Ongoing projects of BioStruct Core and Associated Research Groups:

For detailed information on the currently ongoing research projects please contact the according principal investigators of the BioStruct Research groups.

undefinedAhmadian Group:
Mechanisms of regulation and activation of Rho-effectors

The GTP-binding proteins of the Rho-Family regulate a spectrum of functionally diverse downstream effectors e.g. Rho kinase (ROCK) and thus initiate a variety of cellular processes, ranging from cytoskeleton reorganization to gene transcription. Our interest in the Rho/ROCK pathway as a target for therapeutic interventions comes from the observation on its involvement in tumor invasion and in diseases such as hypertension and bronchial asthma. However, important questions such as (i) How ROCK activation is achieved by RhoA, (ii) how ROCK is integrated in the signaling complexes at the lipid membrane and (iii) how other small GTPases such as Gem/Rad and Rnd3 interact with ROCK and thus antagonize Rho-ROCK signaling, remained to be answered. Our recent data support the notion of multiple effector binding sites in RhoA and strongly indicate the existence of a cooperative binding mechanism for ROCK that may be the molecular basis of Rho-mediated effector activation. To corroborate this working hypothesis we have planned to study comprehensively the structure-function relationship of RhoA interaction with full length and various fragments of ROCK. Furthermore we will analyze ROCK regulation by various small GTPase including RhoA, Gem, Rad and Rnd proteins using biochemical methods and X-Ray crystallography. Finally, we will search for additional ROCK binding partners in a proteomic approach using full length ROCK. Alongside the fundamental signal transduction mechanisms, expected results will be crucial for rational drug development.

BioStruct Fellow: undefinedMr. Ehsan Amin
Topic Supervisor: undefinedPD Dr. Reza Ahmadian

undefinedGohlke Group:
Determinants of selectivity, action, and resistance of antibiotics binding to prokaryotic and eukaryotic ribosomes

Several classes of antibiotics specifically inhibit the activity of ribosomes by binding to the peptidyl transferase center (PTC) and the ribosomal exit tunnel region at adjacent or overlapping sites. Available high-resolution structures of antibiotics bound to ribosomal subunits provide crucial insights regarding the binding sites, binding modes, and mechanism of action for several antibiotics. This information creates possibilities to suggest chemical modifications to achieve higher binding affinity and selectivity of antibiotics or even new classes of anti-bacterial drugs. However, the structure elucidation only provides a static view of the binding processes, but does neither reveal the dynamics governing ribosomal function nor the energetic determinants of antibiotics binding. Molecular dynamics simulations (MD) in combination with free energy calculations are suitable to fill this gap. So far, neither the PTC nor the ribosomal exit tunnel have been investigated in complex with bound antibiotics by these techniques. The goal of this project is to investigate for the first time dynamical and energetic properties of antibiotic-ribosome complexes in atomic detail by MD in combination with free energy calculations. This provides a unique opportunity for a detailed analysis of antibiotics selectivity, action, and resistance in terms of energetics of binding, influences of structural components of more extended binding site regions, and resistance mechanisms resembling allosteric effects.

BioStruct Fellow: undefinedMr. Jagmohan Singh Saini
Topic Supervisor: undefinedProf. Dr. Holger Gohlke

undefinedGroth Group
Structure and regulation of chloroplast F0F1 ATP synthase

F0F1-ATP synthases found in bacteria, mitochondria and in the photosynthetic membranes of chloroplasts or cyanobacteria catalyze the formation of ATP from ADP and inorganic phosphate at the expense of a transmembrane proton gradient. The F0F1 holoenzyme consists of the membrane-embedded F0-domain and the interconnected soluble F1-domain. The energy of the transmembrane proton gradient is used by F0 to translocate protons, which is coupled to ATP synthesis via a rotary catalytic mechanism located on F1. In contrast to bacterial and mitochondrial F-ATPases chloroplast F1 is a latent ATPase requiring activation before catalytic turnover can take place. The activation is regulated via the redox state of two cysteines in the gamma-subunit. Detailed information on the molecular mechanism of this regulation is anticipated from high resolution structures of the oxidized and the reduced F1-complex from the thermophilic cyanobacterium Thermosynechococcus elonagatus BP-1. The structural basis of the redox regulation of photosynthetic F ATPases will be further addressed by structural studies on the isolated, recombinant spinach chloroplast gamma-subunit. We intend to crystallize the isolated gamma-subunit in the reduced and oxidized form as well as a redox-complex of gamma with the natural redox-compound thioredoxin. The second main objective of the project is to solve the structure of the intact F0F1 holoenzyme. Previous structural studies on F0F1 have shown that the F0-F1 contact is fragile. In order to stabilize the F0F1 interaction we will construct chimeric enzymes containing the membrane embedded F0 from Escherichia coli and the catalytic F1 from Thermo synechococcus which are amenable to cysteine-crosslinking.

