Koehler C.,Structural and Computational Biology Unit |
Round A.,CEA Grenoble |
Simader H.,Proteros biostructures |
Suck D.,Structural and Computational Biology Unit |
Svergun D.,German Electron Synchrotron
Nucleic Acids Research | Year: 2013
In the yeast Saccharomyces cerevisiae, the aminoacyl-tRNA synthetases (aaRS) GluRS and MetRS form a complex with the auxiliary protein cofactor Arc1p. The latter binds the N-terminal domains of both synthetases increasing their affinity for the transfer-RNA (tRNA) substrates tRNAMet and tRNA Glu. Until now, structural information was available only on the enzymatic domains of the individual aaRSs but not on their complexes with associated cofactors. We have analysed the yeast Arc1p-complexes in solution by small-angle X-ray scattering (SAXS). The ternary complex of MetRS and GluRS with Arc1p, displays a peculiar extended star-like shape, implying possible flexibility of the complex. We reconstituted in vitro a pentameric complex and demonstrated by electrophoretic mobility shift assay that the complex is active and contains tRNAMet and tRNAGlu, in addition to the three protein partners. SAXS reveals that binding of the tRNAs leads to a dramatic compaction of the pentameric complex compared to the ternary one. A hybrid low-resolution model of the pentameric complex is constructed rationalizing the compaction effect by the interactions of negatively charged tRNA backbones with the positively charged tRNA-binding domains of the synthetases. © 2012 The Author(s). Published by Oxford University Press.
Agency: European Commission | Branch: FP7 | Program: MC-ITN | Phase: FP7-PEOPLE-2011-ITN | Award Amount: 3.76M | Year: 2012
Immune-mediated inflammatory diseases (IMID) are important health challenges in Europe and beyond, afflicting an estimated 5-8% of the total population. IMID with an onset during childhood such as Juvenile Idiopathic Arthritis (JIA) cause particular concern as pediatric patients form an especially vulnerable group. Currently there is no safe and cost-effective cure for JIA and related juvenile IMID. Thus, these children face lifelong treatment with serious consequences both for the patient (high risks of long-term side effects) and for society as a whole (high costs). Translating the progress in molecular medicine into new therapies for JIA has also met with limited success. The route from idea to drug has many hurdles and is a very fragmented process. Translational medicine encompasses the continuum of activities that extend from the conception of an idea to advanced clinical testing and, ultimately, to the development of a new medical technology or drug. This itinerary includes many components that require very different skills such as biomedical research skills, design of pre-clinical and clinical trials, regulatory issues, legal issues, intellectual property rights, communicational skills and more. Such skills are often compartmentalized within three separate domainsacademia, government and industry. Each of these domains has its own set of challenges. If we are to really change the way in which we are thinking and working in the drug development process it will be essential that we start with changing the education process of our students. EUTRAIN brings together leading scientists and institutes in the field of IMID. Its goal is to provide the next generation of researchers with insights, tools and knowledge necessary to bridge the gap between bench and bedside in IMID. In doing this EUTRAIN addresses two specific needs: the need for novel therapeutic approaches for IMID and the need for novel approaches in translational medicine.
Koster H.,University of Marburg |
Craan T.,University of Marburg |
Brass S.,University of Marburg |
Herhaus C.,Merck KGaA |
And 4 more authors.
Journal of Medicinal Chemistry | Year: 2011
Druglike molecules are defined by Lipinski's rule of 5, to characterize fragment thresholds, they have been reduced from 5 to 3 (Astex's rule of 3). They are applied to assemble fragment libraries, and providers use them to select fragments for commercial offer. We question whether these rules are too stringent to compose fragment libraries with candidates exhibiting sufficient room for chemical subsequent growing and merging modifications as appropriate functional groups for chemical transformations are required. Usually these groups exhibit properties as hydrogen bond donors/acceptors and provide entry points for optimization chemistry. We therefore designed a fragment library (364 entries) without strictly applying the rule of 3. For initial screening for endothiapepsin binding, we performed a biochemical cleavage assay of a fluorogenic substrate at 1 mM. "Hits were defined to inhibit the enzyme by at least 40%. Fifty-five hits were suggested and subsequently soaked into endothiapepsin crystals. Eleven crystal structures could be determined covering fragments with diverse binding modes: (i) direct binding to the catalytic dyad aspartates, (ii) water-mediated binding to the aspartates, (iii) no direct interaction with the dyad. They occupy different specificity pockets. Only 4 of the 11 fragments are consistent with the rule of 3. Restriction to this rule would have limited the fragment hits to a strongly reduced variety of chemotypes. © 2011 American Chemical Society.
Kiefersauer R.,Max Planck Institute For Biochemie |
Kiefersauer R.,Proteros biostructures |
Grandl B.,Max Planck Institute For Biochemie |
Grandl B.,Proteros biostructures |
And 5 more authors.
Acta Crystallographica Section D: Biological Crystallography | Year: 2014
A method and the design of instrumentation, and its preliminary practical realisation, including test experiments, with the object of inducing phase changes of biomolecular crystals by controlled dehydration through heating with infrared (IR) light are described. The aim is to generate and select crystalline phases through transformation in the solid state which have improved order (higher resolution in X-ray diffraction experiments) and reduced mosaic spread (more uniformly aligned mosaic blocks) for diffraction data collection and analysis. The crystal is heated by pulsed and/or constant IR laser irradiation. Loss of crystal water following heating and its reabsorption through equilibration with the environment is measured optically by a video system. Heating proved superior to traditional controlled dehydration by humidity change for the test cases CODH (carbon monoxide dehydrogenase) and CLK2 (a protein kinase). Heating with IR light is experimentally simple and offers an exploration of a much broader parameter space than the traditional method, as it allows the option of varying the rate of phase changes through modification of the IR pulse strength, width and repeat frequency. It impacts the crystal instantaneously, isotropically and homogeneously, and is therefore expected to cause less mechanical stress. © 2014 International Union of Crystallography.
