FLI Structural Biology / Bioinformatics Journal Club

Structural Biology / Bioinformatics Journal Club

of the

at the Leibniz Institute for Age Research - Fritz Lipmann Institute

Guests are welcome.

Thursday, January 26, 2012, 11:00
Leibniz Institute for Age Research - Fritz Lipmann Institute
Beutenbergstr. 11, D-07745 Jena
Villa

Presenter: Steffen Kemmerzehl (FLI; Than Lab)

Ujwal R, Bowie JU.
Crystallizing membrane proteins using lipidic bicelles.
Methods. 2011;55(4):337-41. [PDF]

Crystallization of membrane proteins remains a significant challenge. For proteins resistant to the traditional approach of directly crystallizing from detergents, lipidic phase crystallization can be a powerful tool. Bicelles are an excellent medium for crystallizing membrane proteins in a lipidic environment. They can be described as bilayer discs formed by the mixture of a long-chain phospholipid and an amphiphile in an aqueous medium. Membrane proteins can be readily reconstituted into bicelles, where they are maintained in a native-like bilayer environment. Importantly, membrane proteins have been shown to be fully functional in bicelles under physiological conditions. Protein-bicelle mixtures can be manipulated with almost the same ease as detergent-solubilized membrane proteins, making bicelles compatible with standard equipment including high-throughput crystallization robots. A numbe of membrane proteins have now been successfully crystallized using the bicelle method, including bacteriorhodopsin, β2 adrenergic receptor, voltage-dependent anion channel, xanthorhodopsin and rhomboid protease. Because of the success with a variety of membrane proteins and the ease of implementation, bicelles should be a part of every membrane protein crystallographer's arsenal.

Thursday, January 19, 2012, 11:00
Leibniz Institute for Age Research - Fritz Lipmann Institute
Beutenbergstr. 11, D-07745 Jena
Villa

Presenter: Ramadurai Ramachandran (FLI; Görlach Lab)

Linser R, Bardiaux B, Higman V, Fink U, Reif B.
Structure calculation from unambiguous long-range amide and methyl 1H-1H distance restraints for a microcrystalline protein with MAS solid-state NMR spectroscopy.
J Am Chem Soc. 2011;133:5905-12. [PDF]
Magic-angle spinning (MAS) solid-state NMR becomes an increasingly important tool for the determination of structures of membrane proteins and amyloid fibrils. Extensive deuteration of the protein allows multidimensional experiments with exceptionally high sensitivity and resolution to be obtained. Here we present an experimental strategy to measure highly unambiguous spatial correlations for distances up to 13 Å. Two complementary three-dimensional experiments, or alternatively a four-dimensional experiment, yield highly unambiguous cross-peak assignments, which rely on four encoded chemical shift dimensions. Correlations to residual aliphatic protons are accessible via synchronous evolution of the (15)N and (13)C chemical shifts, which encode valuable amide-methyl distance restraints. On average, we obtain six restraints per residue. Importantly, 50% of all restraints correspond to long-range distances between residues i and j with |i - j| > 5, which are of particular importance in structure calculations. Using ARIA, we calculate a high-resolution structure for the microcrystalline 7.2 kDa α-spectrin SH3 domain with a backbone precision of ∼1.1 Å.

Thursday, January 5, 2012, 11:00
Leibniz Institute for Age Research - Fritz Lipmann Institute
Beutenbergstr. 11, D-07745 Jena
Villa

Presenter: Sven Dahms (FLI; Than Lab)

Xue Y, Lee S, Ha Y.
Crystal structure of amyloid precursor-like protein 1 and heparin complex suggests a dual role of heparin in E2 dimerization.
Proc Natl Acad Sci U S A. 2011;108(39):16229-34. [PDF]

Mutations in amyloid precursor protein (APP) are associated with familial Alzheimer's disease. Recent development suggests that homo- and heterodimerization of APP and APP-like proteins (APLPs), which are enhanced by heparan sulfate binding, may play a role in signal transduction and cell adhesion. Despite efforts to model heparin binding based on known apo crystal structures, the mechanism of heparin-induced APP/APLP dimerization has not been established experimentally. Here we report the crystal structure of a complex between heparin and the E2 domain of APLP1, which harbors the conserved high affinity heparin binding site of the full-length molecule. Within the asymmetric E2:heparin complex, the polysaccharide is snugly bound inside a narrow groove between the two helical subdomains of one protein protomer. The nonreducing end of the sugar is positioned near the protein's 2-fold axis, making contacts with basic residues from the second protomer. The inability of the E2 dimer to accommodate two heparin molecules near its symmetry axis explains the observed 21 binding stoichiometry, which is confirmed by isothermal titration calorimetric experiment carried out in solution. We also show that, at high concentrations, heparin can destabilize E2 dimer, probably by forcing into the unoccupied binding site observed in the 21 complex. The binding model suggested by the crystal structure may facilitate the design of heparin mimetics that are capable of modulating APP dimerization in cells.

Thursday, November 17, 2011, 10:45
Leibniz Institute for Age Research - Fritz Lipmann Institute
Beutenbergstr. 11, D-07745 Jena
Villa

Presenter: Rolf Huehne (FLI; Sühnel Lab)

Bult CJ, Drabkin HJ, Evsikov A, Natale D, Arighi C, Roberts N, Ruttenberg A, D'Eustachio P, Smith B, Blake JA, Wu C.
The representation of protein complexes in the Protein Ontology (PRO).
BMC Bioinformatics. 2011 Sep 19;12:371. doi: 10.1186/1471-2105-12-371. [PDF]

BACKGROUND: Representing species-specific proteins and protein complexes in ontologies that are both human- and machine-readable facilitates the retrieval, analysis, and interpretation of genome-scale data sets. Although existing protin-centric informatics resources provide the biomedical research community with well-curated compendia of protein sequence and structure, these resources lack formal ontological representations of the relationships among the proteins themselves. The Protein Ontology (PRO) Consortium is filling this informatics resource gap by developing ontological representations and relationships among proteins and their variants and modified forms. Because proteins are often functional only as members of stable protein complexes, the PRO Consortium, in collaboration with existing protein and pathway databases, has launched a new initiative to implement logical and consistent representation of protein complexes.

DESCRIPTION: We describe here how the PRO Consortium is meeting the challenge of representing species-specific protein complexes, how protein complex representation in PRO supports annotation of protein complexes and comparative biology, and how PRO is being integrated into existing community bioinformatics resources. The PRO resource is accessible at http://pir.georgetown.edu/pro/.

CONCLUSION:

PRO is a unique database resource for species-specific protein complexes. PRO facilitates robust annotation of variations in composition and function contexts for protein complexes within an between species.

Thursday, November 3, 2011, 11:00
Leibniz Institute for Age Research - Fritz Lipmann Institute
Beutenbergstr. 11, D-07745 Jena
Villa

Presenter: Sandra Hoefgen (FLI; Than Lab)

Lockard MA, Listwan P, Pedelacq JD, Cabantous S, Nguyen HB, Terwilliger TC, Waldo, GS.
A high-throughput immobilized bead screen for stable proteins and multi-protein complexes.
Protein Eng Des Sel. 2011;24(7):565-78. [PDF]

We describe an in vitro colony screen to identify Escherichia coli expressing soluble proteins and stable, assembled multiprotein complexes. Proteins with an N-terminal 6His tag and C-terminal green fluorescent protein (GFP) S11 tag are fluorescently labeled in cells by complementation with a coexpressed GFP 1-10 fragment. After partial colony lysis, the fluorescent soluble proteins or complexes diffuse through a supporting filtration membrane and are captured on Talon(®) resin metal affinity beads immobilized in agarose. Images of the fluorescent colonies convey total expression and the level of fluorescence bound to the beads indicates how much protein is soluble. Both pieces of information can be used together when selecting clones. After the assay, colonies can be picked and propagated, eliminating the need to make replica plates. We used the method to screen a DNA fragment library of the human protein p85 and preferentially obtained clones expressing the full-length 'breakpoint cluster region-homology' and NSH2 domains. The assay also distinguished clones expressing stable multi-protein complexes from those that are unstable due to missing subunits. Clones expressing stable, intact heterotrimeric E.coli YheNML complexes were readily identified in libraries dominated by complexes of YheML missing the N subunit.

Thursday, October 6, 2011, 11:00
Leibniz Institute for Age Research - Fritz Lipmann Institute
Beutenbergstr. 11, D-07745 Jena
Villa

Presenter: Peter Bellstedt (FLI; Görlach Lab)

Cheng LK, Unrau PJ.
Closing the circle: replicating RNA with RNA.
Cold Spring Harb Perspect Biol. 2010 Oct;2(10):a002204. Epub 2010 Jun 1 [PDF]

How life emerged on this planet is one of the most important and fundamental questions of science. Although nearly all details concerning our origins have been lost in the depths of time, there is compelling evidence to suggest that the earliest life might have exploited the catalytic and self-recognition properties of RNA to survive. If an RNA based replicating system could be constructed in the laboratory, it would be much easier to understand the challenges associated with the very earliest steps in evolution and provide important insight into the establishment of the complex metabolic systems that now dominate this planet. Recent progress into the selection and characterization of ribozymes that promote nucleotide synthesis and RNA polymerization are discussed and outstanding problems in the field of RNA-mediated RNA replication are summarized.

Friday, July 8, 2011, 11:00
Leibniz Institute for Age Research - Fritz Lipmann Institute
Beutenbergstr. 11, D-07745 Jena
Biocomputing-Verwaltungsgebäude, Seminarraum 101

Presenter: Christoph Wiedemann (FLI; Görlach Lab)

Grote A, Hiller K, Scheer M, Münch R, Nörtemann B, Hempel DC, Jahn D.
JCat: a novel tool to adapt codon usage of a target gene to its potential expression host.
Nucleic Acids Res. 2005;33(Web Server issue):W526-31. [PDF]

A novel method for the adaptation of target gene codon usage to most sequenced prokaryotes and selected eukaryotic gene expression hosts was developed to improve heterologous protein production. In contrast to existing tools, JCat (Java Codon Adaptation Tool) does not require the manual definition of highly expressed genes and is, therefore, a very rapid and easy method. Further options of JCat for codon adaptation include the avoidance of unwanted cleavage sites for restriction enzymes and Rho-independent transcription terminators. The output of JCat is both graphically and as Codon Adaptation Index (CAI) values given for the pasted sequence and the newly adapted sequence. Additionally, a list of genes in FASTA-format can be uploaded to calculate CAI values. In one example, all genes of the genome of Caenorhabditis elegans were adapted to Escherichia coli codon usage and further optimized to avoid commonly used restriction sites. In a second example, the Pseudomonas aeruginosa exbD gene codon usage was adapted to E.coli codon usage with parallel avoidance of the same restriction sites. For both, the degree of introduced changes was documented and evaluated. JCat is integrated into the PRODORIC database that hosts all required information on the various organisms to fulfill the requested calculations. JCat is freely accessible at http://www.prodoric.de/JCat.

Friday, June 24, 2011, 11:00
Leibniz Institute for Age Research - Fritz Lipmann Institute
Beutenbergstr. 11, D-07745 Jena
Biocomputing-Verwaltungsgebäude, Seminarraum 101

Presenter: Rolf Huehne (FLI; Sühnel Lab)

Prilusky J, Hodis E, Canner D, Decatur WA, Oberholser K, Martz E, Berchanski A, Harel M, Sussman JL.
Proteopedia: A status report on the collaborative, 3D web-encyclopedia of proteins and other biomolecules.
J Struct Biol. 2011 Apr 23. [Epub ahead of print] [PDF]

Proteopedia is a collaborative, 3D web-encyclopedia of protein, nucleic acid and other biomolecule structures. Created as a means for communicating biomolecule structures to a diverse scientific audience, Proteopedia (http://www.proteopedia.org) presents structural annotation in an intuitive, interactive format and allows members of the scientific community to easily contribute their own annotations. Here, we provide a status report on Proteopedia by describing advances in the web resource since its inception three and a half years ago, focusing on features of potential direct use to the scientific community. We discuss its progress as a collaborative 3D-encyclopedia of structures as well as its use as a complement to scientific publications and PowerPoint presentations. We also describe Proteopedia's use for 3D visualization in structure-related pedagogy.

Friday, June 10, 2011, 11:00
Leibniz Institute for Age Research - Fritz Lipmann Institute
Beutenbergstr. 11, D-07745 Jena
Biocomputing-Verwaltungsgebäude, Seminarraum 101

Presenter: Sandra Hoefgen (FLI; Than Lab)

Renzi F, Zhang X, Rice WJ, Torres-Arancivia C, Gomez-Llorente Y, Diaz R, Ahn K, Li Y, Sisodia SS, Ubarretxena-Belandia I.
Structure of {gamma}-secretase and its trimeric pre-activation intermediate by single-particle electron microscopy.
J Biol Chem. 2011 Mar 17. [Epub ahead of print] [PDF]

The γ-secretase membrane protein complex is responsible for proteolytic maturation of signaling precursors and catalyzes the final step in the production of the amyloid β-peptides implicated in the pathogenesis of Alzheimer's disease. The incorporation of presenilin enhancer 2 (PEN-2) into a pre-activation intermediate, composed of the catalytic subunit presenilin, and the accessory proteins anterior pharynx defective 1 and nicastrin, triggers the endoproteolysis of presenilin and results in an active tetrameric γ-secretase. We have determined the three-dimensional reconstruction of a matured and catalytically active γ-secretase using single-particle electron cryo-microscopy. γ-Secretase has a cup-like shape with a lateral belt of about 40-50 Å in height that encloses a water accessible internal chamber. Active site labeling with a gold-coupled transition state analog inhibitor suggests that the γ-secretase active site faces this chamber. Comparison with the structure of a trimeric pre-activation intermediate suggests that the incorporation of PEN-2 might contribute to the maturation of the active site architecture.

Friday, May 27, 2011, 11:00
Leibniz Institute for Age Research - Fritz Lipmann Institute
Beutenbergstr. 11, D-07745 Jena
Biocomputing-Verwaltungsgebäude, Seminarraum 101

Presenter: Sven Dahms (FLI; Than Lab)

Lee S, Xue Y, Hu J, Wang Y, Liu X, Demeler B, Ha Y.
The E2 domains of APP and APLP1 share a conserved mode of dimerization.
Biochemistry. 2011 May 16. [Epub ahead of print] [PDF]

Amyloid precursor protein (APP) is genetically linked to Alzheimer's disease. APP is a type I membrane protein, and its oligomeric structure is potentially important because this property may play a role in its function, or affect the processing of the precursor by the secretases to generate amyloid ß-peptide. Several independent studies have shown that APP can form dimers in the cell, but how it dimerizes remains controversial. At least three regions of the precursor, including a centrally located and conserved domain called E2, have been proposed to contribute to dimerization. Here we report two new crystal structures of E2,one from APP, and the other from APLP1, a mammalian APP homolog. Comparison with an earlier APP structure, which was solved in a different space group, shows that the E2 domains share a conserved and anti-parallel mode of dimerization. Biophysical measurements in solution show that heparin binding induces E2dimerization. The 2.1Å resolution electron density map also reveals phosphate ions that are bound to the protein surface. Mutational analysis shows that protein residues interacting with the phosphate ions are also involved in heparin binding. The locations of two of these residues, Arg-369 and His-433, at the dimeric interface suggest a mechanism for heparin-induced protein dimerization.

Friday, May 20, 2011, 11:00
Leibniz Institute for Age Research - Fritz Lipmann Institute
Beutenbergstr. 11, D-07745 Jena
Biocomputing-Verwaltungsgebäude, Seminarraum 101

Presenter: Peter Bellstedt (FLI; Görlach Lab)

Theis T, Ganssle P, Kervern G, Knappe S, Kitching J, Ledbetter MP, Budker D, Pines.
Parahydrogen-enhanced zero-field nuclear magnetic resonance.

Nat Physics 2011;19(2):162-71. [PDF ]

Nuclear magnetic resonance, conventionally detected in magnetic fields of several tesla, is a powerful analytical tool for the determination of molecular identity, structure and function. With the advent of prepolarization methods and detection schemes using atomic magnetometers or superconducting quantum interference devices, interest in NMR in fields comparable to the Earth’s magnetic field and below (down to zero field) has been revived. Despite the use of superconducting quantum interference devices or atomic magnetometers, low-field NMR typically suffers from low sensitivity compared with conventional high-field NMR. Here we demonstrate direct detection of zero-field NMR signals generated through parahydrogen-induced polarization, enabling high-resolution NMR without the use of any magnets. The sensitivity is sufficient to observe spectra exhibiting 13C–1H scalar nuclear spin–spin couplings (known as J couplings) in compounds with 13C in natural abundance, without the need for signal averaging. The resulting spectra showdistinct features that aid chemical fingerprinting.

Friday, May 06, 2011, 11:00
Leibniz Institute for Age Research - Fritz Lipmann Institute
Beutenbergstr. 11, D-07745 Jena
Biocomputing-Verwaltungsgebäude, Seminarraum 101

Presenter: Steffen Kemmerzehl (FLI; Than Lab)

Zhan C, Patskovsky Y, Yan Q, Li Z, Ramagopal U, Cheng H, Brenowitz M, Hui X, Nathenson SG, Almo SC.
Decoy Strategies: the structure of TL1A:DCr3 complex.

Structure. 2011;19(2):162-71. [PDF]

Decoy Receptor 3 (DcR3), a secreted member of the Tumor Necrosis Factor (TNF) receptor superfamily, neutralizes three different TNF ligands: FasL, LIGHT, and TL1A. Each of these ligands engages unique signaling receptors which direct distinct and critical immune responses. We report the crystal structures of the unliganded DcR3 ectodomain and its complex with TL1A, as well as complementary mutagenesis and biochemical studies. These analyses demonstrate that DcR3 interacts with invariant backbone and side-chain atoms in the membrane-proximal half of TL1A which supports recognition of its three distinct TNF ligands. Additional features serve as antideterminants that preclude interaction with other members of the TNF superfamily. This mode of interaction is unique among characterized TNF:TNFR family members and provides a mechanistic basis for the broadened specificity required to support the decoy function of DcR3, as well as for the rational manipulation of specificity and affinity of DcR3 and its ligands.

