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72 results on '"MESH: Crystallization"'

1. Nucleation of protein mesocrystals via oriented attachment

Author

Nani Van Gerven, Rick R. M. Joosten, Wai Li Ling, Alexander E. S. Van Driessche, Nico A. J. M. Sommerdijk, Mike Sleutel, Maria Bacia, Institut des Sciences de la Terre (ISTerre), Institut national des sciences de l'Univers (INSU - CNRS)-Institut de recherche pour le développement [IRD] : UR219-Université Savoie Mont Blanc (USMB [Université de Savoie] [Université de Chambéry])-Centre National de la Recherche Scientifique (CNRS)-Université Gustave Eiffel-Université Grenoble Alpes (UGA), VIB-VUB Center for Structural Biology [Bruxelles], VIB [Belgium], Department of Chemical Engineering and Chemistry [Eindhoven], Eindhoven University of Technology [Eindhoven] (TU/e), Institut de biologie structurale (IBS - UMR 5075), Centre National de la Recherche Scientifique (CNRS)-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Grenoble Alpes (UGA), Department of Biochemistry, Department of Molecular Biology, Faculty of Science, Radboud University, Radboud Institute for Molecular Life Sciences (RIMLS), 6525GA Nijmegen, the Netherlands, Department of Molecular Biology, Radboud Institute for Molecular Life Sciences (RIMLS), Oncode Institute, Radboud University, Nijmegen, the Netherland, Structural Biology Brussels (SBB), Vrije Universiteit Brussel (VUB), ANR-10-INBS-0005,FRISBI,Infrastructure Française pour la Biologie Structurale Intégrée(2010), ANR-17-EURE-0003,CBH-EUR-GS,CBH-EUR-GS(2017), Centre National de la Recherche Scientifique (CNRS)-PRES Université de Grenoble-Université Joseph Fourier - Grenoble 1 (UJF)-Institut Français des Sciences et Technologies des Transports, de l'Aménagement et des Réseaux (IFSTTAR)-Institut national des sciences de l'Univers (INSU - CNRS)-Institut de recherche pour le développement [IRD] : UR219-Université Savoie Mont Blanc (USMB [Université de Savoie] [Université de Chambéry]), Institut national des sciences de l'Univers (INSU - CNRS)-Institut de recherche pour le développement [IRD] : UR219-Université Grenoble Alpes (UGA)-Université Gustave Eiffel-Centre National de la Recherche Scientifique (CNRS)-Université Savoie Mont Blanc (USMB [Université de Savoie] [Université de Chambéry]), Centre National de la Recherche Scientifique (CNRS)-Université Grenoble Alpes (UGA)-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA), Institute for Complex Molecular Systems, Materials and Interface Chemistry, Physical Chemistry, Department of Bio-engineering Sciences, and Structural Biology Brussels

Subjects
0301 basic medicine, Models, Molecular, Chemistry(all), Nucleation, General Physics and Astronomy, Nanoparticle, Crystallography, X-Ray, 01 natural sciences, law.invention, MESH: Recombinant Proteins, law, Crystallization, Aldose-Ketose Isomerases, MESH: Crystallization, Multidisciplinary, MESH: Kinetics, [SDV.BBM.BS]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Structural Biology [q-bio.BM], Self-assembly, Nanocrystalline material, Recombinant Proteins, MESH: Cryoelectron Microscopy, Protein crystallization, MESH: Models, Molecular, Materials science, Science, Nanotechnology, Physics and Astronomy(all), 010402 general chemistry, Molecular resolution, General Biochemistry, Genetics and Molecular Biology, Article, 03 medical and health sciences, MESH: Nanostructures, Point Mutation, [CHIM]Chemical Sciences, MESH: Particle Size, Particle Size, MESH: Aldose-Ketose Isomerases, MESH: Point Mutation, Biochemistry, Genetics and Molecular Biology(all), Cryoelectron Microscopy, Proteins, General Chemistry, MESH: Crystallography, X-Ray, 0104 chemical sciences, Nanostructures, Kinetics, 030104 developmental biology, Phase transitions and critical phenomena, Nanocrystal, [SDU]Sciences of the Universe [physics], Nanomedicine Radboud Institute for Molecular Life Sciences [Radboudumc 19]
Abstract

Self-assembly of proteins holds great promise for the bottom-up design and production of synthetic biomaterials. In conventional approaches, designer proteins are pre-programmed with specific recognition sites that drive the association process towards a desired organized state. Although proven effective, this approach poses restrictions on the complexity and material properties of the end-state. An alternative, hierarchical approach that has found wide adoption for inorganic systems, relies on the production of crystalline nanoparticles that become the building blocks of a next-level assembly process driven by oriented attachment (OA). As it stands, OA has not yet been observed for protein systems. Here we employ cryo-transmission electron microscopy (cryoEM) in the high nucleation rate limit of protein crystals and map the self-assembly route at molecular resolution. We observe the initial formation of facetted nanocrystals that merge lattices by means of OA alignment well before contact is made, satisfying non-trivial symmetry rules in the process. As these nanocrystalline assemblies grow larger we witness imperfect docking events leading to oriented aggregation into mesocrystalline assemblies. These observations highlight the underappreciated role of the interaction between crystalline nuclei, and the impact of OA on the crystallization process of proteins., Past studies on protein nucleation have focused on the routes that molecules follow towards a crystalline cluster, while possible interactions that may occur between nuclei have not been investigated. Here, the authors show that in the high supersaturation limit such interactions dominate the nucleation process in the form of inter-nucleus docking driving by oriented attachment.

Published
2021

2. IgG Fc domains that bind C1q but not effector Fcγ receptors delineate the importance of complement-mediated effector functions

Author

Bianca Balbino, George Delidakis, Tae Hyun Kang, Makiko Watanabe, Kendra Triplett, Anja Lux, Navin Varadarajan, Odile Richard-Le Goff, Oana I. Lungu, Pierre Bruhns, George Georgiou, Corrine Alford, Gabrielle Romain, Margaret A. Lindorfer, Wissam Charab, Falk Nimmerjahn, Yan Jessie Zhang, Hidetaka Tanno, Wupeng Yan, Nicholas M. Marshall, Moses Donkor, Chang-Han Lee, Jiwon Lee, Ronald P. Taylor, Biliana Todorova, University of Texas at Austin [Austin], University of Houston, Anticorps en Thérapie et Pathologie, Institut Pasteur [Paris]-Institut National de la Santé et de la Recherche Médicale (INSERM), University of Erlangen-Nürnberg [Germany], University of Virginia School of Medicine [US], Université Pierre et Marie Curie - Paris 6 - UFR de Médecine Pierre et Marie Curie (UPMC), Université Pierre et Marie Curie - Paris 6 (UPMC), University of Virginia, Department of Biochemistry and Molecular Genetics (xxx), University of Virginia [Charlottesville], We thank Y. Tanno for assistance with protein expression, A. Bui for assistance with liquid chromatography–tandem mass spectrometry, P. Tucker (University of Texas at Austin) for cancer cell lines, D. Lee (MD Anderson Cancer Center) for patient-derived primary acute lymphocytic leukemia cells, the Macromolecular Crystallography Facility of the University of Texas at Austin, the Berkeley Center for Structural Biology, the Advanced Light Source (supported by the Director, Office of Science, Office of Basic Energy Sciences, of the US Department of Energy under contract DE-AC02-05CH11231), the Proteomics Facility at the University of Texas at Austin (supported by grant RP110782 from the Cancer Prevention Research Training Program), and A. Nicola and the Plate-Forme d'Imagerie Dynamique (Institut Pasteur, Paris) for help with the bioluminescence experiments. Supported by the Clayton Foundation, the Institut Pasteur (P.B. laboratory), the Institut National de la Santé et de la Recherche Médicale (P.B. laboratory), the European Research Council Seventh Frame-work Program (ERC-2013-CoG 616050 for the P.B. laboratory), the Pasteur–Paris University International PhD program (B.B.), the Cancer Prevention Research Training Program (RP140108 to M.D., RP160015 to H.T., and RP130570 to N.V.), the American Cancer Society (123506-PF-13-354-01-CDD to N.M.), Uehara Memorial Foundation (H.T.), Japan Society for the Promotion of Science (H.T.), Deutsche Forschungsgemeinschaft (CRC1181-A07 to F.N.), the US National Institutes of Health (R01CA174385 to N.V., and R01 GM104896 to Y.J.Z.) and the Welch Foundation (F-1778 to Y.J.Z.). The content is solely the responsibility of the authors and does not necessarily represent the official views of the Cancer Prevention and Research Institute of Texas., European Project: 616050,EC:FP7:ERC,ERC-2013-CoG,MYELOSHOCK(2014), Friedrich-Alexander Universität Erlangen-Nürnberg (FAU), Institut Pasteur [Paris] (IP)-Institut National de la Santé et de la Recherche Médicale (INSERM), University of Virginia, Martin, Marie, and Role of myeloid cells, their mediators and their antibody receptors in allergic shock (anaphylaxis) using humanized mouse models and clinical samples - MYELOSHOCK - - EC:FP7:ERC2014-09-01 - 2019-08-31 - 616050 - VALID

Subjects
Cytotoxicity, Immunologic, 0301 basic medicine, Cellular immunity, MESH: Complement C1q, MESH: Immunotherapy, Crystallography, X-Ray, Mass Spectrometry, MESH: Antibodies, Monoclonal, Mice, Tandem Mass Spectrometry, Neoplasms, Immunology and Allergy, MESH: Animals, MESH: Neoplasms, Cytotoxicity, Receptor, MESH: Phagocytosis, MESH: Crystallization, MESH: Immunoglobulin G, biology, Effector, Antibodies, Monoclonal, MESH: Enzyme-Linked Immunosorbent Assay, MESH: Chromatography, Gel, Precursor Cell Lymphoblastic Leukemia-Lymphoma, Burkitt Lymphoma, 3. Good health, Cell biology, MESH: Surface Plasmon Resonance, Chromatography, Gel, [SDV.IMM]Life Sciences [q-bio]/Immunology, Immunotherapy, Lymphoma, Large B-Cell, Diffuse, Antibody, Crystallization, Lymphoma, B-Cell, MESH: Cell Line, Tumor, [SDV.IMM] Life Sciences [q-bio]/Immunology, Immunology, Enzyme-Linked Immunosorbent Assay, In Vitro Techniques, MESH: Receptors, IgG, MESH: Immunoglobulin Fc Fragments, 03 medical and health sciences, Classical complement pathway, Immune system, Phagocytosis, Cell Line, Tumor, Animals, Humans, MESH: Cytotoxicity, Immunologic, MESH: Mice, MESH: Lymphoma, B-Cell, MESH: In Vitro Techniques, MESH: Mass Spectrometry, MESH: Precursor Cell Lymphoblastic Leukemia-Lymphoma, MESH: Humans, Complement C1q, Receptors, IgG, MESH: Tandem Mass Spectrometry, Surface Plasmon Resonance, MESH: Crystallography, X-Ray, Immunoglobulin Fc Fragments, Complement system, MESH: Burkitt Lymphoma, 030104 developmental biology, Immunoglobulin G, biology.protein, MESH: Lymphoma, Large B-Cell, Diffuse, MESH: Chromatography, Liquid, Chromatography, Liquid
Abstract

International audience; Engineered crystallizable fragment (Fc) regions of antibody domains, which assume a unique and unprecedented asymmetric structure within the homodimeric Fc polypeptide, enable completely selective binding to the complement component C1q and activation of complement via the classical pathway without any concomitant engagement of the Fcγ receptor (FcγR). We used the engineered Fc domains to demonstrate in vitro and in mouse models that for therapeutic antibodies, complement-dependent cell-mediated cytotoxicity (CDCC) and complement-dependent cell-mediated phagocytosis (CDCP) by immunological effector molecules mediated the clearance of target cells with kinetics and efficacy comparable to those of the FcγR-dependent effector functions that are much better studied, while they circumvented certain adverse reactions associated with FcγR engagement. Collectively, our data highlight the importance of CDCC and CDCP in monoclonal-antibody function and provide an experimental approach for delineating the effect of complement-dependent effector-cell engagement in various therapeutic settings.

Published
2017

3. Camelid nanobodies used as crystallization chaperones for different constructs of PorM, a component of the type IX secretion system from Porphyromonas gingivalis

Author

Christian Cambillau, Christine Kellenberger, Alain Roussel, Aline Desmyter, Anaïs Gaubert, Jennifer Roche, Philippe Leone, Thi Trang Nhung Trinh, Yoan Duhoo, Architecture et fonction des macromolécules biologiques (AFMB), Institut National de la Recherche Agronomique (INRA)-Aix Marseille Université (AMU)-Centre National de la Recherche Scientifique (CNRS), Centre National de la Recherche Scientifique (CNRS)-Aix Marseille Université (AMU)-Institut National de la Recherche Agronomique (INRA), Architecture et fonction des macromolécules biologiques ( AFMB ), and Centre National de la Recherche Scientifique ( CNRS ) -Aix Marseille Université ( AMU ) -Institut National de la Recherche Agronomique ( INRA )

Subjects
MESH: Sequence Homology, Amino Acid, [SDV.NEU.NB]Life Sciences [q-bio]/Neurons and Cognition [q-bio.NC]/Neurobiology, Plasma protein binding, MESH: Amino Acid Sequence, Biochemistry, MESH: Recombinant Proteins, [SDV.MHEP.MI]Life Sciences [q-bio]/Human health and pathology/Infectious diseases, MESH: Animals, [ SDV.BIBS ] Life Sciences [q-bio]/Quantitative Methods [q-bio.QM], Peptide sequence, MESH: Bacterial Proteins, MESH : Porphyromonas gingivalis, MESH : Protein Conformation, alpha-Helical, [SDV.MHEP.ME]Life Sciences [q-bio]/Human health and pathology/Emerging diseases, [ SDV.MHEP.ME ] Life Sciences [q-bio]/Human health and pathology/Emerging diseases, [SDV.BBM.BS]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Structural Biology [q-bio.BM], MESH: Escherichia coli, MESH : Amino Acid Sequence, MESH : Protein Binding, MESH: Camelus, [SDV.BIBS]Life Sciences [q-bio]/Quantitative Methods [q-bio.QM], MESH : Sequence Homology, Amino Acid, MESH : Genetic Vectors, [SDV.BBM.BC]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biomolecules [q-bio.BM], [ SDV.MHEP.MI ] Life Sciences [q-bio]/Human health and pathology/Infectious diseases, MESH : Crystallization, [ SDV.NEU.NB ] Life Sciences [q-bio]/Neurons and Cognition [q-bio.NC]/Neurobiology, [SDV.MP.VIR]Life Sciences [q-bio]/Microbiology and Parasitology/Virology, MESH: Camelids, New World, MESH: Porphyromonas gingivalis, MESH: Models, Molecular, Camelus, MESH: Gene Expression, MESH : Cloning, Molecular, Biophysics, MESH: Sequence Alignment, [ SDV.MP.VIR ] Life Sciences [q-bio]/Microbiology and Parasitology/Virology, MESH: Single-Domain Antibodies, Microbiology, 03 medical and health sciences, Bacterial Proteins, Genetics, MESH: Protein Binding, Secretion, Molecular replacement, Protein Interaction Domains and Motifs, 5fwo, MESH: Cloning, Molecular, Amino Acid Sequence, Porphyromonas gingivalis, MESH: Protein Conformation, alpha-Helical, [ SDV.IMM.II ] Life Sciences [q-bio]/Immunology/Innate immunity, MESH: Protein Interaction Domains and Motifs, MESH : Molecular Chaperones, Periplasmic space, [SDV.MP.BAC]Life Sciences [q-bio]/Microbiology and Parasitology/Bacteriology, MESH : Camelids, New World, MESH : Gene Expression, 030104 developmental biology, MESH: Binding Sites, Protein Conformation, beta-Strand, PorM, Molecular Chaperones, type IX secretion system, 0301 basic medicine, Models, Molecular, Protein Conformation, alpha-Helical, MESH : Escherichia coli, [SDV.BIO]Life Sciences [q-bio]/Biotechnology, Gene Expression, [ SDV.MP.BAC ] Life Sciences [q-bio]/Microbiology and Parasitology/Bacteriology, Crystallography, X-Ray, [SDV.IMM.II]Life Sciences [q-bio]/Immunology/Innate immunity, MESH : Bacterial Secretion Systems, Research Communications, [ SDV.BBM.BC ] Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biomolecules [q-bio.BM], Structural Biology, MESH: Genetic Vectors, 5lmj, MESH : Bacterial Proteins, Cloning, Molecular, MESH: Bacterial Secretion Systems, Bacterial Secretion Systems, MESH : Protein Conformation, beta-Strand, MESH: Crystallization, 5lmw, MESH: Kinetics, MESH : Sequence Alignment, MESH : Camelus, Condensed Matter Physics, Recombinant Proteins, [SDV.BBM.BS]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biomolecules [q-bio.BM], MESH : Single-Domain Antibodies, Thermodynamics, MESH: Protein Conformation, beta-Strand, nb02, MESH : Kinetics, nb01, MESH: Thermodynamics, MESH: Molecular Chaperones, Crystallization, Camelids, New World, Protein Binding, crystallization chaperones, MESH : Recombinant Proteins, MESH : Models, Molecular, Genetic Vectors, Context (language use), Biology, MESH : Peptide Library, Peptide Library, Escherichia coli, Animals, nb130, [SDV.BBM.BC]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biochemistry [q-bio.BM], Peptide library, MESH : Thermodynamics, nb19, Binding Sites, Sequence Homology, Amino Acid, [ SDV.SP.PHARMA ] Life Sciences [q-bio]/Pharmaceutical sciences/Pharmacology, [ SDV.BIO ] Life Sciences [q-bio]/Biotechnology, Single-Domain Antibodies, biology.organism_classification, MESH: Crystallography, X-Ray, Kinetics, [SDV.SP.PHARMA]Life Sciences [q-bio]/Pharmaceutical sciences/Pharmacology, MESH : Animals, MESH: Peptide Library, MESH : Crystallography, X-Ray, camelid nanobodies, Sequence Alignment, 5lz0, MESH : Binding Sites, MESH : Protein Interaction Domains and Motifs, [ SDV.BBM.BS ] Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biomolecules [q-bio.BM]
Abstract

PorM is a membrane protein that is involved in the assembly of the type IX secretion system (T9SS) inPorphyromonas gingivalis, a major bacterial pathogen that is responsible for periodontal disease in humans. In the context of structural studies of PorM to better understand T9SS assembly, four camelid nanobodies were selected, produced and purified, and their specific interaction with the N-terminal or C-terminal part of the periplasmic domain of PorM was investigated. Diffracting crystals were also obtained, and the structures of the four nanobodies were solved by molecular replacement. Furthermore, two nanobodies were used as crystallization chaperones and turned out to be valuable tools in the structure-determination process of the periplasmic domain of PorM.

Published
2017

4. Structure and allosteric inhibition mechanism of excitatory amino acid transporter 1

Author

Canul-Tec, Juan, Assal, Reda, Cirri, Erica, Legrand, Pierre, Brier, Sébastien, Chamot-Rooke, Julia, Reyes, Nicolas, Mécanismes moléculaires du transport membranaire - Molecular mechanisms of membrane transport, Institut Pasteur [Paris]-Centre National de la Recherche Scientifique (CNRS), Synchrotron SOLEIL (SSOLEIL), Centre National de la Recherche Scientifique (CNRS), Spectrométrie de Masse pour la Biologie – Mass Spectrometry for Biology (UTechS MSBio), Institut Pasteur [Paris]-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS), The work was funded by the ERC Starting grant 309657 (N.R.). Further support from G5 Institut Pasteur funds (N.R.), CACSICE grant (ANR-11-EQPX-008), and CNRS UMR3528 (N.R., J.C.-R.) is acknowledged., We thank O. Boudker for comments on the manuscript and discussion on consensus mutagenesis, P. V. Krasteva for comments on the manuscript, A. Haouz and the staff at the crystallogenesis core facility of the Institut Pasteur for assistance with crystallization screens, Staff at Synchrotron SOLEIL and the European Synchrotron Radiation Facility for assistance with data collection, D. O’Brien for discussion of HDX results., ANR-11-EQPX-0008,CACSICE,Centre d'analyse de systèmes complexes dans les environnements complexes(2011), European Project: 309657,EC:FP7:ERC,ERC-2012-StG_20111109,HEAATS(2012), Mécanismes moléculaires du transport membranaire, Institut Pasteur [Paris]-Centre National de la Recherche Scientifique ( CNRS ), Synchrotron SOLEIL ( SSOLEIL ), Centre National de la Recherche Scientifique ( CNRS ), Spectrométrie de Masse pour la Biologie – Mass Spectrometry for Biology ( UTechS MSBio ), ANR-11-EQPX-0008/11-EQPX-0008,CACSICE,Centre d'analyse de systèmes complexes dans les environnements complexes ( 2011 ), European Project : 309657,EC:FP7:ERC,ERC-2012-StG_20111109,HEAATS ( 2012 ), Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS), and Institut Pasteur [Paris] (IP)-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS)

Subjects
MESH: Mass Spectrometry, [ SDV.BBM.BP ] Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biophysics, MESH: Humans, MESH : Models, Molecular, MESH : Allosteric Site, MESH : Humans, MESH : Protein Domains, Molecular neuroscience, MESH: Crystallography, X-Ray, MESH: Allosteric Regulation, MESH : Allosteric Regulation, MESH: Excitatory Amino Acid Transporter 1, [SDV.BBM.BP]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biophysics, MESH : Mass Spectrometry, MESH: Deuterium Exchange Measurement, MESH : Crystallization, Membrane proteins, MESH : Deuterium Exchange Measurement, MESH: Protein Domains, Permeation and transport, MESH : Crystallography, X-Ray, MESH: Allosteric Site, MESH : Excitatory Amino Acid Transporter 1, MESH: Models, Molecular, MESH: Crystallization
Abstract

International audience; Human members of the solute carrier 1 (SLC1) family of transporters take up excitatory neurotransmitters in the brain and amino acids in peripheral organs. Dysregulation of the function of SLC1 transporters is associated with neurodegenerative disorders and cancer. Here we present crystal structures of a thermostabilized human SLC1 transporter, the excitatory amino acid transporter 1 (EAAT1), with and without allosteric and competitive inhibitors bound. The structures reveal architectural features of the human transporters, such as intra- and extracellular domains that have potential roles in transport function, regulation by lipids and post-translational modifications. The coordination of the allosteric inhibitor in the structures and the change in the transporter dynamics measured by hydrogen-deuterium exchange mass spectrometry reveal a mechanism of inhibition, in which the transporter is locked in the outward-facing states of the transport cycle. Our results provide insights into the molecular mechanisms underlying the function and pharmacology of human SLC1 transporters.

