11:00                    Registration at the lecture hall 6B, building 26.11.O0

12:30                    Welcome by Michael Piper,
                              Rector of the Heinrich-Heine University Düsseldorf
                              Welcome by Prof. Detlev Riesner
                              Welcome by Lutz Schmitt, Chairman of BioStruct

13:15 - 15:00       Bernadette Byrne,  ‘Production of membrane proteins for structural
                                                                           and functional studies’

15:00 - 15:30      Tea/Coffee break

15:30 - 15:50      Simon Sindbert,
                              ‘Single molecule FRET accurately measures structure, dynamics and
                                   heterogeneities of an RNA four-way junction‘
15:55 - 16:15      Jonathan Mueller,
                              ‘A heterodimer of human 3 -phospho-adenosine-5 -phosphosulphate
                                   (PAPS) synthases is a new sulphate activating complex‘

16:15 - 16:30      Tea/Coffee break

16:30 - 18:15       Ben Schuler,   ‘Structure and dynamics in protein folding from single
                                                                   molecule fluorescence spectroscopy’

09:00 - 10:45       Eckhard Hofmann,   ‘Introduction into X-ray crystallography’

10:45 - 11:15      Tea/Coffee break

11:15 - 11:35      Badri Nath Dubey,
                              ‘Insights into structure-function relationships in GTPase- effector
                                   interaction: a progress report‘
11:35 - 11:55      Chunmao He,
                              ‘Structural features required for the protein-based sensing of nitric oxide -
                                   Insight from the spectroscopic investigations of ferrous nitrophorins‘
11:55 - 12:15      Arpita Roychoudhury,
                              ‘Stabilization of membrane proteins using compatible solutes

12:15 - 13:00      Lunch
13:00 - 14:00      Poster session (odd numbers)

14:00 - 15:45       Helmut Grubmüller, ‘Biomolecular nanomachines at work:
                                  Computer simulation of conformational dynamics and single molecule experiments ’

15:45 - 16:15      Tea/Coffee break

16:15 - 18:00       Daniel Müller,  ‘AFM: A nanotool complementing structural biology of
                                                                    biological membranes’


              19:00      Congress Dinner at Restaurant "Brauerei Schiffchen"

09:00 - 10:45       Michael Sattler,  ‘Biomolecular NMR-spectroscopy for structural
                                                                       analysis of protein complexes in solution’

10:45 - 11:15      Tea/Coffee break

11:15 - 11:35      Hoa Quynh Do,
                              ‘Mobility and topology of VPU and CD4 proteins by solid state
                                   NMR spectroscopy‘
11:40 - 12:00      Sven Schünke,
                              ‘Structural insights into conformational changes of a cyclic nucleotide-
                                   activated ion channel binding domain in solution‘

12:00 - 13:00      Lunch
13:00 - 14:00      Poster session (even numbers)

14:00 - 15:45       Eckhard Bill,   ‘Radicals coordinated to transition metal ions - Where
                                                                  are the electrons ? (EPR and Mössbauer Spectroscopy)’

15:45 - 16:15      Tea/Coffee break

16:15 - 18:00       Andrew Leslie,   ‘Challenges in structural molecular biology’

              19:00      BBQ at the Botanical Garden

09:00 - 10:45       Holger Stark, ‘Studying structure dynamics of large macromolecules
                                                                  by cryo-EM’

10:45 - 11:15      Tea/Coffee break

11:15 - 11:35      Harish Thakur,
                               ‘Purification and characterization of the centrosomal protein TACC3‘
11:40 - 12:00      Justin Lecher,
                               ‘Structural characterisation of the C39 peptidase like domain of the
                                     ABC-transporter HlyB‘

12:00 - 13:00      Lunch

13:00 - 14:45       Clemens Glaubitz, ‘Biophysical studies on membrane proteins by
                                                                            solid-state NMR’

14:45 - 15:15      Best Poster Award
                              and Closure by Dieter Willbold, Chairman of BioStruct

