freund C. (2009) Proline-rich Philipps-Universität Marburg sequence recognition: II. Proteomics analysis of Tsg101 ubiquitin-E2-like variant (UEV) in- Christian P.R. Hackenberger teractions. Mol Cell Proteomics 8: 2474-2486 FU Berlin

FMP authors Group members

116 RESEARCH GROUPS // CHEMICAL BIOLOGY RESEARCH GROUPS // CHEMICAL BIOLOGY 117 CHEMICAL BIOLOGY

SCREENING UNIT

GROUP LEADER Jens Peter von Kries

Figure 1. In the absence of the compound pitstop, red Figure 2. In the absence of an inhibitor, b-Catenin (green fluorescent vesicles mark functional endocytosis (left, fluorescence) mainly localizes in the nuclei of colon can- nuclei stained blue). In the presence of pitstop clathrin, cer cells (left image), while application of the compound mediated transport is blocked. blocks nuclear localization and b-Catenin localizes at cellular borderlines in adherens junctions (right image).

The Unit serves as an open access technology platform for au- Specific inhibition of Clathrin mediated is deactivated by mutation in many human to develop novel diagnostic tools for the endocytosis tumors (Cancer Research, 2011). Specificity early detection of pancreatic tumors, since tomated screening, either with compound libraries such as the Cellular transport from the cell’s outside of compounds was verified in vitro using Met receptors are specifically overexpressed ChemBioNet collection (30.000 cpds) plus FMP libraries (total of border to intracellular compartments, me- various cellular reporter systems and gene in pancreatic tumors. diated by membrane vesicles and the pro- expression profile analysis. The compounds 60.000 cpds), or with genome-wide RNAi libraries (human, mouse, tein clathrin, plays a key role in the entry were also validated in vivo using Xenopus Novel inhibitors for cytochrome P-450 nematodes). Beside the support in assay development, process of pathogens (HIV, bacteria), in the nervous double-axis formation assay, by inhibit- proteins in Mycobacterium tuberculo- system for synaptic signaling, and for re- ing the of growth of human tumor cells in sis, in Trypanosoma cruzi or for Bacillus automation, screening and automated data analysis, the Unit cycling of trans-membrane receptors. In mouse xenografts, and in ApcMin mice megaterium identifies novel screening technologies (impedance measurements, collaboration with the research group of (multiple intestinal neoplasia, Min), which One focus of the Screening Unit is the Volker Haucke (FU & FMP, Berlin) we iden- represent an animal model for the develop- search for inhibitors of CYP proteins from High-Content-Screening, AlphaScreen, capillary electrophoresis) tified compounds named ‘pitstops’, which ment of human colon cancer. tuberculosis bacteria and parasites like and implements these for service. Three modules of the Unit cur- specifically interfere with the function of the Trypanosoma. We established an absorp- protein clathrin and thereby also interfere Inhibitors for Met-receptor mediated tion scan test to identify compounds which JENS PETER VON KRIES rently support projects: the screening module, the compound with HIV infection (Cell, accepted 2011). metastasis of tumor cells interact with the cytochrome P-450 system

management module (Edgar Specker), and the process automation For identification of inhibitors we screened Growth of tumors at distant sites (metastasis) of enzymes and thereby generate specific 1987 Diploma in biology at the the ChemBioNet collection of 17.000 com- is the process responsible for over 90% of changes in the absorption profiles of the University Hospital Hamburg-Eppendorf module (Martin Neuenschwander). Compound management with pounds using an ELISA for quantification of cancer deaths. The Screening Unit focused systems. Using this method we identified (Prof. Strätling) automated freezers actually provides a storage capacity for more the binding of clathrin to amphiphysin. its research on the inhibition of Met-recep- specific ligands and inhibitors for enzymes 1991 Ph.D. at the University Hospital tors induced scattering of tumor cells in vitro from Mycobacterium tuberculosis (J Biol than 200.000 probes. Furthermore this module collects unique Hamburg-Eppendorf (Prof. Strätling) Wnt-signaling inhibitors interfering in vitro as a cellular model for metastasis. A cellular Chem 2009), from Trypanosoma cruzi (J

compounds donated from academic chemists and monitors quality and in vivo with growth of tumor cells assay with a human tumor cell line was opti- Med Chem 2009) and for Bacillus megate- 1995 – 2000 Research on proteins Canonical Wnt signaling is deregulated mized for HTS in 384 well format using fluo- rium (ChemBioChem 2011). involved in origin of cancer in the lab by LCMS analysis. in several types of human cancer, where rescent staining of DNA, of actin filaments of W. Birchmeier it plays a central role in tumor cell growth and cytoplasm. Automated object identifi- 2000 – 2003 Head of Screening Unit and progression. The last step of the sig- cation of colonies (unscattered, –HGF) and of Semaia Pharmaceuticals nal cascade consists of gene activation by scattered cells (+HGF) was optimized for

b-Catenin in complex with LEF/TCF tran- High Content Screening (MolDiaPacra, EU Since 2003 Head of Screening Unit at FMP scription factors. In collaboration with Ste- funded, SFMET, EU funded). Moreover, we fan Krauss (University of Oslo) we set up a added a label-free test for scattering using High-Content Screen with automated mi- the impedance measurement procedure. croscopes using a GFP reporter cell line, While imaging allowed us to identify Met- which produces Green Fluorescent Protein induced scattering in the hours range, the under the control of complex formation impedance measurements identified sig- by endogenous b-Catenin and TCF. The nificant alterations on a scale of minutes. Screening Unit identified two novel small Inhibitors of the phosphatase Shp2, which is molecules that specifically inhibit the ca- a key factor mediating Met signaling, were nonical Wnt pathway at the level of the profiled for specificity against several other destruction complex for b-Catenin, which human phosphatases. The results may help

118 RESEARCH GROUPS // CHEMICAL BIOLOGY RESEARCH GROUPS // CHEMICAL BIOLOGY 119 From left to right: Edgar Specker, Martin Neuenschwander