Published
2017

5. Crystallization via tubing microfluidics permits both in situ and ex situ X-ray diffraction

Author

Tiphaine Huet, Leonard M. G. Chavas, Nathalie Ferte, Charline J. J. Gerard, Romain Grossier, Nadine Candoni, Laurent Vuillard, Gilles Ferry, Stéphane Veesler, Jean A. Boutin, Centre Interdisciplinaire de Nanoscience de Marseille (CINaM), Aix Marseille Université (AMU)-Centre National de la Recherche Scientifique (CNRS), Institut de Recherches Servier, Centre de Recherches de Croissy, Synchrotron SOLEIL (SSOLEIL), Centre National de la Recherche Scientifique (CNRS), and Centre National de la Recherche Scientifique (CNRS)-Aix Marseille Université (AMU)

Subjects
In situ, Materials science, Microfluidics, Biophysics, MESH: crystallization, 02 engineering and technology, 010402 general chemistry, 01 natural sciences, Biochemistry, Research Communications, law.invention, Crystal, X-Ray Diffraction, Structural Biology, law, Genetics, Crystallization, MESH: microfluidic, Diffractometer, [SDV.BBM.BS]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Structural Biology [q-bio.BM], A protein, Biomolecules (q-bio.BM), 021001 nanoscience & nanotechnology, Condensed Matter Physics, 0104 chemical sciences, MESH: crystallography, Quantitative Biology - Biomolecules, Chemical engineering, FOS: Biological sciences, X-ray crystallography, [PHYS.COND.CM-MS]Physics [physics]/Condensed Matter [cond-mat]/Materials Science [cond-mat.mtrl-sci], 0210 nano-technology
Abstract

A microfluidic platform was used to address the problems of obtaining diffraction-quality crystals and crystal handling during transfer to the X-ray diffractometer. Crystallization conditions of a protein of pharmaceutical interest were optimized and X-ray data were collected both in situ and ex situ.

Published
2017

6. The tyrosine kinase inhibitor imatinib mesylate suppresses uric acid crystal-induced acute gouty arthritis in mice

Author

Dongmin Kang, Stephan Rogalla, Bianca Balbino, Riccardo Sibilano, Harini Raghu, Philipp Starkl, Christopher H. Contag, Stephen J. Galli, Steven Sensarn, William H. Robinson, Laurent L. Reber, Jeremy Sokolove, Mindy Tsai, Nicolas Gaudenzio, Department of Pathology [Stanford], Stanford Medicine, Stanford University-Stanford University, Sean N. Parker Center for Allergy and Asthma Research [Stanford], Anticorps en thérapie et pathologie - Antibodies in Therapy and Pathology, Institut Pasteur [Paris]-Institut National de la Santé et de la Recherche Médicale (INSERM), Université Pierre et Marie Curie - Paris 6 (UPMC), Stanford School of Medicine [Stanford], Molecular Imaging Program at Stanford (MIPS), EWHA Womans University (EWHA), Veterans Affairs Palo Alto Healthcare System [Palo Alto, CA, États-Unis], Department of Microbiology and Immunology [Stanford], L.L.R. acknowledges support from the Arthritis National Research Foundation (ANRF), National Institutes of Health grant K99 AI110645, the European Commission (Marie Skłodowska-Curie Individual Fellowship H2020-MSCA-IF-2014 656086) and the Institut National de la Santé et de la Recherche Médicale (INSERM), P.S. was supported by a Max Kade Fellowship of the Max Kade Foundation and the Austrian Academy of Sciences and a Schroedinger Fellowship of the Austrian Science Fund (FWF): J3399-B21, B.B. was supported by a stipend from the Pasteur - Paris University (PPU) International Ph.D. Program, R.S. was supported by the Lucile Packard Foundation for Children’s Health and the Stanford NIH/NCRR CTSA award number UL1 RR025744, N.G. was the recipient of a fellowship from the French 'Fondation pour la Recherche Médicale FRM', S.R. was supported in part by the George Will Foundation, C.H.C. acknowledges the Chambers Family Foundation Fund for Excellence in Pediatric Research, and the Child Health Research Institute at Stanford for their generous support, S.J.G. acknowledges support from National Institutes of Health grants U19 AI104209, NS 080062, and R01 AR067145 and the Department of Pathology, Stanford University School of Medicine., We thank Dr. Rajadas and the Stanford Biomaterials and Advanced Drug Delivery (BioADD) Laboratory core for the production of the imatinib-loaded PLGA nanoparticles., HAL UPMC, Gestionnaire, Institut Pasteur [Paris] (IP)-Institut National de la Santé et de la Recherche Médicale (INSERM), and Veterans Affairs Palo Alto Healthcare System

Subjects
0301 basic medicine, Gout, MESH: Protein Kinase Inhibitors / administration & dosage, lcsh:Medicine, Arthritis, MESH: Uric Acid / chemistry, Pharmacology, Pathology and Laboratory Medicine, Mouse models, Tyrosine-kinase inhibitor, Mice, chemistry.chemical_compound, 0302 clinical medicine, Medicine and Health Sciences, Nanotechnology, MESH: Animals, lcsh:Science, Musculoskeletal System, Immune Response, Routes of Administration, MESH: Crystallization, [SDV.MHEP.RSOA] Life Sciences [q-bio]/Human health and pathology/Rhumatology and musculoskeletal system, Multidisciplinary, Arthritis, Gouty, Animal Models, Protein-Tyrosine Kinases, 3. Good health, Experimental Organism Systems, [SDV.MHEP.RSOA]Life Sciences [q-bio]/Human health and pathology/Rhumatology and musculoskeletal system, Rheumatoid arthritis, [SDV.SP.PHARMA] Life Sciences [q-bio]/Pharmaceutical sciences/Pharmacology, Physical Sciences, Imatinib Mesylate, Legs, Engineering and Technology, Anatomy, Crystallization, Tyrosine kinase, Injections, Intraperitoneal, MESH: Injections, Intraperitoneal, Research Article, medicine.drug, medicine.medical_specialty, Materials by Structure, medicine.drug_class, Inflammatory Diseases, Materials Science, Immunology, Intraperitoneal injections, MESH: Imatinib Mesylate / administration & dosage, Research and Analysis Methods, Crystals, 03 medical and health sciences, MESH: Protein-Tyrosine Kinases / antagonists & inhibitors, Model Organisms, Signs and Symptoms, Rheumatology, MESH: Imatinib Mesylate / pharmacology, Diagnostic Medicine, MESH: Mice, Inbred C57BL, Internal medicine, medicine, Animals, Protein Kinase Inhibitors, MESH: Mice, 030203 arthritis & rheumatology, Inflammation, MESH: Arthritis, Gouty / prevention & control, business.industry, lcsh:R, Limbs (Anatomy), Ankles, Biology and Life Sciences, Imatinib, medicine.disease, MESH: Uric Acid / adverse effects, MESH: Protein Kinase Inhibitors / pharmacology, Uric Acid, Mice, Inbred C57BL, 030104 developmental biology, Endocrinology, Imatinib mesylate, chemistry, [SDV.SP.PHARMA]Life Sciences [q-bio]/Pharmaceutical sciences/Pharmacology, Uric acid, Nanoparticles, lcsh:Q, business
Abstract

International audience; Gouty arthritis is caused by the deposition of monosodium urate (MSU) crystals in joints. Despite many treatment options for gout, there is a substantial need for alternative treatments for patients unresponsive to current therapies. Tyrosine kinase inhibitors have demonstrated therapeutic benefit in experimental models of antibody-dependent arthritis and in rheumatoid arthritis in humans, but to date, the potential effects of such inhibitors on gouty arthritis has not been evaluated. Here we demonstrate that treatment with the tyrosine kinase inhibitor imatinib mesylate (imatinib) can suppress inflammation induced by injection of MSU crystals into subcutaneous air pouches or into the ankle joint of wild type mice. Moreover, imatinib treatment also largely abolished the lower levels of inflammation which developed in IL-1R1-/- or KitW-sh/W-sh mice, indicating that this drug can inhibit IL-1-independent pathways, as well as mast cell-independent pathways, contributing to pathology in this model. Imatinib treatment not only prevented ankle swelling and synovial inflammation when administered before MSU crystals but also diminished these features when administrated after the injection of MSU crystals, a therapeutic protocol more closely mimicking the clinical situation in which treatment occurs after the development of an acute gout flare. Finally, we also assessed the efficiency of local intra-articular injections of imatinib-loaded poly(lactic-co-glycolic acid) (PLGA) nanoparticles in this model of acute gout. Treatment with low doses of this long-acting imatinib:PLGA formulation was able to reduce ankle swelling in a therapeutic protocol. Altogether, these results raise the possibility that tyrosine kinase inhibitors might have utility in the treatment of acute gout in humans.

Published
2017

7. Structural analysis of point mutations at the Vaccinia virus A20/D4 interface

Author

Wim P. Burmeister, Céline Contesto-Richefeu, Christophe N. Peyrefitte, Xavier Brazzolotto, Frédéric Iseni, Nicolas Tarbouriech, Unité de Virologie [Brétigny-sur-Orge] (IRBA), Institut de Recherche Biomédicale des Armées [Brétigny-sur-Orge] (IRBA), Institut de biologie structurale (IBS - UMR 5075 ), Centre National de la Recherche Scientifique (CNRS)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA), Laboratoire de Chimie Bioorganique et Macromoléculaire - Faculté des Sciences et Techniques (LCBM), Université Cadi Ayyad [Marrakech] (UCA), Emerging Pathogens Laboratory, Fondation Mérieux, Institut de Recherche Biomédicale des Armées (IRBA), Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), and Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Centre National de la Recherche Scientifique (CNRS)

Subjects
Models, Molecular, 0301 basic medicine, Protein Conformation, DNA polymerase, viruses, Amino Acid Motifs, Mutant, Gene Expression, MESH: Catalytic Domain, DNA-Directed DNA Polymerase, Crystallography, X-Ray, Biochemistry, Research Communications, MESH: Recombinant Proteins, MESH: Amino Acid Motifs, MESH: Protein Conformation, X-Ray Diffraction, Structural Biology, immune system diseases, Catalytic Domain, hemic and lymphatic diseases, protein–protein interface, MESH: DNA-Directed DNA Polymerase, MESH: Vaccinia virus, Cloning, Molecular, MESH: Uracil-DNA Glycosidase, Polymerase, MESH: Crystallization, biology, [SDV.BBM.BS]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Structural Biology [q-bio.BM], Chemistry, MESH: Protein Multimerization, MESH: X-Ray Diffraction, Condensed Matter Physics, Recombinant Proteins, Crystallization, MESH: Models, Molecular, Plasmids, MESH: Gene Expression, Stereochemistry, Protein subunit, Biophysics, Vaccinia virus, cation–π interaction, DNA replication, Viral Proteins, 03 medical and health sciences, MESH: Plasmids, Genetics, Point Mutation, MESH: Cloning, Molecular, Uracil-DNA Glycosidase, MESH: Point Mutation, DNA synthesis, Point mutation, MESH: Crystallography, X-Ray, Molecular biology, MESH: Viral Proteins, 030104 developmental biology, DNA glycosylase, biology.protein, X-ray structure, Protein Multimerization
Abstract

TheVaccinia viruspolymerase holoenzyme is composed of three subunits: E9, the catalytic DNA polymerase subunit; D4, a uracil-DNA glycosylase; and A20, a protein with no known enzymatic activity. The D4/A20 heterodimer is the DNA polymerase cofactor, the function of which is essential for processive DNA synthesis. The recent crystal structure of D4 bound to the first 50 amino acids of A20 (D4/A201–50) revealed the importance of three residues, forming a cation–π interaction at the dimerization interface, for complex formation. These are Arg167 and Pro173 of D4 and Trp43 of A20. Here, the crystal structures of the three mutants D4-R167A/A201–50, D4-P173G/A201–50and D4/A201–50-W43A are presented. The D4/A20 interface of the three structures has been analysed for atomic solvation parameters and cation–π interactions. This study confirms previous biochemical data and also points out the importance for stability of the restrained conformational space of Pro173. Moreover, these new structures will be useful for the design and rational improvement of known molecules targeting the D4/A20 interface.

Published
2016

8. Design of a Genetically Stable High Fidelity Coxsackievirus B3 Polymerase That Attenuates Virus Growth in Vivo

Author

Marco Vignuzzi, Olve B. Peersen, Seth McDonald, Gonzalo Moratorio, Stéphanie Beaucourt, Andrew Block, Colorado State University [Fort Collins] (CSU), Populations virales et Pathogenèse - Viral Populations and Pathogenesis, Institut Pasteur [Paris]-Centre National de la Recherche Scientifique (CNRS), This work was supported by National Institutes of Health Grant R01 AI059130 (to O. B. P.) and a LabEx Integrative Biology of Emerging Infectious Diseases program grant (to M. V.). The authors declare that they have no conflicts of interest with the contents of this article. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health., ANR-10-LABX-0062,IBEID,Integrative Biology of Emerging Infectious Diseases(2010), Moratorio, Gonzalo. Instituto Pasteur (París). Universidad de la República (Uruguay). Facultad de Ciencias. Centro de Investigaciones Nucleares., and Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS)

Subjects
0301 basic medicine, Models, Molecular, Genetic variants, Polymers, Protein Conformation, MESH: Sequence Homology, Amino Acid, viruses, [SDV]Life Sciences [q-bio], MESH: Amino Acid Sequence, Virus Replication, Biochemistry, chemistry.chemical_compound, MESH: Protein Conformation, Transcription (biology), RNA polymerase, polymerase fidelity, Infectious virus, Polymerase, Enterovirus, MESH: Crystallization, Genetics, viral polymerase, biology, MESH: Kinetics, [SDV.BBM.BS]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Structural Biology [q-bio.BM], Molecular Bases of Disease, Enterovirus B, Human, virology, RNA Replicase, Amino acids, Crystallization, MESH: RNA Replicase, MESH: Biocatalysis, MESH: Models, Molecular, RNA-dependent RNA polymerase, Viral quasispecies, Virus, 03 medical and health sciences, plus-stranded RNA virus, [SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology, Amino Acid Sequence, [SDV.BBM.BC]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biochemistry [q-bio.BM], Molecular Biology, 030102 biochemistry & molecular biology, Sequence Homology, Amino Acid, MESH: Virus Replication, RNA, protein engineering, [SDV.BBM.BM]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Molecular biology, Cell Biology, RNA-Dependent RNA Polymerase, Kinetics, 030104 developmental biology, chemistry, Viral replication, vaccine development, MESH: Enterovirus B, Human, biology.protein, Biocatalysis
Abstract

International audience; Positive strand RNA viruses replicate via a virally encoded RNA-dependent RNA polymerase (RdRP) that uses a unique palm domain active site closure mechanism to establish the canonical two-metal geometry needed for catalysis. This mechanism allows these viruses to evolutionarily fine-tune their replication fidelity to create an appropriate distribution of genetic variants known as a quasispecies. Prior work has shown that mutations in conserved motif A drastically alter RdRP fidelity, which can be either increased or decreased depending on the viral polymerase background. In the work presented here, we extend these studies to motif D, a region that forms the outer edge of the NTP entry channel where it may act as a nucleotide sensor to trigger active site closure. Crystallography, stopped-flow kinetics, quench-flow reactions, and infectious virus studies were used to characterize 15 engineered mutations in coxsackievirus B3 polymerase. Mutations that interfere with the transport of the metal A Mg(2+) ion into the active site had only minor effects on RdRP function, but the stacking interaction between Phe(364) and Pro(357), which is absolutely conserved in enteroviral polymerases, was found to be critical for processive elongation and virus growth. Mutating Phe(364) to tryptophan resulted in a genetically stable high fidelity virus variant with significantly reduced pathogenesis in mice. The data further illustrate the importance of the palm domain movement for RdRP active site closure and demonstrate that protein engineering can be used to alter viral polymerase function and attenuate virus growth and pathogenesis.

Published
2016

9. In Vitro/In Vivo Production of tRNA for X-Ray Studies

Author

Clément, Dégut, Alexandre, Monod, Franck, Brachet, Thibaut, Crépin, Carine, Tisné, Laboratoire de cristallographie et RMN biologiques (LCRB - UMR 8015), Université Paris Descartes - Paris 5 (UPD5)-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS), Université Sorbonne Paris Cité (USPC), Institut de biologie structurale (IBS - UMR 5075 ), Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Centre National de la Recherche Scientifique (CNRS), Centre National de la Recherche Scientifique (CNRS)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA), Centre National de la Recherche Scientifique (CNRS)-Université Paris Descartes - Paris 5 (UPD5), Institut de biologie structurale (IBS - UMR 5075), Institut de Recherche Interdisciplinaire de Grenoble (IRIG), and Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Grenoble Alpes (UGA)-Centre National de la Recherche Scientifique (CNRS)

Subjects
MESH: Ribonucleases, MESH: Hydrogen-Ion Concentration, Magnetic Resonance Spectroscopy, Transcription, Genetic, Molecular Sequence Data, MESH: Base Sequence, Crystallography, X-Ray, MESH: Chromatography, Viral Proteins, MESH: X-Rays, Ribonucleases, RNA, Transfer, MESH: Plasmids, Escherichia coli, MESH: Crystallization, Chromatography, MESH: Molecular Sequence Data, Base Sequence, [SDV.BBM.BS]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Structural Biology [q-bio.BM], MESH: Escherichia coli, MESH: Magnetic Resonance Spectroscopy, X-Rays, MESH: Transcription, Genetic, Reproducibility of Results, DNA-Directed RNA Polymerases, Hydrogen-Ion Concentration, MESH: Crystallography, X-Ray, MESH: RNA, Transfer, MESH: Viral Proteins, MESH: DNA-Directed RNA Polymerases, MESH: Reproducibility of Results, [SDV.BBM.BS]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biomolecules [q-bio.BM], MESH: Nucleic Acid Conformation, Nucleic Acid Conformation, Crystallization, Plasmids
Abstract

International audience; tRNAs occupy a central role in the cellular life, and they are involved in a broad range of biological processes that relies on their interaction with proteins and RNA. Crystallization and structure resolution of tRNA or/and tRNA/partner complexes can yield in valuable information on structural organizations of key elements of cellular machinery. However, crystallization of RNA, is often challenging. Here we review two methods to produce and purify tRNA in quantity and quality to perform X-ray studies.