Title:
Production of membrane proteins for structural and functional studies

Summary:
Despite major advances over the last decade, the production of membrane proteins for structural studies remains problematic. Many membrane proteins are highly unstable in detergent solution. The protein isolation process is often complicated by significant losses through aggregation during purification. It is often difficult to obtain a fully homogeneous preparation and in addition, pure membrane protein often exhibits low conformational and thermal stability. High throughput expression screening has been useful for identifying targets for further study but indications are that expression is not a particularly useful indication of suitability of a particular target for further study. Well expressing targets can aggregate and degrade upon solubilisation and/or purification. Recent development of generic high-throughput methods for screening stability of membrane proteins have complemented the protein-specific approaches and greatly enhanced the rationality of membrane protein target identification [1,2]. One method allows assessment of aggregation and degradation sensitivity of the target protein in crude solubilised samples [1] while the other provides an estimate of thermostability in pure samples using very small amounts of material. One of the key parameters for stability of a membrane protein is the detergent choice. The development of new detergents for membrane protein is an emerging field. Recent work in my group has focussed on the characterisation of a new class of detergents with a tetra-substituted central carbon atom bonded to two hydrophilic groups and two lipophilic chains. Using an assay based on dye binding to membrane proteins to follow protein unfolding at elevated temperature we assessed the stability of a range of membrane proteins in DDM and the novel detergents. In all cases the use of the novel detergents increased the thermostability of the tested proteins. We also showed that one of the novel detergents retained the quaternary structure and functionality after heating in the case of one of the proteins. The results of this study and a number of other methods for the high-throughput stability of membrane proteins will be discussed.

Speaker:
Bernadette Byrne, Imperial College London, United Kingdom

Reviews on the topic:
[1] Kawate, T.; Gouaux, E., Structure 2006, 14 (4), 673-81.
[2] Alexandrov, A. I.; Mileni, M.; Chien, E. Y.; Hanson, M. A.; Stevens, R. C., Structure 2008, 16 (3), 351-9.

Title:
Structure and dynamics in protein folding from single molecule fluorescence spectroscopy

Summary:
The spontaneous self-organization of an unstructured polypeptide into a well-defined three-dimensional structure is one of the most fundamental processes of life, and its complexity poses one of the central challenges for modern biophysics. We use single molecule Förster resonance energy transfer (FRET) to study fundamental aspects of protein folding that have eluded investigation by ensemble experiments. One of the key aspects we address is the role of elementary dynamics that ultimately limit the rate of protein folding reactions, such as chain reconfiguration in the unfolded state. Single molecule FRET can provide both structural and dynamic information along these lines and has allowed us to map long-range intramolecular distances and reconfiguration dynamics in non-native states of proteins, which are difficult to access with classical structural biology methods. Single molecule methodology can also be used to investigate the folding and dynamics of proteins under more complex conditions, e.g. in the context of cellular factors, such as molecular chaperones.

Speaker:
Ben Schuler, Department of Biochemistry, University of Zurich, Switzerland

Title:
Introduction into X-ray crystallography

Summary:
Even in times of whole genome data we are still not able to reliably predict 3D-structures of protein of known sequence. Therefore experimental techniques to obtain this information are central to our understanding of protein (and RNA) structure and function. Of the roughly 67000 strutures deposited in the protein data bank today most have been obtained by three different techniques, namely X-ray crystallography, NMR and electron microscopy. All these techniques will be introduced during this meeting in different lectures. In this lecture I will give a general introduction into X-ray protein crystallography, which was used to determine 85% of the deposited structures to date. The different stages during the stucture solution pipeline will be discussed. In several examples I will highlight the difficulties involved in refinement of protein complexes with previously undetermined heterocompounds.

Speaker:
Eckhard Hofmann, AG Proteinkristallographie, LS Biophysik, Ruhr-Universität Bochum, Germany

Title:
Biomolecular Nanomachines at Work: Computer Simulation of Conformational Dynamics and Single Molecule Experiments

Summary:
Proteins are biological nanomachines. Virtually every function in the cell is carried out by proteins -- ranging from protein synthesis, ATP synthesis, molecular binding and recognition, selective transport, sensor functions, mechanical stability, and many more. The combined interdisciplinary efforts of the past years have revealed how many of these functions are effected on the molecular level. Computer simulations of the atomistic dynamics as well as of single molecule experiments on these macromolecular complexes play a pivotal role in this enterprise, as they offer both unparalleled temporal and spatial resolution. In this talk we address mechanical energy transfer in F-ATP synthase [4], flexible binding and recognition by nuclear pore transporters [3], and the mechanical properties of viral capsids [1,2]. Close contact with experiments is achieved through the simulation of atomic force and single molecule spectroscopy experiments [5-7].