Silke Radetzki, Jing Du (guest scientist), Carola Seyffarth

Katy Franke

MEMBERS OF THE GROUP SELECTED PUBLICATIONS COLLABORATIONS EXTERNAL FUNDING

Dr. Simone Gräber Choi M, Eulenberg C, Rolle S, von Kries JP, Fetz V, Knauer SK, von Kries JP, Stauber RH International Deutsche Forschungsgemeinschaft PAKT (FLI-Jena)

Luft FC, Kettritz R (2010) The use of small (2009) Translocation biosensors - Cellular “Synthesis, optimisation, and screening with W. Rosenthal Dr. Martin Neuenschwander Larissa Podust molecule high-throughput screening to system integrators to dissect nuclear export of small molecule libraries targeting 01.2011 – 12.2012 UCSF Dr. Silke Radetzki identify inhibitors of the proteinase 3-NB1 by Chemicogenomics. Sensors 9: 5423-5445 protein-protein interactions” 180.000 Euro

interaction. Clin Exp Immunol 161: 389-396 Michael Waterman FOR 806, TP Z1 (RA 895/5-1) Dr. Edgar Specker Podust, LM, Ouellet, H, von Kries JP, ECRC Galina Lepesheva with J. Rademann, M. Beyermann Johnsen L, Weigel C, von Kries JP, Møller de Montellano, PO(2009) Interaction of with MDC Chris Eckert (technical assistant) Vanderbilt 05.2007 – 05.2010 M, Skarstad K (2010) A novel DNA gyrase mycobacterium tuberculosis CYP130 with 10.2008 – 2011 120.814 Euro Katy Franke (technical assistant), PT, * inhibitor rescues Escherichia coli dnaAcos heterocyclic arylamines. Ermanno Gherardi 360.000 Euro

mutant cells from lethal hyperinitiation. J J Biol Chem 284: 25211-25219 Tom Blundell Bundesministerium für Bildung und Sabrina Kleißle (technical assistant), * BMBF/MDC Antimicrob Chemother 65: 924-30 MRC, Cambridge UK Forschung Konkle, ME, Hargrove, TY, Kleshchenko, RNAi & REMP Keven Mallow (technical assistant) “Screening Unit: Assay development Lisurek M, Rupp B, Wichard J, Neuen- YY, von Kries JP, Ridenour, W, Uddin, MdJ, Stefan Krauss with MDC, J. Rademann screening for lead identification and Andreas Oder (technical assistant) schwander M, von Kries JP, Frank R, Caprioli, RM, Marnett, LJ, Nes, WD, Villalta, University Oslo 11.2008 – 2009 optimization” Rademann J, Kühne R (2010) Design of F, Waterman, MR, Lepesheva, GI (2009) 800.000 Euro Carola Seyffarth (technical assistant) Stefan Knapp 01GU0514 – KR chemical libraries with potentially bioactive Indomethacin amides as a novel molecular Oxford University with C. Freund, R. Kühne NGFNplus molecules applying a maximum common scaffold for targeting Trypanosoma cruzi 01.2006 – 05.2009 5 Projektförderungen á 30.000 Euro * Part of period reported substructure concept. sterol 14 alpha-Demethylase. National 543.976 Euro 06.2008 – 05.2013 Mol Diversity 14: 401-408 J Med Chem 52: 2846-2853 PT part time Walter Birchmeier Europäische Kommission

EF position funded externally MDC, Berlin (6. Forschungsrahmenprogramm) FMP authors (3rd-party funds) for at least part PL018771 Group members Christian Ottmann of the reporting period with J. Rademann CGC, Dortmund 08.2006 – 01.2010 Current group members (31.12.2010) Thomas Gress 389.800 Euro

Uniklinik, Marburg EU-SFMET FP7-HEALTH-2007-A HGF/SF and MET in metastasis EU-SFMET Coordinator: Ermanno Gherardi (MRC-Cambridge, UK) 04.2008 – 03.2011 223.750 Euro

MDC Screening Unit 04.2008 – 03.2011 223.750 Euro

120 RESEARCH GROUPS // CHEMICAL BIOLOGY RESEARCH GROUPS // CHEMICAL BIOLOGY 121 CHEMICAL BIOLOGY

MEDICINAL CHEMISTRY

GROUP LEADER Jörg Rademann

Figure 3. Scattering of colony-grown pancreatic tumor Figure 1. The concept of Dynamic Ligation Screening: Figure 2. Crystal structure of a Shank3-PDZ-inhibitor, cells induced by addition of the growth factor HGF Site-specific detection of binding fragment by substrate collaboration U. Heinemann, MDC; G. Krause, is blocked by addition of 1.5 µM of a specific Shp2- competition H. Oschkinat, FMP. inhibitor; collaboration W. Birchmeier, MDC.