Published
2016

10. Urate-induced acute renal failure and chronic inflammation in liver-specific Glut9 knockout mice

Author

Preitner Frederic, Laverriere-Loss Alexandra, Metref Salima, Da Costa Anabela, Moret Catherine, Rotman Samuel, Bazin Dominique, Daudon Michel, Sandt Christophe, Dessombz Arnaud, Thorens Bernard, Mouse Metabolic Facility of the Cardiomet Center, University Hospital Lausanne, Center for Integrative Genomics - Institute of Bioinformatics, Génopode (CIG), Swiss Institute of Bioinformatics [Lausanne] (SIB), Université de Lausanne (UNIL)-Université de Lausanne (UNIL), Mouse Pathology Facility, Université de Lausanne (UNIL), Laboratoire de Chimie de la Matière Condensée de Paris (LCMCP), Université Pierre et Marie Curie - Paris 6 (UPMC)-Centre National de la Recherche Scientifique (CNRS)-Collège de France (CdF (institution))-Institut de Chimie du CNRS (INC), CHU Tenon [AP-HP], Sorbonne Université (SU)-Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP), Synchrotron SOLEIL (SSOLEIL), Centre National de la Recherche Scientifique (CNRS), Laboratoire de Physique des Solides (LPS), Centre National de la Recherche Scientifique (CNRS)-Université Paris-Sud - Paris 11 (UP11), Lausanne University Hospital, Université de Lausanne = University of Lausanne (UNIL)-Université de Lausanne = University of Lausanne (UNIL), Université de Lausanne = University of Lausanne (UNIL), Université Pierre et Marie Curie - Paris 6 (UPMC)-Collège de France (CdF (institution))-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS), Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Sorbonne Université (SU), and Université Paris-Sud - Paris 11 (UP11)-Centre National de la Recherche Scientifique (CNRS)

Subjects
Male, MESH: Inflammation, MESH: Hydrogen-Ion Concentration, Physiology, 030232 urology & nephrology, Glucose Transport Proteins, Facilitative, Organic Anion Transporters, Urine, medicine.disease_cause, MESH: Mice, Knockout, MESH: Uric Acid, MESH: Urine, MESH: Inosine, Mice, 0302 clinical medicine, MESH: Animals, MESH: Crystallization, Mice, Knockout, MESH: Organic Anion Transporters, 0303 health sciences, biology, Chemistry, Acute Kidney Injury, Hydrogen-Ion Concentration, MESH: Hyperuricemia, 3. Good health, MESH: Glucose Transport Proteins, Facilitative, Knockout mouse, medicine.symptom, Crystallization, medicine.medical_specialty, Sterile inflammation, MESH: Acute Kidney Injury, Inflammation, Hyperuricemia, Diet, High-Fat, 03 medical and health sciences, Internal medicine, Liver enzyme, medicine, [SDV.MHEP.PHY]Life Sciences [q-bio]/Human health and pathology/Tissues and Organs [q-bio.TO], Animals, MESH: Mice, 030304 developmental biology, Inosine, MESH: Male, Uric Acid, MESH: Diet, High-Fat, Endocrinology, biology.protein, Oxidative stress, SLC2A9
Abstract

International audience; Plasma urate levels are higher in humans than rodents (240-360 vs. ∼30 μM) because humans lack the liver enzyme uricase. High uricemia in humans may protect against oxidative stress, but hyperuricemia also associates with the metabolic syndrome, and urate and uric acid can crystallize to cause gout and renal dysfunctions. Thus, hyperuricemic animal models to study urate-induced pathologies are needed. We recently generated mice with liver-specific ablation of Glut9, a urate transporter providing access of urate to uricase (LG9KO mice). LG9KO mice had moderately high uricemia (∼120 μM). To further increase their uricemia, here we gavaged LG9KO mice for 3 days with inosine, a urate precursor; this treatment was applied in both chow- and high-fat-fed mice. In chow-fed LG9KO mice, uricemia peaked at 300 μM 2 h after the first gavage and normalized 24 h after the last gavage. In contrast, in high-fat-fed LG9KO mice, uricemia further rose to 500 μM. Plasma creatinine strongly increased, indicating acute renal failure. Kidneys showed tubule dilation, macrophage infiltration, and urate and uric acid crystals, associated with a more acidic urine. Six weeks after inosine gavage, plasma urate and creatinine had normalized. However, renal inflammation, fibrosis, and organ remodeling had developed despite the disappearance of urate and uric acid crystals. Thus, hyperuricemia and high-fat diet feeding combined to induce acute renal failure. Furthermore, a sterile inflammation caused by the initial crystal-induced lesions developed despite the disappearance of urate and uric acid crystals.

Published
2013

11. Relation between dynamics, activity and thermal stability within the cholinesterase family

Author

Patrick Masson, Marcus Trapp, Moeava Tehei, Florian Nachon, Judith Peters, Marie Trovaslet, Martin Weik, Institut de biologie structurale (IBS - UMR 5075 ), Centre National de la Recherche Scientifique (CNRS)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA), Institut Laue-Langevin (ILL), Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Centre National de la Recherche Scientifique (CNRS), and ILL

Subjects
MESH: Enzyme Stability, Kinetics, Thermodynamics, Neutron scattering, Toxicology, MESH: Recombinant Proteins, Mice, 03 medical and health sciences, Enzyme Stability, Animals, Cholinesterases, Humans, Neutron, Thermal stability, MESH: Animals, MESH: Mice, 030304 developmental biology, MESH: Crystallization, Neutrons, 0303 health sciences, MESH: Humans, MESH: Butyrylcholinesterase, MESH: Kinetics, [SDV.BBM.BS]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Structural Biology [q-bio.BM], Chemistry, Hydrolysis, Transition temperature, 030302 biochemistry & molecular biology, Dynamics (mechanics), Temperature, General Medicine, MESH: Acetylcholinesterase, Recombinant Proteins, MESH: Temperature, Mean squared displacement, Crystallography, MESH: Neutrons, Neutron backscattering, Butyrylcholinesterase, Acetylcholinesterase, Cholinesterase Inhibitors, MESH: Thermodynamics, Crystallization, MESH: Cholinesterase Inhibitors, MESH: Hydrolysis, MESH: Cholinesterases
Abstract

International audience; Incoherent neutron scattering is one of the most powerful tools for studying dynamics in biological matter. Using the cold neutron backscattering spectrometer IN16 at the Institut Laue Langevin (ILL, Grenoble, France), temperature dependence of cholinesterases' dynamics (human butyrylcholinesterase from plasma: hBChE; recombinant human acetylcholinesterase: hAChE and recombinant mouse acetylcholinesterase: mAChE) was examined using elastic incoherent neutron scattering (EINS). The dynamics was characterized by the averaged atomic mean square displacement (MSD), associated with the sample flexibility at a given temperature. We found MSD values of hAChE above the dynamical transition temperature (around 200K) larger than for mAChE and hBChE, implying that hAChE is more flexible than the other ChEs. Activation energies for thermodynamical transition were extracted through the frequency window model (FWM) (Becker et al. 2004) [1] and turned out to increase from hBChE to mAChE and finally to hAChE, inversely to the MSDs relations. Between 280 and 316K, catalytic studies of these enzymes were carried out using thiocholine esters: at the same temperature, the hAChE activity was systematically higher than the mAChE or hBChE ones. Our results thus suggest a strong correlation between dynamics and activity within the ChE family. We also studied and compared the ChEs thermal inactivation kinetics. Here, no direct correlation with the dynamics was observed, thus suggesting that relations between enzyme dynamics and catalytic stability are more complex. Finally, the possible relation between flexibility and protein ability to grow in crystals is discussed.

Published
2013

12. Dynamics of polymerization shed light on the mechanisms that lead to multiple amyloid structures of the prion protein

Author

Jacques-Damien Arnaud, Viviana Zomosa-Signoret, Erwan Hingant, Maria-Teresa Alvarez-Martinez, Laurent Pujo-Menjouet, Jean-Pierre Liautard, Pascaline Fontes, BioCampus Montpellier (BCM), Université Montpellier 1 (UM1)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université de Montpellier (UM)-Centre National de la Recherche Scientifique (CNRS), Institut Montpelliérain de biologie (IMB), Centre National de la Recherche Scientifique (CNRS)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre régional de lutte contre le cancer-Institut Français de Recherche pour l'Exploitation de la Mer (IFREMER)-Université Montpellier 2 - Sciences et Techniques (UM2)-Institut National de la Recherche Agronomique (INRA)-Université Montpellier 1 (UM1), Université Montpellier 2 - Sciences et Techniques (UM2), Centre d’études d’Agents Pathogènes et Biotechologies pour la Santé (CPBS), Université de Montpellier (UM)-Centre National de la Recherche Scientifique (CNRS), Multi-scale modelling of cell dynamics : application to hematopoiesis (DRACULA), Centre de génétique et de physiologie moléculaire et cellulaire (CGPhiMC), Centre National de la Recherche Scientifique (CNRS)-Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-Centre National de la Recherche Scientifique (CNRS)-Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-Inria Grenoble - Rhône-Alpes, Institut National de Recherche en Informatique et en Automatique (Inria)-Institut National de Recherche en Informatique et en Automatique (Inria)-Institut Camille Jordan [Villeurbanne] (ICJ), École Centrale de Lyon (ECL), Université de Lyon-Université de Lyon-Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université Jean Monnet [Saint-Étienne] (UJM)-Institut National des Sciences Appliquées de Lyon (INSA Lyon), Université de Lyon-Institut National des Sciences Appliquées (INSA)-Institut National des Sciences Appliquées (INSA)-Centre National de la Recherche Scientifique (CNRS)-École Centrale de Lyon (ECL), Université de Lyon-Institut National des Sciences Appliquées (INSA)-Institut National des Sciences Appliquées (INSA)-Centre National de la Recherche Scientifique (CNRS), Institut Camille Jordan [Villeurbanne] (ICJ), EC ImmunoPrion Project no. 023144, Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Montpellier 1 (UM1)-Centre National de la Recherche Scientifique (CNRS)-Université de Montpellier (UM), Centre National de la Recherche Scientifique (CNRS)-Université Jean Monnet [Saint-Étienne] (UJM)-École Centrale de Lyon (ECL), Université de Lyon-Université de Lyon-Institut National des Sciences Appliquées de Lyon (INSA Lyon), Université de Lyon-Institut National des Sciences Appliquées (INSA)-Institut National des Sciences Appliquées (INSA)-Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Centre National de la Recherche Scientifique (CNRS)-Université Jean Monnet [Saint-Étienne] (UJM)-École Centrale de Lyon (ECL), Université de Lyon-Inria Grenoble - Rhône-Alpes, Institut National de Recherche en Informatique et en Automatique (Inria)-Institut National de Recherche en Informatique et en Automatique (Inria)-Centre de génétique et de physiologie moléculaire et cellulaire (CGPhiMC), Université de Lyon-Université de Lyon-Centre National de la Recherche Scientifique (CNRS), Université de Lyon, Université Montpellier 1 (UM1)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Université Montpellier 1 (UM1)-Institut National de la Recherche Agronomique (INRA)-Université Montpellier 2 - Sciences et Techniques (UM2)-Institut Français de Recherche pour l'Exploitation de la Mer (IFREMER)-Centre régional de lutte contre le cancer-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-Centre National de la Recherche Scientifique (CNRS)-Inria Grenoble - Rhône-Alpes, Institut National de Recherche en Informatique et en Automatique (Inria)-Institut National de Recherche en Informatique et en Automatique (Inria)-Institut Camille Jordan (ICJ), Université de Lyon-Institut National des Sciences Appliquées de Lyon (INSA Lyon), Université de Lyon-Institut National des Sciences Appliquées (INSA)-Institut National des Sciences Appliquées (INSA)-Université Jean Monnet - Saint-Étienne (UJM)-Centre National de la Recherche Scientifique (CNRS)-École Centrale de Lyon (ECL), Université de Lyon-Institut National des Sciences Appliquées (INSA)-Institut National des Sciences Appliquées (INSA)-Université Jean Monnet - Saint-Étienne (UJM)-Centre National de la Recherche Scientifique (CNRS), and Institut Camille Jordan (ICJ)

Subjects
MESH: Signal Transduction, Time Factors, Nucleation, MESH: Cricetinae, Plaque, Amyloid, Biochemistry, Analytical Chemistry, Strain, MESH: Recombinant Proteins, 0302 clinical medicine, Cricetinae, MESH: Animals, MESH: Molecular Dynamics Simulation, MESH: Crystallization, 0303 health sciences, MESH: Kinetics, [SDV.BBM.BS]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Structural Biology [q-bio.BM], Chemistry, MESH: Protein Multimerization, Dynamics (mechanics), Fibrillogenesis, Recombinant Proteins, Dynamics, [SDV.BBM.BS]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biomolecules [q-bio.BM], MESH: Plaque, Amyloid, medicine.anatomical_structure, Prion, Crystallization, Signal Transduction, Amyloid, Prions, Biophysics, Buffers, Molecular Dynamics Simulation, Models, Biological, 03 medical and health sciences, MESH: Prions, medicine, Animals, Humans, Prion protein, Molecular Biology, 030304 developmental biology, MESH: Humans, MESH: Time Factors, MESH: Models, Biological, Biochemistry of Alzheimer's disease, Kinetics, Polymerization, MESH: Buffers, Protein Multimerization, Heterogeneity, Nucleus, 030217 neurology & neurosurgery
Abstract

International audience; It is generally accepted that spongiform encephalopathies result from the aggregation into amyloid of a ubiquitous protein, the so-called prion protein. As a consequence, the dynamics of amyloid formation should explain the characteristics of the prion diseases: infectivity as well as sporadic and genetic occurrence, long incubation time, species barriers and strain specificities. The success of this amyloid hypothesis is due to the good qualitative agreement of this hypothesis with the observations. However, a number of difficulties appeared when comparing quantitatively the in vitro experimental results with the theoretical models, suggesting that some differences should hide important discrepancies. We used well defined quantitative models to analyze the experimental results obtained by in vitro polymerization of the recombinant hamster prion protein. Although the dynamics of polymerization resembles a simple nucleus-dependent fibrillogenesis, neither the initial concentration dependence nor off-pathway hypothesis fit with experimental results. Furthermore, seeded polymerization starts after a long time delay suggesting the existence of a specific mechanism that takes place before nucleus formation. On the other hand, polymerization dynamics reveals a highly stochastic mechanism, the origin of which appears to be caused by nucleation heterogeneity. Moreover, the specific structures generated during nucleation are maintained during successive seeding although a clear improvement of the dynamics parameters (polymerization rate and lag time) is observed. We propose that an additional on-pathway reaction takes place before nucleation and it is responsible for the heterogeneity of structures produced during prion protein polymerization in vitro. These amyloid structures behave like prion strains. A model is proposed to explain the genesis of heterogeneity among prion amyloid.

Published
2011

13. Crystallization and diffraction analysis of the SARS coronavirus nsp10-nsp16 complex

Author

Claire Debarnot, Bruno Canard, Isabelle Varlet, François Ferron, Laure Gluais, Mickaël Bouvet, Etienne Decroly, Julien Lescar, Nicolas Papageorgiou, Isabelle Imbert, Centre de recherche en neurobiologie - neurophysiologie de Marseille (CRN2M), Aix Marseille Université (AMU)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Architecture et fonction des macromolécules biologiques (AFMB), Centre National de la Recherche Scientifique (CNRS)-Aix Marseille Université (AMU)-Institut National de la Recherche Agronomique (INRA), Division Eau et Environnement (LCPC/EAU), Laboratoire Central des Ponts et Chaussées (LCPC)-PRES Université Nantes Angers Le Mans (UNAM), and Institut National de la Recherche Agronomique (INRA)-Aix Marseille Université (AMU)-Centre National de la Recherche Scientifique (CNRS)

Subjects
Methyltransferase, viruses, Molecular Sequence Data, Biophysics, MESH: SARS Virus, RNA-dependent RNA polymerase, Biology, Viral Nonstructural Proteins, Crystallography, X-Ray, Biochemistry, law.invention, 03 medical and health sciences, Structural Biology, law, MESH: Methyltransferases, Genetics, Viral structural protein, Humans, Cap formation, MESH: Cloning, Molecular, Crystallization, Cloning, Molecular, [SDV.BBM.BC]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biochemistry [q-bio.BM], skin and connective tissue diseases, Gene, 030304 developmental biology, MESH: Crystallization, 0303 health sciences, Messenger RNA, MESH: Humans, MESH: Molecular Sequence Data, 030302 biochemistry & molecular biology, fungi, RNA virus, Methyltransferases, Condensed Matter Physics, biology.organism_classification, RNA-Dependent RNA Polymerase, MESH: Crystallography, X-Ray, Virology, 3. Good health, [SDV.BBM.BC]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biomolecules [q-bio.BM], body regions, Severe acute respiratory syndrome-related coronavirus, Crystallization Communications, MESH: Viral Nonstructural Proteins, MESH: RNA Replicase
Abstract

International audience; To date, the SARS coronavirus is the only known highly pathogenic human coronavirus. In 2003, it was responsible for a large outbreak associated with a 10% fatality rate. This positive RNA virus encodes a large replicase polyprotein made up of 16 gene products (nsp1-16), amongst which two methyltransferases, nsp14 and nsp16, are involved in viral mRNA cap formation. The crystal structure of nsp16 is unknown. Nsp16 is an RNA-cap AdoMet-dependent (nucleoside-2'-O-)-methyltransferase that is only active in the presence of nsp10. In this paper, the expression, purification and crystallization of nsp10 in complex with nsp16 are reported. The crystals diffracted to a resolution of 1.9 Å resolution and crystal structure determination is in progress.

Published
2011

14. Crystallization and preliminary X-ray diffraction studies of delta-toxin from Clostridium perfringens

Author

Jessica Huyet, Ajit K. Basak, Michel R. Popoff, Maryse Gilbert, Birkbeck College [University of London], Bactéries anaérobies et Toxines, Institut Pasteur [Paris], The authors wish to thank Dr Robert Sarra for his assistance with ESI-MS and CD spectroscopy, and the beamline staff at ID29, ESRF, Grenoble for assistance with data collection., and Institut Pasteur [Paris] (IP)

Subjects
Clostridium perfringens, Bacterial Toxins, Molecular Sequence Data, Biophysics, MESH: Rabbits, Enterotoxin, Crystallography, X-Ray, medicine.disease_cause, Biochemistry, delta-toxin, law.invention, 03 medical and health sciences, Structural Biology, law, Genetics, medicine, Animals, Humans, MESH: Animals, 030304 developmental biology, MESH: Crystallization, chemistry.chemical_classification, MESH: Clostridium perfringens, 0303 health sciences, MESH: Humans, MESH: Molecular Sequence Data, biology, [SDV.BBM.BS]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Structural Biology [q-bio.BM], Toxin, 030302 biochemistry & molecular biology, Hemolysin, Condensed Matter Physics, biology.organism_classification, MESH: Crystallography, X-Ray, Amino acid, Lytic cycle, chemistry, MESH: Bacterial Toxins, Crystallization Communications, [SDV.TOX]Life Sciences [q-bio]/Toxicology, Recombinant DNA, Rabbits, Crystallization, haemolysins, Bacteria
Abstract

International audience; Clostridium perfringens is a Gram-positive anaerobic bacterium that is responsible for a wide range of diseases in humans and both wild and domesticated animals, including birds. C. perfringens is notable for its ability to produce a plethora of toxins, e.g. phospholipases C (alpha-toxin), pore-forming toxins (epsilon-toxin, beta-toxin and enterotoxin) and binary toxins (iota-toxin). Based on alpha-, beta-, epsilon- and iota-toxin production, the bacterium is classified into five different toxinotypes (A-E). Delta-toxin, which is a 32.6 kDa protein with 290 amino acids, is one of three haemolysins released by type C and possibly by type B strains of C. perfringens. This toxin is immunogenic and lytic to erythrocytes from the even-toed ungulates sheep, goats and pigs, and is cytotoxic to other cell types such as rabbit macrophages, human monocytes and blood platelets from goats, rabbits, guinea pigs and humans. The recombinant delta-toxin has been cloned, expressed, purified and crystallized in two different crystal forms by the hanging-drop vapour-diffusion method. Of these two different crystal forms, only the form II crystal diffracted to atomic resolution (dmin=2.4 Å), while the form I crystal diffracted to only 15 Å resolution. The form II crystals belonged to space group P2(1)2(1)2, with one molecule in the crystallographic asymmetric unit and unit-cell parameters a=49.66, b=58.48, c=112.93 Å.

Published
2011

15. Prion fibrils of Ure2p assembled under physiological conditions contain highly ordered, natively folded modules

Author

Anja Böckmann, Carole Gardiennet, Beat H. Meier, Luc Bousset, Yannick Sourigues, Antoine Loquet, Ronald Melki, Birgit Habenstein, Christian Wasmer, Anne K. Schütz, Institut de biologie et chimie des protéines [Lyon] (IBCP), Centre National de la Recherche Scientifique (CNRS)-Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon, Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-Centre National de la Recherche Scientifique (CNRS), Laboratoire d'Enzymologie et Biochimie Structurales (LEBS), Centre National de la Recherche Scientifique (CNRS), Physical Chemistry [ETH Zürich], Department of Chemistry and Applied Biosciences [ETH Zürich] (D-CHAB), and Eidgenössische Technische Hochschule - Swiss Federal Institute of Technology [Zürich] (ETH Zürich)- Eidgenössische Technische Hochschule - Swiss Federal Institute of Technology [Zürich] (ETH Zürich)

Subjects
Hot Temperature, Magnetic Resonance Spectroscopy, Saccharomyces cerevisiae Proteins, MESH: Amino Acids, MESH: Protein Structure, Quaternary, Prions, animal diseases, [SDV]Life Sciences [q-bio], Structural diversity, MESH: Protein Structure, Secondary, MESH: Pliability, Saccharomyces cerevisiae, MESH: Hot Temperature, 010402 general chemistry, Fibril, 01 natural sciences, Protein Structure, Secondary, 03 medical and health sciences, MESH: Protein Structure, Tertiary, Molecular level, Protein structure, MESH: Saccharomyces cerevisiae Proteins, MESH: Prions, Structural Biology, Amino Acids, Pliability, Protein Structure, Quaternary, Molecular Biology, 030304 developmental biology, MESH: Crystallization, 0303 health sciences, Glutathione Peroxidase, Chemistry, MESH: Magnetic Resonance Spectroscopy, Nuclear magnetic resonance spectroscopy, MESH: Saccharomyces cerevisiae, 0104 chemical sciences, Protein Structure, Tertiary, nervous system diseases, NMR spectra database, Crystallography, Order (biology), Solid-state nuclear magnetic resonance, MESH: Glutathione Peroxidase, Crystallization
Abstract

International audience; The difference between the prion and the non-prion form of a protein is given solely by its three-dimensional structure, according to the prion hypothesis. It has been shown that solid-state NMR can unravel the atomic-resolution three-dimensional structure of prion fragments but, in the case of Ure2p, no highly resolved spectra are obtained from the isolated prion domain. Here, we demonstrate that the spectra of full-length fibrils of Ure2p interestingly lead to highly resolved solid-state NMR spectra. Prion fibrils formed under physiological conditions are therefore well-ordered objects on the molecular level. Comparing the full-length NMR spectra with the corresponding spectra of the prion and globular domains in isolation reveals that the globular part in particular shows almost perfect structural order. The NMR linewidths in these spectra are as narrow as the ones observed in crystals of the isolated globular domain. For the prion domain, the spectra reflect partial disorder, suggesting structural heterogeneity, both in isolation and in full-length Ure2p fibrils, although to different extents. The spectral quality is surprising in the light of existing structural models for Ure2p and in comparison to the corresponding spectra of the only other full-length prion fibrils (HET-s) investigated so far. This opens the exciting perspective of an atomic-resolution structure determination of the fibrillar form of a prion whose assembly is not accompanied by significant conformational changes and documents the structural diversity underlying prion propagation.