Speaker:
Helmut Grubmüller, Max-Planck-Institut für biophysiklaische Chemie, Abteilung Theoretische und Computergestützte Biophysik, Göttingen, Germany

References:
[1] Zink M, Grubmüller H. Primary changes of the mechanical properties of Southern Bean Mosaic Virus upon calcium removal. Biophys. J. 98: 687-695 (2010)
[2] Zink M, Grubmüller H. Mechanical properties of the icosahedral shell of southern bean mosaic virus: a molecular dynamics study. Biophys J. 96(4): 1350-63 (2009)
[3] Zachariae U, Grubmuller H. Importin-ß: Structural and Dynamic Determinants of a Molecular Spring.Structure 16: 906-915 (2008)
[4] Böckmann R, Grubmüller H. Nanoseconds molecular dynamics simulation of primary mechanical energy transfer steps in F1-ATP synthase. Nature Struct. Biol. 9: 198-202 (2002)
[5] Lange OF, Lakomek NA, Farès C, Schroder GF, Walter KFA, Becker S, Meiler J, Grubmuller H, Griesinger C, de Groot BL. Recognition Dynamics Up to Microseconds Revealed from an RDC-Derived Ubiquitin Ensemble in Solution. Science 320: 1471-1475 (2008)
[6] Sieber JJ, Willig KI, Kutzner C, Gerding-Reimers C, Harke B, Donnert G, Rammner B, Eggeling C, Hell SW, Grubmüller H, Lang T. Anatomy and Dynamics of a Supramolecular Membrane Protein Cluster. Science317: 1072-1076 (2007)
[7] Grubmüller H, Heymann B, Tavan P. Ligand Binding: Molecular Mechanics Calculation of the Streptavidin-Biotin Rupture Force. Science 271: 997-999 (1996)

Title:
AFM: A nanotool complementing structural biology of biological membranes

Summary:
Cellular membranes are vital for life. They confine cells and cytosolic compartments and are involved in virtually every cellular process. Cellular membranes form cellular contacts and focal adhesions, anchor the cytoskeleton, generate energy gradients, transform energy, transduce signals, move cells, and actively form compartments to assemble different membrane proteins into functional entities. But how do cellular membranes perform these tasks? What do the machineries of cellular membranes look like, and how are they controlled and guided? Atomic force microscopy (AFM) allows the observation of biological surfaces in their native environment at a signal-to-noise ratio superior to that of any optical microscopic technique. With a spatial resolution approaching ≈1 nm, AFM can identify the supramolecular assemblies, characteristic structure, and functional conformation of native membrane proteins. In recent years, AFM has evolved from imaging applications to a multifunctional ‘laboratory on a tip’ that allows observation and manipulation of the machineries of cellular membranes. In the force spectroscopy mode, AFM detects interactions between two single cells at molecular resolution. Force spectroscopy can also be used to probe the local elasticity, chemical groups, and receptor sites of live cells. Other applications locate molecular interactions driving membrane protein folding, assembly, and their switching between functional states. It is also possible to examine the energy landscape of biomolecular reactions, as well as reaction pathways, associated lifetimes, and free energy. In this seminar, we provide a flavor of the fascinating opportunities offered by the use of AFM as a nanobiotechnological tool in modern membrane biology.

Speaker:
Daniel J. Müller, ETH Zürich, Department of Biosystems Science and Engineering, Basel, Switzerland

Reviews on the topic:
‘Force probing surfaces of living cells to molecular resolution’ D.J. Müller, J. Helenius, D. Alsteens, Y.F. Dufrene, Nature Chemical Biology (2009) 5, 383-390.
‘New frontiers in atomic force microscopy: Analyzing interactions from single-molecules to cells’ D.J. Müller, M. Krieg, D. Alsteens & Y.F. Dufrene, Current Opinion in Biotechnology (2009) 20, 4-13.
‘Single-cell force spectroscopy’ J. Helenius, C.P. Heisenberg, H.E. Gaub & D.J. Müller, Journal of Cell Science (2008) 121, 1785-1791.
‘Vertebrate membrane proteins: Structure, function and insights from biophysical approaches’ D.J. Müller, N. Wu & K. Palczewski, Pharmacological Reviews (2008) 60, 43-78.
‘Atomic force microscopy as a multifunctional molecular toolbox in nanobiotechnology’ D.J. Müller & Y. Dufrene, Nature Nanotechnology (2008) 3, 261-269.