Friday, April 29, 2011, 11:00
Leibniz Institute for Age Research - Fritz Lipmann Institute
Beutenbergstr. 11, D-07745 Jena
Biocomputing-Verwaltungsgebäude, Seminarraum 101

Presenter: Torsten Thalheim, (FLI; Sühnel Lab)

Greene CS, White BC, Moore JH.
Ant Colony Optimization for Genome-Wide Genetic Analysis.
Lect Notes Comput Sci. 2008;5217:37-47. [PDF]

In human genetics it is now feasible to measure large numbers of DNA sequence variations across the human genome. Given current knowledge about biological networks and disease processes it seems likely that disease risk can best be modeled by interactions between biological components, which can be examined as interacting DNA sequence variations. The machine learning challenge is to effectively explore interactions in these datasets to identify combinations of variations which are predictive of common human diseases. Ant colony optimization (ACO) is a promising approach to this problem. The goal of this study is to examine the usefulness of ACO for problems in this domain and to develop a prototype of an expert knowledge guided probabilistic search wrapper. We show that an ACO approach is not successful in the absence of expert knowledge but is successful when expert knowledge is supplied through the pheromone updating rule.

Friday, April 15, 2011, 11:00
Leibniz Institute for Age Research - Fritz Lipmann Institute
Beutenbergstr. 11, D-07745 Jena
Biocomputing-Verwaltungsgebäude, Seminarraum 101

Presenter: Manuel Than (FLI; Than Lab)

Chapman HN et al.
Femtosecond X-ray protein nanocrystallography.
Nature 2011; 470(7332):73-7. [PDF]

X-ray crystallography provides the vast majority of macromolecular structures, but the success of the method relies on growing crystals of sufficient size. In conventional measurements, the necessary increase in X-ray dose to record data from crystals that are too small leads to extensive damage before a diffraction signal can be recorded. It is particularly challenging to obtain large, well-diffracting crystals of membrane proteins, for which fewer than 300 unique structures have been determined despite their importance in all living cells. Here we present a method for structure determination where single-crystal X-ray diffraction 'snapshots' are collected from a fully hydrated stream of nanocrystals using femtosecond pulses from a hard-X-ray free-electron laser, the Linac Coherent Light Source. We prove this concept with nanocrystals of photosystem I, one of the largest membrane protein complexes. More than 3,000,000 diffraction patterns were collected in this study, and a three-dimensional data set was assembled from individual photosystem I nanocrystals (∼200 nm to 2 μm in size). We mitigate the problem of radiation damage in crystallography by using pulses briefer than the timescale of most damage processes. This offers a new approach to structure determination of macromolecules that do not yield crystals of sufficient size for studies using conventional radiation sources or are particularly sensitive to radiation damage.

Friday, April 01, 2011, 11:00
Leibniz Institute for Age Research - Fritz Lipmann Institute
Beutenbergstr. 11, D-07745 Jena
Biocomputing-Verwaltungsgebäude, Seminarraum 101

Presenter: Jürgen Sühnel (FLI; Sühnel Lab)

Partridge L.
The new biology of ageing.
Philos Trans R Soc Lond B Biol Sci. 2010 ;365(1537):147-54. [PDF]

Human life expectancy in developed countries has increased steadily for over 150 years, through improvements in public health and lifestyle. More people are hence living long enough to suffer age-related loss of function and disease, and there is a need to improve the health of older people. Ageing is a complex process of damage accumulation, and has been viewed as experimentally and medically intractable. This view has been reinforced by the realization that ageing is a disadvantageous trait that evolves as a side effect of mutation accumulation or a benefit to the young, because of the decline in the force of natural selection at later ages. However, important recent discoveries are that mutations in single genes can extend lifespan of laboratory model organisms and that the mechanisms involved are conserved across large evolutionary distances, including to mammals. These mutations keep the animals functional and pathology-free to later ages, and they can protect against specific ageing-related diseases, including neurodegenerative disease and cancer. Preliminary indications suggest that these new findings from the laboratory may well also apply to humans. Translating these discoveries into medical treatments poses new challenges, including changing clinical thinking towards broad-spectrum, preventative medicine and finding novel routes to drug development.

Friday, March 19, 2011, 11:00
Leibniz Institute for Age Research - Fritz Lipmann Institute
Beutenbergstr. 11, D-07745 Jena
Biocomputing-Verwaltungsgebäude, Seminarraum 101

Presenter: Matthias Görlach (FLI; Görlach Lab)

Olzscha H, Schermann SM, Woerner AC, Pinkert S, Hecht MH, Tartaglia GG, Vendruscolo M, Hayer-Hartl M, Hartl FU, Vabulas RM.
Amyloid-like aggregates sequester numerous metastable proteins with essential cellular functions.
Cell 2011;144(1):67-78. [PDF]

Protein aggregation is linked with neurodegeneration and numerous other diseases by mechanisms that are not well understood. Here, we have analyzed the gain-of-function toxicity of artificial β sheet proteins that were designed to form amyloid-like fibrils. Using quantitative proteomics, we found that the toxicity of these proteins in human cells correlates with the capacity of their aggregates to promote aberrant protein interactions and to deregulate the cytosolic stress response. The endogenous proteins that are sequestered by the aggregates share distinct physicochemical properties: They are relatively large in size and significantly enriched in predicted unstructured regions, features that are strongly linked with multifunctionality. Many of the interacting proteins occupy essential hub positions in cellular protein networks, with key roles in chromatin organization, transcription, translation, maintenance of cell architecture and protein quality control. We suggest that amyloidogenic aggregation targets a metastable subproteome, thereby causing multifactorial toxicity and, eventually, the collapse of essential cellular functions.

Friday, March 05, 2011, 11:00
Leibniz Institute for Age Research - Fritz Lipmann Institute
Beutenbergstr. 11, D-07745 Jena
Biocomputing-Verwaltungsgebäude, Seminarraum 101

Presenter: Christoph Wiedemann (FLI; Görlach Lab)

Singh Y, Sharpe PC, Hoang HN, Lucke AJ, McDowall AW, Bottomley SP, Fairlie DP.
Amyloid formation from an α-helix peptide bundle is seeded by 3(10)-helix aggregates.
Chemistry. 2011;17(1):151-60. [PDF]

Transformation of proteins and peptides to fibrillar aggregates rich in β sheets underlies many diseases, but mechanistic details of these structural transitions are poorly understood. To simulate aggregation, four equivalents of a water-soluble, α-helical (65 %) amphipathic peptide (AEQLLQEAEQLLQEL) were assembled in parallel on an oxazole-containing macrocyclic scaffold. The resulting 4α-helix bundle is monomeric and even more α helical (85 %), but it is also unstable at pH 4 and undergoes concentration-dependent conversion to β-sheet aggregates and amyloid fibrils. Fibrils twist and grow with time, remaining flexible like rope (>1 μm long, 5-50 nm wide) with multiple strings (2 nm), before ageing to matted fibers. At pH 7 the fibrils revert back to soluble monomeric 4α-helix bundles. During α→β folding we were able to detect soluble 3(10) helices in solution by using 2D-NMR, CD and FTIR spectroscopy. This intermediate satisfies the need for peptide elongation, from the compressed α helix to the fully extended β strand/sheet, and is driven here by 3(10) -helix aggregation triggered in this case by template-promoted helical bundling and by hydrogen-bonding glutamic acid side chains. A mechanism involving α⇌α(4) ⇌(3(10) )(4) ⇌(3(10) )(n) ⇌(β)(n) ⇋m(β)(n) equilibria is plausible for this peptide and also for peptides lacking hydrogen-bonding side chains, with unfavourable equilibria slowing the α→β conversion.

Friday, February 18, 2011, 11:00
Leibniz Institute for Age Research - Fritz Lipmann Institute
Beutenbergstr. 11, D-07745 Jena
Biocomputing-Verwaltungsgebäude, Seminarraum 101

Presenter: Dirk Röser (FLI; Than Lab)

Schlebach JP, Kim MS, Joh NH, Bowie JU, Park C.
Probing membrane protein unfolding with pulse proteolysis.

J Mol Biol. 2011;406(4):545-51. [PDF]

Technical challenges have greatly impeded the investigation of membrane protein folding and unfolding. To develop a new tool that facilitates the study of membrane proteins, we tested pulse proteolysis as a probe for membrane protein unfolding. Pulse proteolysis is a method to monitor protein folding and unfolding, which exploits the significant difference in proteolytic susceptibility between folded and unfolded proteins. This method requires only a small amount of protein and, in many cases, may be used with unpurified proteins in cell lysates. To evaluate the effectiveness of pulse proteolysis as a probe for membrane protein unfolding, we chose Halobacterium halobium bacteriorhodopsin (bR) as a model system. The denaturation of bR in SDS has been investigated extensively by monitoring the change in the absorbance at 560 nm (A(560)). In this work, we demonstrate that denaturation of bR by SDS results in a significant increase in its susceptibility to proteolysis by subtilisin. When pulse proteolysis was applied to bR incubated in varying concentrations of SDS, the remaining intact protein determined by electrophoresis shows a cooperative transition. The midpoint of the cooperative transition (C(m)) shows excellent agreement with that determined by A(560). The C(m) values determined by pulse proteolysis for M56A and Y57A bRs are also consistent with the measurements made by A(560). Our results suggest that pulse proteolysis is a quantitative tool to probe membrane protein unfolding. Combining pulse proteolysis with Western blotting may allow the investigation of membrane protein unfolding in situ without overexpression or purification.

Related work:

Park C, Marqusee S.
Pulse proteolysis: a simple method for quantitative determination of protein stability and ligand binding.

Nat Methods. 2005;2(3):207-12. [PDF]

Thermodynamic stability is fundamental to the biology of proteins. Information on protein stability is essential for studying protein structure and folding and can also be used indirectly to monitor protein-ligand or protein-protein interactions. While clearly valuable, the experimental determination of a protein's stability typically requires biophysical instrumentation and substantial quantities of purified protein, which has limited the use of this technique as a general laboratory method. We report here a simple new method for determining protein stability by using pulse proteolysis with varying concentrations of denaturant. Pulse proteolysis is designed to digest only the unfolded proteins in an equilibrium mixture of folded and unfolded proteins that relaxes on a time scale longer than the proteolytic pulse. We used this method to study the stabilities of Escherichia coli ribonuclease H and its variants, both in purified form and directly from cell lysates. The DeltaG(unf) degrees values obtained by this technique were in agreement with those determined by traditional methods. We also successfully used this method to monitor the binding of maltose-binding protein to maltose, as well as to rapidly screen cognate ligands for this protein. The simplicity of pulse proteolysis suggests that it is an excellent strategy for the high-throughput determination of protein stability in protein engineering and drug discovery applications.

Friday, February 04, 2011, 11:00
Leibniz Institute for Age Research - Fritz Lipmann Institute
Beutenbergstr. 11, D-07745 Jena
Biocomputing-Verwaltungsgebäude, Seminarraum 101

Presenter: Ramadurai Ramachandran (FLI; Görlach Lab)

Robustelli P, Kohlhoff K, Cavalli A, Vendruscolo M.
Using NMR chemical shifts as structural restraints in molecular dynamics simulations of proteins.

Structure. 2010;18(8):923-33. [PDF]

Friday, January 21, 2011, 11:00
Leibniz Institute for Age Research - Fritz Lipmann Institute
Beutenbergstr. 11, D-07745 Jena
Biocomputing-Verwaltungsgebäude, Seminarraum 101

Presenter: Oliver Ohlenschläger (FLI; Görlach Lab)

Nucci NC, Pometun MS , Wand AJ .
Site-resolved measurement of water-protein interactions by solution NMR.
Nat Struct Mol Biol. 2011 Jan 2. [Epub ahead of print] [PDF]

The interactions of biological macromolecules with water are fundamental to their structure, dynamics and function. Historically, characterization of the location and residence times of hydration waters of proteins in solution has been quite difficult. Confining proteins within the nanoscale interior of a reverse micelle slows water dynamics, allowing global protein-water interactions to be detected using nuclear magnetic resonance techniques. Complications that normally arise from hydrogen exchange and long-range dipolar coupling are overcome by the nature of the reverse micelle medium. Characterization of the hydration of ubiquitin demonstrates that encapsulation within a reverse micelle allows detection of dozens of hydration waters. Comparison of nuclear Overhauser effects obtained in the laboratory and rotating frames indicate a considerable range of hydration water dynamics is present on the protein surface. In addition, an unprecedented clustering of different hydration-dynamics classes of sites is evident.

Hilser VJ.
Finding the wet spots.
Nature. 2011;469(7329):166-7 [PDF]

Friday, January 7, 2011, 11:00
Leibniz Institute for Age Research - Fritz Lipmann Institute
Beutenbergstr. 11, D-07745 Jena
Biocomputing-Verwaltungsgebäude, Seminarraum 101

Presenter: Andre Mischo (FLI; Görlach Lab)

Neumann H, Wang K, Davis L, Garcia-Alai M, Chin JW.

Encoding multiple unnatural amino acids via evolution of a quadruplet-decoding ribosome. Nature. 2010;464(7287):441-4. [PDF]

The in vivo, genetically programmed incorporation of designer amino acids allows the properties of proteins to be tailored with molecular precision. The Methanococcus jannaschii tyrosyl-transfer-RNA synthetase-tRNA(CUA) (MjTyrRS-tRNA(CUA)) and the Methanosarcina barkeri pyrrolysyl-tRNA synthetase-tRNA(CUA) (MbPylRS-tRNA(CUA)) orthogonal pairs have been evolved to incorporate a range of unnatural amino acids in response to the amber codon in Escherichia coli. However, the potential of synthetic genetic code expansion is generally limited to the low efficiency incorporation of a single type of unnatural amino acid at a time, because every triplet codon in the universal genetic code is used in encoding the synthesis of the proteome. To encode efficiently many distinct unnatural amino acids into proteins we require blank codons and mutually orthogonal aminoacyl-tRNA synthetase-tRNA pairs that recognize unnatural amino acids and decode the new codons. Here we synthetically evolve an orthogonal ribosome (ribo-Q1) that efficiently decodes a series of quadruplet codons and the amber codon, providing several blank codons on an orthogonal messenger RNA, which it specifically translates. By creating mutually orthogonal aminoacyl-tRNA synthetase-tRNA pairs and combining them with ribo-Q1 we direct the incorporation of distinct unnatural amino acids in response to two of the new blank codons on the orthogonal mRNA. Using this code, we genetically direct the formation of a specific, redox-insensitive, nanoscale protein cross-link by the bio-orthogonal cycloaddition of encoded azide- and alkyne-containing amino acids. Because the synthetase-tRNA pairs used have been evolved to incorporate numerous unnatural amino acids, it will be possible to encode more than 200 unnatural amino acid combinations using this approach. As ribo-Q1 independently decodes a series of quadruplet codons, this work provides foundational technologies for the encoded synthesis and synthetic evolution of unnatural polymers in cells.

Wang K, Neumann H, Peak-Chew JW.
Evolved orthogonal ribosomes enhance the efficiency of genetic code expansion.
Nat Biotech. 2007;25(7):770-7. [PDF]

In vivo incorporation of unnatural amino acids by amber codon suppression is limited by release factor-1-mediated peptide chain termination. Orthogonal ribosome-mRNA pairs function in parallel with, but independent of, natural ribosomes and mRNAs. Here we show that an evolved orthogonal ribosome (ribo-X) improves tRNA(CUA)-dependent decoding of amber codons placed in orthogonal mRNA. By combining ribo-X, orthogonal mRNAs and orthogonal aminoacyl-tRNA synthetase/tRNA pairs in Escherichia coli, we increase the efficiency of site-specific unnatural amino acid incorporation from approximately 20% to >60% on a single amber codon and from <1% to >20% on two amber codons. We hypothesize that these increases result from a decreased functional interaction of the orthogonal ribosome with release factor-1. This technology should minimize the functional and phenotypic effects of truncated proteins in experiments that use unnatural amino acid incorporation to probe protein function in vivo.

Friday, December 17, 2010, 11:00
Leibniz Institute for Age Research - Fritz Lipmann Institute
Beutenbergstr. 11, D-07745 Jena
Biocomputing-Verwaltungsgebäude, Seminarraum 101

Presenter: Jean A. Nzigou Mandouckou (FLI; Than Lab)

Lin SC, Lo YC, Wu H.
Helical assembly in the MyD88-IRAK4-IRAK2 complex in TLR/IL-1 signalling.
Nature 2010;465:885-90. [PDF]

MyD88, IRAK4 and IRAK2 are critical signalling mediators of the TLR/IL1-R superfamily. Here we report the crystal structure of the MyD88-IRAK4-IRAK2 death domain (DD) complex, which surprisingly reveals a left-handed helical oligomer that consists of 6 MyD88, 4 IRAK4 and 4 IRAK2 DDs. Assembly of this helical signalling tower is hierarchical, in which MyD88 recruits IRAK4 and the MyD88-IRAK4 complex recruits the IRAK4 substrates IRAK2 or the related IRAK1. Formation of these Myddosome complexes brings the kinase domains of IRAKs into proximity for phosphorylation and activation. Composite binding sites are required for recruitment of the individual DDs in the complex, which are confirmed by mutagenesis and previously identified signalling mutations. Specificities in Myddosome formation are dictated by both molecular complementarity and correspondence of surface electrostatics. The MyD88-IRAK4-IRAK2 complex provides a template for Toll signalling in Drosophila and an elegant mechanism for versatile assembly and regulation of DD complexes in signal transduction.

Friday, December 3, 2010, 11:00
Leibniz Institute for Age Research - Fritz Lipmann Institute
Beutenbergstr. 11, D-07745 Jena
Biocomputing-Verwaltungsgebäude, Seminarraum 101

Presenter: Miriam Küster (FLI; Than Lab)

Mukai Y, Nakamura T, Yoshikawa M, Yoshioka Y, Tsunoda S, Nakagawa S, Yamagata Y, Tsutsumi Y.
Solution Structure of the TNF-TNFR2 Complex
Sci Signal. 2010;3(148):ra83. [PDF]

Tumor necrosis factor (TNF) is an inflammatory cytokine that has important roles in various immune responses, which are mediated through its two receptors, TNF receptor 1 (TNFR1) and TNFR2. Antibody-based therapy against TNF is used clinically to treat several chronic autoimmune diseases; however, such treatment sometimes results in serious side effects, which are thought to be caused by the blocking of signals from both TNFRs. Therefore, knowledge of the structural basis for the recognition of TNF by each receptor would be invaluable in designing TNFR-selective drugs. Here, we solved the 3.0 angstrom resolution structure of the TNF-TNFR2 complex, which provided insight into the molecular recognition of TNF by TNFR2. Comparison to the known TNFR1 structure highlighted several differences between the ligand-binding interfaces of the two receptors. Additionally, we also demonstrated that TNF-TNFR2 formed aggregates on the surface of cells, which may be required for signal initiation. These results may contribute to the design of therapeutics for autoimmune diseases.