BioStruct Fellow: undefinedMs. Kerstin Foerster
Topic Supervisor: undefinedProf. Dr. Georg Groth

undefinedHeise Group:
Studying Structure, dynamics and topology of membrane proteins by solid-state NMR-spectroscopy

The study of membrane proteins in a native-like lipid bilayer environment remains a major challenge, as such systems often are not easily crystallizable nor solubilizable, conditions which are mandatory for structure determination by X-ray crystallography and solution NMR-spectroscopy, respectively. In the last decade, high-resolution solid-state NMR-spectroscopy has developed into a powerful tool for the investigation of immobilized proteins such as amyloid fibrils and membrane proteins. We will apply solid-state NMR-spectroscopy towards the characterization of structure, dynamics and topology of integral membrane proteins involved in signal transduction processes.

BioStruct Fellow: undefinedMs. Claudia Beumer
Topic Supervisor: undefinedProf. Dr. Henrike Heise

undefinedOesterhelt Group:
Investigation of structure function relationship of membrane proteins using atomic force microscopy

The atomic force microscope is a powerful tool to learn about the intra- and intermolecular interactions of proteins. The focus of the project is on the investigation of membrane proteins by (I) AFM based unfolding and (II) protein-ligand force measurements. By unfolding, forces stabilizing sensory rhodopsin II from Natromonas pharaonis (NpSRII) are going to be measured under different conditions of biological relevance, i.e. alone as well as in complex with its transducer, both in the absence and presence of blue light. Whereas light activation in the presence of the transducer triggers a signalling cascade, which initiates the photophobic response of the bacterium, SRII acts as a proton pump in its absence. Changes in the forces of sensory rhodopsin II in the different conditions indicate differences in molecular interactions, which may lead to a further understanding of the molecular mechanism and recognition processes. In a second part, the lantibiotic nisin, which is covalently bound to the tip via a PEG-linker, is going to be presented to the functional ABC-transporter NisT embedded in an inside-out-vesicle. Here, the amount of force peaks in the retraction cycle and rate-dependant unfolding forces might give insights in the energy landscape of this ligand-receptor relationship.

BioStruct Fellow: undefinedMr. Mario Schneider
Topic Supervisor: undefinedProf. Dr. Filipp Oesterhelt

undefinedSchmitt Group:
Biochemical and structural characterisation of opine dehydrogenases

Opine dehydrogenases (OpDHs) terminate anaerobic glycolysis in many marine invertebrates. OpDHs catalyse the reversible, reductive condensation of an α-keto acid with the amino group of an amino acid, using NADH as a co-substrate. The resulting products are called opines. The octopine dehydrogenase (OcDH) from Pecten maximus, a member of the OpDH family, only uses L-arginine as its amino acid substrate. During the reductive condensation reaction of L-arginine with pyruvate, a second chiral centre is formed. This capability to synthesise a product (opine) with one, new chiral centre with perfect selectivity, from one chiral and one achiral precursor, potentially makes OpDHs biotechnologically important candidates for an enzyme-based chiral synthesis. The aim of this project is to alter the substrate specificity of OcDH by site-directed mutagenesis in order to create novel opines. Furthermore, mutants will be created to change the stereoselectivity. These OcDH mutants will be characterised via biochemical methods as well as by x-ray crystallography. Solving these structures as apo or substrate bound will provide further insights into substrate recognition, binding and the underlying reaction mechanisms. Other enzymes of this family are the alanopine and strombine dehydrogenase from Arenicola marina. Interestingly, their substrates differ only in one methyl chain but initial experiments revealed a significant difference in their kinetics. Thus the reaction mechanism of these two enzymes will be characterised using the same experimental approach outlined above.