Title:
Biomolecular NMR-spectroscopy for structural analysis of protein complexes in solution

Summary:
We are studying the three-dimensional structures and dynamics of protein complexes in solution to understand mechanisms of protein and RNA recognition. Biomoelcular NMR provides very useful and efficient tools to study these protein complexes. The utility of solution state NMR for studying the structure, molecular interactions and conformational dynamics of biological macromolecules will be outlined, including optimizations required for analysis of high-molecular weight systems. For structural analysis of multi-domain proteins and protein complexes, we have developed an efficient protocol for determining the quaternary arrangement of multimeric protein complexes in solution. Here, we also combine advanced solution state NMR methods with Small Angle X-ray and/or Neutron Scattering (SAXS/SANS) experiments. The use of solution techniques is important as many regulatory protein complexes involve weak and transient domain interactions with considerable dynamics. The different techniques and methods will be demonstrated with our on-going study of the 3′ splice site recognition complex that play a critical role in the regulation of pre-mRNA splicing and other examples.

Speaker:
Michael Sattler
Institute of Structural Biology, Helmholtz Zentrum München, Neuherberg, Germany;
Biomolecular NMR, Technische Universität München, Garching, Germany;
Bayerisches NMR Zentrum (BNMRZ), TU München, Garching, Germany

Title:
Radicals Coordinated to Transition Metal Ions - Where are the electrons ? (EPR and Mössbauer Spectroscopy)

Summary:
Organic radicals can be integral parts of the reaction cycle of metallo-enzymes, particularly when it comes to oxidative reactions. The archetype of such an process is the interaction of iron with peroxides, as it is catalyzed by peroxidases and oxygenases. The catalytic reaction leads to formation of highly reactive [Fe=O]3+ species with formal oxidation states (+4) and (+5) of the iron center. Spectroscopic studies of the heme-peroxidases, however, established that both the metal and the porphyrin ligand are oxidized, resulting in an (oxo)iron(IV) and a porphyrin π-cation radical, and not an (oxo)iron(V) complex. In contrast, other non-heme enzymes with ‘innocent’ and redox-inactive ligands appear to form the [Fe=O]3+ species without ligand radicals. The true nature of the key intermediates matters. In Mülheim we systematically studied the electronic structure of such species mostly using synthetic models. The experimental techniques of choice are 57Fe-Mössbauer spectroscopy, multi-frequency electron-paramagnetic-resonance (EPR); magnetic susceptibility measurements, and magnetic circular-dichroism (MCD) spectroscopy. The experimental data are usually para-meterized within the common theoretical framework of a spin-Hamiltonian description. In this phenomenological concept, which is an application of ligand-field theory, the paramag-netic centers are described in terms of electronic interaction energies like zero-field or Zee-man splitting or spin coupling. Fortunately, spin-Hamiltonian parameters are also 'a nice place to rest' for theoreticians using quantum chemical methods, like density functional theory (DFT), to describe the electronic structure of paramagnetic compounds. It will be shown how to derive 'chemical information' from the data about metal valence state, coor-dination symmetry, ligand strength, covalence of bonds and interactions between metal sites to understand the electronic structure of reactive transition metal centers.

Speaker:
Eckhard Bill, MPI for Bioanorganic Chemistry, Mülheim/Ruhr, Germany

Title:
Challenges in Structural Molecular Biology

Summary:
Structural biology, in particular macromolecular crystallography, has had an enormous impact on our basic understanding of molecular biology. In my lecture I will outline some of the technical advances that have enabled the successful structure determination of increasingly challenging targets. Two specific examples of macromolecular machines, ATP synthase and the bacterial ribosome, will be discussed. Membrane proteins are very under-represented in the Protein Data Bank. The particular challenges of working with membrane proteins will be described, with examples from the fields of ABC transporters and G-protein coupled receptors. The combined use of different technologies, in particular electron microscopy and crystallography, has provided unique insights particularly in the field of virus structure, while electron tomography offers the ability to place macromolecular complexes in their cellular context.