Friday, November 19, 2010, 11:00
Leibniz Institute for Age Research - Fritz Lipmann Institute
Beutenbergstr. 11, D-07745 Jena
Biocomputing-Verwaltungsgebäude, Seminarraum 101

Presenter: Ina Könnig (FLI; Than Lab)

Tarasaka JW, Puljung MC, Olivier NB, Flynn GE, Zagotta WN.
Mapping the structure and conformational movements of proteins with transition metal ion FRET.
Nat Methods.. 2009;(7):532-7. [PDF]

Visualizing conformational dynamics in proteins has been difficult, and the atomic-scale motions responsible for the behavior of most allosteric proteins are unknown. Here we report that fluorescence resonance energy transfer (FRET) between a small fluorescent dye and a nickel ion bound to a dihistidine motif can be used to monitor small structural rearrangements in proteins. This method provides several key advantages over classical FRET, including the ability to measure the dynamics of close-range interactions, the use of small probes with short linkers, a low orientation dependence, and the ability to add and remove unique tunable acceptors. We used this 'transition metal ion FRET' approach along with X-ray crystallography to determine the structural changes of the gating ring of the mouse hyperpolarization-activated cyclic nucleotide-regulated ion channel HCN2. Our results suggest a general model for the conformational switch in the cyclic nucleotide-binding site of cyclic nucleotide-regulated ion channels.

Friday, November 5, 2010, 11:00
Leibniz Institute for Age Research - Fritz Lipmann Institute
Beutenbergstr. 11, D-07745 Jena
Biocomputing-Verwaltungsgebäude, Seminarraum 101

Presenter: Rolf Hühne (FLI; Sühnel Lab)

Wermter J, Tomanek K, Hahn U.
High-performance gene name normalization with GeNo.
Bioinformatics. 2009;25(6):815-21. [PDF]

MOTIVATION: The recognition and normalization of textual mentions of gene and protein names is both particularly important and challenging. Its importance lies in the fact that they constitute the crucial conceptual entities in biomedicine. Their recognition and normalization remains a challenging task because of widespread gene name ambiguities within species, across species, with common English words and with medical sublanguage terms.

RESULTS: We present GeNo, a highly competitive system for gene name normalization, which obtains an F-measure performance of 86.4% (precision: 87.8%, recall: 85.0%) on the BioCreAtIvE-II test set, thus being on a par with the best system on that task. Our system tackles the complex gene normalization problem by employing a carefully crafted suite of symbolic and statistical methods, and by fully relying on publicly available software and data resources, including extensive background knowledge based on semantic profiling. A major goal of our work is to present GeNo's architecture in a lucid and perspicuous way to pave the way to full reproducibility of our results.

AVAILABILITY: GeNo, including its underlying resources, will be available from www.julielab.de. It is also currently deployed in the Semedico search engine at www.semedico.org.

Friday, October 29, 2010, 11:00
Leibniz Institute for Age Research - Fritz Lipmann Institute
Beutenbergstr. 11, D-07745 Jena
Biocomputing-Verwaltungsgebäude, Seminarraum 101

Presenter: Sandra Höfgen (FLI; Than Lab)

Alexandrov AI, Mileni M, Chien EY, Hanson MA, Stevens RC.
Microscale fluorescent thermal stability assay for membrane proteins. [PDF]
Structure 2008;16(3):351-9.

Systematic efforts to understand membrane protein stability under a variety of different solution conditions are not widely available for membrane proteins, mainly due to technical problems stemming from the presence of detergents necessary to keep the proteins in the solubilized state and the background that such detergents usually generate during biophysical characterization. In this report, we introduce an efficient microscale fluorescent stability screen using the thiol-specific fluorochrome N-[4-(7-diethylamino-4-methyl-3-coumarinyl)phenyl]maleimide (CPM) for stability profiling of membrane proteins under different solution and ligand conditions. The screen uses the chemical reactivity of the native cysteines embedded in the protein interior as a sensor for the overall integrity of the folded state. The thermal information gained by thorough investigation of the protein stability landscape can be effectively used to guide purification and biophysical characterization efforts including crystallization. To evaluate the method, three different protein families were analyzed, including the Apelin G protein-coupled receptor (APJ).

Ericsson UB, Hallberg BM, Detitta GT, Dekker N, Nordlund P.
Thermofluor-based high-throughput stability optimization of proteins for structural studies. [PDF]
Annal Biochem. 2006;357(2):289-98.

Production of proteins well suited for structural studies is inherently difficult and time-consuming. Protein sample homogeneity, stability, and solubility are strongly correlated with the proteins' probability of yielding crystals, and optimization of these properties will improve success rates of crystallization. In the current study, we applied the thermofluor method as a high-throughput approach for identifying optimal protein formulation for crystallization. The method also allowed optimal stabilizing buffer compositions to be rapidly identified for each protein. Furthermore, the method allowed the identification of potential ligands, physiological or non-physiological, that can be used in subsequent crystallization trials. For this study, the thermally induced melting points were determined in different buffers as well as with additives for a total of 25 Escherichia coli proteins. Crystallization trials were set up together with stabilizing and destabilizing additives identified using thermofluor screening. A twofold increase in the number of crystallization leads was observed when the proteins were cocrystallized with stabilizing additives as compared with experiments without these additives. This suggests that thermofluor constitutes an efficient generic high-throughput method for identification of protein properties predictive of crystallizability.

Friday, October 15, 2010, 11:00
Leibniz Institute for Age Research - Fritz Lipmann Institute
Beutenbergstr. 11, D-07745 Jena
Biocomputing-Verwaltungsgebäude, Seminarraum 101

Presenter: Sven Dahms (FLI; Than Lab)

Egea PF, Stroud RM. M.
Replication of individual DNA molecules under electronic control using a protein nanopore.
Proc Natl Acad Sci U S A. 2010;107(40):17182-7. [PDF]

The structure of the protein-translocating channel SecYEβ from Pyrococcus furiosus at 3.1-Å resolution suggests a mechanism for chaperoning transmembrane regions of a protein substrate during its lateral delivery into the lipid bilayer. Cytoplasmic segments of SecY orient the C-terminal α-helical region of another molecule, suggesting a general binding mode and a promiscuous guiding surface capable of accommodating diverse nascent chains at the exit of the ribosomal tunnel. To accommodate this putative nascent chain mimic, the cytoplasmic vestibule widens, and a lateral exit portal is opened throughout its entire length for partition of transmembrane helical segments to the lipid bilayer. In this primed channel, the central plug still occludes the pore while the lateral gate is opened, enabling topological arbitration during early protein insertion. In vivo, a 15 amino acid truncation of the cytoplasmic C-terminal helix of SecY fails to rescue a secY-deficient strain, supporting the essential role of this helix as suggested from the structure.

Friday, October 1, 2010, 11:00
Leibniz Institute for Age Research - Fritz Lipmann Institute
Beutenbergstr. 11, D-07745 Jena
Biocomputing-Verwaltungsgebäude, Seminarraum 101

Presenter: Peter Bellstedt (FLI; Görlach Lab)

Olasagasti F, Lieberman KR, Benner S, Cherf GM, Dahl JM, Deamer DW, Akeson M.
Replication of individual DNA molecules under electronic control using a protein nanopore.
Nat Nanotechnol. 2010. [Epub ahead of print], [PDF]

Nanopores can be used to analyse DNA by monitoring ion currents as individual strands are captured and driven through the pore in single file by an applied voltage. Here, we show that serial replication of individual DNA templates can be achieved by DNA polymerases held at the α-haemolysin nanopore orifice. Replication is blocked in the bulk phase, and is initiated only after the DNA is captured by the nanopore. We used this method, in concert with active voltage control, to observe DNA replication catalysed by bacteriophage T7 DNA polymerase (T7DNAP) and by the Klenow fragment of DNA polymerase I (KF). T7DNAP advanced on a DNA template against an 80-mV load applied across the nanopore, and single nucleotide additions were measured on the millisecond timescale for hundreds of individual DNA molecules in series. Replication by KF was not observed when this enzyme was held on top of the nanopore orifice at an applied potential of 80 mV. Sequential nucleotide additions by KF were observed upon applying controlled voltage reversals.

Friday, June 18, 2010, 11:00
Leibniz Institute for Age Research - Fritz Lipmann Institute
Beutenbergstr. 11, D-07745 Jena
Biocomputing-Verwaltungsgebäude, Seminarraum 101

Presenter: Manual Than (FLI; Than Lab)

Raines KS, Salha S, Sandberg RL, Jiang H, Rodríguez JA, Fahimian BP, Kapteyn HC, Du J, Miao J.
Three-dimensional structure determination from a single view.
Nature. 2010;463(7278):214-7.[PDF]

The ability to determine the structure of matter in three dimensions has profoundly advanced our understanding of nature. Traditionally, the most widely used schemes for three-dimensional (3D) structure determination of an object are implemented by acquiring multiple measurements over various sample orientations, as in the case of crystallography and tomography, or by scanning a series of thin sections through the sample, as in confocal microscopy. Here we present a 3D imaging modality, termed ankylography (derived from the Greek words ankylos meaning 'curved' and graphein meaning 'writing'), which under certain circumstances enables complete 3D structure determination from a single exposure using a monochromatic incident beam. We demonstrate that when the diffraction pattern of a finite object is sampled at a sufficiently fine scale on the Ewald sphere, the 3D structure of the object is in principle determined by the 2D spherical pattern. We confirm the theoretical analysis by performing 3D numerical reconstructions of a sodium silicate glass structure at 2 A resolution, and a single poliovirus at 2-3 nm resolution, from 2D spherical diffraction patterns alone. Using diffraction data from a soft X-ray laser, we also provide a preliminary demonstration that ankylography is experimentally feasible by obtaining a 3D image of a test object from a single 2D diffraction pattern. With further development, this approach of obtaining complete 3D structure information from a single view could find broad applications in the physical and life sciences.

Friday, June 4, 2010, 11:00
Leibniz Institute for Age Research - Fritz Lipmann Institute
Beutenbergstr. 11, D-07745 Jena
Biocomputing-Verwaltungsgebäude, Seminarraum 101

Presenter: Jürgen Sühnel (FLI; Sühnel Lab)

Kasowski M, Grubert F, Heffelfinger C, Hariharan M, Asabere A, Waszak SM, Habegger L, Rozowsky J, Shi M, Urban AE, Hong MY, Karczewski KJ, Huber W, Weissman SM, Gerstein MB, Korbel JO, Snyder M.
Variation in transcription factor binding among humans.
Science. 2010;328(5975):232-5. [PDF]

Differences in gene expression may play a major role in speciation and phenotypic diversity. We examined genome-wide differences in transcription factor (TF) binding in several humans and a single chimpanzee by using chromatin immunoprecipitation followed by sequencing. The binding sites of RNA polymerase II (PolII) and a key regulator of immune responses, nuclear factor kappaB (p65), were mapped in 10 lymphoblastoid cell lines, and 25 and 7.5% of the respective binding regions were found to differ between individuals. Binding differences were frequently associated with single-nucleotide polymorphisms and genomic structural variants, and these differences were often correlated with differences in gene expression, suggesting functional consequences of binding variation. Furthermore, comparing PolII binding between humans and chimpanzee suggests extensive divergence in TF binding. Our results indicate that many differences in individuals and species occur at the level of TF binding, and they provide insight into the genetic events responsible for these differences.

Zheng W, Zhao H, Mancera E, Steinmetz LM, Snyder M.
Genetic analysis of variation in transcription factor binding in yeast.
Nature. 2010;464(7292):1187-91. [PDF]

Variation in transcriptional regulation is thought to be a major cause of phenotypic diversity. Although widespread differences in gene expression among individuals of a species have been observed, studies to examine the variability of transcription factor binding on a global scale have not been performed, and thus the extent and underlying genetic basis of transcription factor binding diversity is unknown. By mapping differences in transcription factor binding among individuals, here we present the genetic basis of such variation on a genome-wide scale. Whole-genome Ste12-binding profiles were determined using chromatin immunoprecipitation coupled with DNA sequencing in pheromone-treated cells of 43 segregants of a cross between two highly diverged yeast strains and their parental lines. We identified extensive Ste12-binding variation among individuals, and mapped underlying cis- and trans-acting loci responsible for such variation. We showed that most transcription factor binding variation is cis-linked, and that many variations are associated with polymorphisms residing in the binding motifs of Ste12 as well as those of several proposed Ste12 cofactors. We also identified two trans-factors, AMN1 and FLO8, that modulate Ste12 binding to promoters of more than ten genes under alpha-factor treatment. Neither of these two genes was previously known to regulate Ste12, and we suggest that they may be mediators of gene activity and phenotypic diversity. Ste12 binding strongly correlates with gene expression for more than 200 genes, indicating that binding variation is functional. Many of the variable-bound genes are involved in cell wall organization and biogenesis. Overall, these studies identified genetic regulators of molecular diversity among individuals and provide new insights into mechanisms of gene regulation.

Friday, April 23, 2010, 11:00
Leibniz Institute for Age Research - Fritz Lipmann Institute
Beutenbergstr. 11, D-07745 Jena
Biocomputing-Verwaltungsgebäude, Seminarraum 101

Presenter: Dirk Röser (FLI; Than Lab)

Kawate T, Michel JC, Birdsong WT, Gouaux E
Crystal structure of the ATP-gated P2X₄ ion channel in the closed state.
Nature. 2009;460(7255):592-8. [PDF]

P2X receptors are cation-selective ion channels gated by extracellular ATP, and are implicated in diverse physiological processes, from synaptic transmission to inflammation to the sensing of taste and pain. Because P2X receptors are not related to other ion channel proteins of known structure, there is at present no molecular foundation for mechanisms of ligand-gating, allosteric modulation and ion permeation. Here we present crystal structures of the zebrafish P2X(4) receptor in its closed, resting state. The chalice-shaped, trimeric receptor is knit together by subunit-subunit contacts implicated in ion channel gating and receptor assembly. Extracellular domains, rich in beta-strands, have large acidic patches that may attract cations, through fenestrations, to vestibules near the ion channel. In the transmembrane pore, the 'gate' is defined by an approximately 8 A slab of protein. We define the location of three non-canonical, intersubunit ATP-binding sites, and suggest that ATP binding promotes subunit rearrangement and ion channel opening.

Friday, April 09, 2010, 11:00
Leibniz Institute for Age Research - Fritz Lipmann Institute
Beutenbergstr. 11, D-07745 Jena
Biocomputing-Verwaltungsgebäude, Seminarraum 101

Presenter: Ramadurai Ramachandran (FLI, Görlach Lab)

Raman S, Lange OF, Rossi P, Tyka M, Wang X, Aramini J, Liu G, Ramelot TA, Eletsky A, Szyperski T, Kennedy MA, Prestegard J, Montelione GT, Baker D.
NMR structure determination for larger proteins using backbone-only data.
Science. 2010;327(5968):1014-8. [PDF]

Conventional protein structure determination from nuclear magnetic resonance data relies heavily on side-chain proton-to-proton distances. The necessary side-chain resonance assignment, however, is labor intensive and prone to error. Here we show that structures can be accurately determined without nuclear magnetic resonance (NMR) information on the side chains for proteins up to 25 kilodaltons by incorporating backbone chemical shifts, residual dipolar couplings, and amide proton distances into the Rosetta protein structure modeling methodology. These data, which are too sparse for conventional methods, serve only to guide conformational search toward the lowest-energy conformations in the folding landscape; the details of the computed models are determined by the physical chemistry implicit in the Rosetta all-atom energy function. The new method is not hindered by the deuteration required to suppress nuclear relaxation processes for proteins greater than 15 kilodaltons and should enable routine NMR structure determination for larger proteins.

Friday, March 26, 2010, 11:00
Leibniz Institute for Age Research - Fritz Lipmann Institute
Beutenbergstr. 11, D-07745 Jena
Biocomputing-Verwaltungsgebäude, Seminarraum 101

Presenter: Oliver Ohlenschläger (FLI, Görlach Lab)

London N, Movshovitz-Attias D, Schueler-Furman O.
The structural basis of peptide-protein binding strategies.
Structure. 2010; 18(2):188-99. [PDF]

Peptide-protein interactions are very prevalent, mediating key processes such as signal transduction and protein trafficking. How can peptides overcome the entropic cost involved in switching from an unstructured, flexible peptide to a rigid, well-defined bound structure? A structure-based analysis of peptide-protein interactions unravels that most peptides do not induce conformational changes on their partner upon binding, thus minimizing the entropic cost of binding. Furthermore, peptides display interfaces that are better packed than protein-protein interfaces and contain significantly more hydrogen bonds, mainly those involving the peptide backbone. Additionally, "hot spot" residues contribute most of the binding energy. Finally, peptides tend to bind in the largest pockets available on the protein surface. Our study is based on peptiDB, a new and comprehensive data set of 103 high-resolution peptide-protein complex structures. In addition to improved understanding of peptide-protein interactions, our findings have direct implications for the structural modeling, design, and manipulation of these interactions.

Friday, March 19, 2010, 11:00
Leibniz Institute for Age Research - Fritz Lipmann Institute
Beutenbergstr. 11, D-07745 Jena
Biocomputing-Verwaltungsgebäude, Seminarraum 101

Presenter: Rolf Hühne (FLI, Sühnel Lab)

de Magalhães JP, Finch CE, Janssens G.
Next-generation sequencing in aging research: Emerging applications, problems, pitfalls and possible solutions.
Ageing Res Rev. 2009 Nov 10. [Epub ahead of print] [PDF]

Recent technological advances that allow faster and cheaper DNA sequencing are now driving biological and medical research. In this review, we provide an overview of state-of-the-art next-generation sequencing (NGS) platforms and their applications, including in genome sequencing and resequencing, transcriptional profiling (RNA-Seq) and high-throughput survey of DNA-protein interactions (ChIP-Seq) and of the epigenome. Particularly, we focus on how new methods made possible by NGS can help unravel the biological and genetic mechanisms of aging, longevity and age-related diseases. In the same way, however, NGS platforms open discovery not available before, they also give rise to new challenges, in particular in processing, analyzing and interpreting the data. Bioinformatics and software issues plus statistical difficulties in genome-wide studies are discussed, as well as the use of targeted sequencing to decrease costs and facilitate statistical analyses. Lastly, we discuss a number of methods to gather biological insights from massive amounts of data, such as functional enrichment, transcriptional regulation and network analyses. Although in the fast-moving field of NGS new platforms will soon take center stage, the approaches made possible by NGS will be at the basis of molecular biology, genetics and systems biology for years to come, making them instrumental for research on aging.