The group develops novel methods for Fragment-Based Ligand Fragment-based ligand discovery employs tor Leads Versus Emerging and neglected without being cytotoxic. The compounds the iterative assembly of low affinity mo- RNA viruses (SILVER)”, which started in Oc- are currently being validated in animal Discovery. By doing so, we gain a more profound understanding lecular fragments to potent protein ligands tober 2010. models in collaboration with the Birchmeier of the contributions of molecular substructures to the overall with high ligand efficiencies. Essentially, Based on the DLS-methodology, ligands group (MDC) and will be developed further the activity of the obtained protein ligands for various protein targets have been de- at the FMP in a project supported by the activity of protein ligands. Our research delivers protein-specific is enhanced by the cooperative binding of veloped. Initially proteases were selected Pre-GoBIO award of the MDC. A second chemical probes, which are used for the functional and structural the fragments. Classically, low-affinity frag- as targets; subsequently the method was development project funded by a grant ments are identified by biophysical detec- extended to protein tyrosine phosphatases. from the Investitionsbank Berlin deals with characterization of proteins, as well as for imaging purposes. tion methods such as NMR and X-ray crys- As a third target class we work on protein- inhibitors of the transcription factor STAT5, The obtained chemical probes are employed for the validation of tallography. These methods, however, do protein interactions. The initial assay con- which are active against leukaemic cells. not solve the linkage problem, i.e. they do cept has been broadly diversified. In the Ligations of biologically active molecular proteins as potential new drug targets, and in some cases are not disclose how the low-affinity hit frag- beginning, ligated, active fragments were fragments to especially potent, multiva- used as starting points for hit-to-lead development. ments can be combined to obtain higher detected by competition with a fluorogenic lent structures are covered by our project affinity protein ligands. Hitherto, exten- substrate (“substrate competition”). Next, in the SFB 765. There, we develop multiva- Our targets include viral and human proteases, protein tyrosine sive manpower was required in Medicinal the method was extended to binding as- lent, peptide-based probes which recognize phosphatases, and protein-protein interactions. Special attention Chemistry to solve the linkage problem. says. For this the competition of ligation intracellular protein-protein-interaction do- We have developed novel methods for the products with a fluorescently-labelled bind- mains. Polymeric attachment of the peptide is given to bioisosteric fragments and to the cooperative detection and optimization of fragment ing partner was investigated using fluores- fragments enhances the proteolytic stabil- enhancement of fragment-interactions. binders which, for the first time, allow us cence anisotropy (“binding competition”). ity, cell permeability and – due to the multi- to measure the activity of reversibly linked Third, the method was extended to the valent presentation – the biological activity. fragment combinations in a high-through- detection of “dynamic anisotropy enhance- Recently, we were able to demonstrate the put bioassay. The method denominated as ment”, a variation allowing for the selective first potentiation of the efficacy of a peptide JÖRG RADEMANN Dynamic Ligation Screening (DLS) enables identification of cooperatively binding frag- by the polymeric attachment for the multi- the spatially resolved detection of low-affin- ments. Fourth, we have introduced a meth- valent, pro-apoptotic protein-protein inter- 1988 – 1993 Studies in chemistry, 1997 – 1999 Post-Doc at the Carlsberg 2004 – 2010 Professor of Medicinal ity fragments, which subsequently can be od for “dynamic substrate enhancement” action between the Bid-BH3 and Bax. The biochemistry, and economics at the University Laboratory, Copenhagen (Denmark) Chemistry at the FU Berlin linked irreversibly by a chemical reaction which enables us to investigate the selec- polymer conjugated also was proven to be

Konstanz (Germany) and Rutgers Graduate in order to obtain higher affinity ligands. In tive binding contributions of fragments well-suited for imaging purposes, e.g. the 1999 – 2004 DFG Group Leader on Since 2004 Head of Medicinal Chemistry School (USA) the first iterative application of the method, specifically binding to secondary binding monitoring of chloride concentration in en- “Diversity-Oriented Synthesis and Solid at the FMP a low-affinity, peptidic inhibitor of SARS co- sites of structurally similar protein tyrosine dosomal vesicles. 1994 Diploma “Synthesis of O-linked Phase Technology”, Eberhard-Karls Since 2010 Chair of Medicinal rona virus’ main protease was transformed phosphatases. In all cases, our projects in the develop- Glycopeptides”, University Konstanz University Tübingen (Germany) Chemistry at the University Leibzig into a highly active non-peptidic molecule. Bioactive molecules derived from the afore- ment of bioactive chemical probes contain 1994 – 1997 Ph.D. “Solid-Phase Synthesis 2003 Venia Legendi at the University Meanwhile a non-toxic, cellularly active, in- mentioned fragment-based ligand devel- a strong component of synthetic organic of Oligosaccharides” (Prof. Schmidt), Tübingen hibitor of SARS virus replication has been opment have been characterized structur- chemistry, and in many cases they are

University Konstanz developed. These works concerning viral ally and functionally in a number of projects. based on synthetic methodologies in-

protease inhibitors have been funded by For example, we have developed specific vented within the group. the BMBF with a grant of 1.2 million Euro for inhibitors of the phosphotyrosine phos- Beyond our own research, the group of Me- drug discovery of neglected diseases and phatase Shp2 active at nanomolar concen- dicinal Chemistry was responsible for the will be continued at Leipzig University as the trations, which efficiently inhibit the mobility supervision of the Screening Unit and for EU HEALTH project “Small-molecule Inhibi- of cells in several cellular metastasis models the coordination of the ChemBioNet.

122 RESEARCH GROUPS // CHEMICAL BIOLOGY RESEARCH GROUPS // CHEMICAL BIOLOGY 123 From left to right: Boo Geun Kim

Katharina Koschek, Richard Bunnag von Briesen Raz

Kai Holland-Nell, Maria Isabel Fernandez-Bachiller, Katharina Koschek

MEMBERS OF THE GROUP SELECTED PUBLICATIONS COLLABORATIONS

Prof. Taleb Altel, EF, * Franziska Gottschalk (student), PT, * Schmidt M, Isidro-Llobet A, El-Dahshan A, International Deutsche Forschungsgemeinschaft Europäische Kommission

Lisurek M, Tan J, Hilgenfeld R, Rademann J “Synthesis, optimisation, and screening “Small-molecule Inhibitor Leads Versus Dr. Samer Al-Gharabli, EF, * Roland Kersten (student), PT, * Roland Brock (2008) Sensitized detection of inhibitory of small molecule libraries targeting Emerging and neglected RNA viruses Nijmegen, the Netherlands Dr. Read Al-Qawashmeh, EF, * Michael Stolarski (student), PT, * fragments and iterative development of protein-protein interactions” (SILVER)”, darin: “Fragment-based

non-peptidic protease inhibitors by Dynamic Francois Delannoy FOR 806, TP Z1 (RA 895/5-1) discovery of antivirals” Dr. Adeeb El-Dashan, EF, * Stefan Wagner (student), PT, * Ligation Screening. Lille, France with J.P. von Kries HEALTH-2010.2.3.3-2 Dr. Maria Isabel Fernandez-Bachiller, EF, * Franziska Hinterleitner Angew Chem 120: 3319-3323, 05.2007 – 12.2013 2010 – 2014

(technical assistant), EF, * Angew Chem Int Ed 47: 3275-3278 National 285.000 Euro 425.000 Euro Dr. Kai Holland-Nell