Speaker:
Andrew Leslie, MRC Laboratory of Molecular Biology, Cambridge, United Kingdom

Title:
Studying structure dynamics of large macromolecules by cryo-EM

Summary:
Single particle cryo-EM is the method of choice for 3D structure determination of large macromolecular complexes that are difficult to obtain in the amounts and quality needed for X-ray crystallography. Individual molecules are imaged in the electron microscope at low temperature while being embedded in a thin layer of amorphous ice. The 2D projection images can then be used to compute the 3D structure of the macromolecule applying advanced image processing techniques. Single particle cryo-EM is not an ensemble technique and can thus be used to study structural variations of a dynamic macromolecule in solution making use of computational sorting techniques. It is even possible to determine kinetic parameters and to study the conformational landscape as well as the free-energy landscape of macromolecular complexes.

Speaker:
Holger Stark, Max Planck Institute for Biophysical Chemistry, Göttingen, Germany

Title:
Biophysical studies on membrane proteins by solid-state NMR

Summary:
Solid-state NMR allows detailed biophysical and structural studies on membrane proteins within the lipid bilayer. In this presentation the fundamental concepts of solid-state NMR will be introduced. The strength of this approach will be demonstrated by discussing different areas of applications: (i) characterization of small molecules bound to large membrane proteins, (ii) characterization of membrane protein dynamics and (iii) investigations of active sites of membrane proteins. The examples which will be discussed involve GPCRs, retinal proteins and ABC transporter. A methodological outlook covering the area of dynamic nuclear polarization to enhance sensitivity of solid-state NMR by some orders of magnitude will be given.

Speaker:
Clemens Glaubitz, Goethe-University Frankfurt, Institute for Biophysical Chemistry, Centre for Biomolecular Magnetic Resonance, Germany

Title:
Single molecule FRET accurately measures structure, dynamics and heterogeneities of an RNA four-way junction

Summary:
Multiparameter fluorescence detection (MFD) and fluorescence correlation spectroscopy (FCS) is applied to perform single-molecule (sm) FRET studies with an ultimate level of accuracy of 1% of the Förster radius in determining separations1,2,3. First, as a prerequisite to use FRET measurements to accurately predict structures of biomolecules, we characterized dye linkers with different lengths and rigidities to judge their implications on accurate distance determination in nucleic acids via FRET. We demonstrate that only proper consideration of the distribution of the dye positions and of the linker dynamics allows for very high accuracy of FRET based structure determination. Second, the structure of an RNA four-way junction was characterized using 40 Donor-Acceptor pairs. This allowed us to prove the existence of 3 of the 4 possible stacking conformers simultaneously present in equilibrium. Furthermore, we detected Mg2+-concentration dependent dynamics due to interconverting conformers of the junction on the ms time scale via FCS and dynamic photon distribution analysis (PDA)4. These studies show that sm FRET measurements are a valuable tool to complement the structural and dynamic information obtained by X-ray crystallography or NMR spectroscopy as these techniques are limited in detecting minority conformers.

Speaker:
Simon Sindbert, Institute for Physical Chemistry, Molecular Physical Chemistry, Heinrich Heine University Düsseldorf

References
1. Antonik, M.; Felekyan, S.; Gaiduk, A.; Seidel, C. A. M. Journal of Physical Chemistry B 2006, 110, 6970-6978.
2. Kalinin, S.; Felekyan, S.; Valeri, A.; Seidel, C. A. M. Journal of Physical Chemistry B 2008, 112, 8361-8374.
3. Kalinin, S.; Sisamakis, E.; Magennis, S. W.; Felekyan, S.; Seidel, C. A. M. Journal of Physical Chemistry B 2010, 114, 6197-6206
4. Kalinin, S.; Valeri, A.; Antonik, M.; Felekyan, S.; Seidel, C. A. M. Journal of Physical Chemistry B 2010, 114, PMID: 20486698, published online

Title:
A heterodimer of human 3 -phospho-adenosine-5 -phosphosulphate (PAPS) synthases is a new sulphate activating complex