Friday, March 5, 2010, 11:00
Leibniz Institute for Age Research - Fritz Lipmann Institute
Beutenbergstr. 11, D-07745 Jena
Biocomputing-Verwaltungsgebäude, Seminarraum 101

Presenter: Sven Dahms (FLI, Than Lab)

Sjöblom B, Polentarutti M, Djinovic-Carugo K.
Structural study of X-ray induced activation of carbonic anhydrase.
Proc Natl Acad Sci U S A. 2009;106(26):10609-13. [PDF]

Carbonic anhydrase, a zinc metalloenzyme, catalyzes the reversible hydration of carbon dioxide to bicarbonate. It is involved in processes connected with acid-base homeostasis, respiration, and photosynthesis. More than 100 distinct human carbonic anhydrase II (HCAII) 3D structures have been generated in last 3 decades [Liljas A, et al. (1972) Nat New Biol 235:131-137], but a structure of an HCAII in complex with CO(2) or HCO(3)(-) has remained elusive. Here, we report previously undescribed structures of HCAII:CO(2) and HCAII:HCO(3)(-) complexes, together with a 3D molecular film of the enzymatic reaction observed successively in the same crystal after extended exposure to X-ray. We demonstrate that the unexpected enzyme activation was caused in an X-ray dose-dependent manner. Although X-ray damage to macromolecular samples has long been recognized [Ravelli RB, Garman EF (2006) Curr Opin Struct Biol 16:624-629], the detailed structural analysis reports on X-ray-driven reactions have been very rare in literature to date. Here, we report on enzyme activation and the associated chemical reaction in a crystal at 100 K. We propose mechanisms based on water photoradiolysis and/or electron radiolysis as the main cause of enzyme activation.

Friday, February 5, 2010, 11:00
Leibniz Institute for Age Research - Fritz Lipmann Institute
Beutenbergstr. 11, D-07745 Jena
Biocomputing-Verwaltungsgebäude, Seminarraum 101

Presenter: Miriam Küster (FLI, Than Lab)

Baumgartner R, Meder G, Briand C, Decock A, D'arcy A, Hassiepen U, Morse R, Renatus M.
The crystal structure of caspase-6, a selective effector of axonal degeneration.
Biochem J. 2009;423(3):429-39. [PDF]

Neurodegenerative diseases pose one of the most pressing unmet medical needs today. It has long been recognized that caspase-6 may play a role in several neurodegenerative diseases for which there are currently no disease-modifying therapies. Thus it is a potential target for neurodegenerative drug development. In the present study we report on the biochemistry and structure of caspase-6. As an effector caspase, caspase-6 is a constitutive dimer independent of the maturation state of the enzyme. The ligand-free structure shows caspase-6 in a partially mature but latent conformation. The cleaved inter-domain linker remains partially inserted in the central groove of the dimer, as observed in other caspases. However, in contrast with the structures of other caspases, not only is the catalytic machinery misaligned, but several structural elements required for substrate recognition are missing. Most importantly, residues forming a short anti-parallel beta-sheet abutting the substrate in other caspase structures are part of an elongation of the central alpha-helix. Despite the dramatic structural changes that are required to adopt a canonical catalytically competent conformation, the pre-steady-state kinetics exhibit no lag phase in substrate turnover. This suggests that the observed conformation does not play a regulatory role in caspase-6 activity. However, targeting the latent conformation in search for specific and bio-available caspase-6 inhibitors might offer an alternative to active-site-directed approaches.

Friday, January 22, 2010, 11:00
Leibniz Institute for Age Research - Fritz Lipmann Institute
Beutenbergstr. 11, D-07745 Jena
Biocomputing-Verwaltungsgebäude, Seminarraum 101

Presenter: Jean A. Nzigou Mandouckou (FLI, Than Lab)

Okada C, Yamashita E, Lee SJ, Shibata S, Katahira J, Nakagawa A, Yoneda Y, Tsukihara T.
A high-resolution structure of the pre-microRNA nuclear export machinery.
Science. 2009, 326(5957):1275-9. [PDF]

Nuclear export of microRNAs (miRNAs) by exportin-5 (Exp-5) is an essential step in miRNA biogenesis. Here, we present the 2.9 angstrom structure of the pre-miRNA nuclear export machinery formed by pre-miRNA complexed with Exp-5 and a guanine triphosphate (GTP)-bound form of the small nuclear guanine triphosphatase (GTPase) Ran (RanGTP). The x-ray structure shows that Exp-5:RanGTP recognizes the 2-nucleotide 3' overhang structure and the double-stranded stem of the pre-miRNA. Exp-5:RanGTP shields the pre-miRNA stem from degradation in a baseball mitt-like structure where it is held by broadly distributed weak interactions, whereas a tunnel-like structure of Exp-5 interacts strongly with the 2-nucleotide 3' overhang through hydrogen bonds and ionic interactions. RNA recognition by Exp-5:RanGTP does not depend on RNA sequence, implying that Exp-5:RanGTP can recognize a variety of pre-miRNAs.

Friday, January 08, 2010, 11:00
Leibniz Institute for Age Research - Fritz Lipmann Institute
Beutenbergstr. 11, D-07745 Jena
Biocomputing-Verwaltungsgebäude, Seminarraum 101

Presenter: Andre Mischo (FLI, Görlach Lab)

Levin EJ, Quick M, Zhou M.
Crystal structure of a bacterial homologue of the kidney urea transporter.
Nature. 2009 462(7274):757-61. [PDF]

Urea is highly concentrated in the mammalian kidney to produce the osmotic gradient necessary for water re-absorption.Free diffusion of urea across cellmembranes is slow owing to its high polarity, and specialized urea transporters have evolved to achieve rapid and selective urea permeation. Here we present the 2.3A° structure of a functional urea transporter from the bacterium Desulfovibrio vulgaris. The transporter is a homotrimer, and each subunit contains a continuous membrane-spanning pore formed by the two homologous halves of the protein. The pore contains a constricted selectivity filter that can accommodate several dehydrated urea molecules in single file. Backbone and side-chain oxygen atoms provide continuous coordination of urea as it progresses through the filter, and well-placed a-helix dipoles provide further compensation for dehydration energy. These results establish that the urea transporter operates by a channel-like mechanism and reveal the physical and chemical basis of urea selectivity.

Friday, December 18, 2009, 11:00
Leibniz Institute for Age Research - Fritz Lipmann Institute
Beutenbergstr. 11, D-07745 Jena
Biocomputing-Verwaltungsgebäude, Seminarraum 101

Presenter: Christian Herbst (FLI, Görlach Lab)

Nieuwkoop AJ, Wylie BJ, Franks WT, Shah GJ, Rienstra CM.
Atomic resolution protein structure determination by three-dimensional transferred echo double resonance solid-state nuclear magnetic resonance spectroscopy.
J Chem Phys. 2009 Sep 7;131(9):095101. [PDF]

We show that quantitative internuclear (15)N-(13)C distances can be obtained in sufficient quantity to determine a complete, high-resolution structure of a moderately sized protein by magic-angle spinning solid-state NMR spectroscopy. The three-dimensional ZF-TEDOR pulse sequence is employed in combination with sparse labeling of (13)C sites in the beta1 domain of the immunoglobulin binding protein G (GB1), as obtained by bacterial expression with 1,3-(13)C or 2-(13)C-glycerol as the (13)C source. Quantitative dipolar trajectories are extracted from two-dimensional (15)N-(13)C planes, in which approximately 750 cross peaks are resolved. The experimental data are fit to exact theoretical trajectories for spin clusters (consisting of one (13)C and several (15)N each), yielding quantitative precision as good as 0.1 A for approximately 350 sites, better than 0.3 A for another 150, and approximately 1.0 A for 150 distances in the range of 5-8 A. Along with isotropic chemical shift-based (TALOS) dihedral angle restraints, the distance restraints are incorporated into simulated annealing calculations to yield a highly precise structure (backbone RMSD of 0.25+/-0.09 A), which also demonstrates excellent agreement with the most closely related crystal structure of GB1 (2QMT, bbRMSD 0.79+/-0.03 A). Moreover, side chain heavy atoms are well restrained (0.76+/-0.06 A total heavy atom RMSD). These results demonstrate for the first time that quantitative internuclear distances can be measured throughout an entire solid protein to yield an atomic-resolution structure.

Friday, December 11, 2009, 11:00
Leibniz Institute for Age Research - Fritz Lipmann Institute
Beutenbergstr. 11, D-07745 Jena
Biocomputing-Verwaltungsgebäude, Seminarraum 101

Presenter: Sandra Höfgen (FLI, Than Lab)

Dashivets T, Wood N, Hergersberg C, Buchner J, Haslbeck M.
Rapid matrix-assisted refolding of histidine-tagged proteins.
Chembiochem. 2009;10:869-76. [PDF]

The formation of inclusion bodies (IBs)--amorphous aggregates of misfolded insoluble protein--during recombinant protein expression, is still one of the biggest bottlenecks in protein science. We have developed and analyzed a rapid parallel approach for matrix-assisted refolding of recombinant His(6)-tagged proteins. Efficiencies of matrix-assisted refolding were screened in a 96-well format. The developed methodology allowed the efficient refolding of five different test proteins, including monomeric and oligomeric proteins. Compared to refolding in-solution, the matrix-assisted refolding strategy proved equal or better for all five proteins tested. Interestingly, specifically oligomeric proteins displayed significantly higher levels of refolding compared to refolding in-solution. Mechanistically, matrix-assisted folding seems to differ from folding in-solution, as the reaction proceeds more rapidly and shows a remarkably different concentration dependence--it allows refolding at up to 1000-fold higher protein concentration than folding in-solution.

Friday, December 4, 2009, 11:00
Leibniz Institute for Age Research - Fritz Lipmann Institute
Beutenbergstr. 11, D-07745 Jena
Biocomputing-Verwaltungsgebäude, Seminarraum 101

Presenter: Matthias Görlach (FLI, Görlach Lab)

Shechner DM, Grant RA, Bagby SC, Koldobskaya Y, Piccirilli JA, Bartel DP.
Crystal structure of the catalytic core of an RNA-polymerase ribozyme.
Science 2009; 326:1271-1275. [PDF]

Primordial organisms of the putative RNA world would have required polymerase ribozymes able to replicate RNA. Known ribozymes with polymerase activity best approximating that needed for RNA replication contain at their catalytic core the class I RNA ligase, an artificial ribozyme with a catalytic rate among the fastest of known ribozymes. Here we present the 3.0 angstrom crystal structure of this ligase. The architecture resembles a tripod, its three legs converging near the ligation junction. Interacting with this tripod scaffold through a series of 10 minor-groove interactions (including two A-minor triads) is the unpaired segment that contributes to and organizes the active site. A cytosine nucleobase and two backbone phosphates abut the ligation junction; their location suggests a model for catalysis resembling that of proteinaceous polymerases.

Friday, November 27, 2009, 11:00
Leibniz Institute for Age Research - Fritz Lipmann Institute
Beutenbergstr. 11, D-07745 Jena
Biocomputing-Verwaltungsgebäude, Seminarraum 101

Presenter: Michaela Carella (FLI, Görlach Lab)

Rohs R, West SM, Sosinsky A, Liu P, Mann RS, Honig B.
The role of DNA shape in protein-DNA recognition.
Nature. 2009; 461(7268):1248-53. [PDF]

The recognition of specific DNA sequences by proteins is thought to depend on two types of mechanism: one that involves the formation of hydrogen bonds with specific bases, primarily in the major groove, and one involving sequence-dependent deformations of the DNA helix. By comprehensively analysing the three-dimensional structures of protein-DNA complexes, here we show that the binding of arginine residues to narrow minor grooves is a widely used mode for protein-DNA recognition. This readout mechanism exploits the phenomenon that narrow minor grooves strongly enhance the negative electrostatic potential of the DNA. The nucleosome core particle offers a prominent example of this effect. Minor-groove narrowing is often associated with the presence of A-tracts, AT-rich sequences that exclude the flexible TpA step. These findings indicate that the ability to detect local variations in DNA shape and electrostatic potential is a general mechanism that enables proteins to use information in the minor groove, which otherwise offers few opportunities for the formation of base-specific hydrogen bonds, to achieve DNA-binding specificity.

Friday, November 13, 2009, 11:00
Leibniz Institute for Age Research - Fritz Lipmann Institute
Beutenbergstr. 11, D-07745 Jena
Biocomputing-Verwaltungsgebäude, Seminarraum 101

Presenter: Peter Bellstedt (FLI, Görlach Lab)

Hawkins ME.
Fluorescent pteridine probes for nucleic acid analysis.
Methods Enzymol. 2008;450:201-31. [PDF]

This chapter is focused on the fluorescent pteridine guanine analogs, 3MI and 6MI and on the pteridine adenine analog, 6MAP. A brief overview of commonly used methods to fluorescently label oligonucleotides reveals the role the pteridines play in the extensive variety of available probes. We describe the fluorescence characteristics of the pteridine probes as monomers and incorporated into DNA and review a variety of applications including changes in fluorescence intensity, anisotropies, time resolved studies, two photon excitation and single molecule detection.

Rist M, Marino J.
Association of an RNA kissing complex analyzed using 2-aminopurine fluorescence.
Nucleic Acids Res. 2001 Jun 1;29(11):2401-8. [PDF]

The fluorescent probe, 2-aminopurine-2'-O-methyl riboside (2-AP) has been selectively incorporated at adenosine positions in stem-loops (so called R1inv and R2inv), derived from the ColE1 plasmid encoded RNA I and RNA II transcripts, that interact to form stable loop-loop kissing complexes and bind the RNA one modulator (Rom) protein, such that fluorescence-detected stopped-flow and equilibrium methods could be used to study the detailed mechanism of this RNA-RNA interaction. Formation of loop-loop kissing complexes between R1inv and R2inv hairpins, substituted with 2-AP at positions in the complementary loops, results in a 5-10-fold fluorescence emission decrease (F(max) = 370 nm), which provides a sensitive measure for the binding reaction. The 2-AP substituted complexes are found to have equilibrium binding properties (average K(D) = 2.6 +/- 1.7 nM) and affinity for Rom (average K(D) = 60 +/- 24 nM) that are similar to complexes formed with equivalent unlabeled hairpins. Using stopped-flow experiments, it was found that the 2-AP probes experienced at least three different microenvironments during association of the RNA complex, thus suggesting a kinetic intermediate in the kissing pathway. In contrast, dissociation of the complex was found to fit a single exponential decay (average k(off) = 8.9 x 10(-5) s(-1)). Consistent with these observations, a two-step mechanism for RNA loop-loop complex association is proposed in which the complementary loops of R1inv and R2inv first base pair to form the loop-loop helix (average k(1) = 0.13 microM(-1)s(-1)) in the initial encounter reaction, and subsequently isomerize to the final tertiary fold in a second slower step (average k(2) = 0.09 s(-1)), where the helical stacking around the junctions is optimized.

Friday, July 24, 2009, 11:00
Leibniz Institute for Age Research - Fritz Lipmann Institute
Beutenbergstr. 11, D-07745 Jena
Biocomputing-Verwaltungsgebäude, Seminarraum 101

Presenter: Christian Herbst (FLI, Görlach Lab)

Agarwal V, Reif B
Residual methyl protonation in perdeuterated proteins for multi-dimensional correlation experiments in MAS solid-state NMR spectroscopy.
J Magn Reson. 2008, 194(1):16-24. [PDF]

NMR studies involving perdeuterated proteins focus in general on exchangeable amide protons. However, non-exchangeable sites contain as well a small amount of protons as the employed precursors for protein biosynthesis are not completely proton depleted. The degree of methyl group protonation is in the order of 9% for CD2H using >97% deuterium enriched glucose. We show in this manuscript that this small amount of residual protonation is sufficient to perform 2D and 3D MAS solid-state NMR experiments. In particular, we suggest a HCCH-TOBSY type experiment which we successfully employ to assign the methyl resonances in aliphatic side chains in a perdeuterated sample of the SH3 domain of chicken alpha-spectrin.

Chevelkov V, Rehbein K, Diehl A, Reif B.
Ultrahigh resolution in proton solid-state NMR spectroscopy at high levels of deuteration.
Angew Chem Int Ed Engl. 2006,45(23):3878-81. [PDF]

Friday, July 10, 2009, 11:00
Leibniz Institute for Age Research - Fritz Lipmann Institute
Beutenbergstr. 11, D-07745 Jena
Biocomputing-Verwaltungsgebäude, Seminarraum 101

Presenter: Dirk Röser (FLI, Than Lab)

Osenkowski P, Ye W, Wang R, Wolfe MS, Selkoe DJ.
Direct and potent regulation of gamma-secretase by its lipid microenvironment.
J Biol Chem. 2008; 283(33):22529-40. [PDF]

Gamma-Secretase is an unusual and ubiquitous aspartyl protease with an intramembrane catalytic site that cleaves many type-I integral membrane proteins, most notably APP and Notch. Several reports suggest that cleavage of APP to produce the Abeta peptide is regulated in part by lipids. As gamma-secretase is a multipass protein complex with 19 transmembrane domains, it is likely that the local lipid composition of the membrane can regulate gamma-activity. To determine the direct contribution of the lipid microenvironment to gamma-secretase activity, we purified the human protease from overexpressing mammalian cells, reconstituted it in vesicles of varying lipid composition, and examined the effects of individual phospholipids, sphingolipids, cholesterol, and complex lipid mixtures on substrate cleavage. A conventional gamma-activity assay was modified to include a detergent-removal step to facilitate proteoliposome formation, and this increased baseline activity over 2-fold. Proteoliposomes containing sphingolipids significantly increased gamma-secretase activity over a phosphatidylcholine-only baseline, whereas the addition of phosphatidylinositol significantly decreased activity. Addition of soluble cholesterol in the presence of phospholipids and sphingolipids robustly increased the cleavage of APP- and Notch-like substrates in a dose-dependent manner. Reconstitution of gamma-secretase in complex lipid mixtures revealed that a lipid raft-like composition supported the highest level of activity compared with other membrane compositions. Taken together, these results demonstrate that membrane lipid composition is a direct and potent modulator of gamma-secretase and that cholesterol, in particular, plays a major regulatory role.