Kevin Mallow (technical assistant), EF, * Ahsanullah, Schmieder P, Kühne R, Walter Birchmeier Deutsche Forschungsgemeinschaft Support for Training and Career Dr. Boo Geun Kim, EF, * Rademann, J (2009) Metal-free, MDC, Berlin “Reaktive Intermediate in polymeren Development of Researchers (Marie Curie) Dr. Ludmilla Perepelittchenko Dr. Samuel Beligny, EF, * regioselective triazole ligations deliver Gelen, ihre Anwendung in parallelen “Design, synthesis, identification (technical assistant) Udo Heinemann locked cis-peptide mimetics. Synthesen von Proteaseinhibitoren and evalution of novel non-peptidic Dr. Vera Martos, EF, * MDC, Berlin Angew Chem 121: 5143-5147; sowie deren biochemische Evaluierung“ inhibitorrs of main protease employing Ahsanullah (doctoral student), EF, PT, * * Part of period reported Angew Chem Int Ed 48: 5042-5045 Rolf Hilgenfeld RA 895/2-6 dynamic ligation screening (DLS)”

University of Lübeck 04.2008 – 03.2009 PISARSCoV/PIEF-GA-2009-237062 Richard Bunnag von Briesen Raz PT part time Schmidt M, El-Dahshan A, Keller S, 32.000 Euro 03.2009 – 02.2011 (doctoral student), EF, PT, * Rademann J (2009) Selective identification EF position funded externally 170.418 Euro of cooperatively binding fragments in a Bundesministerium für Bildung und Stefanie Grosskopf (3rd-party funds) for at least part EXTERNAL FUNDING high-throughput ligation assay enables the Forschung Fonds der Chemischen Industrie (doctoral student), EF, PT, * of the reporting period evolution of a picomolar caspase-3 inhibi- DAAD, PhD-Stipendium Research Award Deutsche Forschungsgemeinschaft André Horatscheck Current group members (31.12.2010) tor. Angew Chem Int Ed 48: 6346-6349 A/06/19232 Chemierelevante Forschung und Lehre “Modulation of PDZ-domain-mediated (doctoral student), EF, PT, * with Absanullah 10.000 Euro Ahsanullah, Rademann J (2010) Cyclative protein-protein interactions” 04.2007 – 03.2010 Katharina Koschek cleavage of azido-peptidyl-phosphoranes FOR 806, TP 5 (RA 895/5-2) Land Berlin 35.1000 Euro (doctoral student), EF, PT, * delivers locked cis-triazolyl-cyclopeptides with H. Oschkinat, G. Krause “Entwicklung eines STAT5-Aktivier-

as privileged protein binders. 02.2010 – 01.2013 Europäische Kommission Teilprojekt ungsinhibitors“ Sina Meyer (doctoral student), EF, PT, * Angew Chem Int E 49: 5378-5382 342.000 Euro des Integrated Project MolDiag-Paca IBB 10143721

Samina Nazir (doctoral student), EF, PT, * “Novel molecular diagnostic tools 10.2009 – 03.2012 Weinert S, Jabs S, Supanchart C, Sch- Deutsche Forschungsgemeinschaft for the prevention and diagnosis of 319.003 Euro Johannes Preidl (doctoral student), PT, * weizer M, Gimber N, Richter M, Rademann “Peptid- und Kohlenhydrat-Polymere pancreatic cancer” J, Stauber T, Kornak U, Jentsch TJ (2010) zur Modulation multivalenter Protein- Alexander v. Humboldt-Stiftung Martin Richter (doctoral student), EF, PT, * PL018771 Lysosomal pathology and osteopetrosis Liganden-Interaktionen“ Forschungsstipendium von with J.P. von Kries Jörn Saupe (doctoral student), EF, PT, * upon loss of H+-driven lysosomal Cl- accu- SFB 765 B4 Prof. Dr. Pradeep Kumar 08.2006 – 01.2010 mulation. Science 328: 1401-1403< 01.2008 – 12.2011 03.2010. – 05.2010, 9.450 Euro Marco Schmidt (doctoral student), EF, PT, * 389.800 Euro 482.500 Euro Postdok-Stipendium von Viviane Uryga-Polowy (doctoral student), PT, * FMP authors Dr. Vera Martos Swantje Behnken (student), PT, * Group members 09.2010 – 08.2012, 73.200 Euro

124 RESEARCH GROUPS // CHEMICAL BIOLOGY RESEARCH GROUPS // CHEMICAL BIOLOGY 125 CHEMICAL BIOLOGY

PROTEIN CHEMISTRY

GROUP LEADER Dirk Schwarzer

Figure 1. Selected PTMs found on several proteins including histones. a) Lysine Acetylation b) Lysine methylation and c) Phosphorylation of Serine residues.

Our group is interested in developing and utilizing chemical tools All eukaryotes package their DNA into chro- time. This approach is based on specific In order to identify these binding proteins, matin, a complex of DNA and dedicated NMR characteristics that lysine residues dis- we synthesize suitable proteomic baits that to study the physiological function of post-translational protein packing proteins - so-called histones. Chro- play upon acetylation: in 15N-HSQC experi- allow for the isolation of such binding pro- modifications (PTMs). A central goal is the development of probes matin occupies an important role in the reg- ments the backbone amide resonance of teins from cellular extracts. ulation of gene activity and histones have Lys residues experiences a chemical shift which can be used to monitor the dynamics of protein modifica- a dedicated function in this process. These upon acetylation and the unique signal of New Tools for Chromatin Biochemistry tions, or which can serve as baits to trap, isolate and identify regulatory processes are mediated in part the Ne amide affords a new signal. Both sig- Histone-derived peptides are very useful by a multitude of posttranslational protein nals can be used for monitoring acetylation probes for analyzing the function of histone interaction partners of PTMs. Our focus is on investigating the modifications (PTMs) found on the histone and deacetylation reactions. Our approach PTMs, but there are limits with respect to physiological function of PTMs found on histone proteins. His- proteins, and there is a current effort in the includes a chemical synthesis scheme for biological questions that require larger biomedical sciences to uncover the crosstalk the incorporation of stable, NMR-active chromatin structures. This problem can be tones package DNA into chromatin, and histone PTMs are known between histone PTMs and gene activity. isotopes at selected lysine positions within addressed with chemoselective ligation to play important roles in gene regulation. Deciphering the com- synthetic histone tails. These NMR report- techniques such as native chemical ligation Histone Acetyltion ers enable time-resolved observations of (NCL) or expressed protein ligations (EPL) DIRK SCHWARZER plex relation between histone PTMs and gene activity is important Reversible acetylation of lysine residues con- multiple lysine acetylation events in paral- that allow the ligation of synthetic peptides