BioStruct Fellow: undefinedMr. Michael Lenders
Topic Supervisor: undefinedProf. Dr. Lutz Schmitt

Schröder Group:
From Heterogeneity to Resolution Improvement in Single-Particle Cryo-EM

Single-particle cryo-electron microscopy (CryoEM) is an emerging technique to determine the structure of large macromolecular assemblies such as protein complexes. CryoEM takes projection images of individual particles under different views, from which a three-dimensional density map can be reconstructed. This requires averaging over a usually large number of particles. Unfortunately, large protein complexes tend to be flexible and conformationally heterogeneous, which can severely limit the achievable resolution of the reconstruction. We will develop algorithms to exploit knowledge on this heterogeneity during the density reconstruction process with the goal of improving the resolution of the density.

BioStruct Fellow: position open
Topic Supervisor: undefinedJun.-Prof. Dr. Gunnar Schröder

undefinedSeidel Group:
Analysis of the three-dimensional architecture and dynamics of helical structures in biomolecules using single molecule fluorescence spectroscopy with multiparameter fluorescence detection

Many biomolecules, e.g. DNA, RNA or membrane proteins, have helical structure elements and it is often difficult to analyze their structural dynamics with techniques such as NMR or X-ray crystallography. Fluorescence spectroscopy of single molecules using Multiparameter Fluorescence Detection (MFD) was chosen to analyze molecules for following reasons:

    As single molecules are measured, subpopulations are made visible and, therefore, one has direct access to molecular dynamics and mechanisms.
    The technique allows for direct measurements of distances between two positions in a molecule via Fluorescence Resonance Energy Transfer (FRET).
    Only small amounts of sample are needed. Typically, concentrations in the fM-range.
    The technique has a high sensitivity and specificity.
    Also, compared to e.g. X-ray and NMR, it is inexpensive.

The obtained data is compared and complemented with data from e.g. X-ray crystallography, NMR, bioinformatics and computer simulations and, finally, a unified structural model of the molecule is established. As a first model system for analysis non-coding RNA was chosen because:

    There is very little known about RNA/Protein interactions.
    One has the possibility of a controlled buildup of the single RNA stemloops and can, thus, analyze the regulation of the RNA's self-expression.
    They are simple to synthesize and, therefore, easily accessible.

Transmembrane helices of membrane proteins, e.g. ABC-transporters, would also be suitable for examination with this technique. They are difficult to produce in high concentrations and are hard to crystallize, which makes them hard to analyze with NMR and X-ray crystallography, respectively.

BioStruct Fellow: undefinedMs. Katherina Hemmen
Topic Supervisor: undefinedProf. Dr. Claus Seidel

undefinedWillbold Group:
Structural Studies of a Cyclic Nucleotide-Activated Potassium Channel Binding Domain in Solution by Nuclear Magnetic Resonance (NMR) Spectroscopy

Ion channels activated by cyclic nucleotides play key roles in neuronal excitability and signal transduction of visual and olfactory neurons. They belong to two subfamilies: Cyclic nucleotide-gated (CNG) channels, and hyperpolarization-activated and cyclic nucleotidegated (HCN) channels (Kaupp & Seifert, 2001, 2002). Both channel types share a carboxyterminal cyclic nucleotide-binding domain (CNBD). HCN channels are activated by hyperpolarization and their activity is modulated by cyclic nucleotides. In contrast, CNG channels are voltage independent and require cyclic nucleotides to open. Binding of cyclic nucleotides promotes the opening of the channel. Probably, a conformational change in the CNBD is propagated to the pore. However, the mechanism by which the binding of cyclic nucleotides to the CNBD leads to the opening of the channel is not well understood. Therefore we initiated the structural characterization of the CNBD in solution by Nuclear Magnetic Resonance (NMR) spectroscopy in order to get insights into the "gating" mechanism of such channel types.

BioStruct Fellow: undefinedMs. Claudia Börger
Topic Supervisor: undefinedProf. Dr. Dieter Willbold

For detailed information on the research projects please contact the according principal investigators of the BioStruct Core Research groups. For contact data please click BioStruct Scientists...