Published
2009

16. Crystal growth of proteins, nucleic acids, and viruses in gels

Author

Bernard Lorber, Sarah Sanglier, Pascale Schellenberger, M.C. Robert, Noelle Potier, Abel Moreno, Claude Sauter, Anne Théobald-Dietrich, Richard Giegé, B. Capelle, Architecture et Réactivité de l'ARN (ARN), Institut de biologie moléculaire et cellulaire (IBMC), Université de Strasbourg (UNISTRA)-Centre National de la Recherche Scientifique (CNRS)-Université de Strasbourg (UNISTRA)-Centre National de la Recherche Scientifique (CNRS)-Centre National de la Recherche Scientifique (CNRS), Institut de minéralogie et de physique des milieux condensés (IMPMC), Université Pierre et Marie Curie - Paris 6 (UPMC)-IPG PARIS-Université Paris Diderot - Paris 7 (UPD7)-Centre National de la Recherche Scientifique (CNRS), Laboratoire de Spectrométrie de Masse BioOrganique [Strasbourg] (LSMBO), Département Sciences Analytiques et Interactions Ioniques et Biomoléculaires (DSA-IPHC), Institut Pluridisciplinaire Hubert Curien (IPHC), Université de Strasbourg (UNISTRA)-Institut National de Physique Nucléaire et de Physique des Particules du CNRS (IN2P3)-Centre National de la Recherche Scientifique (CNRS)-Université de Strasbourg (UNISTRA)-Institut National de Physique Nucléaire et de Physique des Particules du CNRS (IN2P3)-Centre National de la Recherche Scientifique (CNRS)-Institut Pluridisciplinaire Hubert Curien (IPHC), Université de Strasbourg (UNISTRA)-Institut National de Physique Nucléaire et de Physique des Particules du CNRS (IN2P3)-Centre National de la Recherche Scientifique (CNRS)-Université de Strasbourg (UNISTRA)-Institut National de Physique Nucléaire et de Physique des Particules du CNRS (IN2P3)-Centre National de la Recherche Scientifique (CNRS), Institut de Chimie de Strasbourg, Université de Strasbourg (UNISTRA)-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS), Chimie de la matière complexe (CMC), Université Pierre et Marie Curie - Paris 6 (UPMC)-Université Paris Diderot - Paris 7 (UPD7)-Institut de Physique du Globe de Paris (IPG Paris)-Centre National de la Recherche Scientifique (CNRS), Université de Strasbourg (UNISTRA)-Université de Haute-Alsace (UHA) Mulhouse - Colmar (Université de Haute-Alsace (UHA))-Institut National de Physique Nucléaire et de Physique des Particules du CNRS (IN2P3)-Centre National de la Recherche Scientifique (CNRS)-Université de Strasbourg (UNISTRA)-Université de Haute-Alsace (UHA) Mulhouse - Colmar (Université de Haute-Alsace (UHA))-Institut National de Physique Nucléaire et de Physique des Particules du CNRS (IN2P3)-Centre National de la Recherche Scientifique (CNRS)-Institut Pluridisciplinaire Hubert Curien (IPHC), and Université de Strasbourg (UNISTRA)-Université de Haute-Alsace (UHA) Mulhouse - Colmar (Université de Haute-Alsace (UHA))-Institut National de Physique Nucléaire et de Physique des Particules du CNRS (IN2P3)-Centre National de la Recherche Scientifique (CNRS)-Université de Strasbourg (UNISTRA)-Université de Haute-Alsace (UHA) Mulhouse - Colmar (Université de Haute-Alsace (UHA))-Institut National de Physique Nucléaire et de Physique des Particules du CNRS (IN2P3)-Centre National de la Recherche Scientifique (CNRS)

Subjects
02 engineering and technology, 01 natural sciences, law.invention, Crystal, chemistry.chemical_compound, law, Nucleic Acids, Agarose, MESH: Proteins, MESH: Animals, Crystallization, ComputingMilieux_MISCELLANEOUS, MESH: Crystallization, Gel, Silica, Lipid phase, 021001 nanoscience & nanotechnology, Virus, Viruses, [SDV.MP.VIR]Life Sciences [q-bio]/Microbiology and Parasitology/Virology, 0210 nano-technology, Macromolecule, MESH: Gels, Biophysics, Crystal growth, [SDV.BC]Life Sciences [q-bio]/Cellular Biology, 010402 general chemistry, MESH: Viruses, Capillary tube, [CHIM.ANAL]Chemical Sciences/Analytical chemistry, Animals, Humans, [SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology, Cellulose, Molecular Biology, Counter-diffusion, MESH: Humans, Protein, Proteins, DNA, Crystallisation, MESH: Nucleic Acids, 0104 chemical sciences, Nucleoprotein, Crystallography, chemistry, Chemical engineering, Nucleic acid, RNA, Gels
Abstract

International audience; Medium-sized single crystals with perfect habits and no defect producing intense and well-resolved diffraction patterns are the dream of every protein crystallographer. Crystals of biological macromolecules possessing these characteristics can be prepared within a medium in which mass transport is restricted to diffusion. Chemical gels (like polysiloxane) and physical gels (such as agarose) provide such an environment and are therefore suitable for the crystallisation of biological macromolecules. Instructions for the preparation of each type of gel are given to urge crystal growers to apply diffusive media for enhancing crystallographic quality of their crystals. Examples of quality enhancement achieved with silica and agarose gels are given. Results obtained with other substances forming gel-like media (such as lipidic phases and cellulose derivatives) are presented. Finally, the use of gels in combination with capillary tubes for counter-diffusion experiments is discussed. Methods and techniques implemented with proteins can also be applied to nucleic acids and nucleoprotein assemblies such as viruses.

Published
2009

17. Crystallization and preliminary X-ray analysis of aD-Ala:D-Ser ligase associated with VanG-type vancomycin resistance

Author

Patrice Courvalin, Djalal Meziane-Cherif, Frederick Saul, Patrick Weber, Ahmed Haouz, Cristallogenèse et Diffraction des Rayons X (Plate-forme/PF6), Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS), Agents antibactériens, Institut Pasteur [Paris] (IP), and Immunologie structurale

Subjects
MESH: Enterococcus faecalis, D-Ala:D-Ser ligase, [SDV]Life Sciences [q-bio], Biophysics, Crystallography, X-Ray, Biochemistry, Enterococcus faecalis, law.invention, Crystal, chemistry.chemical_compound, Bacterial Proteins, Structural Biology, law, Genetics, VanG, Molecule, MESH: Cloning, Molecular, Cloning, Molecular, Peptide Synthases, Crystallization, MESH: Bacterial Proteins, MESH: Crystallization, chemistry.chemical_classification, DNA ligase, Dipeptide, biology, Condensed Matter Physics, biology.organism_classification, MESH: Crystallography, X-Ray, MESH: Peptide Synthases, Crystallography, chemistry, Crystallization Communications, vancomycin resistance, biological sciences, health occupations, MESH: Vancomycin Resistance, bacteria, Peptidoglycan, Single crystal
Abstract

International audience; Acquired VanG-type resistance to vancomycin in Enterococcus faecalis BM4518 arises from inducible synthesis of peptidoglycan precursors ending in d-alanyld-serine, to which vancomycin exhibits low binding affinity. VanG, a d-alanine: d-serine ligase, catalyzes the ATP-dependent synthesis of the d-Ala-d-Ser dipeptide, which is incorporated into the peptidoglycan synthesis of VanG-type vancomycin-resistant strains. Here, the purification, crystallization and preliminary crystallographic analysis of VanG in complex with ADP are reported. The crystal belonged to space group P3 1 21, with unit-cell parameters a = b = 116.1, c = 177.2 Å , and contained two molecules in the asymmetric unit. A complete data set has been collected to 2.35 Å resolution from a single crystal under cryogenic conditions using synchrotron radiation.

Published
2009

18. Structural plasticity and catalysis regulation of a thermosensor histidine kinase

Author

Diego de Mendoza, Pedro M. Alzari, Daniela Albanesi, Ahmed Haouz, Alejandro Buschiazzo, María C. Mansilla, Mariana Martín, Felipe Trajtenberg, Biochimie Structurale, Centre National de la Recherche Scientifique (CNRS)-Institut Pasteur [Paris], Instituto de Biología Molecular y Celular de Rosario [Rosario] (IBR), Consejo Nacional de Investigaciones Científicas y Técnicas [Buenos Aires] (CONICET)-Universidad Nacional de Rosario [Santa Fe], Protein Crystallography / Cristalografía de Proteínas [Montevideo], Institut Pasteur de Montevideo, Réseau International des Instituts Pasteur (RIIP)-Réseau International des Instituts Pasteur (RIIP), Cristallogenèse et Diffraction des Rayons X (Plate-forme/PF6), Institut Pasteur [Paris]-Centre National de la Recherche Scientifique (CNRS), This work was supported by grants from Agencia Nacional de Investigación e Innovación, Uruguay, Agence Nationale de la Recherche, France, Institut Pasteur (France), Consejo Nacional de Investigaciones Científicas y Técnicas, Argentina, Agencia de Promoción Científica y Tecnológica, Argentina, and ECOS France-Argentine Action Grant A05B02. D.A. is a Marie Curie Incoming International Fellow (European Union), F.T. is a fellow from Agencia Nacional de Investigación e Innovación, and D.d.M. is an International Research Scholar from the Howard Hughes Medical Institute., and Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS)

Subjects
Models, Molecular, Histidine Kinase, Protein Conformation, MESH: Protein Structure, Secondary, Plasma protein binding, Crystallography, X-Ray, Protein Structure, Secondary, Phosphotransferase, MESH: Protein Structure, Tertiary, Protein structure, Adenosine Triphosphate, MESH: Protein Conformation, MESH: Structure-Activity Relationship, MESH: Adenosine Triphosphate, MESH: Bacterial Proteins, MESH: Crystallization, 0303 health sciences, Multidisciplinary, 030302 biochemistry & molecular biology, Temperature, MESH: Chromatography, Gel, Biological Sciences, MESH: Amino Acid Substitution, Transmembrane protein, MESH: Temperature, Biochemistry, MESH: Histidine Kinase, Chromatography, Gel, Crystallization, MESH: Models, Molecular, Bacillus subtilis, Protein Binding, MESH: Mutation, Phosphatase, Biology, Catalysis, 03 medical and health sciences, Structure-Activity Relationship, Bacterial Proteins, [CHIM.CRIS]Chemical Sciences/Cristallography, MESH: Protein Binding, [SDV.BBM.BC]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biochemistry [q-bio.BM], MESH: Protein Kinases, Histidine, 030304 developmental biology, Binding Sites, Histidine kinase, MESH: Bacillus subtilis, MESH: Catalysis, MESH: Crystallography, X-Ray, [SDV.MP.BAC]Life Sciences [q-bio]/Microbiology and Parasitology/Bacteriology, Protein Structure, Tertiary, Response regulator, Amino Acid Substitution, MESH: Binding Sites, Mutation, Biophysics, Protein Kinases
Abstract

Temperature sensing is essential for the survival of living cells. A major challenge is to understand how a biological thermometer processes thermal information to optimize cellular functions. Using structural and biochemical approaches, we show that the thermosensitive histidine kinase, DesK, from Bacillus subtilis is cold-activated through specific interhelical rearrangements in its central four-helix bundle domain. As revealed by the crystal structures of DesK in different functional states, the plasticity of this helical domain influences the catalytic activities of the protein, either by modifying the mobility of the ATP-binding domains for autokinase activity or by modulating binding of the cognate response regulator to sustain the phosphotransferase and phosphatase activities. The structural and biochemical data suggest a model in which the transmembrane sensor domain of DesK promotes these structural changes through conformational signals transmitted by the membrane-connecting two-helical coiled-coil, ultimately controlling the alternation between output autokinase and phosphatase activities. The structural comparison of the different DesK variants indicates that incoming signals can take the form of helix rotations and asymmetric helical bends similar to those reported for other sensing systems, suggesting that a similar switching mechanism could be operational in a wide range of sensor histidine kinases.

Published
2009

19. Biostructural analysis of the metal-sensor domain of CnrX from Cupriavidus metallidurans CH34

Author

Guillaume Pompidor, Stéphanie Ramella-Pairin, Richard Kahn, Antoine P. Maillard, Beate Bersch, Eric Girard, Jacques Covès, Institut de biologie structurale (IBS - UMR 5075 ), Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Centre National de la Recherche Scientifique (CNRS), Pathogenèse bactérienne et réponses cellulaires (PBRC), Biologie du Cancer et de l'Infection (BCI ), Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut National de la Santé et de la Recherche Médicale (INSERM)-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Centre National de la Recherche Scientifique (CNRS)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA), Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), and Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG)

Subjects
MESH: Signal Transduction, MESH: Sequence Analysis, DNA, Magnetic Resonance Spectroscopy, Dimer, [SDV]Life Sciences [q-bio], MESH: Amino Acid Sequence, chemistry.chemical_compound, MESH: Structure-Activity Relationship, X-Ray Diffraction, Cloning, Molecular, Peptide sequence, Protein secondary structure, MESH: Bacterial Proteins, MESH: Crystallization, 0303 health sciences, MESH: Gene Expression Regulation, Bacterial, Cupriavidus, MESH: Periplasm, MESH: X-Ray Diffraction, General Medicine, Nuclear magnetic resonance spectroscopy, MESH: Metals, Heavy, Biochemistry, visual_art, Periplasm, visual_art.visual_art_medium, Crystallization, Dimerization, Signal Transduction, Molecular Sequence Data, Size-exclusion chromatography, Biology, Microbiology, Metal, Structure-Activity Relationship, 03 medical and health sciences, Bacterial Proteins, Metals, Heavy, Drug Resistance, Bacterial, MESH: Drug Resistance, Bacterial, MESH: Cloning, Molecular, Amino Acid Sequence, Molecular Biology, 030304 developmental biology, MESH: Cupriavidus, MESH: Molecular Sequence Data, 030306 microbiology, Cupriavidus metallidurans, MESH: Magnetic Resonance Spectroscopy, Gene Expression Regulation, Bacterial, Sequence Analysis, DNA, Periplasmic space, biology.organism_classification, Crystallography, chemistry, MESH: Dimerization
Abstract

International audience; In Cupriavidus metallidurans CH34, the proteins CnrX, CnrY, and CnrH regulate the expression of the cnrCBA operon that codes for a cation-efflux pump involved in cobalt and nickel resistance. The periplasmic part of CnrX can be defined as the metal sensor in the signal transduction complex composed of the membrane-bound anti-sigma factor CnrY and the extra-cytoplasmic function sigma factor CnrH. A soluble form of CnrX was overproduced and purified. This protein behaves as a dimer in solution as judged from gel filtration, sedimentation velocity experiments, and NMR. Native crystals diffracting to 2.3 A using synchrotron radiation were obtained using the hanging-drop vapor-diffusion method. They belong to the primitive monoclinic space group P2(1), with unit cell parameters a = 31.87, b = 74.80, c = 93.67 A, beta = 90.107 degrees. NMR data and secondary structure prediction suggest that this protein is essentially formed by helices.

Published
2009

20. Structural and functional characterization of a putative polysaccharide deacetylase of the human parasite Encephalitozoon cuniculi

Author

Urch, Jonathan E, Hurtado-Guerrero, Ramon, Brosson, Damien, Liu, Zhanliang, Eijsink, Vincent G H, Texier, Catherine, van Aalten, Daan M F, College of Life Sciences (Division of Molecular Microbiology), University of Dundee, Laboratoire Microorganismes : Génome et Environnement (LMGE), Université Blaise Pascal - Clermont-Ferrand 2 (UBP)-Centre National de la Recherche Scientifique (CNRS)-Université d'Auvergne - Clermont-Ferrand I (UdA), Biotechnology and Food Science (Department of Chemistry), Center for Molecular Microbiology, and Université Blaise Pascal - Clermont-Ferrand 2 (UBP)-Université d'Auvergne - Clermont-Ferrand I (UdA)-Centre National de la Recherche Scientifique (CNRS)

Subjects
MESH: Amidohydrolases, carbohydrates, macromolecular substances, peptidoglycan, Crystallography, X-Ray, chitin, Article, MESH: Chitin, Amidohydrolases, Cell Line, MESH: Recombinant Proteins, MESH: Dogs, Dogs, glycobiology, Polysaccharides, parasitic diseases, Animals, Humans, MESH: Animals, MESH: Cloning, Molecular, Cloning, Molecular, protein structure, MESH: Crystallization, MESH: Humans, fungi, MESH: Crystallography, X-Ray, Recombinant Proteins, deacetylase, MESH: Cell Line, carbohydrates (lipids), MESH: Polysaccharides, [SDV.MP]Life Sciences [q-bio]/Microbiology and Parasitology, cell wall, MESH: Encephalitozoon cuniculi, Crystallization, Encephalitozoon cuniculi
Abstract

International audience; The microsporidian Encephalitozoon cuniculi is an intracellular eukaryotic parasite considered to be an emerging opportunistic human pathogen. The infectious stage of this parasite is a unicellular spore that is surrounded by a chitin containing endospore layer and an external proteinaceous exospore. A putative chitin deacetylase (ECU11_0510) localizes to the interface between the plasma membrane and the endospore. Chitin deacetylases are family 4 carbohydrate esterases in the CAZY classification, and several bacterial members of this family are involved in evading lysis by host glycosidases, through partial de-N-acetylation of cell wall peptidoglycan. Similarly, ECU11_0510 could be important for E. cuniculi survival in the host, by protecting the chitin layer from hydrolysis by human chitinases. Here, we describe the biochemical, structural, and glycan binding properties of the protein. Enzymatic analyses showed that the putative deacetylase is unable to deacetylate chitooligosaccharides or crystalline beta-chitin. Furthermore, carbohydrate microarray analysis revealed that the protein bound neither chitooligosaccharides nor any of a wide range of other glycans or chitin. The high resolution crystal structure revealed dramatic rearrangements in the positions of catalytic and substrate binding residues, which explain the loss of deacetylase activity, adding to the unusual structural plasticity observed in other members of this esterase family. Thus, it appears that the ECU11_0510 protein is not a carbohydrate deacetylase and may fulfill an as yet undiscovered role in the E. cuniculi parasite.