for understanding eukaryotic genomes. To this end we combine stitutes an important histone modification, lel, as well as in a continuous and quantita- with recombinant protein fragments. Modi- 1993 – 1999 Studied chemistry at the which is generally associated with active tive manner. We demonstrate the feasibility fied regions of histones can be synthesized Philipps-Universität in Marburg methods from chemistry, biochemistry, and molecular biology gene transcription. Over 20 acetylation sites of this approach by analyzing the catalytic with the desired PTM patterns and ligated to 2002 Ph.D. at the Philipps-Universität to probe a wide range of chromatin factors, including chromatin have been identified on histone proteins to properties of HATs and HDACs at individual the remaining unmodified histone fragment (Prof. Marahiel), Marburg date. The acetylation level at individual ly- lysine residues within histone-derived pep- in order to provide homogenously modified binding proteins and the enzymes that install and remove histone sine residues is determined by the balanced tide substrates. full-length “designer histones” for biochem- 2003 – 2006 Post-Doc at the Johns- modifications. action of histone acetyl-transferases (HATs) ical investigations. We are developing, in Hopkins University (Prof. Cole) and histone deacetylases (HDACs). HDACs Histone Phosphorylation collaboration with Christian Freund (Protein

2006 – 2007 Post-Doc at the Universität have gained considerable attention recently Phosphorylation of serine and threonine engineering, FMP) and Michael Beyermann (Prof. Mootz), Dortmund because they have been identified as prom- residues is another long-known histone (Peptide Synthesis, FMP), such chemoselec-

ising drug targets. Therapeutic interven- modification. In contrast to acetylation and tive ligation methods that should provide Since 2007 Head of am Emmy-Noether tions of aberrant HDAC activities, which are methylation of Lys residues, the physiologi- easy access to “designer histone”. Group at the FMP in Berlin believed to result in the down-regulation cal function of histone phosphorylation is of pro-apoptotic genes in cancer, depend only poorly understood and is puzzling be- on the discovery of new drugs targeting cause these modifications are associated HDACs. A central requirement for HDAC-re- with condensed inactive chromatin and lated drug discoveries are sophisticated as- with relaxed and transcriptionally active says, which allow for the analysis of the site chromatin regions. We address this ambi- preferences of individual HDACs. Our group guity in collaboration with Eberhard Krause is interested in developing such assays. (Mass Spectrometry, FMP) using mass spec- In collaboration with Philipp Selenko (In-cell trometry. According to the “histone code NMR, FMP) we have developed a method theory”, histone modifications constitute for monitoring histone deacetylation and recognition sites for dedicated binding acetylation reactions at multiple sites in real proteins, which are recruited to chromatin.

126 RESEARCH GROUPS // CHEMICAL BIOLOGY RESEARCH GROUPS // CHEMICAL BIOLOGY 127 From left to right: Falko Schirmeister (intern), Rebecca Klingberg, Jan-Oliver Jost

Alexander Dose

Diana Lang (group E. Krause)

MEMBERS OF THE GROUP SELECTED PUBLICATIONS COLLABORATIONS

Alexander Dose (doctoral student), EF, PT, * Tsai WW, Wang Z, Yiu TT, Akdemir KC, Xia International W, Winter S, Tsai CY, Shi X, Schwarzer D, Jan-Oliver Jost (doctoral student), EF, PT, * Michael S. Cosgrove, Plunkett W, Aronow B, Gozani O, Fischle Syracuse University, New York, USA Rebecca Klingberg W, Hung MC, Patel DJ, Barton MC (2010)

(doctoral student), EF, PT, * TRIM24 links a noncanonical histone signa- National ture to breast cancer. Nature 486: 927-932 Till Teschke (doctoral student), PT, * Wolfgang Fischle, Liokatis S, Dose A, Schwarzer D, Selenko Bernhard Geltinger (technical assistant), * MPI Göttingen P (2010) Simultaneous detection of protein phosphorylation and acetylation by high- * Part of period reported resolution NMR spectroscopy. J Am Chem EXTERNAL FUNDING Soc 132: 14704-14705 PT part time Schwarzer D (2010) Chemical tools in chro- Deutsche Forschungsgemeinschaft EF position funded externally matin research. J Pep Sci 16: 530-537 “Entwicklung und Nutzung chemischer (3rd-party funds) for at least part Werkzeuge zur Untersuchung von Histon- of the reporting period Ludwig C, Schwarzer D, Zettler J, Garbe Modifikationen und den zugehörigen D, Janning P, Czeslik C, Mootz HD (2009) Current group members (31.12.2010) Enzymen“ Semisynthesis of proteins using split inteins. SCH 1163 3-1 (1. Förderabschnitt) Methods Enzymol 462: 77-96 08.2007 – 07.2010 Schwarzer D (2009) Hacking the genetic 603.208 Euro code of mammalian cells. Figure 2. Chemo-selective ligation techniques like Chembiochem 10: 1602-1604 NCL allow the linkage of recombinant and synthetic protein fragments. Since the synthetic fragment is not limited to the canonical 20 amino acids of the ribo- somal code, any non-natural or modified amino acid (X) FMP authors of interest can be introduced into the protein. Group members

128 RESEARCH GROUPS // CHEMICAL BIOLOGY RESEARCH GROUPS // CHEMICAL BIOLOGY 129 ADMINISTRATIVE INDEX AND TECHNICAL SERVICES

DIRECTORATE A Ahsanullah...... 124 Courault, Christian...... 76 Friedl, Sabine...... 76 Prof. Hartmut Oschkinat Grischa Nikolenko Eva Lojek Abel, Sabine...... 100 Cremer, Nils...... 36 Furkert, Jens...... 18,84,85 Acting Director since 01.01.2009 General Administration Technical assistance Abu Ajaj, Khaled...... 96 Fürl, Stephanie...... 88 Dr. Almut Caspary Katrin Schubert Annika Eggert Agarwal, Vipin...... 52 D Scientific coordination General Administration Technical assistance Akbey, Ümit...... 10,36 da Costa Goncalves, Andrey Christian.....76 G Albert, Gesa...... 40 Däubler, Gregor...... 68 Gajera, Chandresh...... 68 Katharina Schulz Gabriele Schumacher Julia Hagemeister Al-Gharabli, Samer...... 124 Dabrowski, Sebastian...... 72 Ge, Feng...... 36 Scientific coordination Secretary Technical assistance Alken, Martina...... 85 Dasari, Muralidhar...... 52 Geelhaar, Andrea...... 76