Friday, June 26, 2009, 11:00
Leibniz Institute for Age Research - Fritz Lipmann Institute
Beutenbergstr. 11, D-07745 Jena
Biocomputing-Verwaltungsgebäude, Seminarraum 101

Presenter: Ramadurai Ramachandran (FLI, Görlach Lab)

Wylie BJ, Schwieters CD, Oldfield E, Rienstra CM.
Protein structure refinement using 13C alpha chemical shift tensors.
J Am Chem Soc. 2009, 131(3):985-92. [PDF]

We have obtained the (13)C alpha chemical shift tensors for each amino acid in the protein GB1. We then developed a CST force field and incorporated this into the Xplor-NIH structure determination program. GB1 structures obtained by using CST restraints had improved precision over those obtained in the absence of CST restraints and were also more accurate. When combined with isotropic chemical shifts, distance, and vector angle restraints, the root-mean squared error with respect to existing X-ray structures was better than approximately 1.0 A. These results are of broad general interest since they show that chemical shift tensors can be used in protein structure refinement, improving both structural accuracy and precision, opening up the way to accurate de novo structure determination.

Friday, June 12, 2009, 11:00
Leibniz Institute for Age Research - Fritz Lipmann Institute
Beutenbergstr. 11, D-07745 Jena
Biocomputing-Verwaltungsgebäude, Seminarraum 101

Presenter: Jessica Meinhardt (FLI, Görlach Lab)

Maji SK, Schubert D, Rivier C, Lee S, Rivier JE, Riek R.
Amyloid as a depot for the formulation of long-acting drugs.
PLoS Biol. 2008 Feb;6(2):e17. [PDF]

Amyloids are highly organized protein aggregates that are associated with both neurodegenerative diseases such as Alzheimer disease and benign functions like skin pigmentation. Amyloids self-polymerize in a nucleation-dependent manner by recruiting their soluble protein/peptide counterpart and are stable against harsh physical, chemical, and biochemical conditions. These extraordinary properties make amyloids attractive for applications in nanotechnology. Here, we suggest the use of amyloids in the formulation of long-acting drugs. It is our rationale that amyloids have the properties required of a long-acting drug because they are stable depots that guarantee a controlled release of the active peptide drug from the amyloid termini. This concept is tested with a family of short- and long-acting analogs of gonadotropin-releasing hormone (GnRH), and it is shown that amyloids thereof can act as a source for the sustained release of biologically active peptides.

Friday, May 29, 2009, 11:00
Leibniz Institute for Age Research - Fritz Lipmann Institute
Beutenbergstr. 11, D-07745 Jena
Biocomputing-Verwaltungsgebäude, Seminarraum 101

Presenter: Jean A. Nzigou Mandouckou (FLI, Than Lab)

Oganesyan N, Ankoudinova I, Kim SH, Kim R.
Effect of osmotic stress and heat shock in recombinant protein overexpression and crystallization.
Protein Expr Purif. 2007, 280-5. Epub 2006 Oct 10. [PDF]

Overexpressed recombinant proteins in bacteria often tend to misfold and accumulate as soluble aggregates and/or inclusion bodies. A strategy for improving the level of expression of recombinant proteins in a soluble native form is to increase the cellular concentration of osmolytes or of chaperones. This can be accomplished by growing the bacterial cells in the presence of high salt, sorbitol, and betaine as well as exposing the cells to a heat shock step. Our results suggest that by growing the cells under varied conditions one may be able to express targets as soluble proteins (from previously insoluble targets) and to improve the chances of their crystallization.

Diamant S, Eliahu N, Rosenthal D, Goloubinoff P.
Chemical chaperones regulate molecular chaperones in vitro and in cells under combined salt and heat stresses.
J Biol Chem. 2001, 276(43):39586-91. Epub 2001 Aug 21. {PDF]

Salt and heat stresses, which are often combined in nature, induce complementing defense mechanisms. Organisms adapt to high external salinity by accumulating small organic compounds known as osmolytes, which equilibrate cellular osmotic pressure. Osmolytes can also act as "chemical chaperones" by increasing the stability of native proteins and assisting refolding of unfolded polypeptides. Adaptation to heat stress depends on the expression of heat-shock proteins, many of which are molecular chaperones, that prevent protein aggregation, disassemble protein aggregates, and assist protein refolding. We show here that Escherichia coli cells preadapted to high salinity contain increased levels of glycine betaine that prevent protein aggregation under thermal stress. After heat shock, the aggregated proteins, which escaped protection, were disaggregated in salt-adapted cells as efficiently as in low salt. Here we address the effects of four common osmolytes on chaperone activity in vitro. Systematic dose responses of glycine betaine, glycerol, proline, and trehalose revealed a regulatory effect on the folding activities of individual and combinations of chaperones GroEL, DnaK, and ClpB. With the exception of trehalose, low physiological concentrations of proline, glycerol, and especially glycine betaine activated the molecular chaperones, likely by assisting local folding in chaperone-bound polypeptides and stabilizing the native end product of the reaction. High osmolyte concentrations, especially trehalose, strongly inhibited DnaK-dependent chaperone networks, such as DnaK+GroEL and DnaK+ClpB, likely because high viscosity affects dynamic interactions between chaperones and folding substrates and stabilizes protein aggregates. Thus, during combined salt and heat stresses, cells can specifically control protein stability and chaperone-mediated disaggregation and refolding by modulating the intracellular levels of different osmolytes.

de Marco A, Vigh L, Diamant S, Goloubinoff P.
Native folding of aggregation-prone recombinant proteins in Escherichia coli by osmolytes, plasmid- or benzyl alcohol-overexpressed molecular chaperones.
Cell Stress Chaperones. 2005,10:329-39. [PDF]

When massively expressed in bacteria, recombinant proteins often tend to misfold and accumulate as soluble and insoluble nonfunctional aggregates. A general strategy to improve the native folding of recombinant proteins is to increase the cellular concentration of viscous organic compounds, termed osmolytes, or of molecular chaperones that can prevent aggregation and can actively scavenge and convert aggregates into natively refoldable species. In this study, metal affinity purification (immobilized metal ion affinity chromatography [IMAC]), confirmed by resistance to trypsin digestion, was used to distinguish soluble aggregates from soluble nativelike proteins. Salt-induced accumulation of osmolytes during induced protein synthesis significantly improved IMAC yields of folding-recalcitrant proteins. Yet, the highest yields were obtained with cells coexpressing plasmid-encoded molecular chaperones DnaK-DnaJ-GrpE, ClpB, GroEL-GroES, and IbpA/B. Addition of the membrane fluidizer heat shock-inducer benzyl alcohol (BA) to the bacterial medium resulted in similar high yields as with plasmid-mediated chaperone coexpression. Our results suggest that simple BA-mediated induction of endogenous chaperones can substitute for the more demanding approach of chaperone coexpression. Combined strategies of osmolyte-induced native folding with heat-, BA-, or plasmid-induced chaperone coexpression can be thought to optimize yields of natively folded recombinant proteins in bacteria, for research and biotechnological purposes.

Friday, May 15, 2009, 11:00
Leibniz Institute for Age Research - Fritz Lipmann Institute
Beutenbergstr. 11, D-07745 Jena
Biocomputing-Verwaltungsgebäude, Seminarraum 101

Presenter: Miriam Küster (FLI, Than Lab)

Scott FL, Stec B, Pop C, Dobaczewska MK, Lee JJ, Monosov E, Robinson H, Salvesen GS, Schwarzenbacher R, Riedl SJ.
The Fas-FADD death domain complex structure unravels signalling by receptor clustering.
Nature. 2009, 457:1019-22. [PDF]

The death inducing signalling complex (DISC) formed by Fas receptor, FADD (Fas-associated death domain protein) and caspase 8 is a pivotal trigger of apoptosis. The Fas-FADD DISC represents a receptor platform, which once assembled initiates the induction of programmed cell death. A highly oligomeric network of homotypic protein interactions comprised of the death domains of Fas and FADD is at the centre of DISC formation. Thus, characterizing the mechanistic basis for the Fas-FADD interaction is crucial for understanding DISC signalling but has remained unclear largely because of a lack of structural data. We have successfully formed and isolated the human Fas-FADD death domain complex and report the 2.7 A crystal structure. The complex shows a tetrameric arrangement of four FADD death domains bound to four Fas death domains. We show that an opening of the Fas death domain exposes the FADD binding site and simultaneously generates a Fas-Fas bridge. The result is a regulatory Fas-FADD complex bridge governed by weak protein-protein interactions revealing a model where the complex itself functions as a mechanistic switch. This switch prevents accidental DISC assembly, yet allows for highly processive DISC formation and clustering upon a sufficient stimulus. In addition to depicting a previously unknown mode of death domain interactions, these results further uncover a mechanism for receptor signalling solely by oligomerization and clustering events.

Friday, May 08, 2009, 11:00
Leibniz Institute for Age Research - Fritz Lipmann Institute
Beutenbergstr. 11, D-07745 Jena
Biocomputing-Verwaltungsgebäude, Seminarraum 101

Presenter: Andre Mischo (FLI, Görlach Lab)

Showalter SA, Bruschweiler-Li L, Johnson E, Zhang F, Brüschweiler R.
Quantitative lid dynamics of MDM2 reveals differential ligand binding modes of the p53-binding cleft.
J Am Chem Soc. 2008, 130:6472-8. Epub 2008 Apr 25. [PDF]

The oncoprotein MDM2 regulates the activity and stability of the tumor suppressor p53 through protein-protein interaction involving their N-terminal domains. The N-terminal lid of MDM2 has been implicated in p53 regulation; however, due to its flexible nature, limited data are available concerning its role in ligand binding. The quantitative dynamics study using NMR reported here shows, for the first time, that the lid in apo-MDM2 slowly interconverts between a "closed" state that is associated with the p53-binding cleft and an "open" state that is highly flexible. Our results reveal that apo-MDM2 predominantly populates the closed state, whereas the p53-bound MDM2 exclusively populates the open state. Unlike p53 binding, the small molecule MDM2 antagonist nutlin-3 binds to the cleft essentially without perturbing the closed lid state. The lid dynamics thereby represents a signature for the experimental and virtual screening of therapeutic antagonists that target the p53-MDM2 interaction.

Friday, April 16, 2009, 11:00
Leibniz Institute for Age Research - Fritz Lipmann Institute
Beutenbergstr. 11, D-07745 Jena
Biocomputing-Verwaltungsgebäude, Seminarraum 101

Presenter: Maik Friedel (FLI, Sühnel Lab)

Parker SC, Hansen L, Abaan HO, Tullius TD, Margulies EH.
Local DNA Topography Correlates with Functional Noncoding Regions of the Human Genome.
Science. 2009 Mar 12. [Epub ahead of print] [PDF]

The three-dimensional molecular structure of DNA, specifically the shape of the backbone and grooves of genomic DNA, can be dramatically affected by nucleotide changes, which can cause differences in protein binding affinity and phenotype. We developed an algorithm to measure constraint on the basis of similarity of DNA topography among multiple species using hydroxyl radical cleavage patterns to interrogate the solvent accessible surface area of DNA. This algorithm found that 12% of bases in the human genome are evolutionarily constrained-double the number detected by nucleotide sequence-based algorithms. Topography-informed constrained regions correlated with functional noncoding elements, including enhancers, better than regions identified solely on the basis of nucleotide sequence. These results support that the molecular shape of DNA is under selection and can identify evolutionary history.

Friday, March 27, 2009, 11:00
Leibniz Institute for Age Research - Fritz Lipmann Institute
Beutenbergstr. 11, D-07745 Jena
Biocomputing-Verwaltungsgebäude, Seminarraum 101

Presenter: Sven Dahms (FLI, Than Lab)

Cherezov V, Rosenbaum DM, Hanson MA, Rasmussen SG, Thian FS, Kobilka TS, Choi HJ, Kuhn P, Weis WI, Kobilka BK, Stevens RC.
High-resolution crystal structure of an engineered human beta2-adrenergic G protein-coupled receptor.
Science. 2007, 318(5854):1258-65. [PDF]

Heterotrimeric guanine nucleotide-binding protein (G protein)-coupled receptors constitute the largest family of eukaryotic signal transduction proteins that communicate across the membrane. We report the crystal structure of a human beta2-adrenergic receptor-T4 lysozyme fusion protein bound to the partial inverse agonist carazolol at 2.4 angstrom resolution. The structure provides a high-resolution view of a human G protein-coupled receptor bound to a diffusible ligand. Ligand-binding site accessibility is enabled by the second extracellular loop, which is held out of the binding cavity by a pair of closely spaced disulfide bridges and a short helical segment within the loop. Cholesterol, a necessary component for crystallization, mediates an intriguing parallel association of receptor molecules in the crystal lattice. Although the location of carazolol in the beta2-adrenergic receptor is very similar to that of retinal in rhodopsin, structural differences in the ligand-binding site and other regions highlight the challenges in using rhodopsin as a template model for this large receptor family.

Warne T, Serrano-Vega MJ, Baker JG, Moukhametzianov R, Edwards PC, Henderson R, Leslie AG, Tate CG, Schertler GF.
Structure of a beta1-adrenergic G-protein-coupled receptor.
Nature. 2008, 454(7203):486-91. [PDF]

G-protein-coupled receptors have a major role in transmembrane signalling in most eukaryotes and many are important drug targets. Here we report the 2.7 A resolution crystal structure of a beta(1)-adrenergic receptor in complex with the high-affinity antagonist cyanopindolol. The modified turkey (Meleagris gallopavo) receptor was selected to be in its antagonist conformation and its thermostability improved by earlier limited mutagenesis. The ligand-binding pocket comprises 15 side chains from amino acid residues in 4 transmembrane alpha-helices and extracellular loop 2. This loop defines the entrance of the ligand-binding pocket and is stabilized by two disulphide bonds and a sodium ion. Binding of cyanopindolol to the beta(1)-adrenergic receptor and binding of carazolol to the beta(2)-adrenergic receptor involve similar interactions. A short well-defined helix in cytoplasmic loop 2, not observed in either rhodopsin or the beta(2)-adrenergic receptor, directly interacts by means of a tyrosine with the highly conserved DRY motif at the end of helix 3 that is essential for receptor activation.

Friday, March 6, 2009, 11:00
Leibniz Institute for Age Research - Fritz Lipmann Institute
Beutenbergstr. 11, D-07745 Jena
Biocomputing-Verwaltungsgebäude, Seminarraum 101

Presenter: Oliver Ohlenschläger (FLI, Görlach Lab)

Casini A, Gabbiani C, Mastrobuoni G, Pellicani RZ, Intini FP, Arnesano F, Natile G, Moneti G, Francese S, Messori L
Insights into the molecular mechanisms of protein platination from a case study: the reaction of anticancer platinum(II) iminoethers with horse heart cytochrome c.
Biochemistry. 200;46:12220-30. [PDF]

The interactions of anticancer metallodrugs with proteins are attracting a growing interest in the current literature because of their relevant pharmacological and toxicological consequences. To understand in more depth the nature of those interactions, we have investigated the reactions of four anticancer platinum(II) iminoether complexes, namely, trans- and cis-EE (trans- and cis-[PtCl2{(E)-HN=C(OCH3)CH3}2], respectively) and trans- and cis-Z (trans- and cis-[PtCl2(NH3){(Z)-HN=C(OCH3)CH3}], respectively), with horse heart cytochrome c (cyt c). Our investigation was performed using mainly electrospray ionization mass spectrometry (ESI MS) but was also supported by NMR, inductively coupled plasma optical emission spectroscopy (ICP OES), and absorption electronic spectroscopy. ESI MS spectra clearly revealed the formation of a variety of platinum-protein adducts predominantly corresponding to monoplatinated cyt c species. From a careful analysis of the major ESI MS peaks, specific information on the nature of the protein-bound metallic fragments and on the underlying metallodrug-cyt c reactions was gained for the various cases. We found that trans-EE produces a major cyt c adduct (12 667 Da) that is different from that produced by either cis-EE or by trans-Z and cis-Z (12 626 Da). In particular, occurrence of extensive hydrolysis/aminolysis (the latter fostered by ammonium carbonate buffer) of the iminoether ligands and formation of the corresponding amides/amidines has been unambiguously documented. The reactivity of the iminoether ligands is greatly enhanced by the presence of cyt c as inferred from comparative NMR solution studies. Additional ESI MS measurements recorded on enzymatically cleaved samples of platinated cyt c adducts, together with NMR investigation of the cyt c/trans-EE adduct, strongly suggest that protein platination primarily occurs at Met 65. The biological and pharmacological implications of the described protein platination processes are discussed.

Casini A, Guerri A, Gabbiani C, Messori L.
Biophysical characterisation of adducts formed between anticancer metallodrugs and selected proteins: new insights from X-ray diffraction and mass spectrometry studies.
J Inorg Biochem. 2008;102:995-1006. [PDF]

There is considerable interest today for the reactions of anticancer metallodrugs with proteins as these interactions might feature processes that are crucial for the biodistribution, the toxicity and even the mechanism of action of this important group of anticancer agents. We survey here the results of research activities carried out in our "Laboratory of Metals in Medicine" (Department of Chemistry, University of Florence) during the last three years, concerning the molecular characterisation of adducts formed between platinum, ruthenium and gold metallodrugs and a few model proteins. Valuable structural and functional information on these adducts could be derived from several biophysical studies mainly relying on the application of X-ray diffraction and ESI MS techniques. The value and the limitations of both approaches are outlined through a number of examples. Remarkably, the structural and functional information achieved on the respective metallodrug-protein adducts allowed us to identify some general trends in the reactivity of anticancer metallodrugs with protein targets.

Friday, February 6, 2009, 11:00
Leibniz Institute for Age Research - Fritz Lipmann Institute
Beutenbergstr. 11, D-07745 Jena
Biocomputing-Verwaltungsgebäude, Seminarraum 101

Presenter: Jürgen Sühnel (FLI, Sühnel Lab)

Keating KS, Flores SC, Gerstein MB, Kuhn LA.
StoneHinge: Hinge prediction by network analysis of individual protein structures.
Protein Sci. 2009;18:359-371. [PDF]

Hinge motions are important for molecular recognition, and knowledge of their location can guide the sampling of protein conformations for docking. Predicting domains and intervening hinges is also important for identifying structurally self-determinate units and anticipating the influence of mutations on protein flexibility and stability. Here we present StoneHinge, a novel approach for predicting hinges between domains using input from two complementary analyses of noncovalent bond networks: StoneHingeP, which identifies domain-hinge-domain signatures in ProFlex constraint counting results, and StoneHingeD, which does the same for DomDecomp Gaussian network analyses. Predictions for the two methods are compared to hinges defined in the literature and by visual inspection of interpolated motions between conformations in a series of proteins. For StoneHingeP, all the predicted hinges agree with hinge sites reported in the literature or observed visually, although some predictions include extra residues. Furthermore, no hinges are predicted in six hinge-free proteins. On the other hand, StoneHingeD tends to overpredict the number of hinges, while accurately pinpointing hinge locations. By determining the consensus of their results, StoneHinge improves the specificity, predicting 11 of 13 hinges found both visually and in the literature for nine different open protein structures, and making no false-positive predictions. By comparison, a popular hinge detection method that requires knowledge of both the open and closed conformations finds 10 of the 13 known hinges, while predicting four additional, false hinges.