Agency: European Commission | Branch: FP7 | Program: CP-FP | Phase: HEALTH-2007-2.3.1-1 | Award Amount: 7.69M | Year: 2009
Multi-drug-resistant bacterial infections are increasing at an alarming pace in both developing and developed countries and in both community and nosocomial settings. The few antimicrobial agents that have been launched during the last decade (e.g. linezolid, daptomycin) have a good activity against Gram-positive bacteria. However, multi-drug-resistant bacteria are often found among the Gram-negative group. To tackle this problem, the AntiPathoGN project proposes a novel strategy for the discovery of new antimicrobial drug targets in a number of Gram-negative bacteria. This strategy is based on a comparative, system-level analysis of molecular processes involved in pathogenicity, drug resistance, cell division and/or growth of selected pathogenic Gram-negative bacteria through a combination of computational biology, interactome discovery, in-vivo protein-blocking and structural biology techniques. This comparative analysis, at one organization level above genomics, shall permit the discovery of new potential drug targets, relevant to both species-specific and broad-spectrum antimicrobial strategies. In addition, the AntiPathoGN project will pursue the identification of novel antibacterial compounds acting against previously validated targets by screening purpose-specific libraries of products derived from natural resources and from synthetic compounds. An important aspect of the project is that more than one approach is planned for the key objectives of target identification, target validation, target characterisation, and hit finding. The AntiPathoGN consortium is composed of 1 university bringing expertises in computational biology and microbiology, 3 research centres with expertises in computational and experimental interactomics, structural biology and clinical microbiology, and 7 SMEs with records on recombinant-antibody technology, structural biology, drug discovery and clinical research. The AntiPathoGN project fully addresses topic HEALTH-2007-2.3.1-1.
Neumann L.,Proteros biostructures |
Von Knig K.,Proteros biostructures |
Ullmann D.,Proteros biostructures
Methods in Enzymology | Year: 2011
Abstract Parameters such as residence time, kinetic selectivity, and thermodynamic signature are more and more under debate as optimization objectives within fragment-based lead discovery. However, broad implementation of these parameters is hampered by the lack of technologies that give rapid access to binding kinetics and thermodynamic information for large amounts of compoundtarget interactions. Here, the authors describe a technologythe reporter displacement assaythat is capable of opening this bottleneck and of supporting data-driven design of lead compounds with tailor-made residence time, kinetic selectivity, and thermodynamic signature. © 2011 Elsevier Inc.
Rajagopala S.V.,J. Craig Venter Institute |
Uetz P.,Proteros biostructures
Methods in Molecular Biology | Year: 2011
The yeast two-hybrid (Y2H) system is a powerful tool to identify binary protein-protein interactions. Here, we describe array-based two-hybrid methods that use defined libraries of open reading frames (ORFs) and pooled prey library screenings that use random genomic or cDNA libraries. The array-based Y2H system is well-suited for interactome studies of existing ORFeomes or subsets thereof, preferentially in a recombination-based cloning system. Array-based Y2H screens efficiently reduce false positives by using built-in controls, retesting, and evaluation of background activation. Hands-on time and the amount of used resources grow exponentially with the number of tested proteins; this is a disadvantage for large genome sizes. For large genomes, random library screen may be more efficient in terms of time and resources, but not as comprehensive as array screens, and it requires significant sequencing capacity. Furthermore, multiple variants of the Y2H vector systems detect markedly different subsets of interactions in the same interactome. Hence, only multiple variations of the Y2H systems ensure comprehensive coverage of an interactome. © 2011 Springer Science+Business Media, LLC.
Rajagopala S.V.,J. Craig Venter Institute |
Casjens S.,University of Utah |
Uetz P.,Virginia Commonwealth University |
Uetz P.,Proteros biostructures
BMC Microbiology | Year: 2011
Background: Bacteriophage lambda is a model phage for most other dsDNA phages and has been studied for over 60 years. Although it is probably the best-characterized phage there are still about 20 poorly understood open reading frames in its 48-kb genome. For a complete understanding we need to know all interactions among its proteins. We have manually curated the lambda literature and compiled a total of 33 interactions that have been found among lambda proteins. We set out to find out how many protein-protein interactions remain to be found in this phage. Results: In order to map lambda's interactions, we have cloned 68 out of 73 lambda open reading frames (the "ORFeome") into Gateway vectors and systematically tested all proteins for interactions using exhaustive array-based yeast two-hybrid screens. These screens identified 97 interactions. We found 16 out of 30 previously published interactions (53%). We have also found at least 18 new plausible interactions among functionally related proteins. All previously found and new interactions are combined into structural and network models of phage lambda. Conclusions: Phage lambda serves as a benchmark for future studies of protein interactions among phage, viruses in general, or large protein assemblies. We conclude that we could not find all the known interactions because they require chaperones, post-translational modifications, or multiple proteins for their interactions. The lambda protein network connects 12 proteins of unknown function with well characterized proteins, which should shed light on the functional associations of these uncharacterized proteins. © 2011Rajagopala et al; licensee BioMed Central Ltd.
Proteros biostructures | Date: 2012-10-30
Scientific apparatus, namely, apparatus for the dispensing of solutions onto the surface of protein crystals for protein structure analysis. Scientific research and development; technological research and development in the field of crystallography, X-ray structure determination, proteins and protein crystals; industrial analysis and research services, namely, research related to drug development, proteins, protein structures and protein crystals.