Published
2009

21. Microfluidic chips for the crystallization of biomacromolecules by counter-diffusion and on-chip crystal X-ray analysis

Author

Richard Giegé, Bernard Gauthier-Manuel, Claude Sauter, Jean-Luc Ferrer, Wilhelm Pfleging, Chantal Khan Malek, Gaël Thuillier, Kaouthar Dhouib, Bernard Lorber, Roland Duffait, Jeremy Ohana, Lilian Jacquamet, Rosaria Ferrigno, Anne Théobald-Dietrich, Architecture et réactivité de l'ARN (ARN), Université Louis Pasteur - Strasbourg I-Centre National de la Recherche Scientifique (CNRS), Franche-Comté Électronique Mécanique, Thermique et Optique - Sciences et Technologies (UMR 6174) (FEMTO-ST), Université de Technologie de Belfort-Montbeliard (UTBM)-Ecole Nationale Supérieure de Mécanique et des Microtechniques (ENSMM)-Université de Franche-Comté (UFC), Université Bourgogne Franche-Comté [COMUE] (UBFC)-Université Bourgogne Franche-Comté [COMUE] (UBFC)-Centre National de la Recherche Scientifique (CNRS), Université de Franche-Comté (UFC)-Centre National de la Recherche Scientifique (CNRS)-Ecole Nationale Supérieure de Mécanique et des Microtechniques (ENSMM)-Université de Technologie de Belfort-Montbeliard (UTBM), Centre National de la Recherche Scientifique (CNRS)-Université Louis Pasteur - Strasbourg I, Institut für Angewandte Materialien (IAM), Karlsruher Institut für Technologie (KIT), Centre de Transfert des Micro et Nanotechnologies (CTMN), CTMN, INL - Lab-On-Chip et Instrumentation (INL - LOCI), Institut des Nanotechnologies de Lyon (INL), Centre National de la Recherche Scientifique (CNRS)-Institut National des Sciences Appliquées de Lyon (INSA Lyon), Université de Lyon-Institut National des Sciences Appliquées (INSA)-Université de Lyon-Institut National des Sciences Appliquées (INSA)-École Centrale de Lyon (ECL), Université de Lyon-Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-École supérieure de Chimie Physique Electronique de Lyon (CPE)-Centre National de la Recherche Scientifique (CNRS)-Institut National des Sciences Appliquées de Lyon (INSA Lyon), Université de Lyon-École supérieure de Chimie Physique Electronique de Lyon (CPE), Institut de biologie structurale (IBS - UMR 5075 ), Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Centre National de la Recherche Scientifique (CNRS), École Centrale de Lyon (ECL), Université de Lyon-Université de Lyon-Centre National de la Recherche Scientifique (CNRS)-Institut National des Sciences Appliquées de Lyon (INSA Lyon), Université de Lyon-Institut National des Sciences Appliquées (INSA)-Institut National des Sciences Appliquées (INSA)-École supérieure de Chimie Physique Electronique de Lyon (CPE)-Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-École Centrale de Lyon (ECL), Université de Lyon, Institut de biologie structurale (IBS - UMR 5075), Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Grenoble Alpes (UGA)-Centre National de la Recherche Scientifique (CNRS), Université de Technologie de Belfort-Montbeliard (UTBM)-Ecole Nationale Supérieure de Mécanique et des Microtechniques (ENSMM)-Centre National de la Recherche Scientifique (CNRS)-Université de Franche-Comté (UFC), Université Bourgogne Franche-Comté [COMUE] (UBFC)-Université Bourgogne Franche-Comté [COMUE] (UBFC), Université de Lyon-Université de Lyon-Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-École Supérieure de Chimie Physique Électronique de Lyon (CPE)-Institut National des Sciences Appliquées de Lyon (INSA Lyon), Université de Lyon-Institut National des Sciences Appliquées (INSA)-Institut National des Sciences Appliquées (INSA)-Centre National de la Recherche Scientifique (CNRS)-École Centrale de Lyon (ECL), Université de Lyon-Institut National des Sciences Appliquées (INSA)-Institut National des Sciences Appliquées (INSA)-Centre National de la Recherche Scientifique (CNRS), Centre National de la Recherche Scientifique (CNRS)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), and Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)

Subjects
Diffraction, Materials science, Macromolecular Substances, Microfluidics, Biomedical Engineering, Bioengineering, Nanotechnology, Crystal growth, 02 engineering and technology, [SDV.BC]Life Sciences [q-bio]/Cellular Biology, 010402 general chemistry, Crystallography, X-Ray, 01 natural sciences, Biochemistry, law.invention, Crystal, law, MESH: Dimethylpolysiloxanes, Polymethyl Methacrylate, Dimethylpolysiloxanes, Crystallization, MESH: Polymethyl Methacrylate, MESH: Crystallization, chemistry.chemical_classification, Laser ablation, Biomolecule, [SDV.BBM.BM]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Molecular biology, General Chemistry, Polymer, MESH: Macromolecular Substances, Microfluidic Analytical Techniques, 021001 nanoscience & nanotechnology, MESH: Crystallography, X-Ray, 0104 chemical sciences, chemistry, MESH: Microfluidic Analytical Techniques, 0210 nano-technology
Abstract

International audience; Microfluidic devices were designed to perform on micromoles of biological macromolecules and viruses the search and the optimization of crystallization conditions by counter-diffusion, as well as the on-chip analysis of crystals by X-ray diffraction. Chips composed of microchannels were fabricated in poly-dimethylsiloxane (PDMS), poly-methyl-methacrylate (PMMA) and cyclo-olefin-copolymer (COC) by three distinct methods, namely replica casting, laser ablation and hot embossing. The geometry of the channels was chosen to ensure that crystallization occurs in a convection-free environment. The transparency of the materials is compatible with crystal growth monitoring by optical microscopy. The quality of the protein 3D structures derived from on-chip crystal analysis by X-ray diffraction using a synchrotron radiation was used to identify the most appropriate polymers. Altogether the results demonstrate that for a novel biomolecule, all steps from the initial search of crystallization conditions to X-ray diffraction data collection for 3D structure determination can be performed in a single chip.

Published
2009

22. Crystallization and preliminary crystallographic studies of putative threonyl-tRNA synthetases from Aeropyrum pernix and Sulfolobus tokodaii

Author

Akio Takénaka, Takeshi Sekiguchi, Yu Ichiro Miyashita, K. Suzuki, Masataka Yogiashi, M.M. Hoque, Ella Czarina Magat Juan, Satoru Shimizu, Masaru Tsunoda, Anne Catherine Dock-Bregeon, Yoshiteru Sato, Dino Moras, Institut de génétique et biologie moléculaire et cellulaire (IGBMC), Centre National de la Recherche Scientifique (CNRS)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Louis Pasteur - Strasbourg I, Peney, Maité, and Université Louis Pasteur - Strasbourg I-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)

Subjects
Models, Molecular, Protein Conformation, Aeropyrum, Archaeal Proteins, 030303 biophysics, Biophysics, Sulfolobus tokodaii, Aminoacylation, MESH: Amino Acid Sequence, Crystallography, X-Ray, Biochemistry, Sulfolobus, 03 medical and health sciences, Protein structure, MESH: Protein Conformation, Species Specificity, Structural Biology, MESH: Aeropyrum, Genetics, Threonine-tRNA Ligase, Aeropyrum pernix, MESH: Species Specificity, Amino Acid Sequence, 030304 developmental biology, MESH: Crystallization, MESH: Threonine-tRNA Ligase, 0303 health sciences, [SDV.BIBS] Life Sciences [q-bio]/Quantitative Methods [q-bio.QM], biology, MESH: Archaeal Proteins, Condensed Matter Physics, biology.organism_classification, MESH: Crystallography, X-Ray, [SDV.BIBS]Life Sciences [q-bio]/Quantitative Methods [q-bio.QM], Crystallography, Threonine—tRNA ligase, Crystallization Communications, MESH: Sulfolobus, Transfer RNA, Crystallization, MESH: Models, Molecular
Abstract

International audience; Threonyl-tRNA synthetase (ThrRS) plays an essential role in protein synthesis by catalyzing the aminoacylation of tRNA(Thr) and editing misacylation. ThrRS generally contains an N-terminal editing domain, a catalytic domain and an anticodon-binding domain. The sequences of the editing domain in ThrRSs from archaea differ from those in bacteria and eukaryotes. Furthermore, several creanarchaea including Aeropyrum pernix K1 and Sulfolobus tokodaii strain 7 contain two genes encoding either the catalytic or the editing domain of ThrRS. To reveal the structural basis for this evolutionary divergence, the two types of ThrRS from the crenarchaea A. pernix and S. tokodaii have been overexpressed in Eschericha coli, purified and crystallized by the hanging-drop vapour-diffusion method. Diffraction data were collected and the structure of a selenomethionine-labelled A. pernix type-1 ThrRS crystal has been solved using the MAD method.

Published
2008

23. Nuclear receptor ligand-binding domains: reduction of helix H12 dynamics to favour crystallization

Author

Yvan Boublik, Jean-François Guichou, Virginie Nahoum, William Bourguet, Efrén Pérez, Fabien Quillard, Pierre Germain, Alexandra Lipski, Angel R. de Lera, Centre de Biochimie Structurale [Montpellier] (CBS), Centre National de la Recherche Scientifique (CNRS)-Université de Montpellier (UM)-Institut National de la Santé et de la Recherche Médicale (INSERM), Centre de recherche en Biologie Cellulaire (CRBM), Université Montpellier 2 - Sciences et Techniques (UM2)-Centre National de la Recherche Scientifique (CNRS)-Université de Montpellier (UM)-Université Montpellier 1 (UM1), Departamento de Química Orgánica, Universidade de Vigo, Institut de génétique et biologie moléculaire et cellulaire (IGBMC), Université Louis Pasteur - Strasbourg I-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Peney, Maité, Institut National de la Santé et de la Recherche Médicale (INSERM)-Université de Montpellier (UM)-Centre National de la Recherche Scientifique (CNRS), and Université de Montpellier (UM)-Centre National de la Recherche Scientifique (CNRS)

Subjects
Stereochemistry, Biophysics, Receptors, Cytoplasmic and Nuclear, MESH: Protein Structure, Secondary, Fluorescence Polarization, Plasma protein binding, Retinoid X receptor, MESH: Receptors, Cytoplasmic and Nuclear, Ligands, MESH: Retinoid X Receptor alpha, Biochemistry, Protein Structure, Secondary, law.invention, 03 medical and health sciences, MESH: Protein Structure, Tertiary, 0302 clinical medicine, Protein structure, Structural Biology, law, Predictive Value of Tests, Genetics, [SDV.BBM] Life Sciences [q-bio]/Biochemistry, Molecular Biology, MESH: Ligands, Humans, MESH: Protein Binding, [SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology, Binding site, Crystallization, 030304 developmental biology, Fluorescent Dyes, MESH: Crystallization, 0303 health sciences, MESH: Fluorescence Polarization, Binding Sites, Retinoid X Receptor alpha, MESH: Humans, Retinoid X receptor alpha, Chemistry, Condensed Matter Physics, MESH: Fluorescent Dyes, MESH: Predictive Value of Tests, Protein Structure, Tertiary, Nuclear receptor, MESH: Binding Sites, Crystallization Communications, 030220 oncology & carcinogenesis, Helix, Protein Binding
Abstract

International audience; Crystallization trials of the human retinoid X receptor alpha ligand-binding domain (RXRalpha LBD) in complex with various ligands have been carried out. Using fluorescence anisotropy, it has been found that when compared with agonists these small-molecule effectors enhance the dynamics of the RXRalpha LBD C-terminal helix H12. In some cases, the mobility of this helix could be dramatically reduced by the addition of a 13-residue co-activator fragment (CoA). In keeping with these observations, crystals have been obtained of the corresponding ternary RXRalpha LBD-ligand-CoA complexes. In contrast, attempts to crystallize complexes with a highly mobile H12 remained unsuccessful. These experimental observations substantiate the previously recognized role of co-regulator fragments in facilitating the crystallization of nuclear receptor LBDs.

Published
2008

24. Meriolins (3-(pyrimidin-4-yl)-7-azaindoles): synthesis, kinase inhibitory activity, cellular effects, and structure of a CDK2/cyclin A/meriolin complex

Author

Olivier Lozach, Annie Valette, Laurent Meijer, Monique Clément, François Liger, Aude Echalier, Yoan Ferandin, Karima Bettayeb, Jonathan C. Morris, Bernard Marquet, Jane A. Endicott, Benoît Joseph, Molécules et cibles thérapeutiques (MCT), Station biologique de Roscoff [Roscoff] (SBR), Université Pierre et Marie Curie - Paris 6 (UPMC)-Centre National de la Recherche Scientifique (CNRS)-Université Pierre et Marie Curie - Paris 6 (UPMC)-Centre National de la Recherche Scientifique (CNRS)-Centre National de la Recherche Scientifique (CNRS), Laboratoire de Biologie Cellulaire et Moléculaire du Contrôle de la Prolifération (LBCMCP), Université Toulouse III - Paul Sabatier (UT3), Université de Toulouse (UT)-Université de Toulouse (UT)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Centre de Biologie Intégrative (CBI), Université de Toulouse (UT)-Université de Toulouse (UT)-Centre National de la Recherche Scientifique (CNRS)-Centre National de la Recherche Scientifique (CNRS), Centre d'études et de recherches appliquées à la gestion (CERAG), Université Pierre Mendès France - Grenoble 2 (UPMF)-Centre National de la Recherche Scientifique (CNRS), Institut de Mathématiques (UNINE), Université de Neuchâtel (UNINE), Centre de recherche en gestion (CRG), École polytechnique (X)-Centre National de la Recherche Scientifique (CNRS), Institut de Chimie et Biochimie Moléculaires et Supramoléculaires (ICBMS), Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-École Supérieure de Chimie Physique Électronique de Lyon (CPE)-Institut National des Sciences Appliquées de Lyon (INSA Lyon), Université de Lyon-Institut National des Sciences Appliquées (INSA)-Institut National des Sciences Appliquées (INSA)-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Centre de Biologie Intégrative (CBI), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Centre National de la Recherche Scientifique (CNRS)-Centre National de la Recherche Scientifique (CNRS), Centre National de la Recherche Scientifique (CNRS)-Université Pierre Mendès France - Grenoble 2 (UPMF), Centre National de la Recherche Scientifique (CNRS)-École polytechnique (X), Université de Lyon-Université de Lyon-Institut National des Sciences Appliquées de Lyon (INSA Lyon), Institut National des Sciences Appliquées (INSA)-Université de Lyon-Institut National des Sciences Appliquées (INSA)-Institut de Chimie du CNRS (INC)-École Supérieure Chimie Physique Électronique de Lyon-Centre National de la Recherche Scientifique (CNRS), Molécules et cibles thérapeutiques ( MCT ), Centre National de la Recherche Scientifique ( CNRS ), Laboratoire d'économie et de sociologie du travail ( LEST ), Aix Marseille Université ( AMU ) -Centre National de la Recherche Scientifique ( CNRS ), Biologie cellulaire et moléculaire du contrôle de la prolifération ( BCMCP ), Université Paul Sabatier - Toulouse 3 ( UPS ) -Centre National de la Recherche Scientifique ( CNRS ), Centre d'études et de recherches appliquées à la gestion ( CERAG ), Université Pierre Mendès France - Grenoble 2 ( UPMF ) -Centre National de la Recherche Scientifique ( CNRS ), Institut de Mathématiques ( UNINE ), Université de Neuchâtel, Centre de recherche en gestion ( CRG ), École polytechnique ( X ) -Centre National de la Recherche Scientifique ( CNRS ), Institut de Chimie et Biochimie Moléculaires et Supramoléculaires ( ICBMS ), Université Claude Bernard Lyon 1 ( UCBL ), Université de Lyon-Université de Lyon-Institut National des Sciences Appliquées de Lyon ( INSA Lyon ), Université de Lyon-Institut National des Sciences Appliquées ( INSA ) -Institut National des Sciences Appliquées ( INSA ) -École Supérieure Chimie Physique Électronique de Lyon-Centre National de la Recherche Scientifique ( CNRS ), Laboratoire d'économie et de sociologie du travail (LEST), Aix Marseille Université (AMU)-Centre National de la Recherche Scientifique (CNRS), and Université de Lyon-Institut National des Sciences Appliquées (INSA)-Institut National des Sciences Appliquées (INSA)-Institut de Chimie du CNRS (INC)-École Supérieure Chimie Physique Électronique de Lyon-Centre National de la Recherche Scientifique (CNRS)

Subjects
Models, Molecular, MESH : Cell Line, Indoles, DYRK1A, Cyclin A, MESH : Pyrimidines, MESH: Spheroids, Cellular, Apoptosis, Crystallography, X-Ray, 01 natural sciences, MESH: Bicyclo Compounds, Heterocyclic, MESH : Bicyclo Compounds, Heterocyclic, MESH: Structure-Activity Relationship, MESH : Indoles, Drug Discovery, MESH : Structure-Activity Relationship, MESH : Cyclin A, MESH: Crystallization, MESH: Indoles, 0303 health sciences, MESH : Cell Survival, biology, Chemistry, Kinase, MESH : Drug Screening Assays, Antitumor, MESH: Drug Screening Assays, Antitumor, MESH : Cyclin-Dependent Kinase 2, 3. Good health, MESH : Antineoplastic Agents, MESH : Cyclin-Dependent Kinase 9, Biochemistry, MESH: Cell Survival, MESH : Crystallization, Molecular Medicine, Crystallization, MESH: Models, Molecular, MESH: Cell Line, Tumor, Cell Survival, MESH : Models, Molecular, MESH: Cyclin-Dependent Kinase 2, Antineoplastic Agents, MESH: Cyclin-Dependent Kinase 9, Cell Line, Structure-Activity Relationship, 03 medical and health sciences, Cyclin-dependent kinase, Cell Line, Tumor, Spheroids, Cellular, Humans, Structure–activity relationship, [SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology, [ SDV.BBM ] Life Sciences [q-bio]/Biochemistry, Molecular Biology, 030304 developmental biology, Aza Compounds, MESH: Humans, 010405 organic chemistry, MESH : Cell Line, Tumor, MESH: Apoptosis, Cyclin-Dependent Kinase 2, Cyclin-dependent kinase 2, MESH : Humans, MESH: Cyclin A, Bridged Bicyclo Compounds, Heterocyclic, MESH: Crystallography, X-Ray, Cyclin-Dependent Kinase 9, In vitro, 0104 chemical sciences, MESH: Cell Line, Pyrimidines, MESH: Aza Compounds, MESH: Pyrimidines, biology.protein, MESH : Spheroids, Cellular, MESH: Antineoplastic Agents, Cyclin-dependent kinase 9, Drug Screening Assays, Antitumor, MESH : Aza Compounds, MESH : Crystallography, X-Ray, MESH : Apoptosis
Abstract

We report the synthesis and biological characterization of 3-(pyrimidin-4-yl)-7-azaindoles (meriolins), a chemical hybrid between the natural products meridianins and variolins, derived from marine organisms. Meriolins display potent inhibitory activities toward cyclin-dependent kinases (CDKs) and, to a lesser extent, other kinases (GSK-3, DYRK1A). The crystal structures of 1e (meriolin 5) and variolin B (Bettayeb, K.; Tirado, O. M.; Marionneau-Lambert, S.; Ferandin, Y.; Lozach, O.; Morris, J.; Mateo-Lozano, S.; Drückes, P.; Schächtele, C.; Kubbutat, M.; Liger, F.; Marquet, B.; Joseph, B.; Echalier, A.; Endicott, J.; Notario, V.; Meijer, L. Cancer Res. 2007, 67, 8325-8334) in complex with CDK2/cyclin A reveal that the two inhibitors are orientated in very different ways inside the ATP-binding pocket of the kinase. A structure-activity relationship provides further insight into the molecular mechanism of action of this family of kinase inhibitors. Meriolins are also potent antiproliferative and proapoptotic agents in cells cultured either as monolayers or in spheroids. Proapoptotic efficacy of meriolins correlates best with their CDK2 and CDK9 inhibitory activity. Meriolins thus constitute a promising class of pharmacological agents to be further evaluated against the numerous human diseases that imply abnormal regulation of CDKs including cancers, neurodegenerative disorders, and polycystic kidney disease.

Published
2008

25. tRNA-dependent asparagine formation in prokaryotes: Characterization, isolation and structural and functional analysis of a ribonucleoprotein particle generating Asn-tRNAAsn

Author

Bernard Lorber, Hubert Dominique Becker, Catherine Birck, Marzanna Deniziak, Hervé Roy, Mickaël Blaise, Daniel Kern, Marc Bailly, Architecture et réactivité de l'ARN (ARN), Université Louis Pasteur - Strasbourg I-Centre National de la Recherche Scientifique (CNRS), Institut de Recherche en Infectiologie de Montpellier (IRIM), Université de Montpellier (UM)-Centre National de la Recherche Scientifique (CNRS), Institut de Génétique et de Biologie Moléculaire et Cellulaire (IGBMC), Université de Strasbourg (UNISTRA)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Institut de génétique et biologie moléculaire et cellulaire (IGBMC), Université Louis Pasteur - Strasbourg I-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), and Centre National de la Recherche Scientifique (CNRS)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Louis Pasteur - Strasbourg I

Subjects
MESH: Asparagine, Light, Operon, Nitrogenous Group Transferases, MESH: RNA, Transfer, Asn, [SDV]Life Sciences [q-bio], MESH: Nitrogenous Group Transferases, General Biochemistry, Genetics and Molecular Biology, 03 medical and health sciences, Scattering, Radiation, TRNA aminoacylation, Transfer RNA Aminoacylation, MESH: Scattering, Radiation, Molecular Biology, ComputingMilieux_MISCELLANEOUS, 030304 developmental biology, Glutamine amidotransferase, Ribonucleoprotein, MESH: Crystallization, MESH: Prokaryotic Cells, 0303 health sciences, RNA, Transfer, Asn, biology, 030302 biochemistry & molecular biology, MESH: Ultracentrifugation, Ribonucleoprotein particle, [SDV.BBM.BM]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Molecular biology, Thermus thermophilus, biology.organism_classification, MESH: Light, MESH: Ribonucleoproteins, MESH: Transfer RNA Aminoacylation, Prokaryotic Cells, Ribonucleoproteins, Biochemistry, Transfer RNA, Asparagine, Crystallization, Ultracentrifugation
Abstract

In some living organisms the 20 aa-tRNA species participating in protein synthesis are not charged by a complete set of 20 aminoacyl-tRNA synthetases. In prokaryotes, the deficiency of asparaginyl- and/or glutaminyl-tRNA synthetases is compensated by another aminoacyl-tRNA synthetase of relaxed specificity that mischarges the orphan tRNA and by an enzyme that converts the amino acid into that homologous to the tRNA. In Thermus thermophilus Asn-tRNA(Asn) is formed indirectly via a two-step pathway whereby tRNA(Asn) is mischarged with Asp that will subsequently be amidated into Asn by an amidotransferase. The non-discriminating aspartyl-tRNA synthetase, the trimeric GatCAB tRNA-dependent amidotransferase and the tRNA(Asn) promoting this pathway assemble into a ribonucleoprotein particle termed transamidosome. This article deals with the methods and techniques employed to clone the genes encoding the enzymes and the tRNA involved in this pathway, to express them in Escherichia coli, to isolate them on a large scale, and to transcribe and produce mg quantities of pure tRNA(Asn)in vitro. The approaches designed especially for this system include (i) clustering of the ORFs encoding the subunits of the heterotrimeric GatCAB that are sprinkled in the genome into an artificial operon, and (ii) the self-cleavage of the tRNA(Asn) transcript starting with U in 5' position through fusion with a hammerhead ribozyme. Further, the crystallization of the free enzymes is described and the characterization of their assembly with tRNA(Asn) into a ribonucleoprotein particle, as well as the investigation of the catalytic mechanism of Asn-tRNA(Asn) formation by the complex are reported.