Birkmann Group:
Structural analysis of membrane-anchored prion proteins and protein protein interaction within the membrane

Prion diseases like Creutzfeldt Jakob disease are an unique group of transmissible neurodegenerative diseases which can also occur spontaneously or have a genetic background. The infectious particles are termed prions. The main component of prions is a misfolded conformer (PrPSc) of a normal glycosylated cell surface protein, the cellular prion protein (PrPC), whose function is unknown in detail (for review see: Prusiner 2007, Fields Virol. 3059-91). During replication of prions, PrPC is converted into PrPSc. We investigated the conversion process by in vitro studies with several biophysical methods using recombinant PrP and natural PrPSc. We identified intermediates and precursor states during the conversion process and investigated the kinetics of spontaneous as well as seeded fibrillogenesis (for review see: Birkmann et al., 2008, Prion, 2 (2), 67-72). The non-infectious PrPC undergoes several posttranslational modifications, in particular attachment of two N-linked glycans and a glycosylphosphatidylinositol (GPI)-anchor, by which it is linked to the plasma membrane on the exterior cell surface. We studied the interaction of posttranslationally modified PrPC from Chinese hamster ovary cell culture (CHO-PrP) with model membranes in vitro, i.e. either with lipid vesicles in solution or lipid bilayers bound on a chip surface (Elfrink et al., Biol. Chem. 2007: 388(1):79-89). The equilibrium and mechanism of PrPC-association with model membranes was analyzed by surface plasmon resonance (SPR) and the ratio between free and membrane-attached CHO-PrPC was quantified. It is not known in which part of the cell PrPC and PrPSc interact and where the conversion takes place. One hypothesis is that the interaction takes place at the cell membrane. Within this project we aim at the structural and biophysical analysis of PrPC in the membrane.

BioStruct Fellow: undefinedMr. Jendrik Marbach
Topic Supervisor: undefinedDr. Eva Birkmann

undefinedBode Group:
Analysis of the structure of the molecular interaction between HCV NS5B polymerase and c-Src

The hepatitis C virus (HCV) is worldwide a major cause of chronic liver disease with a high tendency to establish a persistent infection. To replicate in its host cell the virus has evolved mechanisms to utilize and control cellular molecules or pathways required for virus genome replication. We could demonstrate that functional c-Src is required for sufficient replication of HCV (unpublished data). A more detailed analysis of the interaction of c-Src with HCV encoded proteins by immunoprecipitation studies or by pull-down experiments using GST-tagged proteins revealed that c-Src physically interacts with the RNA-dependent RNA polymerase NS5B of HCV. In particular the isolated SH3 domain of c-Src strongly interacts with NS5B. Further studies suggested that in part also the SH2 but not the SH1 domain of c-Src interacts with NS5B (unpublished data). We are currently focusing on the exact localization of the regions of NS5B required for this interaction and on the evaluation of the differential contributions of the SH2 and SH3 domain of c-Src to the interaction with NS5B. Based on these data, the aim of the proposed project is to resolve the structure of the interaction of the isolated SH3 (and/or SH2) domain of c-Src with the respective region of NS5B by NMR studies. For this purpose GST-fusion proteins comprising the respective domains of c-Src and NS5B will be expressed and purified. A second part of the project aims to analyze the structure of the complex of the whole proteins of c-Src and NS5B using X-ray analysis.

BioStruct Fellow: undefinedMs. Sabine Eisenbürger
Topic Supervisor: undefinedPD Dr. med. Johannes Bode

undefinedBott / Brocker Group:
Investigating structure, dynamics and mechanism of a membrane-bound sensor kinase using solid-state NMR and X-ray crystallography

Corynebacterium glutamicum is of outstanding importance in industrial biotechnology as a platform for the production of amino acids and several other products. We are studying metabolic, regulatory and signal transduction networks of this bacterium as a basis for metabolic engineering and the definition of optimal cultivation conditions in production processes. In this context, we investigate two-component signal transduction systems (TCS), which are composed of a membrane-bound sensor kinase and a soluble response regulator. Sensing of a specific stimulus by the dimeric sensor kinase leads to autophosphorylation of a conserved histidine residue, with one monomer phosphorylating the other. Subsequently the phosphoryl group is transferred to a conserved aspartate residue of the cognate response regulator, which thereby is activated or inactivated. Although thousands of studies were performed on TCS, no complete structure of a membrane-bound sensor kinase is available up to now. Only few structures of individual domains of sensor kinases have been published, either alone or in complex with a stimulatory ligand or in complex with the cognate response regulator. In the planned work, we want to investigate structure and dynamics of a sensor kinase by using solid-state NMR and X-ray crystallography and thereby get hints on the mechanism by which the external signal is transferred across the cytoplasmic membrane. Knowledge of this mechanism will certainly be relevant for many TCS and help to manipulate these systems in the field of biotechnology.