Dr. Britta Horstmann Al-Qawashmeh, Read...... 124 Dathe, Margitta...... 102,104 Geltinger, Bernhard...... 129 Safety Officer Public relations Computer Services Altel, Taleb...... 124 Davies, Victoria Ann...... 36 Georgi, Monika...... 112

Dr. Jens Furkert Asami, Sam...... 52 de Palma, Gregorio...... 36,37 Gerth, Fabian...... 40,41 Silke Oßwald Thomas Jahn Dekowski, Brigitte...... 108,109 Gimber, Niclas...... 68 Public relations Nework administration Technical Service del Vecchio, Giovanna...... 72 Gödde, Kathrin...... 68 B Dr. Anne Höner Ingrid Hermann Bagheri, Mojtaba...... 104 Diehl, Anne...... 36 Göritz, Petra...... 68 Hans-Jürgen Mevert EU-liasion officer System administration Ball, Linda...... 36 Döpfert, Jörg...... 60,61 Göttert, Henrijke...... 76

Marco Mussehl Ballaschk, Martin...... 56 Dorn, Matthias...... 56 Gottschalk, Franziska...... 124 Dr. Maxine Saborowski Hans-Werner Pisarz Bardiaux, Benjamin...... 36 Dose, Alexander...... 22,129 Götz, Frank...... 76,77 PhD-programme coordinator Service engineer Holger Panzer Beerbaum, Monika...... 56,57 Dreißigacker, Marianne...... 96,130 Goyette, Sandy...... 64 Dr. Birgit Oppmann Ingo Breng Michael Uschner Behnken, Swantje...... 124 Gräber, Simone...... 120

Coordination Net for Drug Discovery Service engineer Bekei, Beata...... 22,64–66 Grantcharova, Evelina...... 76 Stephanie Wendt E and Development Berlin-Brandenburg Beligny, Samuel...... 124 Eckert, Chris...... 120 Grosskopf, Stefanie...... 124 Alexander Heyne (DDDNet) Roy Wolschke Bellmann, Christian...... 72 Eggert, Annika...... 130 Großmann, Solveig...... 76 Student Bengtsson, Luiza...... 68 Ehrlich, Angelika...... 100 Grzesik, Paul...... 17,44 Heidemarie Petschick Roy Moritz Björn Schümann Berg, Johanna...... 72 Eichhorn, Inga...... 72,116 Günther, Sebastian...... 40 Secretary Student Pascal Schulz Bergsdorf, Eun-Yeong...... 68 Eichhorst, Jenny...... 92 Gusewski, Sandra...... 112 Alexandra Kiesling Beulshausen, Sven...... 76 Eilemann, Barbara...... 72 Secretary Offices Library Beyermann, Michael...... 6,55,98,127 Eisenmenger, Frank...... 48 H Billig, Gwendolyn...... 27–31,68 Eisermann, Beate...... 76 Händler, Kristian...... 36 Andrea Steuer Dr. Michael Beyermann Binolfi, Andres...... 64,65 El-Dashan, Adeeb...... 76,124 Haas, Ann-Karin...... 16–18,43–45 Administration Department of NMR-supported Marianne Dreißigacker Blank, Sindy...... 40 Ellermann, Thomas...... 130 Hagemeister, Julia...... 130 Structural Biology Frank Schilling Blasig, Ingolf...... 70 Erdmann, Natalja...... 36 Hagen, Volker...... 106 Head Marianne Dreißigacker Bogum, Jana...... 76,77 Ezerski, Verena...... 76 Hahn, Aaron...... 56

Department of Peptide, Bordag, Natalie...... 112 Handel, Lieselotte...... 11,36,37 Thomas Ellermann Chemistry and Biochemistry Breng, Ingo...... 130 Haseloff, Reiner...... 72 General Administration F Briand, Benoit...... 100 Faelber, Katja...... 52 Heidenreich, Matthias...... 29–31,68 Dr. Dietmar Zimmer Silvia Mauks Bröcker, Jana...... 112 Fast, Alexander...... 68 Heinrich, Nadja...... 100 Scientific Coordination Personnel Manager Büssow, Cindy...... 76 Faust, Dörthe...... 76,77 Heise, Ivonne...... 72 Department of Physiology and Fernandez-Bachiller, Maria Isabel....124,125 Helmbrecht, Tolga...... 56 Marina Spors Pathology of Ion Transport Fidzinski, Pawel...... 68 Hermann, Ingrid...... 130 Personnel Manager C Camponeschi, Francesca...... 36 Fiedler, Sebastian...... 112 Heyne, Alexander...... 130 Christel Otto DNA Sequencing Service Carbone, Anna...... 79,81 Fink, Uwe...... 52 Hiller, Matthias...... 36

General Administration Caspary, Almut...... 130 Fischermeier, Elisabeth...... 112 Hinterleitner, Franziska...... 124 Dr. Eberhard Klauschenz For a full account of all 2009/2010 Castro Villela, Victor Manuel...... 72 Frank, Ronald...... 6,94 Hohensee, Svea...... 92 Claudia Messing publications, grants, collaborations, patents, Chevelkov, Veniamin...... 52 Franke, Katy...... 120,121 Holland-Nell, Kai...... 124,125 General Administration Animal Housing innovations, teaching activities as well as Choudhury, Anup...... 36 Franks, Trent...... 36 Holtmann, Jan Hendrik...... 36 Mathias Schmidt Dr. Regina Richter a series of image films please refer to the Christian, Frank...... 76 Freund, Christian...... 38,47,115,127 Höner, Anne...... 96,130 General Administration Head Data CD enclosed at the back of the report. Cording, Jimmi...... 72 Freyert, Inga...... 68 Hoppmann, Christian...... 100