Friday, January 23, 2009, 11:00
Leibniz Institute for Age Research - Fritz Lipmann Institute
Beutenbergstr. 11, D-07745 Jena
Biocomputing-Verwaltungsgebäude, Seminarraum 101

Presenter: Manual Than (FLI, Than Lab)

Yamasaki M, Li W, Johnson DJ, Huntington JA.
Crystal structure of a stable dimer reveals the molecular basis of serpin polymerization.
Nature. 2008;455(7217):1255-8. [PDF]

Repeating intermolecular protein association by means of beta-sheet expansion is the mechanism underlying a multitude of diseases including Alzheimer's, Huntington's and Parkinson's and the prion encephalopathies. A family of proteins, known as the serpins, also forms large stable multimers by ordered beta-sheet linkages leading to intracellular accretion and disease. These 'serpinopathies' include early-onset dementia caused by mutations in neuroserpin, liver cirrhosis and emphysema caused by mutations in alpha(1)-antitrypsin (alpha(1)AT), and thrombosis caused by mutations in antithrombin. Serpin structure and function are quite well understood, and the family has therefore become a model system for understanding the beta-sheet expansion disorders collectively known as the conformational diseases. To develop strategies to prevent and reverse these disorders, it is necessary to determine the structural basis of the intermolecular linkage and of the pathogenic monomeric state. Here we report the crystallographic structure of a stable serpin dimer which reveals a domain swap of more than 50 residues, including two long antiparallel beta-strands inserting in the centre of the principal beta-sheet of the neighbouring monomer. This structure explains the extreme stability of serpin polymers, the molecular basis of their rapid propagation, and provides critical new insights into the structural changes which initiate irreversible beta-sheet expansion.

Friday, December 19, 2008, 11:00
Leibniz Institute for Age Research - Fritz Lipmann Institute
Beutenbergstr. 11, D-07745 Jena
Biocomputing-Verwaltungsgebäude, Seminarraum 101

Presenter: Rolf Hühne (FLI, Sühnel Lab)

Kumar P, Ziegler A, Ziegler J, Uchanska-Ziegler B, Ziegler A
Grasping molecular structures through publication-integrated 3D models.
Trends Biochem Sci. 2008 ;33:408-12. [PDF]

Although the need for communicating 3D data using simple and intuitive means extends to disciplines as diverse as biology, engineering sciences and the visual arts, none of the currently available molecular-visualization programs depicting potentially highly complex structures are compatible with the portable document format (PDF), the current gold standard of electronic publishing. Therefore, it is highly desirable for authors to be able to provide their readers with a basic 3D display of structures that can be accessed without the need for specialized visualization software. Here, we describe how an interactive 3D model of a molecular complex can be embedded directly into a PDF, thus providing readers with important and educational visual information that would otherwise be more difficult to disseminate.

Friday, December 5, 2008, 11:00
Leibniz Institute for Age Research - Fritz Lipmann Institute
Beutenbergstr. 11, D-07745 Jena
Biocomputing-Verwaltungsgebäude, Seminarraum 101

Presenter: Dirk Röser (FLI, Than Lab)

Drew D, Newstead S, Sonoda Y, Kim H, von Heijne G, Iwata S.
GFP-based optimization scheme for the overexpression and purification of eukaryotic membrane proteins in Saccharomyces cerevisiae.
Nat Protoc. 2008;3(5):784-98. [PDF]

It is often difficult to produce eukaryotic membrane proteins in large quantities, which is a major obstacle for analyzing their biochemical and structural features. To date, yeast has been the most successful heterologous overexpression system in producing eukaryotic membrane proteins for high-resolution structural studies. For this reason, we have developed a protocol for rapidly screening and purifying eukaryotic membrane proteins in the yeast Saccharomyces cerevisiae. Using this protocol, in 1 week many genes can be rapidly cloned by homologous recombination into a 2 micro GFP-fusion vector and their overexpression potential determined using whole-cell and in-gel fluorescence. The quality of the overproduced eukaryotic membrane protein-GFP fusions can then be evaluated over several days using confocal microscopy and fluorescence size-exclusion chromatography (FSEC). This protocol also details the purification of targets that pass our quality criteria, and can be scaled up for a large number of eukaryotic membrane proteins in either an academic, structural genomics or commercial environment.

Drew D, Lerch M, Kunji E, Slotboom DJ, de Gier JW.
Optimization of membrane protein overexpression and purification using GFP fusions.
Nat Methods. 2006 Apr;3(4):303-13. [PDF]

Friday, November 28, 2008, 11:00
Leibniz Institute for Age Research - Fritz Lipmann Institute
Beutenbergstr. 11, D-07745 Jena
Biocomputing-Verwaltungsgebäude, Seminarraum 101

Presenter: Maik Friedel (FLI, Sühnel Lab)

Verstrepen KJ, Jansen A, Lewitter F, Fink GR.
Intragenic tandem repeats generate functional variability.
Nat Genet. 2005;37:986-90. [PDF]

Tandemly repeated DNA sequences are highly dynamic components of genomes. Most repeats are in intergenic regions, but some are in coding sequences or pseudogenes. In humans, expansion of intragenic triplet repeats is associated with various diseases, including Huntington chorea and fragile X syndrome. The persistence of intragenic repeats in genomes suggests that there is a compensating benefit. Here we show that in the genome of Saccharomyces cerevisiae, most genes containing intragenic repeats encode cell-wall proteins. The repeats trigger frequent recombination events in the gene or between the gene and a pseudogene, causing expansion and contraction in the gene size. This size variation creates quantitative alterations in phenotypes (e.g., adhesion, flocculation or biofilm formation). We propose that variation in intragenic repeat number provides the functional diversity of cell surface antigens that, in fungi and other pathogens, allows rapid adaptation to the environment and elusion of the host immune system.

Friday, November 14, 2008, 11:00
Leibniz Institute for Age Research - Fritz Lipmann Institute
Beutenbergstr. 11, D-07745 Jena
Biocomputing-Verwaltungsgebäude, Seminarraum 101

Presenter: Thomas Seiboth (FLI, Görlach Lab)

Vallurupalli P, Hansen DF, Kay LE.
Structures of invisible, excited protein states by relaxation dispersion NMR spectroscopy.
Proc Natl Acad Sci U S A. 2008, 105:11766-71. [PDF], Supporting Information [PDF]

Molecular function is often predicated on excursions between ground states and higher energy conformers that can play important roles in ligand binding, molecular recognition, enzyme catalysis, and protein folding. The tools of structural biology enable a detailed characterization of ground state structure and dynamics; however, studies of excited state conformations are more difficult because they are of low population and may exist only transiently. Here we describe an approach based on relaxation dispersion NMR spectroscopy in which structures of invisible, excited states are obtained from chemical shifts and residual anisotropic magnetic interactions. To establish the utility of the approach, we studied an exchanging protein (Abp1p SH3 domain)-ligand (Ark1p peptide) system, in which the peptide is added in only small amounts so that the ligand-bound form is invisible. From a collection of (15)N, (1)HN, (13)C(alpha), and (13)CO chemical shifts, along with (1)HN-(15)N, (1)H(alpha)-(13)C(alpha), and (1)HN-(13)CO residual dipolar couplings and (13)CO residual chemical shift anisotropies, all pertaining to the invisible, bound conformer, the structure of the bound state is determined. The structure so obtained is cross-validated by comparison with (1)HN-(15)N residual dipolar couplings recorded in a second alignment medium. The methodology described opens up the possibility for detailed structural studies of invisible protein conformers at a level of detail that has heretofore been restricted to applications involving visible ground states of proteins.

Friday, October 24, 2008, 11:00
Leibniz Institute for Age Research - Fritz Lipmann Institute
Beutenbergstr. 11, D-07745 Jena
Biocomputing-Verwaltungsgebäude, Seminarraum 101

Presenter: Christian Herbst (FLI, Görlach Lab)

Linser R, Fink U, Reif B.
Proton-detected scalar coupling based assignment strategies in MAS solid-state NMR spectroscopy applied to perdeuterated proteins.
J Magn Reson. 2008, 193 :89-93. [PDF]

Assignment of proteins in MAS (magic angle spinning) solid-state NMR relies so far on correlations among heteronuclei. This strategy is based on well dispersed resonances in the (15)N dimension. In many complex cases like membrane proteins or amyloid fibrils, an additional frequency dimension is desirable in order to spread the amide resonances. We show here that proton detected HNCO, HNCA, and HNCACB type experiments can successfully be implemented in the solid-state. Coherences are sufficiently long lived to allow pulse schemes of a duration greater than 70 ms before incrementation of the first indirect dimension. The achieved resolution is comparable to the resolution obtained in solution-state NMR experiments. We demonstrate the experiments using a triply labeled sample of the SH3 domain of chicken alpha-spectrin, which was re-crystallized in H(2)O/D(2)O using a ratio of 1/9. We employ paramagnetic relaxation enhancement (PRE) using EDTA chelated Cu(II) to enable rapid data acquisition.

Friday, October 10, 2008, 11:00
Leibniz Institute for Age Research - Fritz Lipmann Institute
Beutenbergstr. 11, D-07745 Jena
Biocomputing-Verwaltungsgebäude, Seminarraum 101

Presenter: Yvonne Ihle (FLI, Görlach Lab)

Hart JM, Kennedy SD, Mathews DH, Turner DH.
NMR-assisted prediction of RNA secondary structure: identification of a probable pseudoknot in the coding region of an R2 retrotransposon.
J Am Chem Soc. 2008, 130: 10233-10239. [PDF]. Supporting information [PDF].

As the rate of functional RNA sequence discovery escalates, high-throughput techniques for reliable structural determination are becoming crucial for revealing the essential features of these RNAs in a timely fashion. Computational predictions of RNA secondary structure quickly generate reasonable models but suffer from several approximations, including overly simplified models and incomplete knowledge of significant interactions. Similar problems limit the accuracy of predictions for other self-folding polymers, including DNA and peptide nucleic acid (PNA). The work presented here demonstrates that incorporating unassigned data from simple nuclear magnetic resonance (NMR) experiments into a dynamic folding algorithm greatly reduces the potential folding space of a given RNA and therefore increases the confidence and accuracy of modeling. This procedure has been packaged into an NMR-assisted prediction of secondary structure (NAPSS) algorithm that can produce pseudoknotted as well as non-pseudoknotted secondary structures. The method reveals a probable pseudoknot in the part of the coding region of the R2 retrotransposon from Bombyx mori that orchestrates second-strand DNA cleavage during insertion into the genome.

Friday, September 26, 2008, 11:00
Leibniz Institute for Age Research - Fritz Lipmann Institute
Beutenbergstr. 11, D-07745 Jena
Biocomputing-Verwaltungsgebäude, Seminarraum 101

Presenter: Vladimir Shelest (HKI, Department of Molecular and Applied Microbiology )

Fourier transformation and its application in the spectral analysis of DNA sequences

Related information:

Li W.

The study of correlation structures of DNA sequences: a critical review.

Comput Chem. 1997, 21:257-271. [PDF]

Lobzin V, Chechetkin VR.
Order and correlations in genomic DNA sequences. The spectral approach.
Physics - Uspekhi 2000, 43:55 - 78. [PDF]

Friday, September 12, 2008, 11:00
Leibniz Institute for Age Research - Fritz Lipmann Institute
Beutenbergstr. 11, D-07745 Jena
Biocomputing-Verwaltungsgebäude, Seminarraum 101

Presenter: Andre Mischo (FLI, Görlach Lab)

Okazaki K, Takada S.

Dynamic energy landscape view of coupled binding and protein conformational change: induced-fit versus population-shift mechanisms.

Proc Natl Acad Sci U S A. 2008, 105:11182-7. [PDF]

Allostery, the coupling between ligand binding and protein conformational change, is the heart of biological network and it has often been explained by two representative models, the induced-fit and the population-shift models. Here, we clarified for what systems one model fits better than the other by performing molecular simulations of coupled binding and conformational change. Based on the dynamic energy landscape view, we developed an implicit ligand-binding model combined with the double-basin Hamiltonian that describes conformational change. From model simulations performed for a broad range of parameters, we uncovered that each of the two models has its own range of applicability, stronger and longer-ranged interaction between ligand and protein favors the induced-fit model, and weaker and shorter-ranged interaction leads to the population-shift model. We further postulate that the protein binding to small ligand tends to proceed via the population-shift model, whereas the protein docking to macromolecules such as DNA tends to fit the induced-fit model.

Friday, July 18, 2008, 11:00
Leibniz Institute for Age Research - Fritz Lipmann Institute
Beutenbergstr. 11, D-07745 Jena
Biocomputing-Verwaltungsgebäude, Seminarraum 101

Presenter: Michela Carelle (FLI, Görlach Lab)

Forneris F, Mattevi A.
Enzymes without borders: mobilizing substrates, delivering products.Science. 2008, 321:213-6. [PDF]

Many cellular reactions involve both hydrophobic and hydrophilic molecules that reside within the chemically distinct environments defined by the phospholipid-based membranes and the aqueous lumens of cytoplasm and organelles. Enzymes performing this type of reaction are required to access a lipophilic substrate located in the membranes and to catalyze its reaction with a polar, water-soluble compound. Here, we explore the different binding strategies and chemical tricks that enzymes have developed to overcome this problem. These reactions can be catalyzed by integral membrane proteins that channel a hydrophilic molecule into their active site, as well as by water-soluble enzymes that are able to capture a lipophilic substrate from the phospholipid bilayer. Many chemical and biological aspects of this type of enzymology remain to be investigated and will require the integration of protein chemistry with membrane biology.

Friday, July 04, 2008, 11:00
Leibniz Institute for Age Research - Fritz Lipmann Institute
Beutenbergstr. 11, D-07745 Jena
Biocomputing-Verwaltungsgebäude, Seminarraum 101

Presenter: Miriam Küster (FLI, Than Lab)

Kwon HJ, Lagace TA, McNutt MC, Horton JD, Deisenhofer J.
Molecular basis for LDL receptor recognition by PCSK9.
Proc Natl Acad Sci U S A. 2008 105(6):1820-5. [PDF]

Proprotein convertase subtilisin/kexin type 9 (PCSK9) posttranslationally regulates hepatic low-density lipoprotein receptors (LDLRs) by binding to LDLRs on the cell surface, leading to their degradation. The binding site of PCSK9 has been localized to the epidermal growth factor-like repeat A (EGF-A) domain of the LDLR. Here, we describe the crystal structure of a complex between PCSK9 and the EGF-A domain of the LDLR. The binding site for the LDLR EGF-A domain resides on the surface of PCSK9's subtilisin-like catalytic domain containing Asp-374, a residue for which a gain-of-function mutation (Asp-374-Tyr) increases the affinity of PCSK9 toward LDLR and increases plasma LDL-cholesterol (LDL-C) levels in humans. The binding surface on PCSK9 is distant from its catalytic site, and the EGF-A domain makes no contact with either the C-terminal domain or the prodomain. Point mutations in PCSK9 that altered key residues contributing to EGF-A binding (Arg-194 and Phe-379) greatly diminished binding to the LDLR's extracellular domain. The structure of PCSK9 in complex with the LDLR EGF-A domain defines potential therapeutic target sites for blocking agents that could interfere with this interaction in vivo, thereby increasing LDLR function and reducing plasma LDL-C levels.

Friday, June 13, 2008, 11:00
Leibniz Institute for Age Research - Fritz Lipmann Institute
Beutenbergstr. 11, D-07745 Jena
Biocomputing-Verwaltungsgebäude, Seminarraum 101

Presenter: Thomas Seiboth (FLI, Görlach Lab)

Tuinstra RL, Peterson FC, Kutlesa S, Elgin ES, Kron MA, Volkman BF.
Interconversion between two unrelated protein folds in the lymphotactin native state.
Proc Natl Acad Sci U S A. 2008 105(13):5057-62. [PDF]

Proteins often have multiple functional states, which might not always be accommodated by a single fold. Lymphotactin (Ltn) adopts two distinct structures in equilibrium, one corresponding to the canonical chemokine fold consisting of a monomeric three-stranded beta-sheet and carboxyl-terminal helix. The second Ltn structure solved by NMR reveals a dimeric all-beta-sheet arrangement with no similarity to other known proteins. In physiological solution conditions, both structures are significantly populated and interconvert rapidly. Interconversion replaces long-range interactions that stabilize the chemokine fold with an entirely new set of tertiary and quaternary contacts. The chemokine-like Ltn conformation is a functional XCR1 agonist, but fails to bind heparin. In contrast, the alternative structure binds glycosaminoglycans with high affinity but fails to activate XCR1. Because each structural species displays only one of the two functional properties essential for activity in vivo, the conformational equilibrium is likely to be essential for the biological activity of lymphotactin. These results demonstrate that the functional repertoire and regulation of a single naturally occurring amino acid sequence can be expanded by access to a set of highly dissimilar native-state structures.

Friday, June 06, 2008, 11:00
Leibniz Institute for Age Research - Fritz Lipmann Institute
Beutenbergstr. 11, D-07745 Jena
Biocomputing-Verwaltungsgebäude, Seminarraum 101

Presenter: Christian Herbst (FLI, Görlach Lab)

Zhou DH, Shea JJ, Nieuwkoop AJ, Franks WT, Wylie BJ, Mullen C, Sandoz D, Rienstra CM.
Solid-state protein-structure determination with proton-detected triple-resonance 3D magic-angle-spinning NMR spectroscopy.
Angew Chem Int Ed Engl. 2007;:8380-3. [PDF]

Friday, May 09, 2008, 11:00
Leibniz Institute for Age Research - Fritz Lipmann Institute
Beutenbergstr. 11, D-07745 Jena
Biocomputing-Verwaltungsgebäude, Seminarraum 101

Presenter: Jessica Meinhardt (FLI, Fändrich / Görlach Labs)

Chimon S, Shaibat MA, Jones CR, Calero DC, Aizezi B, Ishii Y.
Evidence of fibril-like beta-sheet structures in a neurotoxic amyloid intermediate of Alzheimer's beta-amyloid.
Nat Struct Mol Biol. 2007 Dec 2 [PDF]

Diffusible subfibrillar aggregates of amyloid proteins are potent neurotoxins and primary suspects in amyloid diseases including Alzheimer's disease. Despite widespread interest, the molecular structures of the amyloid intermediates and the conformational conversions in amyloid misfolding are poorly understood. Here we present a molecular-level examination of sequence-specific secondary structures and supramolecular structures of a neurotoxic amyloid intermediate of the 40-residue beta-amyloid (Abeta) peptide involved in Alzheimer's disease. Using solid-state NMR and electron microscopy, we show that, before fibrillization, natively unstructured monomeric Abeta is subject to large conformational changes into a spherical amyloid intermediate of 15-35 nm diameter, which has predominantly parallel beta-sheet structures. Structural comparison with Abeta fibrils demonstrates that formation of this beta-sheet intermediate (I(beta)) largely defines conformational transitions in amyloid misfolding. Neurotoxicity assays on PC12 cells show that I(beta) shows higher toxicity than the fibril, indicating that the beta-sheet formation may trigger neurotoxicity.