BioStruct Fellow: undefinedMs. Xenia Schuplezow
Topic Supervisor: undefinedDr. Melanie Brocker & Prof. Dr. Michael Bott

undefinedBrötz-Oesterhelt Group:
Dynamics of proteolytic activation of and substrate degradation by the peptidase ClpP

Acyldepsipeptides of the ADEP-class are novel antibiotics with potent activity against multi-drug resistant bacteria. Their unprecedented mechanism relies on dysregulating the bacterial caseinolytic protease. ClpP forms the proteolytic core of the caseinolytic protease and is activated and controlled by several Clp-ATPases that co-operate with ClpP within the protease complex. By binding to important docking positions at ClpP that are normally occupied by the Clp-ATPases, ADEPs activate ClpP for non-specific protein degradation that is detrimental to the cell. As various attempts to obtain X-ray structures of ClpP/ClpATPase complexes have failed, ADEPs serve as an ideal tool to study the process of ClpP activation as well as substrate degradation by the activated core. In X-ray structures 14 ADEP molecules occupy 7 cavities on each of the two heptameric ClpP rings, which stack to form the secluded proteolytic chamber. The structures also reveal that in the presence of ADEPs the gated entrance pores widen to a diameter that should allows passage of 2-3 protein strands. However, X-ray structures are static and the dynamics of the activation process have not been investigated, so far. Furthermore, we have recently made the intriguing observation that ADEP activated ClpP cannot only degrade unstructured protein substrates, but that the folded cell division protein FtsZ is also a preferred target. Within the BioStruct program we apply to use atomic force microscopy and single molecule FRET to study the dynamics of ClpP in the presence of ADEPs, particularly oligomer formation, catalytic activation, pore opening and substrate passage into the pore.

BioStruct Fellow: Mr. Imran Malik
Topic Supervisor: undefinedProf. Dr. Heike Brötz-Oesterhelt

undefinedHäussinger Group:
Prof. Dr. Dieter Häussinger and PD Dr. Roland Reinehr:
Compatible solute induced structural changes in membrane proteins that correlate with the modulation of protein function

In nature, compatible solutes (organic osmolytes) are used for protecting cells against high osmotic stress. They are known also to stabilize protein structures and support protein folding. Compatible solutes displace the solvent molecules, thereby affecting protein folding.

We want to understand whether biological effects on the cellular level induced by an increase or decrease in the solute level, respectively, may be explained by the direct interaction of the compatible solutes with the membrane proteins under investigation. We will test the effect of betaine and taurine and for comparison the bacterial solute ectoine as well as dimethylthiourea on the structure of bacteriorhodopsine, the bile salt export pump (Bsep), the sodium taurocholate cotransporting peptide (Ntcp) and the volume sensitive chloride channel ICln.

Using Atomic Force Microscopy and Force Spectroscopy we will investigate the effects in presence and absence of the osmolytes: first, stabilization of secondary structure elements of the membrane proteins by forced unfolding experiments. Second, stiffening of the unfolded amino acid chains due to solvent binding by measuring the polymer elasticity. Third, visualizing structural changes in the unfolded parts of the inner and outer loops of the respective proteins by high resolution imaging. Fourth, observing variations in the folding pathway with force guided refolding experiments.