Summary:
3'-Phospho-adenosine-5'-phosphosulphate (PAPS) synthases are fundamental to mammalian sulphate metabolism. These enzymes have recently been linked to a rising number of human diseases. Despite many studies, it is not yet understood how the mammalian PAPS synthases 1 and 2 interact with each other. We provide first evidence for heterodimerisation of these two enzymes by pull-down assays and Förster resonance energy transfer (FRET) measurements. Kinetics of dimer dissociation/association indicates that these heterodimers form as soon as PAPSS1 and -S2 encounter each other in solution. Affinity of the homo- and heterodimers were found to be in the low nanomolar range using anisotropy measurements employing proteins. Within its kinase domain, the PAPS synthase heterodimer displays similar substrate inhibition by adenosine-5 -phosphosulphate (APS) as the homodimers. Due to divergent catalytic efficacies of PAPSS1 and -S2, the heterodimer might be a way of regulating PAPS synthase function within mammalian cells.

Speaker:
Jonathan Mueller, Structural and Medicinal Biochemistry, Centre for Medical Biotechnology, Faculty of Biology and Geography, University of Duisburg-Essen

Title:
Insights into structure-function relationships in GTPase- effector interaction: a progress report

Summary:
Signaling functions of small GTPases are based on the formation of distinct protein-protein complexes. The large number of complex structures affords a unique opportunity to comprehend binding and specificity determining sites of the GTPases. Our investigation has been focused on interacting surfaces of the Rho GTPases and the quantitative analysis of their structures in complex with regulators and effectors. We found striking evidence of common `hot spots for GTPase interaction with diverse partners. In contrast to the regulators (GDIs, GEFs, GAPs), whose mechanisms of action are well defined, the structural features of effectors, their binding mode with the GTPase and the underlying principles of activation are still poorly understood. We have been using a combination of X-ray crystallography and fluorescence spectroscopy to study GTPase-effector interaction in detail, with RhoA as a model. The characterization of novel RhoA binding domains found in Rho kinase, describing the structure-function relationship of RhoA-effector interaction and shedding new light on possible activation mechanisms, involving scaffold proteins and protein kinases, will also be discussed.

Speaker:
Badri Nath Dubey, Institute for Biochemistry & Molekularbiology II, University Hospital Düsseldorf

Title:
Structural features required for the protein-based sensing of nitric oxide - Insight from the spectroscopic investigations of ferrous nitrophorins

Summary:
Nitrophorins (NPs) comprise a class of unique heme proteins from the saliva of the blood feeding insect Rhodnius prolixus, which appear together as a mixture of at least five isoproteins. The purpose of the proteins is to deliver the signaling molecule nitric oxide, which promotes vasodilatation and anti-coagulation in a host upon administration during a blood meal. This study resembles the first detailed spectroscopic investigation of the ferrous NP4 and NP7, which was mainly conducted by the determination of the UV-vis absorption and resonance Raman (RR) spectra. In case of ferrous NP4, the heme showed the expected five-coordination with His as the axial ligand at pH 7.5. Surprisingly, the data obtained for ferrous NP7 indicates a mixed five-coordinate high-spin species at pH 7.5, with both His-on and His-off. Lowering the pH to 4.0 resulted in solely the His-off heme. pH titration indicated that this process has a pKa of 8.9, which is exceptionally high compared to other deoxy heme proteins. In contrast, ferrous NP4 remains a single five-coordinate high-spin heme with His-on even at pH 5.5, which is also the case for other NPs. Several mutants including NP7(E27V), NP2(V24E), and NP4(D70A) were also studied. As a result, the spatial structural arrangement involving Glu27, Phe40, and His60 was found responsible for the unique behavior of NP7. The weakening of Fe-His bond, which is found crucial for the activation of the human ferrous heme protein soluble guanylate cyclase (sGC), which is the central receptor protein for the physiologically important signaling of nitric oxide. However, at present, the structural requirements for the weakening of the Fe-His bond in sGC are unknown. With this study, we present the first detailed elucidation of a structural motif that can accomplish this task.