Published
2008

26. A regular pattern of Ig super-motifs defines segmental flexibility as the elastic mechanism of the titin chain

Author

Petr V. Konarev, Eleonore von Castelmur, Siegfried Labeit, Olga Mayans, Marco Marino, Zöhre Ucurum-Fotiadis, Laurent Kreplak, Dietmar Labeit, Alexandre Urzhumtsev, Dmitri I. Svergun, Institut de génétique et biologie moléculaire et cellulaire (IGBMC), Centre National de la Recherche Scientifique (CNRS)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Louis Pasteur - Strasbourg I, and Université Louis Pasteur - Strasbourg I-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)

Subjects
Models, Molecular, MESH: Immunoglobulins, Amino Acid Motifs, Muscle Proteins, MESH: Rabbits, MESH: Amino Acid Sequence, Crystallography, X-Ray, Sarcomere, Protein filament, MESH: Amino Acid Motifs, MESH: Protein Structure, Tertiary, 0302 clinical medicine, MESH: Structure-Activity Relationship, Connectin, MESH: Animals, Conserved Sequence, MESH: Crystallization, 0303 health sciences, MESH: Muscle, Skeletal, Multidisciplinary, MESH: Conserved Sequence, biology, MESH: Sarcomeres, Chemistry, Small-angle X-ray scattering, MESH: Protein Array Analysis, Molecular spring, Biological Sciences, Tandem Repeat Sequences, Titin, Rabbits, Crystallization, MESH: Models, Molecular, Sarcomeres, Molecular Sequence Data, Protein Array Analysis, Hinge, Immunoglobulins, Structure-Activity Relationship, 03 medical and health sciences, MESH: Muscle Proteins, Animals, Humans, Amino Acid Sequence, Elasticity (economics), Muscle, Skeletal, MESH: Protein Kinases, 030304 developmental biology, MESH: Molecular Sequence Data, MESH: Humans, [SDV.BBM.BM]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Molecular biology, MESH: Crystallography, X-Ray, Elasticity, Protein Structure, Tertiary, MESH: Tandem Repeat Sequences, Crystallography, biology.protein, Biophysics, MESH: Elasticity, Myofibril, Protein Kinases, 030217 neurology & neurosurgery
Abstract

Myofibril elasticity, critical to muscle function, is dictated by the intrasarcomeric filament titin, which acts as a molecular spring. To date, the molecular events underlying the mechanics of the folded titin chain remain largely unknown. We have elucidated the crystal structure of the 6-Ig fragment I65–I70 from the elastic I-band fraction of titin and validated its conformation in solution using small angle x-ray scattering. The long-range properties of the chain have been visualized by electron microscopy on a 19-Ig fragment and modeled for the full skeletal tandem. Results show that conserved Ig–Ig transition motifs generate high-order in the structure of the filament, where conformationally stiff segments interspersed with pliant hinges form a regular pattern of dynamic super-motifs leading to segmental flexibility in the chain. Pliant hinges support molecular shape rearrangements that dominate chain behavior at moderate stretch, whereas stiffer segments predictably oppose high stretch forces upon full chain extension. There, librational entropy can be expected to act as an energy barrier to prevent Ig unfolding while, instead, triggering the unraveling of flanking springs formed by proline, glutamate, valine, and lysine (PEVK) sequences. We propose a mechanistic model based on freely jointed rigid segments that rationalizes the response to stretch of titin Ig-tandems according to molecular features.

Published
2008

27. Crystallization and X-ray analysis of the Schistosoma mansoni guanidino kinase

Author

Colette Dissous, Olivier Marcillat, Ayman M. Awama, Patrice Gouet, Sabine Laurent, Patricia Paracuellos, Institut de Chimie et Biochimie Moléculaires et Supramoléculaires (ICBMS), Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-École Supérieure de Chimie Physique Électronique de Lyon (CPE)-Institut National des Sciences Appliquées de Lyon (INSA Lyon), Université de Lyon-Institut National des Sciences Appliquées (INSA)-Institut National des Sciences Appliquées (INSA)-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS), Institut de biologie et chimie des protéines [Lyon] (IBCP), Université de Lyon-Université de Lyon-Centre National de la Recherche Scientifique (CNRS), Schistosomiase, paludisme et inflammation, Institut Pasteur de Lille, Réseau International des Instituts Pasteur (RIIP)-Réseau International des Instituts Pasteur (RIIP)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université de Lille, Droit et Santé, BioSciences Lyon-Gerland (BLG), École normale supérieure de Lyon (ENS de Lyon)-Institut National de la Recherche Agronomique (INRA)-Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-Hospices Civils de Lyon (HCL)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), The authors thank the ESRF staff at ID29 for technical support and assistance during X-ray data collection., Université de Lyon-Université de Lyon-Institut National des Sciences Appliquées de Lyon (INSA Lyon), Université de Lyon-Institut National des Sciences Appliquées (INSA)-Institut National des Sciences Appliquées (INSA)-École Supérieure Chimie Physique Électronique de Lyon-Centre National de la Recherche Scientifique (CNRS), École normale supérieure - Lyon (ENS Lyon)-Institut National de la Recherche Agronomique (INRA)-Université Claude Bernard Lyon 1 (UCBL), and Université de Lyon-Institut National des Sciences Appliquées (INSA)-Institut National des Sciences Appliquées (INSA)-École Supérieure Chimie Physique Électronique de Lyon-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS)

Subjects
guanidino kinase, MESH: GTP Pyrophosphokinase/isolation & purification, Stereochemistry, Molecular Sequence Data, Biophysics, MESH: Amino Acid Sequence, Crystallography, X-Ray, Biochemistry, law.invention, 03 medical and health sciences, GTP Pyrophosphokinase, Structural Biology, law, parasitic diseases, Genetics, Animals, MESH: Animals, Amino Acid Sequence, Crystallization, [SDV.BBM.BC]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biochemistry [q-bio.BM], Trypanosoma cruzi, X ray analysis, Peptide sequence, MESH: GTP Pyrophosphokinase/chemistry, 030304 developmental biology, MESH: Crystallization, 0303 health sciences, MESH: Molecular Sequence Data, biology, Kinase, 030302 biochemistry & molecular biology, Schistosoma mansoni, Arginine kinase, Condensed Matter Physics, biology.organism_classification, MESH: Crystallography, X-Ray, 3. Good health, [SDV.BBM.BC]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biomolecules [q-bio.BM], Crystallization Communications, biology.protein, MESH: Schistosoma mansoni/enzymology, Monoclinic crystal system, MESH: Schistosoma mansoni/chemistry
Abstract

International audience; The 716-amino-acid guanidino kinase from the parasitic flatworm Schistosoma mansoni results from the fusion of two guanidino kinase subunits. Crystals of this 80 kDa protein have been obtained in the monoclinic space group P2(1), with unit-cell parameters a = 52.7, b = 122.1, c = 63.2 A, beta = 108.5 degrees . Synchrotron data were collected to 2.8 A resolution on ESRF beamline ID29. The structure was solved by the molecular-replacement method, using the 357-amino-acid structure of the arginine kinase from Trypanosoma cruzi as the search model.

Published
2008

28. Expression, purification, crystallization and preliminary crystallographic study of the SRA domain of the human UHRF1 protein

Author

Jean Pierre Samama, Bénédicte Delagoutte, Nada Lallous, Pierre Oudet, Catherine Birck, Peney, Maité, Institut de génétique et biologie moléculaire et cellulaire (IGBMC), Université Louis Pasteur - Strasbourg I-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), and Centre National de la Recherche Scientifique (CNRS)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Louis Pasteur - Strasbourg I

Subjects
medicine.disease_cause, 01 natural sciences, Biochemistry, MESH: CCAAT-Enhancer-Binding Proteins, Histones, chemistry.chemical_compound, MESH: DNA Methylation, Structural Biology, Histone methylation, Histone code, MESH: Peptide Fragments, [INFO.INFO-BI] Computer Science [cs]/Bioinformatics [q-bio.QM], MESH: Crystallization, MESH: Histones, 0303 health sciences, Crystallography, [SDV.BIBS] Life Sciences [q-bio]/Quantitative Methods [q-bio.QM], MESH: Crystallography, MESH: DNA, Condensed Matter Physics, [SDV.BIBS]Life Sciences [q-bio]/Quantitative Methods [q-bio.QM], Crystallization Communications, DNA methylation, Crystallization, Protein Binding, HMG-box, Ubiquitin-Protein Ligases, Biophysics, Biology, 010402 general chemistry, DNA methyltransferase, Histone Deacetylases, 03 medical and health sciences, Histone H2A, Genetics, medicine, Humans, MESH: Protein Binding, Escherichia coli, 030304 developmental biology, MESH: Humans, DNA, DNA Methylation, Molecular biology, Peptide Fragments, 0104 chemical sciences, MESH: Histone Deacetylases, chemistry, CCAAT-Enhancer-Binding Proteins, [INFO.INFO-BI]Computer Science [cs]/Bioinformatics [q-bio.QM]
Abstract

International audience; Human UHRF1 belongs to the unique mammalian family of proteins which contain a SET- and RING finger-associated (SRA) domain. This 180-residue domain has been reported to play key roles in the functions of the protein. It allows UHRF1 to bind methylated DNA, histone deacetylase 1 and DNA methyltransferase 1, suggesting a bridge between DNA methylation and the histone code. No structural data is available for any SRA domain. Native and SeMet-labelled SRA domains of human UHRF1 were overexpressed in Escherichia coli cells, purified to homogeneity and crystallized using the hanging-drop vapour-diffusion method. A complete MAD data set was collected to 2.2 A resolution at 100 K. Crystals of the SeMet-labelled protein belonged to the trigonal space group P3(2)21, with unit-cell parameters a = b = 53.78, c = 162.05 A.

Published
2008

29. [MicroRNAs: biosynthesis: mechanisms of action and biological functions]

Author

Jérôme CAVAILLE, Laboratoire de biologie moléculaire eucaryote (LBME), Centre National de la Recherche Scientifique (CNRS)-Centre de Biologie Intégrative (CBI), Université Toulouse III - Paul Sabatier (UT3), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Centre National de la Recherche Scientifique (CNRS)-Université Toulouse III - Paul Sabatier (UT3), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Centre National de la Recherche Scientifique (CNRS), Institut de Biologie du Développement de Marseille (IBDM), Aix Marseille Université (AMU)-Centre National de la Recherche Scientifique (CNRS), Science et Ingénierie des Matériaux et Procédés (SIMaP), Université Joseph Fourier - Grenoble 1 (UJF)-Institut polytechnique de Grenoble - Grenoble Institute of Technology (Grenoble INP )-Institut National Polytechnique de Grenoble (INPG)-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS), Centre de Recherches sur les Macromolécules Végétales (CERMAV), Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS)-Université Joseph Fourier - Grenoble 1 (UJF), Université de Toulouse (UT)-Université de Toulouse (UT)-Centre National de la Recherche Scientifique (CNRS)-Centre de Biologie Intégrative (CBI), Université de Toulouse (UT)-Université de Toulouse (UT)-Centre National de la Recherche Scientifique (CNRS)-Centre National de la Recherche Scientifique (CNRS), and Université Joseph Fourier - Grenoble 1 (UJF)-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS)

Subjects
MESH: Surface Properties, MESH: Cell Differentiation, MESH: Molecular Conformation, MESH: Materials Testing, MESH: Polystyrenes, Cell Differentiation, MESH: 3' Untranslated Regions, MESH: Macromolecular Substances, MicroRNAs, Protein Biosynthesis, MESH: Protein Biosynthesis, MESH: Nanotubes, Carbon, MESH: Colloids, [SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology, MESH: Particle Size, 3' Untranslated Regions, MESH: MicroRNAs, MESH: Nanotechnology, MESH: Nitrogen, MESH: Crystallization
Abstract

Polymer grafting of polystyrene (PS) on nitrogen-doped multiwall carbon nanotubes (CNx) was successfully obtained by a "grafting from" technique. The production method involves the immobilization of initiators, using wet chemistry, onto the nanotube surface, followed by an in situ surface-initiated polymerization. The polymer-grafting carbon nanotubes synthesis includes the free radical functionalization of CNx and the "controlled/living" Nitroxide Mediated Radical Polymerization (NMRP). The obtained products were studied using several microscopic techniques as scanning electron microscopy (SEM), high-resolution transmission electron microscopy (HRTEM), and electron energy loss spectroscopy (EELS). The characterization also includes thermogravimetric analysis (TGA), Raman spectroscopy, infrared spectroscopy, and electron spin resonance (ESR), among others. The analyzed samples were also compared with solutions fabricated by physical blending of the polymer and CNx nanotubes. These results indicate that the nanotube radical functionalization, the chemical grafting, and the polymerization reaction were obtained over CNx when NMRP method was successfully used, giving rise to a uniform PS layer of several nanometers grafted on the outer surface of the CNx nanotubes. Several properties of the PS-grafted CNx nanotubes were also studied. It is shown that the production method leads to a narrower distribution of the external diameters. Moreover, their solubilization in organic solvents is greatly improved. Finally, the dispersion of PS-grafted CNx into a PS matrix is studied to determine the differences in filler dispersion and interfacial adhesion strength, in comparison with nanocomposites elaborated with as-produced CNx.

Published
2007

30. Expression, purification, crystallization and preliminary crystallographic study of isolated modules of the mouse coactivator-associated arginine methyltransferase 1

Author

Vincent Cura, Nathalie Troffer-Charlier, Dino Moras, Pierre Hassenboehler, Jean Cavarelli, Institut de génétique et biologie moléculaire et cellulaire (IGBMC), Université Louis Pasteur - Strasbourg I-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), and Centre National de la Recherche Scientifique (CNRS)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Louis Pasteur - Strasbourg I

Subjects
MESH: DNA Primers, Protein-Arginine N-Methyltransferases, CARM1, Multiple isomorphous replacement, Protein Conformation, Biophysics, MESH: Protein-Arginine N-Methyltransferase, Spodoptera, Biology, MESH: Base Sequence, Crystallography, X-Ray, Biochemistry, Cell Line, Mice, 03 medical and health sciences, Protein structure, MESH: Protein Conformation, Structural Biology, Coactivator, Genetics, Protein methylation, Animals, MESH: Animals, MESH: Cloning, Molecular, Cloning, Molecular, MESH: Mice, DNA Primers, 030304 developmental biology, MESH: Crystallization, Regulation of gene expression, 0303 health sciences, MESH: Spodoptera, Base Sequence, 030302 biochemistry & molecular biology, Resolution (electron density), [SDV.BBM.BM]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Molecular biology, Condensed Matter Physics, MESH: Crystallography, X-Ray, MESH: Cell Line, Crystallography, Nuclear receptor, Crystallization Communications, Crystallization
Abstract

Coactivator-associated arginine methyltransferase 1 (CARM1) plays a crucial role in gene expression as a coactivator of several nuclear hormone receptors and also of non-nuclear receptor systems. Its recruitment by the transcriptional machinery induces protein methylation, leading to chromatin remodelling and gene activation. CARM1(28-507) and two structural states of CARM1(140-480) were expressed, purified and crystallized. Crystals of CARM1(28-507) belong to space group P6(2)22, with unit-cell parameters a = b = 136.0, c = 125.3 A; they diffract to beyond 2.5 A resolution using synchrotron radiation and contain one monomer in the asymmetric unit. The structure of CARM1(28-507) was solved by multiple isomorphous replacement and anomalous scattering methods. Crystals of apo CARM1(140-480) belong to space group I222, with unit-cell parameters a = 74.6, b = 99.0, c = 207.4 A; they diffract to beyond 2.7 A resolution and contain two monomers in the asymmetric unit. Crystals of CARM1(140-480) in complex with S-adenosyl-L-homocysteine belong to space P2(1)2(1)2, with unit-cell parameters a = 74.6, b = 98.65, c = 206.08 A; they diffract to beyond 2.6 A resolution and contain four monomers in the asymmetric unit. The structures of apo and holo CARM1(140-480) were solved by molecular-replacement techniques from the structure of CARM1(28-507).

Published
2007

31. Tyrosyl-tRNA synthetase: the first crystallization of a human mitochondrial aminoacyl-tRNA synthetase

Author

Luc Bonnefond, Magali Frugier, Richard Giegé, Claude Sauter, Joëlle Rudinger-Thirion, Catherine Florentz, Elodie Touzé, Bernard Lorber, Architecture et réactivité de l'ARN (ARN), and Université Louis Pasteur - Strasbourg I-Centre National de la Recherche Scientifique (CNRS)

Subjects
MESH: DNA Primers, Protein Conformation, MESH: Mitochondria, Biophysics, Biology, Mitochondrion, MESH: Base Sequence, Crystallography, X-Ray, 010402 general chemistry, medicine.disease_cause, 01 natural sciences, Biochemistry, 03 medical and health sciences, chemistry.chemical_compound, Protein structure, MESH: Protein Conformation, Tyrosine-tRNA Ligase, Structural Biology, MESH: Tyrosine-tRNA Ligase, Genetics, medicine, Humans, Molecular replacement, [SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology, Tyrosine, Escherichia coli, DNA Primers, 030304 developmental biology, MESH: Crystallization, 0303 health sciences, MESH: Humans, Base Sequence, Aminoacyl tRNA synthetase, Condensed Matter Physics, MESH: Crystallography, X-Ray, Mitochondria, 0104 chemical sciences, enzymes and coenzymes (carbohydrates), Tyrosine—tRNA ligase, chemistry, Crystallization Communications, Transfer RNA, Crystallization, hormones, hormone substitutes, and hormone antagonists
Abstract

International audience; Human mitochondrial tyrosyl-tRNA synthetase and a truncated version with its C-terminal S4-like domain deleted were purified and crystallized. Only the truncated version, which is active in tyrosine activation and Escherichia coli tRNA(Tyr) charging, yielded crystals suitable for structure determination. These tetragonal crystals, belonging to space group P4(3)2(1)2, were obtained in the presence of PEG 4000 as a crystallizing agent and diffracted X-rays to 2.7 A resolution. Complete data sets could be collected and led to structure solution by molecular replacement.

Published
2007

32. Virology: holed up in a natural crystal

Author

Rey, Félix A., Virologie Structurale, Institut Pasteur [Paris], and Institut Pasteur [Paris] (IP)

Subjects
MESH: Insects, MESH: Protein Conformation, [SDV.MP]Life Sciences [q-bio]/Microbiology and Parasitology, MESH: Inclusion Bodies, Viral, MESH: Animals, MESH: Crystallography, X-Ray, MESH: Viral Proteins, MESH: Models, Molecular, MESH: Nucleopolyhedrovirus, MESH: Crystallization
Published
2007

33. Structure of the prefusion form of the vesicular stomatitis virus glycoprotein G

Author

Félix A. Rey, Stéphane Roche, Stéphane Bressanelli, Yves Gaudin, Synthèse et étude de systèmes à intêret biologique (SEESIB), Université Blaise Pascal - Clermont-Ferrand 2 (UBP)-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS), Institut de génétique humaine (IGH), Université de Montpellier (UM)-Centre National de la Recherche Scientifique (CNRS), Service de Physique Statistique, Magnétisme et Supraconductivité (SPSMS - UMR 9001), Institut Nanosciences et Cryogénie (INAC), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019]), Smurfit Institute of Genetics, Trinity College Dublin, Virologie Structurale, Institut Pasteur [Paris] (IP), Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS), Université Blaise Pascal - Clermont-Ferrand 2 (UBP)-Centre National de la Recherche Scientifique (CNRS)-Institut de Chimie du CNRS (INC), Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Commissariat à l'énergie atomique et aux énergies alternatives (CEA), Institut Pasteur [Paris], and Institut Pasteur [Paris]-Centre National de la Recherche Scientifique (CNRS)

Subjects
Models, Molecular, Protein Folding, Conformational change, MESH: Hydrogen-Ion Concentration, MESH: Protein Structure, Quaternary, Protein Conformation, MESH: Membrane Glycoproteins, MESH: Protein Structure, Secondary, MESH: Amino Acid Sequence, Crystallography, X-Ray, medicine.disease_cause, Membrane Fusion, Protein Structure, Secondary, MESH: Protein Structure, Tertiary, Protein structure, MESH: Protein Conformation, Viral Envelope Proteins, CRYSTAL-STRUCTURE, MESH: Crystallization, 0303 health sciences, Membrane Glycoproteins, Multidisciplinary, RABIES VIRUS, Hydrogen-Ion Concentration, ENVELOPE GLYCOPROTEIN, INFLUENZA HEMAGGLUTININ, Biochemistry, Vesicular stomatitis virus, [SDV.MP.VIR]Life Sciences [q-bio]/Microbiology and Parasitology/Virology, MESH: Viral Fusion Proteins, Protein folding, Crystallization, Hydrophobic and Hydrophilic Interactions, MESH: Models, Molecular, MESH: Vesicular stomatitis-Indiana virus, Viral protein, MESH: Protein Folding, Molecular Sequence Data, Biology, MEMBRANE-FUSION, MESH: Membrane Fusion, Vesicular stomatitis Indiana virus, 03 medical and health sciences, medicine, Amino Acid Sequence, Protein Structure, Quaternary, 030304 developmental biology, MESH: Hydrophobicity, MESH: Molecular Sequence Data, 030306 microbiology, Lipid bilayer fusion, FUSION-INACTIVE CONFORMATION, Viral membrane, Rhabdoviridae, biology.organism_classification, MESH: Crystallography, X-Ray, Protein Structure, Tertiary, LOW-PH, MESH: Viral Envelope Proteins, Biophysics, Viral Fusion Proteins
Abstract

International audience; Glycoprotein G of the vesicular stomatitis virus triggers membrane fusion via a low pH-induced structural rearrangement. Despite the equilibrium between the pre- and postfusion states, the structure of the prefusion form, determined to 3.0 angstrom resolution, shows that the fusogenic transition entails an extensive structural reorganization of G. Comparison with the structure of the postfusion form suggests a pathway for the conformational change. In the prefusion form, G has the shape of a tripod with the fusion loops exposed, which point toward the viral membrane, and with the antigenic sites located at the distal end of the molecule. A large number of G glycoproteins, perhaps organized as in the crystals, act cooperatively to induce membrane merging.