BioStruct Fellow: Ms. Anna Bronder
Topic Supervisor: undefinedProf. Dr. Dieter Häussinger

undefinedHegemann Group
Prof. Dr. Johannes H. Hegemann:
Structural Biology studies of recombinant Chlamydia pneumoniae adhesins OmcB and Pmp21

Chlamydiae are obligate intracellular bacterial pathogens which cause a variety of important human diseases. Chlamydia trachomatis is the most common agent of bacterial sexually transmitted diseases and is responsible for over 90 million new infections every year worldwide. The C. trachomatis ocular serovars are responsible for trachoma, the major cause of preventable blindness in developing countries. Acute C. pneumoniae infections cause pulmonary diseases (e.g. 10 % of all pneumonia incidents worldwide), whereas chronic infections are linked to atherosclerosis and artery disease. The chlamydial infection starts with the adhesion of the bacteria to the human cell by binding of bacterial adhesins to eukaryotic receptors. Next internalisation occurs via receptor-mediated endocytosis. We have identified and characterised the chlamydial cell surface protein OmcB as the bona fide adhesin which binds to heparan sulfate-like glycosaminoglycan (GAG) structures on the human cell surface. We have characterised the GAG-binding properties of OmcB and showed that recombinant OmcB protein or OmcB antibodies reduced binding of bacteria to human cells and subsequent infection proofing that OmcB is essential for the chlamydial infection. Mutational analysis identified the GAG binding domain of OmcB (OmcB-BD) within its N-terminal 60 amino acids. Furthermore single amino acids could be identified which are essential for GAG binding and which may determine cell tropism and disease pattern for different C. trachomatis serovars (systemic versus local infection). Analysis of the three-dimensional structure of OmcB-BD from C. pneumoniae and the 2 C. trachomatis E and L1 will form the basis for a molecular understanding of the interaction of this adhesin with its ligand. Molecular modelling studies will also be done to find novel chlamydial adhesion inhibiting peptides based on these solved structures.

BioStruct Fellow: undefinedMr. Soeren Luczak
Topic Supervisor: undefinedProf. Dr. Johannes H. Hegemann

undefinedJaeger Group
Prof. Dr. Karl-Erich Jaeger and Dr. Ulrich Krauss:
Blue-light activated LOV-photoswitches and their biotechnological applications

This project aims to evaluate the potential of bacterial photoreceptors of the light, oxygen, voltage (LOV)-family to generate genetically-encoded biocatalyst-photoswitches, which allow for the light-dependent modulation of enzyme activities (properties) at will. To provide a rational basis for the engineering of such switches, biomolecular NMR will be used to study the signal-transduction mechanism in bacterial LOV-proteins. The main focus-point will be the unfolding or dissociation of LOV-domain C-terminal helical extensions, which have previously, in plant LOV-systems, been suggested to unfold upon light excitation. This light-dependent conformational change allows in the plant-system to trigger the activity in LOV-associated effector domains.

BioStruct Fellow: undefinedMs. Raj Rani
Topic Supervisor: undefinedProf. Dr. Karl-Erich Jaeger

undefinedJose Group
Determination of the AIDA-I β-barrel crystal structure: getting the clue to the autotransporter secretion pathway.

In the group of Inv. 1, a surface display system for recombinant proteins has been developed which is named Autodisplay. It can be used for the efficient surface display of recombinant proteins, including enzymes, antibodies, receptors or small protein drugs on E. coli. Autodisplay is based on the secretion mechanism of the autotransporter proteins, a large family of self-translocating proteins in gram-negative bacteria. The crucial part of this secretion mechanism is a porin-like β-barrel structure, responsible for outer membrane translocation of the passenger domain. In autodisplay this beta-barrel is derived from the natural E. coli autotransporter protein AIDA-I. In developing and optimizing the autodisplay system, we found a way to overexpress the β-barrel of AIDA-I within the outer membrane of E. coli, up to an amount, which is in the same order of magnitude as the natural outer membrane proteins OmpA or OmpF/C. The aim of the present project is a) to use this method of overexpression for purification of the β-barrel of AIDA-I and to create crystals as the basis for a structural model and b) to verify this model by functional studies.