Friday, April 25, 2008, 11:00
Leibniz Institute for Age Research - Fritz Lipmann Institute
Beutenbergstr. 11, D-07745 Jena
Biocomputing-Verwaltungsgebäude, Seminarraum 101

Presenter: Yvonne Ihle (FLI, Görlach Lab)

Gilbert SD, Rambo RP, Van Tyne D, Batey RT.
Structure of the SAM-II riboswitch bound to S-adenosylmethionine.
Nat Struct Mol Biol. 2008 15:177-82. [PDF: Paper | Supplementary Information]

In bacteria, numerous genes harbor regulatory elements in the 5' untranslated regions of their mRNA, termed riboswitches, which control gene expression by binding small-molecule metabolites. These sequences influence the secondary and tertiary structure of the RNA in a ligand-dependent manner, thereby directing its transcription or translation. The crystal structure of an S-adenosylmethionine-responsive riboswitch found predominantly in proteobacteria, SAM-II, has been solved to reveal a second means by which RNA interacts with this important cellular metabolite. Notably, this is the first structure of a complete riboswitch containing all sequences associated with both the ligand binding aptamer domain and the regulatory expression platform. Chemical probing of this RNA in the absence and presence of ligand shows how the structure changes in response to S-adenosylmethionine to sequester the ribosomal binding site and affect translational gene regulation.

Friday, April 11, 2008, 11:00
Leibniz Institute for Age Research - Fritz Lipmann Institute
Beutenbergstr. 11, D-07745 Jena
Biocomputing-Verwaltungsgebäude, Seminarraum 101

Presenter: Maik Friedel (FLI, Sühnel Lab)

Bock C, Lengauer T.
Computational epigenetics.
Bioinformatics. 2008;24:1-10. [PDF]

Epigenetic research aims to understand heritable gene regulation that is not directly encoded in the DNA sequence. Epigenetic mechanisms such as DNA methylation and histone modifications modulate the packaging of the DNA in the nucleus and thereby influence gene expression. Patterns of epigenetic information are faithfully propagated over multiple cell divisions, which makes epigenetic regulation a key mechanism for cellular differentiation and cell fate decisions. In addition, incomplete erasure of epigenetic information can lead to complex patterns of non-Mendelian inheritance. Stochastic and environment-induced epigenetic defects are known to play a major role in cancer and ageing, and they may also contribute to mental disorders and autoimmune diseases. Recent technical advances such as ChIP-on-chip and ChIP-seq have started to convert epigenetic research into a high-throughput endeavor, to which bioinformatics is expected to make significant contributions. Here, we review pioneering computational studies that have contributed to epigenetic research. In addition, we give a brief introduction into epigenetics-targeted at bioinformaticians who are new to the field-and we outline future challenges in computational epigenetics.

Friday, April 4, 2008, 11:00
Leibniz Institute for Age Research - Fritz Lipmann Institute
Beutenbergstr. 11, D-07745 Jena
Biocomputing-Verwaltungsgebäude, Seminarraum 101

Presenter: Andre Mischo (FLI, Görlach Lab)

Brown C, Kowalczyk AM, Taylor ER, Morgan IM, Gaston K.
P53 represses human papillomavirus type 16 DNA replication via the viral E2 protein.
Virol J. 2008;5:5. [PDF]

BACKGROUND: Human papillomavirus (HPV) DNA replication can be inhibited by the cellular tumour suppressor protein p53. However, the mechanism through which p53 inhibits viral replication and the role that this might play in the HPV life cycle are not known. The papillomavirus E2 protein is required for efficient HPV DNA replication and also regulates viral gene expression. E2 represses transcription of the HPV E6 and E7 oncogenes and can thereby modulate indirectly host cell proliferation and survival. In addition, the E2 protein from HPV 16 has been shown to bind p53 and to be capable of inducing apoptosis independently of E6 and E7. RESULTS: Here we use a panel of E2 mutants to confirm that mutations which block the induction of apoptosis via this E6/E7-independent pathway, have little or no effect on the induction of apoptosis by the E6/E7-dependent pathway. Although these mutations in E2 do not affect the ability of the protein to mediate HPV DNA replication, they do abrogate the repressive effects of p53 on the transcriptional activity of E2 and prevent the inhibition of E2-dependent HPV DNA replication by p53. CONCLUSION: These data suggest that p53 down-regulates HPV 16 DNA replication via the E2 protein.

Friday, February 22, 2008, 11:00
Leibniz Institute for Age Research - Fritz Lipmann Institute
Beutenbergstr. 11, D-07745 Jena
Biocomputing-Verwaltungsgebäude, Seminarraum 101

Presenter: Jürgen Sühnel (FLI, Sühnel Lab)

Kato M, Slack FJ.
microRNAs: small molecules with big roles - C. elegans to human cancer.
Biol Cell. 2008;100:71-81. [PDF]

miRNAs (microRNAs) were first discovered as critical regulators of developmental timing events in Caenorhabditis elegans. Subsequent studies have shown that miRNAs and cellular factors necessary for miRNA biogenesis are conserved in many organisms, suggesting the importance of miRNAs during developmental processes. Indeed, mutations in the miRNA-processing pathway induce pleiotropic defects in development, which accompany perturbation of correct expression of target genes. However, control of gene expression in development is not the only function of miRNAs. Recent work has provided new insights into the role of miRNAs in various biological events, including aging and cancer. C. elegans continues to be helpful in facilitating a further understanding of miRNA function in human diseases.

Friday, February 22, 2008, 11:00
Leibniz Institute for Age Research - Fritz Lipmann Institute
Beutenbergstr. 11, D-07745 Jena
Biocomputing-Verwaltungsgebäude, Seminarraum 101

Presenter: Dirk Röser (FLI, Than Lab)

Feng L, Yan H, Wu Z, Yan N, Wang Z, Jeffrey PD, Shi Y.
Structure of a site-2 protease family intramembrane metalloprotease.
Science. 2007;318(5856):1608-12. [PDF]

Regulated intramembrane proteolysis by members of the site-2 protease (S2P) family is an important signaling mechanism conserved from bacteria to humans. Here we report the crystal structure of the transmembrane core domain of an S2P metalloprotease from Methanocaldococcus jannaschii. The protease consists of six transmembrane segments, with the catalytic zinc atom coordinated by two histidine residues and one aspartate residue approximately 14 angstroms into the lipid membrane surface. The protease exhibits two distinct conformations in the crystals. In the closed conformation, the active site is surrounded by transmembrane helices and is impermeable to substrate peptide; water molecules gain access to zinc through a polar, central channel that opens to the cytosolic side. In the open conformation, transmembrane helices alpha1 and alpha6 separate from each other by 10 to 12 angstroms, exposing the active site to substrate entry. The structure reveals how zinc embedded in an integral membrane protein can catalyze peptide cleavage.

Friday, February 8, 2008, 11:00
Leibniz Institute for Age Research - Fritz Lipmann Institute
Beutenbergstr. 11, D-07745 Jena
Biocomputing-Verwaltungsgebäude, Seminarraum 101

Presenter: Miriam Küster (FLI, Than Lab)

Hampton EN, Knuth MW, Li J, Harris JL, Lesley SA, Spraggon G.
The self-inhibited structure of full-length PCSK9 at 1.9 A reveals structural homology with resistin within the C-terminal domain.
Proc Natl Acad Sci U S A. 2007;104(37):14604-9. Epub 2007 Sep 5. [PDF]

Mutations in proprotein convertase subtilisin/kexin type 9 (PCSK9) are strongly associated with levels of low-density lipoprotein cholesterol in the blood plasma and, thereby, occurrence or resistance to atherosclerosis and coronary heart disease. Despite this importance, relatively little is known about the biology of PCSK9. Here, the crystal structure of a full-length construct of PCSK9 solved to 1.9-A resolution is presented. The structure contains a fully folded C-terminal cysteine-rich domain (CRD), showing a distinct structural similarity to the resistin homotrimer, a small cytokine associated with obesity and diabetes. This structural relationship between the CRD of PCSK9 and the resistin family is not observed in primary sequence comparisons and strongly suggests a distant evolutionary link between the two molecules. This three-dimensional homology provides insight into the function of PCSK9 at the molecular level and will help to dissect the link between PCSK9 and CHD.

Friday, January 25, 2008, 11:00
Leibniz Institute for Age Research - Fritz Lipmann Institute
Beutenbergstr. 11, D-07745 Jena
Biocomputing-Verwaltungsgebäude, Seminarraum 101

Presenter: Frank-Thomas Koch (FLI, Sühnel Lab)

Shamim MT, Anwaruddin M, Nagarajaram HA.
Support Vector Machine-based classification of protein folds using the structural properties of amino acid residues and amino acid residue pairs.

Bioinformatics. 2007;23:3320-7. [PDF]

MOTIVATION: Fold recognition is a key step in the protein structure discovery process, especially when traditional sequence comparison methods fail to yield convincing structural homologies. Although many methods have been developed for protein fold recognition, their accuracies remain low. This can be attributed to insufficient exploitation of fold discriminatory features. RESULTS: We have developed a new method for protein fold recognition using structural information of amino acid residues and amino acid residue pairs. Since protein fold recognition can be treated as a protein fold classification problem, we have developed a Support Vector Machine (SVM) based classifier approach that uses secondary structural state and solvent accessibility state frequencies of amino acids and amino acid pairs as feature vectors. Among the individual properties examined secondary structural state frequencies of amino acids gave an overall accuracy of 65.2% for fold discrimination, which is better than the accuracy by any method reported so far in the literature. Combination of secondary structural state frequencies with solvent accessibility state frequencies of amino acids and amino acid pairs further improved the fold discrimination accuracy to more than 70%, which is approximately 8% higher than the best available method. In this study we have also tested, for the first time, an all-together multi-class method known as Crammer and Singer method for protein fold classification. Our studies reveal that the three multi-class classification methods, namely one versus all, one versus one and Crammer and Singer method, yield similar predictions. AVAILABILITY: Dataset and stand-alone program are available upon request.

Friday, January 11, 2008, 11:00
Leibniz Institute for Age Research - Fritz Lipmann Institute
Beutenbergstr. 11, D-07745 Jena
Biocomputing-Verwaltungsgebäude, Seminarraum 101

Presenter: Sven Dahms (FLI, Than Lab)

Kong GK, Miles LA, Crespi GA, Morton CJ, Ng HL, Barnham KJ, McKinstry WJ, Cappai R, Parker MW.
Copper binding to the Alzheimer's disease amyloid precursor protein.

Eur Biophys J. 2007 Nov 21 [PDF]

Alzheimer's disease is the fourth biggest killer in developed countries. Amyloid precursor protein (APP) plays a central role in the development of the disease, through the generation of a peptide called Abeta by proteolysis of the precursor protein. APP can function as a metalloprotein and modulate copper transport via its extracellular copper binding domain (CuBD). Copper binding to this domain has been shown to reduce Abeta levels and hence a molecular understanding of the interaction between metal and protein could lead to the development of novel therapeutics to treat the disease. We have recently determined the three-dimensional structures of apo and copper bound forms of CuBD. The structures provide a mechanism by which CuBD could readily transfer copper ions to other proteins. Importantly, the lack of significant conformational changes to CuBD on copper binding suggests a model in which copper binding affects the dimerisation state of APP leading to reduction in Abeta production. We thus predict that disruption of APP dimers may be a novel therapeutic approach to treat Alzheimer's disease.

Friday, December 14, 2007, 11:00
Leibniz Institute for Age Research - Fritz Lipmann Institute
Beutenbergstr. 11, D-07745 Jena
Biocomputing-Verwaltungsgebäude, Seminarraum 101

Presenter: Maik Friedel (FLI, Sühnel Lab)

Abouelhoda MI, Kurtz S, Ohlebusch E.
Replacing suffix trees with enhanced suffix arrays
Journal of Discrete Algorithms 2004; 2: 53–86 [PDF]

The suffix tree is one of the most important data structures in string processing and comparative
genomics. However, the space consumption of the suffix tree is a bottleneck in large scale applications
such as genome analysis. In this article, we will overcome this obstacle. We will show how
every algorithm that uses a suffix tree as data structure can systematically be replaced with an algorithm
that uses an enhanced suffix array and solves the same problem in the same time complexity.
The generic name enhanced suffix array stands for data structures consisting of the suffix array and
additional tables. Our new algorithms are not only more space efficient than previous ones, but they
are also faster and easier to implement.

Friday, November 30, 2007, 11:00
Leibniz Institute for Age Research - Fritz Lipmann Institute
Beutenbergstr. 11, D-07745 Jena
Biocomputing-Verwaltungsgebäude, Seminarraum 101

Presenter: Mario Schubert (Guest in the Görlach Lab at the FLI; from the Institute of Molecular Biology and Biophysics at the ETH Zürich )

Schubert M, Lapouge K, Duss O, Oberstrass FC, Jelesarov I, Haas D, Allain FH.
Molecular basis of messenger RNA recognition by the specific bacterial repressing clamp RsmA/CsrA.
Nat Struct Mol Biol. 2007;14:807-13. [PDF]

Proteins of the RsmA/CsrA family are global translational regulators in many bacterial species. We have determined the solution structure of a complex formed between the RsmE protein, a member of this family from Pseudomonas fluorescens, and a target RNA encompassing the ribosome-binding site of the hcnA gene. The RsmE homodimer with its two RNA-binding sites makes optimal contact with an 5'-A/UCANGGANGU/A-3' sequence in the mRNA. When tightly gripped by RsmE, the ANGGAN core folds into a loop, favoring the formation of a 3-base-pair stem by flanking nucleotides. We validated these findings by in vivo and in vitro mutational analyses. The structure of the complex explains well how, by sequestering the Shine-Dalgarno sequence, the RsmA/CsrA proteins repress translation.

Friday, November 23, 2007, 11:00
Leibniz Institute for Age Research - Fritz Lipmann Institute
Beutenbergstr. 11, D-07745 Jena
Biocomputing-Verwaltungsgebäude, Seminarraum 101

Presenter: Thomas Seiboth (FLI, Görlach Lab)

Pervushin K, Vamvaca K, Vögeli B, Hilvert D.

Structure and dynamics of a molten globular enzyme.

Nat Struct Mol Biol. 2007 Nov 11 [PDF]

Although protein dynamics has been recognized as a potentially important contributor to enzyme catalysis, structural disorder is generally considered to reduce catalytic efficiency. This widely held assumption has recently been challenged by the finding that an engineered chorismate mutase combines high catalytic activity with the properties of a molten globule, a loosely packed and highly dynamic conformational ensemble. Taking advantage of the ordering observed upon ligand binding, we have now used NMR spectroscopy to characterize this enzyme in complex with a transition-state analog. The complex adopts a helix-bundle structure, as designed, but retains unprecedented flexibility on the millisecond timescale across its entire length. Moreover, pre-steady-state kinetics data show that binding occurs by an induced-fit mechanism on the same timescale as the enzymatic reaction, linking global conformational plasticity with efficient catalysis.

Friday, November 9, 2007, 11:00
Leibniz Institute for Age Research - Fritz Lipmann Institute
Beutenbergstr. 11, D-07745 Jena
Biocomputing-Verwaltungsgebäude, Seminarraum 101

Presenter: Eberhard Schmitt (FLI, Sühnel Lab)

Geci I, Filichev VV, Pedersen EB.
Stabilization of parallel triplexes by twisted intercalating nucleic acids (TINAs) incorporating 1,2,3-triazole units and prepared by microwave-accelerated click chemistry.
Chemistry. 2007;13:6379-6386. [PDF] Supplementary Material [PDF]

A highly efficient method for postsynthetic modification of unprotected oligonucleotides incorporating internal insertions of (R)-1-O-(4-ethynylbenzyl)glycerol has been developed through the application of click chemistry with water-insoluble pyren-1-yl azide and water-soluble benzyl azide and acceleration by microwave irradiation. The twisted intercalating nucleic acids (TINAs) obtained in these reactions, possessing bulged insertions of (R)-3-O-{4-[1-(pyren-1-yl)-1H-1,2,3-triazol-4-yl]benzyl}glycerol (7), formed parallel triplexes with thermal stabilities of 20.0, 34.0, and 40.0 degrees C at pH 7.2 in the cases of one, two, or three insertions of 7, respectively, separated by three nucleic bases. An oligonucleotide with four insertions of 7--each between three nucleic bases in the sequence--was unable to form complexes with complementary single- or double-stranded DNAs, as a result of self-aggregation of the pyrene moieties. This assumption was supported by the formation of a very strong excimer band at 460 nm in the fluorescence spectra. Molecular modeling of the parallel triplex with bulged insertion of the monomer 7 in the triplex-forming oligonucleotide (TFO) showed that only the pyrene moiety was stacking between the bases of the dsDNA, whereas 1,2,3-triazole did not participate in the triplex stabilization. Thermal denaturation studies of the duplexes and triplexes, as well as the fluorescence properties of TINA-triazole 7, are discussed and compared with previous studies on TINA.