130 ADMINISTRATIVE AND TECHNICAL SERVICES INDEX 131 Horatscheck, André...... 124 Krönke, Nicole...... 68 Michl, Dagmar...... 100 Piotukh, Kirill...... 40 Schmikale, Bernhard...... 100 Tröger, Jessica...... 76 Horstmann, Britta...... 130 Krylova, Oxana...... 104,112 Milic, Jelena...... 76 Pisarz, Hans-Werner...... 130 Schramm, Philipp...... 56,57 Tscheik, Christian...... 72 Hoyer, Inna...... 16–18,44,45 Kublik, Anja...... 72 Morandi, Federica...... 48 Plans, Vanessa...... 68 Schrey, Anna...... 48 Kühne, Ronald...... 5,46 Moritz, Roy...... 130 Plested, Andrew...... 78 Schröder, Leif...... 58,61 U J Kuhnke, Lara...... 48 Motzny, Kathrin...... 40 Preidl, Johannes...... 124 Schröder, Sandra...... 88 Uryga-Polowy, Viviane...... 124 Jabs, Sabrina...... 68 Kunert, Britta...... 36,37 Moutty, Marie...... 76 Preston, Patricia...... 68 Schubert, Katrin...... 130 Uschner, Michael...... 130 Jacso, Tomas...... 52,53 Kunth, Martin...... 60,61 Müller, Matthias...... 48,49 Protze, Jonas ...... 19,44,72 Schülein, Ralf...... 16,18,82 Jahn, Thomas...... 130 Kuropka, Benno...... 116,117 Müller, Sebastian...... 44 Schult, Christoph...... 76 V Jahnke, Nadin...... 112,113 Münzer, Matthias...... 88 Schulz, Katharina...... 85,130 van Rossum, Barth-Jan...... 10,36

Jankowski, Sophie...... 72 Mussehl, Marco...... 130 R Schulz, Pascal...... 130 van Rossum, Marleen...... 64 Rademann, Jörg...... 122 Jehle, Stefan...... 10,36 L Schulz, Vivian...... 76 Verzini, Silvia...... 64 Lalli, Daniela...... 36 Radetzki, Silke...... 36,120,121 Jentsch, Thomas...... 4,27,66 Schumacher, Gabriele...... 130 Veshnyakova, Anna...... 44 Lang, Diana...... 23,116,129 N Raz, Richard Bunnag von Briezen....124,125 Joshi, Mangesh...... 52 Nandel, Fateh Sing...... 112 Schümann, Björn...... 130 Vogelbein, Susanne...... 85 Lange, Sabine...... 116 Rehbein, Kristina...... 36 Jost, Jan-Oliver...... 128,129 Natho, Kristin...... 68 Schümann, Michael...... 116,117 Vogelreiter, Gabriela...... 104 Lange, Sascha...... 10,36 Reif, Bernd...... 50 Junkes, Christof...... 104 Nazir, Samina...... 124 Schütze, Sebastian...... 68,69 von Bock, Anyess...... 68 Lange, Vivien...... 36 Retel, Joren Sebastian...... 36 Jurk, Marcel...... 56 Neagoe, Ioana...... 68 Schwarzer, Dirk...... 24,126 von Kries, Jens-Peter...... 87,118 Lau, Benjamin...... 72 Richter, Martin...... 124 Neuenschwander, Martin...... 120 Schwiebs, Anja...... 88 Voreck, Anja...... 11,36,37 Lauterbach, Ina...... 68 Richter, Regina...... 130 Neumann, Anita...... 76 Seedorff, Sabine...... 56 Vorreiter, Fränze...... 72 K Leben, Rainer...... 68 Ridelis Rivas, Ingrid...... 85 Kahlich, Bettina...... 88 Newie, Inga...... 72 Seidler, Patrick...... 68 Voss, Felizia...... 68 Lehmann, Roland...... 40 Riemann, Katja...... 36 Kaiser, Hermann-Josef...... 68 Nguyen, Thao Thi Bich...... 36 Seiter, Florian...... 36,37 Leibrandt, Benjamin...... 76 Ringling, Martina...... 92,93 Kamdem, Nestor...... 36 Nickeleit, Irina...... 96 Seja, Patricia...... 68 Leidert, Martina...... 36 Röben, Marco...... 56 W Keller, Sandro...... 110 Nikolenko, Grischa...... 130 Selenko, Philipp...... 22,62,127 Wagner, Florian...... 68 Leisle, Lilia...... 68 Rohde, Elvira...... 76 Kemmer, Gerdi...... 112 Nikolenko, Heike...... 104,105 Seyffarth, Carola...... 120,121 Wagner, Stefan...... 124 Leupold, Eik...... 104 Rohleder, Cathleen...... 68 Kern, Christian...... 72 Niquet, Sylvia...... 76 Shahid Shakeel, Ahmad...... 36 Walter, Juliane...... 72 Liebold, Janet...... 68 Rosenthal, Walter...... 74 Kersten, Roland...... 124 Noack, Claudia...... 76 Sieber, Anja...... 112 Walter, Maria...... 72 Linden, Arne...... 36 Rossa, Jan...... 72 Kiesling, Alexandra...... 130 Novarino, Gaia...... 29,68 Siems, Wolf...... 86 Wartenberg, Anne...... 36 Linser, Rasmus...... 52 Rossella, Federica...... 61 Kilic, Funda...... 108 Skroblin, Philipp...... 76 Wartosch, Lena...... 68 Liokatis, Stamatios...... 64 Rülke,Robert...... 88 Kim, Boo Geun...... 124,125 Specker, Edgar...... 118,120 Waziri, Negar...... 72 Lisurek, Michael...... 48,49 O Rupp, Bernd...... 48 Kinne, Anita...... 16,44,45 Oberheide, Karina...... 68 Spitzmaul, Guillermo...... 68 Weinert, Stefanie...... 68 Loening, Nikolaus...... 36 Rutz, Claudia...... 18,85 Kirschner, Aline...... 76 Oder, Andreas...... 120 Spors, Marina...... 130 Wendt, Stephanie...... 130 Lojek, Eva...... 130 Klauschenz, Eberhard...... 130 Oehlke, Johannes...... 102,104 Staat, Christian...... 72,73 Wernick, Stephanie...... 68 Lopez del Amo, Juan Miguel...... 52,53 Kleinau, Gunnar...... 14,44 Opitz, Robert...... 48 Stauber, Tobias ...... 68 Werther, Tobias...... 36 Lorenz, Dorothea...... 92,93 S Kleißle, Sabrina...... 120 Oppmann, Birgit...... 130 Saborowski, Maxine...... 130 Stechmann, Bahne...... 96 Westendorf, Carolin...... 84,85 Ludwig, Carmen...... 68 Klingberg, Rebecca...... 129 Oschkinat, Hartmut...... 4–6,9,34,130 Salazar, Héctor...... 81 Steinhagen, Kerstin...... 52 Wichard, Jörg...... 48 Lukesch, Manja...... 76 Klippel, Stefan...... 40 Oßwald, Silke...... 130 Santos de Freitas, Monica...... 36 Stephanowitz, Heike...... 116,117 Wieczorek, Marek...... 40 Klippenstein, Viktoria...... 79,81 Otto, Christel...... 130 Sargent, Catherine...... 44 Steuer, Andrea...... 130 Wiesner, Burkhard...... 90 Klose, Annerose...... 100 M Saupe, Jörn...... 124 Sticht, Jana...... 40 Wietstruk, Marcus...... 81,82 Klußmann, Enno...... 74 Mainz, Andi...... 52 P Schaal, Janina...... 108 Stolarski, Michael...... 124 Winkler, Franziska...... 44 Köhler, Christian...... 12,36 Malkewitz, Jürgen...... 76 Pál, Balazs...... 68 Schäfer, Gesa...... 76 Sydow, Karl...... 104,105 Wistuba, Sarah...... 36 Körner, Jana...... 36 Mallow, Keven...... 120,124 Panzer, Holger...... 130 Scheinpflug, Kathi...... 104 Sylvester, Marc...... 40 Witte, Christopher...... 61 Koschek, Katharina...... 124 Markovic, Stefan...... 36 Papsdorf, Gisela...... 85 Schilling, Frank...... 130 Wolkenhauer, Jan...... 85 Kosslick, Daniela...... 40 Martos, Vera...... 124 Pareja, Ruth...... 68 Schillinger, Christian...... 44 Wolschke, Roy...... 130 Kosten, Jonas...... 64 Mauch, Philip...... 36 Pawletta, Martyna...... 48 Schlegel, Brigitte...... 56,57 T Kotte, Maria...... 85 Mauks, Silvia...... 130 Pechstein, Arndt...... 36 Schlomann, Uwe...... 72 Tabor, Vedrana...... 76 Z Kotzur, Nico...... 76,108 May Rose, Honor...... 64 Perepelittchenko, Lioudmila...... 96,97,124 Schlundt, Andreas...... 40,41 Teichmann, Anke...... 92 Zampatis, Dimitrios...... 85 Krainer, Georg...... 112 McLean, Martin...... 96 Perneczky, Verena...... 68 Schmeißer, Edelgard...... 96,97 Teschke, Till...... 129 Zapke, Janet...... 36 Krause, Dagmar...... 100 Meineke, Bernhard...... 40 Petschick, Heidi...... 130 Schmidt, Antje...... 16,85 Theillet, Francois-Xavier...... 64 Zillmann, Silke...... 68 Krause, Eberhard...... 114,127 Messing, Claudia...... 130 Pfeffer, Carsten...... 68 Schmidt, Marco...... 124 Thongwichian, Rossukon (Tim)...... 64,65 Zimmer, Dietmar...... 68,130 Krause, Gerd...... 16,42,44 Mevert, Hans-Jürgen ...... 130 Piehl, Christian...... 72 Schmidt, Mathias...... 130 Tourel, Silvain...... 64 Zühlke, Kerstin...... 76 Kreuchwig, Annika...... 44 Meyer, Sina...... 124 Piontek, Jörg...... 72 Schmieder, Peter...... 6,54,99 Trapp, Kristin...... 72 Zwanziger, Denise...... 72