BioStruct Fellow: undefinedMs. Iris Gawarzewski
Topic Supervisor: undefinedProf. Dr. Joachim Jose

undefinedKorth Group
Structural studies on molecular interactions of the Disrupted-in-schizphrenia (DISC1) protein

The molecular causes of schizophrenia are still unknown. Recently, based on several genetic studies, the disrupted-in-schizophrenia 1 (DISC1) protein has come into focus for investigating molecular mechanisms of schizophrenia and other mental diseases. Here, structural and quantitative investigations of DISC1 with its interacting centrosomal molecules NDEL1, NDE1, PDE4B and LIS1 are proposed. All proteins, or soluble representative fragments thereof, as well as mutant / polymorphic counterparts will be expressed in Escherichia coli. Structural studies will be attempted by NMR and crystallography for single and complexed proteins and complemented by binding studies with surface plasmon resonance. These investigations will yield insight into the stochiometry and regulation of the interactions in the DISC1/NDEL1/NDE1/LIS1/PDE4B complex, and, through structural determination of binding interfaces, open novel pharmacological targets for these diseases.

BioStruct Fellow: undefinedMr. A. Sravan K. Yerabham
Topic Supervisor: undefinedProf. Dr. Carsten Korth

undefinedMünk Group
Characterization of the HIV-1 Vif interaction site in anti-viral cytidine deaminases (APOBEC3)

HIV-1 uses its accessory protein Vif to destroy the potent intracellular inhibitors APOBEC3F and -G that are antiviral cytidine deaminases. The interaction of Vif and APOBEC3 is delicate and species-specific: e.g. the Vif protein of SIV of African green monkey fails to bind to human APOBEC3G and in consequence, humans are protected and resistant to this monkey virus. The proposed project will obtain structural and functional data of the Vif:APOBEC3 interaction, design interfering peptides to block Vif and stop the replication of HIV-1. Required work involves the generation of recombinant proteins, NMR, modelling, construction of eukaryotic expression plasmids, work with human cells and viral infection assays.

BioStruct Fellow: undefinedMr. Ananda Ayyappan Jaguva Vasudevan
Topic Supervisor: undefinedProf. Dr. Carsten Münk

undefinedPiekorz Group
Structural and functional characterization of the centrosomal TACC3-chTOG protein complex, a new mitotic cancer target

The centrosome organizes the bipolar mitotic spindle to ensure faithful separation of chromosomes during mitosis. Spindle poles, kinetochores, and various microtubule (MT)-associated proteins are involved in the regulation of MT dynamics. Mitotic spindle assembly is a highly dynamic process and tightly controlled by the cell cycle. On the other hand, alterations in centrosome and mitotic spindle architecture lead to chromosomal instability and aneuploidy with profound consequences for cell cycle progression and cellular survival. The mitotic spindle apparatus is often altered in pathologies from neurological diseases to neoplasia and represents a major cellular target for antitumor therapy. However, commonly used MT-interfering cancer drugs cause chemoresistance and severe side effects. To validate novel antineoplastic targets overexpressed in transformed cells we have identified and functionally characterized members of the transforming acidic coiled-coil (TACC) family as important structural components of the centrosome/spindle apparatus. TACC proteins share a ~200-amino acid C-terminal coiled coil motif (CC) with only limited homology at the N-terminus. Interestingly, more than 70% of all centrosomal proteins contain CC motifs. TACCs interact through the CC motif with the C-terminus of the MT polymerase chTOG which binds to MT ends through multiple N-terminal TOG domains. We and other have shown that (1) TACC3 binding to chTOG is required for centrosomal and spindle localization of chTOG, and hence (2) depletion of TACC3 or chTOG interferes with centrosome integrity, centrosome-dependent assembly of MTs, and spindle stability thereby leading to either mitotic cell death or p53-dependent postmitotic cell cycle arrest. Determination and analysis of the three-dimensional structure of the CC domain of TACC3 alone and in complex with the C-terminus of chTOG as well as biochemical analysis of such a bimolecular interaction will provide (1) novel molecular insight in the interaction of TACC3 with the MT polymerase chTOG and (2) the opportunity to design inhibitors to block TACC3 binding to chTOG as candidate antineoplastic approach.