Friday, October 19, 2007, 11:00
Leibniz Institute for Age Research - Fritz Lipmann Institute
Beutenbergstr. 11, D-07745 Jena
Biocomputing-Verwaltungsgebäude, Seminarraum 101

Presenter: Kerstin Riedel (FLI, Görlach Lab)

Malcom H. Levitt
Symmetry-based pulse sequences in magic-angle spinning solid-state NMR
Encyclopedia of NMR 2002, 9:165 [no PDF file available]

Friday, October 5, 2007, 11:00
Leibniz Institute for Age Research - Fritz Lipmann Institute
Beutenbergstr. 11, D-07745 Jena
Biocomputing-Verwaltungsgebäude, Seminarraum 101

Presenter: Ramadurai Ramachandran (FLI, Görlach Lab)

Jordan J.B. et al.
Three-dimensional 13C-detected CH3-TOCSY using selectively protonated proteins: Facile methyl resonance assignment and protein structure determination.
J. Am. Chem. Soc. 2006, 128:9119-9128. [PDF]

Recent advances in instrumentation and isotope labeling methodology allow proteins up to 100
kDa in size to be studied in detail using NMR spectroscopy. Using 2H/13C/15N enrichment and selective methyl protonation, we show that newly developed 13C direct detection methods can be used to rapidly yield proton and carbon resonance assignments for the methyl groups of Val, Leu, and Ile residues. We present a highly sensitive 13C-detected CH3-TOCSY experiment that, in combination with standard 1Hdetected backbone experiments, allows the full assignment of side chain resonances in methyl-protonated residues. Selective methyl protonation, originally developed by Kay and co-workers (Rosen, M. K.; Gardner, K. H.; Willis, R. C.; Parris, W. E.; Pawson, T.; Kay, L. E. J. Mol. Biol. 1996, 263, 627-636; Gardner, K. G.; Kay, L. E. Annu. Rev. Biophys. Biomol. Struct. 1998, 27, 357-406; Goto, N. K.; Kay, L. E. Curr. Opin. Struct. Biol. 2000, 10, 585-592), improves the nuclear relaxation behavior of larger proteins compared to their fully protonated counterparts, allows significant simplification of spectra, and facilitates NOE assignments. Here, we demonstrate the usefulness of the 13C-detected CH3-TOCSY experiment through studies of (i) a medium-sized protein (CbpA-R1; 14 kDa) with a repetitive primary sequence that yields highly degenerate NMR spectra, and (ii) a larger, bimolecular protein complex (p21-KID/Cdk2; 45 kDa) at low concentration in a high ionic strength solution. Through the analysis of NOEs involving amide and Ile, Leu, and Val methyl protons, we determined the global fold of CbpA-R1, a bacterial protein that mediates the pathogenic effects of Streptococcus pneumoniae, demonstrating that this approach can significantly reduce the time required to determine protein structures by NMR.

Friday, September 21, 2007, 11:00
Leibniz Institute for Age Research - Fritz Lipmann Institute
Beutenbergstr. 11, D-07745 Jena
Biocomputing-Verwaltungsgebäude, Seminarraum 101

Presenter: Oliver Ohlenschläger (FLI, Görlach Lab)

Tidow H, Melero R, Mylonas E, Freund SM, Grossmann JG, Carazo JM, Svergun DI, Valle M, Fersht AR.
Quaternary structures of tumor suppressor p53 and a specific p53 DNA complex.
Proc Natl Acad Sci U S A. 2007 Jul 24;104(30):12324-9. Epub 2007 Jul 9. [PDF]

The homotetrameric tumor suppressor p53 consists of folded core and tetramerization domains, linked and flanked by intrinsically disordered segments that impede structure analysis by x-ray crystallography and NMR. Here, we solved the quaternary structure of human p53 in solution by a combination of small-angle x-ray scattering, which defined its shape, and NMR, which identified the core domain interfaces and showed that the folded domains had the same structure in the intact protein as in fragments. We combined the solution data with electron microscopy on immobilized samples that provided medium resolution 3D maps. Ab initio and rigid body modeling of scattering data revealed an elongated cross-shaped structure with a pair of loosely coupled core domain dimers at the ends, which are accessible for binding to DNA and partner proteins. The core domains in that open conformation closed around a specific DNA response element to form a compact complex whose structure was independently determined by electron microscopy. The structure of the DNA complex is consistent with that of the complex of four separate core domains and response element fragments solved by x-ray crystallography and contacts identified by NMR. Electron microscopy on the conformationally mobile, unbound p53 selected a minor compact conformation, which resembled the closed conformation, from the ensemble of predominantly open conformations. A multipronged structural approach could be generally useful for the structural characterization of the rapidly growing number of multidomain proteins with intrinsically disordered regions.

Friday, September 7, 2007, 11:00
Leibniz Institute for Age Research - Fritz Lipmann Institute
Beutenbergstr. 11, D-07745 Jena
Biocomputing-Verwaltungsgebäude, Seminarraum 101

Presenter: André Mischo (FLI, Görlach Lab)

Yim EK, Lee KH, Myeong J, Tong SY, Um SJ, Park JS.
Novel Interaction between HPV E6 and BARD1 (BRCA1-Associated Ring Domain 1) and Its Biologic Roles.
DNA Cell Biol. 2007 Aug 3; [Epub ahead of print] [PDF]

Human papillomaviruses (HPVs), which are associated with the majority of cervical cancers, encode a transforming protein, E6, which interacts with the p53 tumor suppressor protein. There is a wide effort focused on searching for the target of the involvement of p53-independent HPV 16 E6-interacting proteins. We identified Breast Cancer 1 Gene (BRCA1)-associated ring domain protein 1 (BARD1) as a binding partner of E6 and investigated its biological function in cervical cancer cells. In vivo co-immunoprecipitation assay was performed to determine whether E6-BARD1 interaction occurred. We then used a degradation assay to determine whether E6-mediated inactivation of BARD1 transactivation function was associated with BARD1 degradation. A mutation assay revealed the site of interaction of E6 with BARD1. The effect of BARD1 on p53 transcriptional activity was tested using BARD1 knockdown and overexpression systems. BARD1 was not degraded by E6, and, instead, formed a physical complex with E6. Moreover, the mutations of the metal motif zinc-finger region decreased the ability of E6 to interact with BARD1. Transient transfection of BARD1 increased the p53-mediated activation of p21(WAF1) promoter despite the presence of E6. Additionally, the existence of BARD1 inactivated the expression of E6 in cervical cancer cells. These findings suggest that BARD1 may regulate the transcriptional activities of p53 as tumor suppressors.

Friday, July 20, 2007, 11:00
Leibniz Institute for Age Research - Fritz Lipmann Institute
Beutenbergstr. 11, D-07745 Jena
Biocomputing-Verwaltungsgebäude, Seminarraum 101

Presenter: Jong Kwang Kim (FLI, Wilhelm and Sühnel Labs)

Ye Y, Godzik A.
Comparative analysis of protein domain organization.
Genome Res. 2004,14:343-53. [PDF]

We have developed a set of graph theory-based tools, which we call Comparative Analysis of Protein Domain Organization (CADO), to survey and compare protein domain organizations of different organisms. In the language of CADO, the organization of protein domains in a given organism is shown as a domain graph in which protein domains are represented as vertices, and domain combinations, defined as instances of two domains found in one protein, are represented as edges. CADO provides a new way to analyze and compare whole proteomes, including identifying the consensus and difference of domain organization between organisms. CADO was used to analyze and compare >50 bacterial, archaeal, and eukaryotic genomes. Examples and overviews presented here include the analysis of the modularity of domain graphs and the functional study of domains based on the graph topology. We also report on the results of comparing domain graphs of two organisms, Pyrococcus horikoshii (an extremophile) and Haemophilus influenzae (a parasite with reduced genome) with other organisms. Our comparison provides new insights into the genome organization of these organisms. Finally, we report on the specific domain combinations characterizing the three kingdoms of life, and the kingdom "signature" domain organizations derived from those specific domain combinations.

Friday, June 22, 2007, 11:00
Leibniz Institute for Age Research - Fritz Lipmann Institute
Beutenbergstr. 11, D-07745 Jena
Biocomputing-Verwaltungsgebäude, Seminarraum 101

Presenter: Christine Kamperdick (FLI, Görlach Lab)

Jensen MR, Kristensen SM, Led JJ
Elimination of spin diffusion effects in saturation transfer experiments: application to hydrogen exchange in proteins.
Magn Reson Chem. 2007, 45:257-61.[PDF]

The NMR saturation transfer experiment is widely used to characterize exchange processes in proteins that take place on the ms-s timescale. However, spin diffusion effects are inherently associated with the saturation transfer experiment and may overshadow the effect of the exchange processes of interest. As shown here, the effects from spin diffusion and exchange processes can be separated by varying the field strength of the saturation pulse, thereby allowing correct exchange rates to be obtained. The method is demonstrated using the hydrogen exchange process in the protein Escherichia coli thioredoxin as an example.

Friday, June 8, 2007, 11:00
Leibniz Institute for Age Research - Fritz Lipmann Institute
Beutenbergstr. 11, D-07745 Jena
Biocomputing-Verwaltungsgebäude, Seminarraum 101

Presenter: Yvonne Ihle (FLI, Görlach Lab)

Du Z, Lee JK, Fenn S, Tjhen R, Stroud RM, James TL
X-ray crystallographic and NMR studies of protein–protein and protein–nucleic acid interactions involving the KH domains from human poly(C)-binding protein-2
RNA. 2007, 13, 1-9. [PDF]

Poly(C)-binding proteins (PCBPs) are KH (hnRNP K homology) domain-containing proteins that recognize poly(C) DNA and
RNA sequences in mammalian cells. Binding poly(C) sequences via the KH domains is critical for PCBP functions. To reveal the
mechanisms of KH domain-D/RNA recognition and its functional importance, we have determined the crystal structures of
PCBP2 KH1 domain in complex with a 12-nucleotide DNA corresponding to two repeats of the human C-rich strand telomeric
DNA and its RNA equivalent. The crystal structures reveal molecular details for not only KH1-DNA/RNA interaction but also
protein–protein interaction between two KH1 domains. NMR studies on a protein construct containing two KH domains (KH1 +
KH2) of PCBP2 indicate that KH1 interacts with KH2 in a way similar to the KH1–KH1 interaction. The crystal structures and
NMR data suggest possible ways by which binding certain nucleic acid targets containing tandem poly(C) motifs may induce
structural rearrangement of the KH domains in PCBPs; such structural rearrangement may be crucial for some PCBP functions.

Friday, May 25, 2007, 11:00
Leibniz Institute for Age Research - Fritz Lipmann Institute
Beutenbergstr. 11, D-07745 Jena
Biocomputing-Verwaltungsgebäude, Seminarraum 101

Presenter: Rolf Hühne (FLI, Sühnel Lab)

Spjuth O, Helmus T, Willighagen EL, Kuhn S, Eklund M, Wagener J, Murray-Rust P, Steinbeck C, Wikberg JE.
Bioclipse: an open source workbench for chemo- and bioinformatics.
BMC Bioinformatics. 2007 Feb 22;8:59. [PDF]

BACKGROUND: There is a need for software applications that provide users with a complete and extensible toolkit for chemo- and bioinformatics accessible from a single workbench. Commercial packages are expensive and closed source, hence they do not allow end users to modify algorithms and add custom functionality. Existing open source projects are more focused on providing a framework for integrating existing, separately installed bioinformatics packages, rather than providing user-friendly interfaces. No open source chemoinformatics workbench has previously been published, and no successful attempts have been made to integrate chemo- and bioinformatics into a single framework. RESULTS: Bioclipse is an advanced workbench for resources in chemo- and bioinformatics, such as molecules, proteins, sequences, spectra, and scripts. It provides 2D-editing, 3D-visualization, file format conversion, calculation of chemical properties, and much more; all fully integrated into a user-friendly desktop application. Editing supports standard functions such as cut and paste, drag and drop, and undo/redo. Bioclipse is written in Java and based on the Eclipse Rich Client Platform with a state-of-the-art plugin architecture. This gives Bioclipse an advantage over other systems as it can easily be extended with functionality in any desired direction. CONCLUSION: Bioclipse is a powerful workbench for bio- and chemoinformatics as well as an advanced integration platform. The rich functionality, intuitive user interface, and powerful plugin architecture make Bioclipse the most advanced and user-friendly open source workbench for chemo- and bioinformatics. Bioclipse is released under Eclipse Public License (EPL), an open source license which sets no constraints on external plugin licensing; it is totally open for both open source plugins as well as commercial ones. Bioclipse is freely available at http://www.bioclipse.net.

Friday, May 11, 2007, 11:00
Leibniz Institute for Age Research - Fritz Lipmann Institute
Beutenbergstr. 11, D-07745 Jena
Biocomputing-Verwaltungsgebäude, Seminarraum 101

Presenter: Christian Herbst (FLI, Görlach Lab)

Li Y, Wylie BJ, Rienstra CM.
Selective refocusing pulses in magic-angle spinning NMR: characterization and applications to multi-dimensional protein spectroscopy.
J Magn Reson. 2006, 179, 206-216. [PDF]

Band-selective pulses are frequently used in multi-dimensional NMR in solution, but have been used relatively less often in solid-state NMR applications because of the complications imposed by magic-angle spinning. In this work, we examine the frequency profiles and the refocusing efficiency of several commonly employed selective general rotation pi pulses through experiments and numerical simulations. We demonstrate that highly efficient refocusing of transverse magnetization can be achieved, with experiments that agree well with numerical simulations. We also show that the rotational echo is shifted by a half rotor period if a selective pulse is applied over an integer number of rotor periods. Appropriately synchronizing indirect evolution periods with selective pulses ensures proper phasing of cross peaks in 2D spectra. The improved performance of selective pulses in multi-dimensional protein spectroscopy is demonstrated on the 56-residue beta1 immunoglobulin binding domain of protein G (GB1).

Friday, April 27, 2007, 11:00
Leibniz Institute for Age Research - Fritz Lipmann Institute
Beutenbergstr. 11, D-07745 Jena
Biocomputing-Verwaltungsgebäude, Seminarraum 101

Presenter: Matthias Görlach (FLI, Görlach Lab)

Robertson MP, Scott WG.
The structural basis of ribozyme-catalyzed RNA assembly.
Science 2007, 315, 1549-1553. [PDF]

Life originated, according to the RNA World hypothesis, from self-replicating ribozymes that catalyzed ligation of RNA fragments. We have solved the 2.6 angstrom crystal structure of a ligase ribozyme that catalyzes regiospecific formation of a 5' to 3' phosphodiester bond between the 5'-triphosphate and the 3'-hydroxyl termini of two RNA fragments. Invariant residues form tertiary contacts that stabilize a flexible stem of the ribozyme at the ligation site, where an essential magnesium ion coordinates three phosphates. The structure of the active site permits us to suggest how transition-state stabilization and a general base may catalyze the ligation reaction required for prebiotic RNA assembly.

Friday, April 13, 2007, 11:00
Leibniz Institute for Age Research - Fritz Lipmann Institute
Beutenbergstr. 11, D-07745 Jena
Biocomputing-Verwaltungsgebäude, Seminarraum 101

Presenter: Maik Friedel (FLI, Wilhelm and Sühnel Labs)

Paz A, Mester D, Nevo E, Korol A.
Looking for organization patterns of highly expressed genes: Purine-pyrimidine composition of precursor mRNAs.
J Mol Evol. 2007, 64, 248-60 [PDF]

We analyzed precursor messenger RNAs (pre-mRNAs) of 12 eukaryotic species. In each species, three groups of highly expressed genes, ribosomal proteins, heat shock proteins, and amino-acyl tRNA synthetases, were compared with a control group (randomly selected genes). The purine-pyrimidine (R-Y) composition of pre-mRNAs of the three targeted gene groups proved to differ significantly from the control. The exons of the three groups tested have higher purine contents and R-tract abundance and lower abundance of Y-tracts compared to the control (R-tract-tract of sequential purines with R( n ) >/= 5; Y-tract-tract of sequential pyrimidines with Y( n ) >/= 5). In species widely employing "intron definition" in the splicing process, the Y content of introns of the three targeted groups appeared to be higher compared to the control group. Furthermore, in all examined species, the introns of the targeted genes have a lower abundance of R-tracts compared to the control. We hypothesized that the R-Y composition of the targeted gene groups contributes to high rate and efficiency of both splicing and translation, in addition to the mRNA coding role. This is presumably achieved by (1) reducing the possibility of the formation of secondary structures in the mRNA, (2) using the R-tracts and R-biased sequences as exonic splicing enhancers, (3) lowering the amount of targets for pyrimidine tract binding protein in the exons, and (4) reducing the amount of target sequences for binding of serine/arginine-rich (SR) proteins in the introns, thereby allowing SR proteins to bind to proper (exonic) targets.

Friday, March 23, 2007, 11:00
Leibniz Institute for Age Research - Fritz Lipmann Institute
Beutenbergstr. 11, D-07745 Jena
Biocomputing-Verwaltungsgebäude, Seminarraum 101

Presenter: Manual Than (FLI, Than Lab)

Wang Y, Zhang Y, Ha Y.
Crystal structure of a rhomboid family intramembrane protease.
Nature. 2006, 444, 179-180. [PDF]

Escherichia coli GlpG is an integral membrane protein that belongs to the widespread rhomboid protease family. Rhomboid proteases, like site-2 protease (S2P) and gamma-secretase, are unique in that they cleave the transmembrane domain of other membrane proteins. Here we describe the 2.1 A resolution crystal structure of the GlpG core domain. This structure contains six transmembrane segments. Residues previously shown to be involved in catalysis, including a Ser-His dyad, and several water molecules are found at the protein interior at a depth below the membrane surface. This putative active site is accessible by substrate through a large 'V-shaped' opening that faces laterally towards the lipid, but is blocked by a half-submerged loop structure. These observations indicate that, in intramembrane proteolysis, the scission of peptide bonds takes place within the hydrophobic environment of the membrane bilayer. The crystal structure also suggests a gating mechanism for GlpG that controls substrate access to its hydrophilic active site.

Friday, March 16, 2007, 11:00
Leibniz Institute for Age Research - Fritz Lipmann Institute
Beutenbergstr. 11, D-07745 Jena
Biocomputing-Verwaltungsgebäude, Seminarraum 101

Presenter: Marius Felder (FLI, Sühnel Lab)

Lau NC, Seto AG, Kim J, Kuramochi-Miyagawa S, Nakano T, Bartel DP, Kingston RE.
Characterization of the piRNA complex from rat testes.
Science. 2006, 313, 363-367. [PDF]

Small noncoding RNAs regulate processes essential for cell growth and development, including mRNA degradation, translational repression, and transcriptional gene silencing (TGS). During a search for candidate mammalian factors for TGS, we purified a complex that contains small RNAs and Riwi, the rat homolog to human Piwi. The RNAs, frequently 29 to 30 nucleotides in length, are called Piwi-interacting RNAs (piRNAs), 94% of which map to 100 defined (< or = 101 kb) genomic regions. Within these regions, the piRNAs generally distribute across only one genomic strand or distribute on two strands but in a divergent, nonoverlapping manner. Preparations of piRNA complex (piRC) contain rRecQ1, which is homologous to qde-3 from Neurospora, a gene implicated in silencing pathways. Piwi has been genetically linked to TGS in flies, and slicer activity cofractionates with the purified complex. These results are consistent with a gene-silencing role for piRC in mammals.

FLI Home