Speaker:
Chunmao He, Max-Planck-Insitute for Bioanorganic Chemistry, Mülheim an der Ruhr

Title:
Stabilization of membrane proteins using compatible solutes

Summary:
Mechanical single molecule techniques offer exciting possibilities for investigating protein folding and stability in native environments at sub-nanometer resolutions. The single molecules without inherent symmetry can directly be monitored in their physiological conditions using atomic force microscopy (AFM). Recent developments in AFM enable us to go beyond the ensemble average and measure the behavior of individual molecules. In nature, compatible solutes (organic osmolytes) are used for protecting cells against high osmotic stress. They are compatible with cell metabolism even at molar concentrations. The influence of ectoine (1M) and betaine (1M) on the mechanical properties of bacteriorhodopsin (BR) has been investigated by single molecule force spectroscopy. Unfolding experiments taking BR as a model system revealed that ectoine and betaine increase the tendency of the polypeptide to coil, thus decreasing its persistence length. This behavior can be explained by the theory that the osmolytes are expelled from the protein surface due to the increase in chemical potential of the denatured (stretched) state forcing the protein into a more compact structure. These information and approaches provide basis for our further studies regarding the effects of compatible solutes on other membrane proteins of medical importance which can directly resolve transient intermediate states and multiple reaction pathways, and thus are uniquely powerful in characterizing the complex dynamics of protein folding. Thus, this study is set to provide exciting possibilities in the field of drug development including in vitro rescue of the misfolded proteins and to directly analyze and correlate their structural and functional properties at the sub-molecular level.

Speaker:
Arpita Roychoudhury, Insitute of Physical Biology, Heinrich Heine University Düsseldorf

Title:
Mobility and topology of VPU and CD4 proteins by solid state NMR spectroscopy

Summary:
The viral protein VPU of HIV-1 directly interacts with the human T-cell coreceptor CD4 and subsequently induces the degradation of this protein.1,2 Towards the study of this interaction on a residue-specific level, shorter constructs of these proteins comprising the cytoplasmic domains with and without the transmembrane part, have been expressed and studied in the presence of detergents.3,4 In this contribution, we present the results of initial solid-state MAS NMR studies on liposome-reconstituted constructs of both membrane proteins. Sequential assignments were obtained using standard methods. Utilizing double-quantum buildup characteristics at different temperatures, we could identify different dynamics of transmembrane and cytoplasmic domains, and the overall topology with respect to the membrane was probed utilizing well-established experiments5. Initial results suggest that the transmembrane helices of both proteins are rather rigid, whereas the cytoplasmic domains are flexible.

Speaker:
Hoa Quynh Do, Institute of Physical Biology, Heinrich-Heine University Düsseldorf and Institute of Structural Biology and Biophysics (ISB3), Research Center Jülich

References
1. Willey R.L., Maldarelli F., Martin M.A. and Strebel K., Journal of Virology, 66, 7193-7200 (1992)
2. Chen M.Y., Maldarelli F., Karczewski M.K., Willey R.L. and Strebel K., Journal of Virology, 67, 3877-3884 (1993)
3. Wittlich M., Koenig B. W., Stoldt M., Schmidt H. and Willbold D., FEBS Journal, 276, 6560-6575 (2009)
4. Wittlich M., Thiagarajan P., Koenig B. W., Hartmann R. and Willbold D., Biochimica Et Biophysica Acta-Biomembranes, 1798, 122-127 (2010)
5. Huster D., Yao X.L. and Hong M., Journal of the American Chemical Society, 124, 874-883 (2002)

Title:
Structural insights into conformational changes of a cyclic nucleotide-activated ion channel binding domain in solution

Summary:
Ion channels activated by cyclic nucleotides play crucial roles in neuronal excitability and sensory signaling. These channels are activated by binding of cyclic nucleotides to their intracellular cyclic nucleotide-binding domain (CNBD). Ligand binding to the CNBD promotes the opening of the channel, most probably by propagating a conformational change from the CNBD to the pore. However, the mechanism underlying the channel activation is only poorly understood. To elucidate the mechanism of channel gating knowledge of the structure of ligand-free and ligand-bound CNBDs is required. One member of ion channels activated by cyclic nucleotides represents the prokaryotic K+-selective MloK1 channel that has been identified in Mesorhizobium loti. The MloK1 channel forms homotetramers. Each subunit encompasses six transmembrane segments, a signature sequence for potassium selectivity, and a C-terminal intracellular located CNBD. In this study, the three-dimensional structure of the 15 kDa CNBD was determined by NMR spectroscopy in solution and structural insights into the gating mechanism of the channel will be discussed.