132 INDEX INDEX 133 CAMPUS BERLIN BUCH

Research

Leibniz-Institut für Molekulare Pharmakologie (FMP)

C 81 leibniz-Institut für Molekulare Pharmakologie C 81.1 NMR I C 81.2 NMR 2

Max Delbrück Center for Molecular Medicine (MDC)

C 27 Walter-Friedrich-House C 31 Max-Delbrück-House C 83 Max Delbrück Communications Center C 84 Hermann-von-Helmholtz-House A 10 library B 63 Research services

Shared Facilities by MDC and FMP

C 84.1 Research services C 87 Timoféeff-Ressovsky-Haus

Clinical Research

Common Facilities

A 8 gate House with Café Max A 9 Reception A 13 life Science Learning Lab; CampusInfoCenter A 14 Cafeteria

Guesthouses of the MDC

B 54 Hans-Gummel-Guest House B 61 Kindergarten; Salvadore-Luria-Guest House

Companies

134 CAMPUS BERLIN BUCH CAMPUS BERLIN BUCH 135 IMPRINT

Leibniz-Institut für Molekulare Pharmakologie (FMP) Research Report 2009–2010 im Forschungsverbund Berlin e.V. Editorial Board Campus Berlin-Buch Hartmut Oschkinat, Thomas Jentsch

Robert-Rössle-Str. 10 Coordination 13125 Berlin Silke Oßwald Germany

Author Feature Articles and Interviews Phone + 49 30 947 93 - 104 Russ Hodge Fax + 49 30 947 93 - 109 e-mail [email protected] Editing Arne Linden, Martin McLean, Russ Hodge, Silke Oßwald

Photography Silke Oßwald

3-D Illustrations Barth von Rossum, Annika Kreuchwig

Supplementary information on CD Arne Linden, Alexandra Kiesling

Design and Layout Apfel Zet, Berlin

Further Photography Maj Brit Jansen (p. 5, 34, 66, 70, 86, 122) Kai Bienert (p. 6)

Print NEUNPLUS1 Verlag + Service GmbH, Berlin

136 IMPRINT