BioStruct Fellow: undefinedMr. Harish Thakur
Topic Supervisor: undefinedDr. Roland Piekorz

undefinedPietruszka Group
Rational Design of Aldolases for Organic Synthesis

Biocatalytical approaches towards key building blocks in organic synthesis have emerged as an important tool in the last decade. While the majority of applications were based on hydrolases, other enzyme classes, e. g., oxidoreductases or lyases, moved into the focus of current research projects and beyond: The mild reaction conditions combined with the high stereo-, regio-, and chemoselectivity often lead to economic and ecological advantages of enzymatic conversions, also because reaction pathways are regularly shortened. Aldolases represent a promising class of enzymes catalysing the formation of a C-C-bond between an aldehyde as the electrophile and a second carbonyl compound acting as the nucleophile. They have become an encouraging and sustainable alternative to conventional synthetic methods. However, there are still a number of problems to face, especially concerning enzyme stability and selectivity: While stability issues (solvent, temperature, and substrate tolerance) are of obvious concern, selectivity as the second aspect is not a matter of course. In the current project we will focus on the structural basis and on the understanding of stabilizing aldolases. We are aiming at introducing rational designed new, altered and optimized enzymes into chemoenzymatic sequences. A close co-operation with a computational team (Gohlke) as well as with a group having in-depth expertise in protein crystallography (Groth) is of fundamental importance for the success of the project.

BioStruct Fellow: undefinedMr. Thomas Classen
Topic Supervisor: undefinedProf. Jörg Pietruszka

undefinedSmits Group
Structure of the nisin resistance protein

Lantibiotics are antimicrobial peptides produced by Gram-positive bacteria acting against other Gram-positive bacteria found in the habitat. The best studied lanthibiotic is nisin, which is a 34 amino acid long peptide which comprises five (methyl)lanthionine rings produced by Lactococcus lactis. The potency of nisin to penetrate the membrane and inducing lysis of the target organism is extremely high since only a few nisin molecules are required to induce cell lysis. In contrast to nisin producer strains, the target organisms developed a defense mechanism consisting of the nisin resistance protein (NSR). In vitro studies demonstrated the capability of NSR to cleave nisin between MeLan28 and Ser29. The obtained nisin fragment, which still contains the five characteristic lanthionine rings, is 100 fold less active compared to full-length nisin. The NSR family is the first example for resistance against nisin, although the latter has been used in (food) industry already for several decades.

The goal of this PhD project is to elucidate the structure of NSR with and without bound substrate as well as to identify the molecular mechanism of substrate recognition and cleavage. Techniques used during this protein will include molecular biology, protein biochemistry and purification, fluorescence spectroscopy and X-ray crystallography.

Candidates should have a strong background in protein biochemistry, especially protein expression and purification. Furthermore, an interest in x-ray crystallography is a prerequisite.

Topic Supervisor:
undefinedDr. Sander Smits and undefinedProf. Dr. Lutz Schmitt, Institute of Biochemistry, Heinrich Heine University Düsseldorf

undefinedStoldt Group
β3-Integrin-Dependent Fibrillogenesis of Macromolecular RGD-Ligands in Relation to Biome-chanic Stress

Integrins and their ligands are prominent players regulating interactions of the extracellular environment and cellular functions. Sensing the biomechanic microenvironment integrins bidirectionally transmit conformational changes across the membrane. The resulting signals modulate cytoskeletal functions (outside-in signalling) and vice versa the cytoskeleton regulates the molecular relationship between receptor molecules and their ligands (inside-out signalling). Thus, mechanotransduction of ligand-occupied integrins regulate stable but flexible interactions between neighbouring cells. This is particular true for cells which are exposed to shear forces. The integrins αIIbβ3 and αvβ3 both incorporate the same β-subunit. β3-integrins bind RGD containing ligands e.g. fibronectin (Fn), fibrinogen and von Willebrand Faktor (vWF). Whereas Fn is a core component of extracellular matrices fibrinogen and vWF are plasmatic proteins. Fn and vWF are of a compact structure. They are unfolded by either shear stress or β3-integrins and cell traction forces modulate their fibrillogenesis. In our project we focus on the dynamic of β3-integrin-ligand interaction in response to conformational and functional changes upon exposure to shear stress. In a flow model we use human platelets or cell lines expressing β3-integrins to measure fibrillogenesis of Fn in response to mechanotransduction. Therefore, fluorescently labelled Fn is used to measure e.g. disappearance of fluorescence resonance energy transfer (FRET). To assess the specific role of the β3-subunit, two isoforms (Leu33 and Pro33) are compared because the Pro33 isoform is a risk determinant of acute coronary syndromes due to a prothrombotic characteristics like enhanced platelet adhesion and increased outside-in signaling.

Topic Supervisor: undefinedDr. Volker R. Stoldt

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