Speaker:
Sven Schünke, Institute of Physical Biology, Heinrich-Heine University Düsseldorf and Institute of Structural Biology and Biophysics (ISB3), Research Center Jülich

Title:
Purification and characterization of the centrosomal protein TACC3

Summary:
The mitotic spindle apparatus consists of centrosomes, kinetochores and microtubules (MT). Together with various MT-associated proteins these structures are critically involved in the regulation of MT dynamics and faithful separation of chromosomes during mitosis. This process is tightly regulated during the cell cycle. Alterations in centrosomal 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. Transforming acidic coiled-coil (TACC) proteins have been identified as important structural components of the centrosome/spindle apparatus. These protein family shares a 200 amino acid C-terminal coiled coil motif (CC) with only limited homology at the N-terminus. TACCs interact through their CC motif with the C-terminus of the MT polymerase chTOG which binds to MT ends through multiple N-terminal TOG domains. Depletion of TACC3 or chTOG interferes with centrosome integrity, centrosome-dependent assembly of MTs and spindle stability thereby leading to mitotic cell death or p53-dependent postmitotic cell cycle arrest. The major aim of my project is the determination and analysis of the three-dimensional structure of full length murine TACC3 and its CC domain alone and in complex with the C-terminus of murine chTOG as well as the in-depth biochemical analysis of such a bimolecular interaction. These analysis 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.

Speaker:
Harish Thakur, Institute for Biochemistry & Molekularbiology II, University Hospital Düsseldorf

Title:
Structural characterisation of the C39 peptidase like domain of the ABC-transporter HlyB

Summary:
Haemolysin B (HlyB) from Escherichia coli belongs to the family of bacteriocin-associated ATP-binding cassette (ABC)-transporter. In complex with the outer membrane protein (TolC) and the membrane fusion protein (MFP) HlyD, the ABC-transporter HlyB translocates the toxin HlyA directly over the inner and outer membranes into the medium with no detectable periplasmic intermediates. In general, ABC-transporters consist of two domains, the transmembrane domain (TMD) and the ATP- or nucleotide-binding domain (NBD) [1]. HlyB however, like all members of the bacteriocin ABC-transporter family, features an additional N-terminal C39 peptidase like domain. C39 peptidases are members of the thiol protease family possessing a catalytic triade consisting of a histidin, cystein and an acidic residue. As a domain of bacteriocin ABC-transporters, those peptidases are assumed to cleave the protein or peptide substrate after a consensus Gly-Gly motif [2]. Interestingly, HlyB contains a proteolytically inactive C39 domain [3], as the functional important cystein is mutated to a tyrosine residue. Nevertheless the domain has to play an important role, because deletion of the C39 domain in HlyB abolishes the translocation activity completely [4]. We investigated the C39 domain structurally and solved the solution structure by NMR. Besides the fact that the C39 domain of HlyB contains a tyrosine at the important cystein position, the structure revealed a further distortion of the catalytic triade, stabilized by a tryptophan sandwich. Thereby the domain is proteolytically inactive. Thus, the role of the C39 like domain in the haemolysin transport system may differ from the canonical role known for homologous domains of other bacteriocin transport systems. We will present the solution structure of the isolated C39 domain of HlyB from E. coli and give a detailed view onto the structural rearrangement of the triade. Furthermore we will reveal insights in the interaction with the substrate of the transporter, the bacteriocin HlyA.

Speaker:
Justin Lecher, Institute of Structural Biology and Biophysics (ISB3), Research Center Jülich and Institute of Biochemistry, Heinrich-Heine University Düsseldorf

References
1. Zaitseva J, Oswald C, Jumpertz T, Jenewein S, Wiedenmann A, Holland IB, Schmitt L.; EMBO J. 2006 26;25(14)
2. Havarstein LS, Diep DB, Nes IF; Mol Microbiol.; (1995) 16:229-240
3. Felmlee T, Pellett S, Lee EY, Welch RA; J Bacteriol.; (1985) 163(1): 88-93