Published
2007

34. Mutational analysis of cytochrome b at the ubiquinol oxidation site of yeast complex III

Author

Petra Hellwig, Carola Hunte, Brigitte Meunier, Fraser MacMillan, Raul Covian, Bernard L. Trumpower, Tina Wenz, Department Molecular Membrane Biology, Max-Planck-Institut für Biophysik - Max Planck Institute of Biophysics (MPIBP), Max-Planck-Gesellschaft-Max-Planck-Gesellschaft, Department of Biochemistry, Dartmouth Medical School, Institut de Chimie de Strasbourg, Centre National de la Recherche Scientifique (CNRS)-Université Louis Pasteur - Strasbourg I-Institut de Chimie du CNRS (INC), Institut for Physical & Theorical Chemistry & Center for Biomolecular Magnetic Resonance, Johan W. Goethe University, Goethe-Universität Frankfurt am Main, Centre de génétique moléculaire (CGM), Centre National de la Recherche Scientifique (CNRS), and Université Louis Pasteur - Strasbourg I-Centre National de la Recherche Scientifique (CNRS)

Subjects
MESH: Oxidation-Reduction, MESH: Enzyme Stability, Ubiquinone, DNA Mutational Analysis, Biochemistry, MESH: Aspartic Acid, MESH: Tyrosine, Electron Transport Complex III, chemistry.chemical_compound, 0302 clinical medicine, Cytochrome C1, MESH: Electron Transport Complex III, Enzyme Stability, MESH: Ubiquinone, MESH: DNA Mutational Analysis, Heme, MESH: Crystallization, 0303 health sciences, biology, MESH: Kinetics, Cytochrome b, Cytochrome bc1, Cytochrome c, Cytochromes c, MESH: Cytochromes c, MESH: Glutamic Acid, Cytochromes b, MESH: Cytochromes b, MESH: Saccharomyces cerevisiae, [SDV.BBM.BC]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biomolecules [q-bio.BM], MESH: Mutagenesis, Site-Directed, Crystallization, Oxidation-Reduction, Ubiquinol, MESH: Enzyme Activation, Phenylalanine, Glutamic Acid, Saccharomyces cerevisiae, macromolecular substances, Q cycle, 03 medical and health sciences, MESH: Phenylalanine, [SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology, [SDV.BBM.BC]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biochemistry [q-bio.BM], Molecular Biology, 030304 developmental biology, Aspartic Acid, Binding Sites, Cell Biology, Enzyme Activation, Kinetics, chemistry, MESH: Binding Sites, Coenzyme Q – cytochrome c reductase, Mutagenesis, Site-Directed, biology.protein, Tyrosine, 030217 neurology & neurosurgery
Abstract

The cytochrome bc1 complex is a dimeric enzyme of the inner mitochondrial membrane that links electron transfer from ubiquinol to cytochrome c by a protonmotive Q cycle mechanism in which ubiquinol is oxidized at one center in the enzyme, referred to as center P, and ubiquinone is rereduced at a second center, referred to as center N. To better understand the mechanism of ubiquinol oxidation, we have examined catalytic activities and pre-steady-state reduction kinetics of yeast cytochrome bc1 complexes with mutations in cytochrome b that we expected would affect oxidation of ubiquinol. We mutated two residues thought to be involved in proton conduction linked to ubiquinol oxidation, Tyr132 and Glu272, and two residues proposed to be involved in docking ubiquinol into the center P pocket, Phe129 and Tyr279. Substitution of Phe129 by lysine or arginine yielded a respiration-deficient phenotype and lipid-dependent catalytic activity. Increased bypass reactions were detectable for both variants, with F129K showing the more severe effects. Substitution with lysine leads to a disturbed coordination of a b heme as deduced from changes in the midpoint potential and the EPR signature. Removal of the aromatic side chain in position Tyr279 lowers the catalytic activity accompanied by a low level of bypass reactions. Pre-steady-state kinetics of the enzymes modified at Glu272 and Tyr132 confirmed the importance of their functional groups for electron transfer. Altered center N kinetics and activation of ubiquinol oxidation by binding of cytochrome c in the Y132F and E272D enzymes indicate long range effects of these mutations.

Published
2007

35. An electrodeposition method of calcium phosphate coatings on titanium alloy

Author

Pierre Layrolle, Pierre Weiss, Marco A. Lopez-Heredia, Laboratoire d'ingénierie osteo-articulaire et dentaire (LIOAD), Université de Nantes (UN)-IFR26-Institut National de la Santé et de la Recherche Médicale (INSERM), and Layrolle, Pierre

Subjects
Calcium Phosphates, MESH: Materials Testing, 02 engineering and technology, Electrolyte, 01 natural sciences, Crystallinity, Coating, Coated Materials, Biocompatible, MESH: Coated Materials, Biocompatible, Materials Testing, MESH: Crystallization, Titanium, Magnesium, 021001 nanoscience & nanotechnology, MESH: Calcium Phosphates, MESH: Titanium, 0210 nano-technology, Crystallization, Porosity, Materials science, Surface Properties, Biomedical Engineering, Biophysics, chemistry.chemical_element, Bioengineering, MESH: Bone Substitutes, engineering.material, Calcium, 010402 general chemistry, MESH: Electroplating, Biomaterials, MESH: Porosity, Alloys, MESH: Particle Size, Particle Size, [SDV.IB.BIO]Life Sciences [q-bio]/Bioengineering/Biomaterials, MESH: Surface Properties, Metallurgy, technology, industry, and agriculture, Titanium alloy, equipment and supplies, Electroplating, 0104 chemical sciences, Anode, [SDV.IB.BIO] Life Sciences [q-bio]/Bioengineering/Biomaterials, chemistry, Chemical engineering, Bone Substitutes, engineering, Adsorption, MESH: Adsorption
Abstract

Calcium phosphates coatings were deposited onto titanium alloy discs via en electrodeposition method. Titanium alloy discs were blasted with calcium phosphate particles, then etched in a mixture of nitric and fluoric acids and rinsed in demineralized water. The titanium alloy disc (cathode) and platinum mesh (anode) were immersed in a supersaturated calcium phosphate electrolyte buffered at pH 7.4 and connected to a current generator. The microstructure, chemical composition and crystallinity of the electrodeposited coatings were studied as function of time 10-120 min, temperature 25-80 degrees C, current density 8-120 mA/cm(2), magnesium and hydrogen carbonate amounts (0.1-1 mM). Uniform calcium phosphate coatings were obtained in 30 min but coating thickness increased with deposition time. Raising the temperature of electrolyte resulted in more uniform coatings as ionic mobility increased. Low current density was preferable due to hydrogen gas evolving at the cathode, which disturbed the deposition of calcium phosphate crystals on titanium. The amounts of magnesium and hydrogen carbonate ions affected both the homogeneity and morphology of the coatings. This study showed that the electrodeposition method is efficient for coating titanium with osteoconductive calcium phosphate layers.

Published
2007

36. Calcium-deficient apatite synthesized by ammonia hydrolysis of dicalcium phosphate dihydrate: influence of temperature, time, and pressure

Author

Laetitia Obadia, Thierry Rouillon, Guy Daculsi, Jean-Michel Bouler, Bruno Bujoli, Bouler, Jean Michel, Matériaux d'intérêt biologique, Université de Nantes (UN)-Institut National de la Santé et de la Recherche Médicale (INSERM), Laboratoire de Synthèse Organique (LSO), Université de Nantes (UN)-Centre National de la Recherche Scientifique (CNRS), Chimie Et Interdisciplinarité : Synthèse, Analyse, Modélisation (CEISAM), Université de Nantes - UFR des Sciences et des Techniques (UN UFR ST), and Université de Nantes (UN)-Université de Nantes (UN)-Centre National de la Recherche Scientifique (CNRS)-Institut de Chimie du CNRS (INC)

Subjects
Calcium Phosphates, Hot Temperature, Materials science, MESH: Apatites, Biomedical Engineering, Mineralogy, MESH: Heat, 02 engineering and technology, 010402 general chemistry, 01 natural sciences, Apatite, law.invention, Biomaterials, Crystal, Ammonia, chemistry.chemical_compound, Hydrolysis, law, Apatites, Pressure, MESH: Ammonia, Crystallization, [SDV.IB.BIO]Life Sciences [q-bio]/Bioengineering/Biomaterials, MESH: Crystallization, Rietveld refinement, Factorial experiment, 021001 nanoscience & nanotechnology, MESH: Calcium Phosphates, 0104 chemical sciences, [SDV.IB.BIO] Life Sciences [q-bio]/Bioengineering/Biomaterials, chemistry, visual_art, visual_art.visual_art_medium, Crystallite, 0210 nano-technology, MESH: Pressure, MESH: Hydrolysis, Nuclear chemistry
Abstract

International audience; In this work, calcium-deficient apatites (CDA) were synthesized by ammonia hydrolysis reaction of dicalcium phosphate dihydrate (DCPD; CaHPO4 x 2 H2O) to obtain biphasic calcium phosphates (BCP) without any extraionic substitution. The influence of three parameters was studied: temperature of the reaction (70 and 100 degrees C), time of the reaction (4 and 18 h), and the pressure (open and closed system). Experiments were made according to a factorial design method (FDM) allowing optimization of the number of samples as well as statistical analysis of results. Moreover, the influence of temperature (until 200 degrees C) was investigated. The crystal size of CDA was determined according to the Scherrer's formula and from Rietveld refinements taking the CDA anisotropy into account. The last method seems to be a reliable method to determine crystallite sizes of CDA, since crystallite sizes of CDA along and directions were accessible. The results describe the hydroxyapatite % (HA%) in BCP by a first-order polynomial equation in the experimental area studied and the HA content was found to increase by raising time and temperature of the reaction. Moreover, the type of reaction system (open/closed vessel) appeared to have little influence on HA%.

Published
2007

37. Crystal structures of an Extracytoplasmic Solute Receptor from a TRAP transporter in its open and closed forms reveal a helix-swapped dimer requiring a cation for alpha-keto acid binding

Author

Sophie Gonin, Jérôme Lavergne, David Pignol, Pascal Arnoux, Bénédicte Pierru, Béatrice Alonso, Monique Sabaty, Microbiologie Environnementale et Moléculaire (MEM), Institut de Biosciences et Biotechnologies d'Aix-Marseille (ex-IBEB) (BIAM), Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Aix Marseille Université (AMU)-Centre National de la Recherche Scientifique (CNRS)-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Aix Marseille Université (AMU)-Centre National de la Recherche Scientifique (CNRS), Service de Biochimie et Toxicologie Nucléaire (SBTN), Commissariat à l'énergie atomique et aux énergies alternatives (CEA), Aix Marseille Université (AMU)-Centre National de la Recherche Scientifique (CNRS)-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), and Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)

Subjects
Conformational change, Stereochemistry, Dimer, MESH: Biological Transport, MESH: Keto Acids, MESH: Protein Structure, Secondary, ATP-binding cassette transporter, MESH: Carrier Proteins, Rhodobacter sphaeroides, Crystallography, X-Ray, Protein Structure, Secondary, MESH: Membrane Transport Proteins, 03 medical and health sciences, chemistry.chemical_compound, Bacterial Proteins, Structural Biology, Cations, [SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology, Binding site, MESH: Cations, lcsh:QH301-705.5, MESH: Bacterial Proteins, 030304 developmental biology, MESH: Crystallization, 0303 health sciences, biology, Chemistry, Membrane transport protein, 030302 biochemistry & molecular biology, Membrane Transport Proteins, RNA-Binding Proteins, Biological Transport, Periplasmic space, MESH: Rhodobacter sphaeroides, MESH: Transcription Factors, MESH: Crystallography, X-Ray, Keto Acids, Transport protein, Crystallography, MESH: RNA-Binding Proteins, lcsh:Biology (General), MESH: Dimerization, biology.protein, Carrier Proteins, Crystallization, Dimerization, Alpha helix, Transcription Factors, Research Article
Abstract

Background The import of solutes into the bacterial cytoplasm involves several types of membrane transporters, which may be driven by ATP hydrolysis (ABC transporters) or by an ion or H+ electrochemical membrane potential, as in the tripartite ATP-independent periplasmic system (TRAP). In both the ABC and TRAP systems, a specific periplasmic protein from the ESR family (Extracytoplasmic Solute Receptors) is often involved for the recruitment of the solute and its presentation to the membrane complex. In Rhodobacter sphaeroides, TakP (previously named SmoM) is an ESR from a TRAP transporter and binds α-keto acids in vitro. Results We describe the high-resolution crystal structures of TakP in its unliganded form and as a complex with sodium-pyruvate. The results show a limited "Venus flytrap" conformational change induced by substrate binding. In the liganded structure, a cation (most probably a sodium ion) is present and plays a key role in the association of the pyruvate to the protein. The structure of the binding pocket gives a rationale for the relative affinities of various ligands that were tested from a fluorescence assay. The protein appears to be dimeric in solution and in the crystals, with a helix-swapping structure largely participating in the dimer formation. A 30 Å-long water channel buried at the dimer interface connects the two ligand binding cavities of the dimer. Conclusion The concerted recruitment by TakP of the substrate group with a cation could represent a first step in the coupled transport of both partners, providing the driving force for solute import. Furthermore, the unexpected dimeric structure of TakP suggests a molecular mechanism of solute uptake by the dimeric ESR via a channel that connects the binding sites of the two monomers.

Published
2007

38. Crystal structure of AFV3-109, a highly conserved protein from crenarchaeal viruses

Author

Sophie Quevillon-Cheruel, David Prangishvili, Herman van Tilbeurgh, Patrick Forterre, Jenny Keller, Nicolas Leulliot, Stéphanie Porciero, Christian Cambillau, Valérie Campanacci, Diego Cortez, Institut de biochimie et biophysique moléculaire et cellulaire (IBBMC), Université Paris-Sud - Paris 11 (UP11)-Centre National de la Recherche Scientifique (CNRS), Architecture et fonction des macromolécules biologiques (AFMB), Institut National de la Recherche Agronomique (INRA)-Aix Marseille Université (AMU)-Centre National de la Recherche Scientifique (CNRS), Biologie Moléculaire du Gène chez les Extrêmophiles (BMGE), Institut Pasteur [Paris], Institut de génétique et microbiologie [Orsay] (IGM), Centre National de la Recherche Scientifique (CNRS)-Aix Marseille Université (AMU)-Institut National de la Recherche Agronomique (INRA), and Institut Pasteur [Paris] (IP)

Subjects
Archaeal Viruses, Models, Molecular, Viral protein, Sequence analysis, Molecular Sequence Data, Bicaudaviridae, MESH: Sequence Alignment, Sequence alignment, MESH: Amino Acid Sequence, medicine.disease_cause, Crystallography, X-Ray, lcsh:Infectious and parasitic diseases, Conserved sequence, 03 medical and health sciences, Open Reading Frames, Virology, MESH: Archaeal Viruses, medicine, lcsh:RC109-216, [SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology, Amino Acid Sequence, Peptide sequence, Conserved Sequence, MESH: Capsid Proteins, 030304 developmental biology, MESH: Crystallization, Genetics, 0303 health sciences, MESH: Conserved Sequence, MESH: Molecular Sequence Data, biology, 030306 microbiology, Research, Crenarchaeota, Lipothrixviridae, MESH: Crenarchaeota, MESH: Open Reading Frames, biology.organism_classification, MESH: Crystallography, X-Ray, Infectious Diseases, [SDV.MP]Life Sciences [q-bio]/Microbiology and Parasitology, MESH: Dimerization, Capsid Proteins, Crystallization, Dimerization, Sequence Alignment, MESH: Models, Molecular
Abstract

The extraordinary morphologies of viruses infecting hyperthermophilic archaea clearly distinguish them from bacterial and eukaryotic viruses. Moreover, their genomes code for proteins that to a large extend have no related sequences in the extent databases. However, a small pool of genes is shared by overlapping subsets of these viruses, and the most conserved gene, exemplified by the ORF109 of the Acidianus Filamentous Virus 3, AFV3, is present on genomes of members of three viral familes, the Lipothrixviridae, Rudiviridae, and "Bicaudaviridae", as well as of the unclassified Sulfolobus Turreted Icosahedral Virus, STIV. We present here the crystal structure of the protein (Mr = 13.1 kD, 109 residues) encoded by the AFV3 ORF 109 in two different crystal forms at 1.5 and 1.3 Å resolution. The structure of AFV3-109 is a five stranded β-sheet with loops on one side and three helices on the other. It forms a dimer adopting the shape of a cradle that encompasses the best conserved regions of the sequence. No protein with a related fold could be identified except for the ortholog from STIV1, whose structure was deposited at the Protein Data Bank. We could clearly identify a well bound glycerol inside the cradle, contacting exclusively totally conserved residues. This interaction was confirmed in solution by fluorescence titration. Although the function of AFV3-109 cannot be deduced directly from its structure, structural homology with the STIV1 protein, and the size and charge distribution of the cavity suggested it could interact with nucleic acids. Fluorescence quenching titrations also showed that AFV3-109 interacts with dsDNA. Genomic sequence analysis revealed bacterial homologs of AFV3-109 as a part of a putative previously unidentified prophage sequences in some Firmicutes.

Published
2007

39. The Tim21 binding domain connects the preprotein translocases of both mitochondrial membranes

Author

Reinhard Albrecht, Jan Brix, Agnieszka Chacinska, Rudolf Volkmer, Sergio A. Cadamuro, Peter Rehling, Bernard Guiard, Nikolaus Pfanner, Kornelius Zeth, Abteilung Membranbiochemie [Martinsried], Max-Planck-Institut für Biochemie (MPIB), Max-Planck-Gesellschaft-Max-Planck-Gesellschaft, Abteilung Protein Evolution [Tübingen], Max-Planck-Institut für Entwicklungsbiologie, Institute of Molecular Medicine and Cell Research (ZBMZ), University of Freiburg [Freiburg], Max-Planck-Gesellschaft, Charité - UniversitätsMedizin = Charité - University Hospital [Berlin], Centre de génétique moléculaire (CGM), Centre National de la Recherche Scientifique (CNRS), Deutsche Forschungsgemeinschaft, the Sonderforschungsbereich 388, Gottfried Wilhelm Leibniz Program, Max Planck Research Award, Alexander von Humboldt Foundation, Bundesministerium fur Bildung und Forschung, the Fonds der Chemischen Industrie, and Charité - Universitätsmedizin Berlin / Charite - University Medicine Berlin

Subjects
MESH: Protein Precursors, MESH: Protein Structure, Secondary, MESH: Amino Acid Sequence, Biochemistry, Mitochondrial Membrane Transport Proteins, Protein Structure, Secondary, MESH: Membrane Transport Proteins, MESH: Protein Structure, Tertiary, 0302 clinical medicine, MESH: Saccharomyces cerevisiae Proteins, MESH: Structure-Activity Relationship, MESH: Mitochondrial Membranes, Mitochondrial Precursor Protein Import Complex Proteins, Translocase, MESH: Crystallization, 0303 health sciences, MESH: Models, Chemical, MESH: Saccharomyces cerevisiae, [SDV.BBM.BC]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biomolecules [q-bio.BM], MESH: Repressor Proteins, Mitochondrial Membranes, ATP–ADP translocase, Intermembrane space, Crystallization, Binding domain, Saccharomyces cerevisiae Proteins, Translocase of the outer membrane, Molecular Sequence Data, Scientific Report, TIM/TOM complex, MESH: Carrier Proteins, Saccharomyces cerevisiae, Biology, 03 medical and health sciences, Mitochondrial membrane transport protein, Structure-Activity Relationship, Genetics, [SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology, Amino Acid Sequence, Protein Precursors, [SDV.BBM.BC]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biochemistry [q-bio.BM], Molecular Biology, 030304 developmental biology, Binding Sites, MESH: Molecular Sequence Data, Membrane Transport Proteins, Protein Structure, Tertiary, Repressor Proteins, Models, Chemical, MESH: Binding Sites, Translocase of the inner membrane, biology.protein, Biophysics, Carrier Proteins, 030217 neurology & neurosurgery
Abstract

Proteins destined for the mitochondrial matrix are imported by the translocase of the outer membrane--the TOM complex--and the presequence translocase of the inner membrane--the TIM23 complex. At present, there is no structural information on components of the presequence translocase. Tim21, a subunit of the presequence translocase consisting of a membrane anchor and a carboxy-terminal domain exposed to the intermembrane space, directly connects the TOM and TIM23 complexes by binding to the intermembrane space domain of the Tom22 receptor. We crystallized the binding domain of Tim21 of Saccharomyces cerevisiae and determined its structure at 1.6 A resolution. The Tim21 structure represents a new alpha/beta-mixed protein fold with two alpha-helices flanked by an extended eight-stranded beta-sheet. We also identified a core sequence of Tom22 that binds to Tim21. Furthermore, negatively charged amino-acid residues of Tom22 are important for binding to Tim21. Here we suggest a mechanism for the TOM-TIM interaction.

Published
2006

40. Synthesis of an X-ray opaque biodegradable copolyester by chemical modification of poly (epsilon-caprolactone)

Author

Michel Vert, Jean Coudane, Benjamin Nottelet, Institut des Biomolécules Max Mousseron [Pôle Chimie Balard] (IBMM), and Centre National de la Recherche Scientifique (CNRS)-Institut de Chimie du CNRS (INC)-Université de Montpellier (UM)-Ecole Nationale Supérieure de Chimie de Montpellier (ENSCM)

Subjects
Time Factors, MESH: Iodine, Biocompatible Materials, 02 engineering and technology, 01 natural sciences, law.invention, Crystallinity, chemistry.chemical_compound, Lactones, MESH: Biocompatible Materials, law, Copolymer, Crystallization, MESH: Crystallization, Molecular Structure, Viscosity, Hydrolysis, MESH: Molecular Weight, 021001 nanoscience & nanotechnology, Copolyester, Lithium diisopropylamide, MESH: Hexanoic Acids, MESH: Polyesters, Polyester, Mechanics of Materials, Polycaprolactone, Thermodynamics, MESH: Thermodynamics, 0210 nano-technology, MESH: Hydrolysis, MESH: Lactones, Iodine, Materials science, Polyesters, MESH: Viscosity, MESH: Molecular Structure, Biophysics, Bioengineering, 010402 general chemistry, Biomaterials, MESH: X-Rays, Polymer chemistry, [SDV.IB.BIO]Life Sciences [q-bio]/Bioengineering/Biomaterials, Caproates, X-Rays, MESH: Time Factors, Chemical modification, 0104 chemical sciences, Molecular Weight, chemistry, Ceramics and Composites
Abstract

International audience; Poly (epsilon-caprolactone-co-alpha-iodo-epsilon-caprolactone) was synthesized by binding iodine to PCL chain bearing carbanionic site on alpha-position of carbonyl groups using lithium diisopropylamide. Copolyesters containing from 10% to 25% of iodo-units were thus obtained. Viscoelastic properties (modulus, loss angle), thermal properties (T(m), T(c), DeltaH(m)), crystallinity and in vitro degradability of this new type of copolymers were measured. Their opacity to X-rays was assessed, and appeared high enough to be of interest for biomedical applications.

Published
2006

41. Comparison of hydrogen determination with X-ray and neutron crystallography in a human aldose reductase-inhibitor complex

Author

Isabelle Hazemann, Andre Mitschler, Alberto Podjarny, Flora Meilleur, Matthew P. Blakeley, Dean A. A. Myles, Institut de génétique et biologie moléculaire et cellulaire (IGBMC), Centre National de la Recherche Scientifique (CNRS)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Louis Pasteur - Strasbourg I, and Université Louis Pasteur - Strasbourg I-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)

Subjects
Models, Molecular, MESH: Deuterium, Protein Conformation, Neutron diffraction, Ab initio, Crystallography, X-Ray, 01 natural sciences, law.invention, MESH: Aldehyde Reductase, MESH: Protein Conformation, law, Crystallization, MESH: Crystallization, 0303 health sciences, 010304 chemical physics, Molecular Structure, Chemistry, MESH: Hydrogen, General Medicine, Aldose reductase inhibitor, MESH: Neutron Diffraction, Neutron Diffraction, Protons, MESH: NADP, MESH: Models, Molecular, medicine.drug, inorganic chemicals, MESH: Molecular Structure, Biophysics, Protonation, 03 medical and health sciences, Aldehyde Reductase, 0103 physical sciences, medicine, Humans, Neutron, MESH: Hydrogen Bonding, 030304 developmental biology, Aldose reductase, Binding Sites, MESH: Humans, Hydrogen Bonding, [SDV.BBM.BM]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Molecular biology, Deuterium, MESH: Crystallography, X-Ray, Crystallography, MESH: Binding Sites, MESH: Protons, NADP, Hydrogen
Abstract

International audience; Protonation states determination by neutron (2.2 A at room temperature) and X-ray (0.66 A at 100 K) crystallographic studies were compared for a medium size enzyme, human aldose reductase (MW=36 kDa), complexed with its NADP+ coenzyme and a selected inhibitor of therapeutic interest. The neutron resolution could be achieved only with the ab initio fully deuterated protein and the subsequent crystallization in D2O of the complex. We used the largest good-quality crystal (1.00x0.67x0.23 mm, i.e. volume of 0.15 mm3) that we were able to grow so far. Both studies enable the determination of protonation states, with a clear advantage for neutrons in the case of less-ordered atoms (B>5 A2). Hydrogen atoms are best determined by a complementary analysis of the Fourier maps obtained from both methods.

Published
2006

42. Anisotropic lattice distortions in biogenic calcite induced by intra-crystalline organic molecules

Author

Moshe Kapon, Andrew N. Fitch, Emil Zolotoyabko, Frédéric Marin, Noam Adir, Boaz Pokroy, Department of Materials Engineering, Technion - Israel Institute of Technology [Haifa], European Synchrotron Radiation Facility ( ESRF ), Biogéosciences [Dijon] ( BGS ), AgroSup Dijon - Institut National Supérieur des Sciences Agronomiques, de l'Alimentation et de l'Environnement-Université de Bourgogne ( UB ) -Centre National de la Recherche Scientifique ( CNRS ), Faculty of Chemistry, Work supported by ESRF, by the Israel Science Foundation founded by the Israel Academy of Science and Humanities (Grant No. 15/03- 12.6) and by the Technion V.P.R. Fund (Grant No. 2004116), European Synchrotron Radiation Facility (ESRF), Biogéosciences [UMR 6282] [Dijon] (BGS), Centre National de la Recherche Scientifique (CNRS)-Université de Bourgogne (UB)-AgroSup Dijon - Institut National Supérieur des Sciences Agronomiques, de l'Alimentation et de l'Environnement, and Université de Bourgogne (UB)-AgroSup Dijon - Institut National Supérieur des Sciences Agronomiques, de l'Alimentation et de l'Environnement-Centre National de la Recherche Scientifique (CNRS)

Subjects
Diffraction, Biomineralization, MESH : Calcium Carbonate, MESH: Bivalvia, 02 engineering and technology, Crystallography, X-Ray, 01 natural sciences, law.invention, chemistry.chemical_compound, Structural Biology, law, MESH : Bivalvia, Ostrea, MESH : Anisotropy, MESH: Animals, Organic Chemicals, Crystallization, Anisotropy, MESH: Crystallization, Calcite, MESH: Ostrea, Synchrotron radiation, Crystal growth and nucleation, MESH : Organic Chemicals, 021001 nanoscience & nanotechnology, MESH: Calcium Carbonate, MESH : Crystallization, X-ray crystallography, 0210 nano-technology, Materials science, MESH : Crassostrea, 010402 general chemistry, Calcium Carbonate, Animals, Crassostrea, Intra-crystalline organic molecules, [SDV.IB.BIO]Life Sciences [q-bio]/Bioengineering/Biomaterials, Biogenic crystals, MESH : Ostrea, MESH: Organic Chemicals, MESH: Crystallography, X-Ray, [ SDV.IB.BIO ] Life Sciences [q-bio]/Bioengineering/Biomaterials, Bivalvia, 0104 chemical sciences, X-ray diffraction, Crystallography, MESH: Crassostrea, chemistry, MESH: Anisotropy, MESH : Animals, MESH : Crystallography, X-Ray, Single crystal, Powder diffraction
Abstract

9 pages; International audience; We have performed precise structural measurements on five different calcitic seashells by high-resolution X-ray powder diffraction on a synchrotron beam line and by laboratory single crystal X-ray diffraction. The unit cell parameters a and c of biogenic calcite were found to be systematically larger than those measured in the non-biogenic calcite. The maximum lattice distortion (about 2.10(-3)) was detected along the c-axis. Under heat treatment above 200 degrees C, a pronounced lattice relaxation was observed, which allowed us to conclude that anisotropic lattice swelling in biogenic calcite is induced by organic macromolecules incorporated within the single crystal calcitic prisms during biomineralization. This conclusion is supported by the results of crystallization experiments in the presence of specific protein extracted from one of the shells.

Published
2006

43. Novel 1alpha,25-dihydroxyvitamin D3 analogues with the side chain at C12

Author

Natacha Rochel, Dino Moras, Xosé C. Gonzalez-Avion, Antonio Mourino, Institut de génétique et biologie moléculaire et cellulaire (IGBMC), Université Louis Pasteur - Strasbourg I-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), and Centre National de la Recherche Scientifique (CNRS)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Louis Pasteur - Strasbourg I

Subjects
Transcriptional Activation, Spectrometry, Mass, Electrospray Ionization, medicine.medical_treatment, MESH: Receptors, Calcitriol, Ligands, Transfection, 01 natural sciences, Chemical synthesis, Calcitriol receptor, MESH: Spectrometry, Mass, Electrospray Ionization, Steroid, 03 medical and health sciences, Transactivation, Structure-Activity Relationship, MESH: Structure-Activity Relationship, Calcitriol, Drug Discovery, Chlorocebus aethiops, Vitamin D and neurology, medicine, MESH: Ligands, Animals, Combinatorial Chemistry Techniques, Humans, MESH: Animals, 030304 developmental biology, MESH: Crystallization, 0303 health sciences, MESH: Humans, 010405 organic chemistry, Chemistry, MESH: Transfection, [SDV.BBM.BM]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Molecular biology, Ligand (biochemistry), MESH: Cercopithecus aethiops, In vitro, 0104 chemical sciences, 3. Good health, MESH: COS Cells, Nuclear receptor, Biochemistry, MESH: Trans-Activation (Genetics), MESH: Calcitriol, COS Cells, Molecular Medicine, Receptors, Calcitriol, MESH: Combinatorial Chemistry Techniques, Crystallization
Abstract

International audience; The plethora of actions of 1alpha,25(OH)2D3 in various systems suggested wide clinical applications of vitamin D nuclear receptor (VDR) ligands in treatments of inflammation, dermatological indication, osteoporosis, cancers, and autoimmune diseases. More than 3000 vitamin D analogues have been synthesized in order to reduce the calcemic side effects while maintaining the transactivation potency of the natural ligand. In light of the crystal structures of the vitamin D nuclear receptor (VDR), novel analogues of the hormone 1alpha,25(OH)2D3 with side chains attached to C-12 were synthesized via the convergent Wittig-Horner approach. Among the compounds studied, the analogue 2b showed the highest binding affinity for VDR and was the most potent at inducing VDR transcriptional activity in a transient transfection assay (20% of the transactivation activity of the natural ligand).

Published
2006

44. Purification, crystallization and preliminary X-ray analysis of the outer membrane complex HasA-HasR from Serratia marcescens

Author

Frédéric Huché, Philippe Delepelaire, Wolfram Welte, Cécile Wandersman, Génétique et biochimie des microorganismes (GBM), Institut Pasteur [Paris]-Centre National de la Recherche Scientifique (CNRS), Fachbereich Biologie, University of Konstanz, and Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS)

Subjects
Serratia, Macromolecular Substances, Molecular Sequence Data, Biophysics, MESH: Carrier Proteins, Heme, MESH: Amino Acid Sequence, Biology, medicine.disease_cause, Crystallography, X-Ray, Biochemistry, 03 medical and health sciences, chemistry.chemical_compound, MESH: Serratia, Bacterial Proteins, Structural Biology, Genetics, medicine, [SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology, Amino Acid Sequence, Escherichia coli, Peptide sequence, MESH: Bacterial Proteins, 030304 developmental biology, MESH: Crystallization, 0303 health sciences, MESH: Molecular Sequence Data, 030302 biochemistry & molecular biology, Membrane Proteins, MESH: Macromolecular Substances, Condensed Matter Physics, biology.organism_classification, MESH: Crystallography, X-Ray, Membrane, Membrane protein, chemistry, Crystallization Communications, MESH: Heme, Serratia marcescens, MESH: Membrane Proteins, Bacterial outer membrane, Carrier Proteins, Crystallization
Abstract

International audience; Serratia marcescens is able to acquire iron using its haem-acquisition system (;has'), which contains an outer membrane receptor HasR and a soluble haemophore HasA. After secretion, HasA binds free haem in the extracellular medium or extracts it from haemoproteins and delivers it to the receptor. Here, the crystallization of a HasA-HasR complex is reported. HasA and HasR have been overexpressed in Escherichia coli and the complex formed and crystallized. Small platelets and bunches of needles of dimensions 0.01 x 0.1 x 1 mm were obtained. A native data set has been collected to 6.8 A.

Published
2006

45. High-resolution crystal structure of AKR11C1 from Bacillus halodurans: an NADPH-dependent 4-hydroxy-2,3-trans-nonenal reductase

Author

Tobias Marquardt, Fritz K. Winkler, Alberto Podjarny, Antonietta Gasperina, Dirk Kostrewa, Rajkumar Balakrishnan, Xiao-Dan Li, Christian Kambach, Ganesaratnam K. Balendiran, Institut de génétique et biologie moléculaire et cellulaire (IGBMC), Centre National de la Recherche Scientifique (CNRS)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Louis Pasteur - Strasbourg I, and Université Louis Pasteur - Strasbourg I-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)

Subjects
Models, Molecular, Arginine, MESH: Sequence Homology, Amino Acid, Aldo-Keto Reductases, Bacillus, MESH: Amino Acid Sequence, Reductase, Crystallography, X-Ray, MESH: Benzaldehydes, Substrate Specificity, Structural Biology, Nonenal, Cloning, Molecular, MESH: Crystallization, chemistry.chemical_classification, 0303 health sciences, biology, MESH: Kinetics, Chemistry, Biochemistry, Benzaldehydes, Crystallization, MESH: NADP, MESH: Models, Molecular, Stereochemistry, Molecular Sequence Data, Glyceraldehyde, Cofactor, Catalysis, MESH: Alcohol Oxidoreductases, 03 medical and health sciences, Oxidoreductase, Aldehyde Reductase, MESH: Bacillus, MESH: Cloning, Molecular, Amino Acid Sequence, Molecular Biology, 030304 developmental biology, Aldo-keto reductase, Aldehydes, Binding Sites, MESH: Molecular Sequence Data, Sequence Homology, Amino Acid, 030306 microbiology, Active site, MESH: Glyceraldehyde, [SDV.BBM.BM]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Molecular biology, biology.organism_classification, MESH: Catalysis, MESH: Crystallography, X-Ray, Alcohol Oxidoreductases, Kinetics, MESH: Binding Sites, biology.protein, Bacillus halodurans, MESH: Aldehydes, MESH: Substrate Specificity, NADP
Abstract

International audience; Aldo-keto reductase AKR11C1 from Bacillus halodurans, a new member of aldo-keto reductase (AKR) family 11, has been characterized structurally and biochemically. The structures of the apo and NADPH bound form of AKR11C1 have been solved to 1.25 A and 1.3 A resolution, respectively. AKR11C1 possesses a novel non-aromatic stacking interaction of an arginine residue with the cofactor, which may favor release of the oxidized cofactor. Our biochemical studies have revealed an NADPH-dependent activity of AKR11C1 with 4-hydroxy-2,3-trans-nonenal (HNE). HNE is a cytotoxic lipid peroxidation product, and detoxification in alkaliphilic bacteria, such as B.halodurans, plays a crucial role in survival. AKR11C1 could thus be part of the detoxification system, which ensures the well being of the microorganism. The very poor activity of AKR11C1 on standard, small substrates such as benzaldehyde or DL-glyeraldehyde is consistent with the observed, very open active site lacking a binding pocket for these substrates. In contrast, modeling of HNE with its aldehyde function suitably positioned in the active site suggests that its elongated hydrophobic tail occupies a groove defined by hydrophobic side-chains. Multiple sequence alignment of AKR11C1 with the highly homologous iolS and YqkF proteins shows a high level of conservation in this putative substrate-binding site. We suggest that AKR11C1 is the first structurally characterized member of a new class of AKRs with specificity for substrates with long aliphatic tails.

Published
2005

46. Caspartin and calprismin, two proteins of the shell calcitic prisms of the Mediterranean fan mussel Pinna nobilis

Author

Pierre Layrolle, Christoplie Riondet, Nathalie Guichard, Gérard Alcaraz, Peter Westbroek, M. Stigter, Reinout Amons, Arnaud Hecker, Frédéric Marin, Gilles Luquet, Biogéosciences [UMR 6282] [Dijon] (BGS), Université de Bourgogne (UB)-AgroSup Dijon - Institut National Supérieur des Sciences Agronomiques, de l'Alimentation et de l'Environnement-Centre National de la Recherche Scientifique (CNRS), IsoTis, Sylvius Laboratories, Universiteit Leiden [Leiden], Institut de génétique et microbiologie [Orsay] (IGM), Université Paris-Sud - Paris 11 (UP11)-Centre National de la Recherche Scientifique (CNRS), Phytopharmacie et Biochimie des Iteractions Cellulaires (PBIC), Etablissement National d'Enseignement Supérieur Agronomique de Dijon (ENESAD)-Institut National de la Recherche Agronomique (INRA)-Université de Bourgogne (UB), Leiden Institute of Chemistry, Biogéosciences [Dijon] ( BGS ), Université de Bourgogne ( UB ) -AgroSup Dijon - Institut National Supérieur des Sciences Agronomiques, de l'Alimentation et de l'Environnement-Centre National de la Recherche Scientifique ( CNRS ), Institut de génétique et microbiologie [Orsay] ( IGM ), Université Paris-Sud - Paris 11 ( UP11 ) -Centre National de la Recherche Scientifique ( CNRS ), Phytopharmacie et Biochimie des Iteractions Cellulaires ( PBIC ), Etablissement National d'Enseignement Supérieur Agronomique de Dijon ( ENESAD ) -Institut National de la Recherche Agronomique ( INRA ) -Université de Bourgogne ( UB ), Centre National de la Recherche Scientifique (CNRS)-Université de Bourgogne (UB)-AgroSup Dijon - Institut National Supérieur des Sciences Agronomiques, de l'Alimentation et de l'Environnement, Layrolle, Pierre, Biogéosciences [UMR 6282] (BGS), and Université de Bourgogne (UB)-Centre National de la Recherche Scientifique (CNRS)

Subjects
MESH : Molecular Sequence Data, MESH : Calcium Carbonate, MESH: Bivalvia, MESH: Electrophoresis, MESH: Amino Acid Sequence, 01 natural sciences, Biochemistry, chemistry.chemical_compound, MESH : Bivalvia, MESH: Animals, MESH: Crystallization, Calcite, Immunoassay, 0303 health sciences, biology, MESH : Amino Acid Sequence, Immunogold labelling, MESH : Immunoassay, MESH: Calcium Carbonate, MESH : Crystallization, Crystallization, MESH: Immunoassay, Electrophoresis, Amino Acid Sequence, Animals, Bivalvia, Calcium Carbonate, Glycoproteins, Molecular Sequence Data, MESH: Glycoproteins, 010402 general chemistry, Biomaterials, 03 medical and health sciences, [SDV.IB.BIO]Life Sciences [q-bio]/Bioengineering/Biomaterials, Molecular Biology, 030304 developmental biology, Antiserum, MESH: Molecular Sequence Data, MESH : Electrophoresis, Cell Biology, Mussel, [ SDV.IB.BIO ] Life Sciences [q-bio]/Bioengineering/Biomaterials, biology.organism_classification, MESH : Glycoproteins, 0104 chemical sciences, [SDV.IB.BIO] Life Sciences [q-bio]/Bioengineering/Biomaterials, Calcium carbonate, chemistry, Polyclonal antibodies, biology.protein, Biomatériaux, MESH : Animals, Pinna nobilis, Cysteine
Abstract

We used the combination of preparative electrophoresis and immunological detection to isolate two new proteins from the shell calcitic prisms of Pinna nobilis, the Mediterranean fan mussel. The amino acid composition of these proteins was determined. Both proteins are soluble, intracrystalline, and acidic. The 38-kDa protein is glycosylated; the 17-kDa one is not. Ala, Asx, Thr, and Pro represent the dominant residues of the 38-kDa protein, named calprismin. An N-terminal sequence was obtained from calprismin. This sequence, which comprises a pattern of 4 cysteine residues, is not related to any known protein. The second protein, named caspartin, exhibits an unusual amino acid composition, since Asx constitutes by far the main amino acid residue. Preliminary sequencing surprisingly suggests that the first 75 N-terminal residues are all Asp. Caspartin self-aggregates spontaneously into multimers. In vitro tests show that it inhibits the precipitation of calcium carbonate. Furthermore, it strongly interferes with the growth of calcite crystals. A polyclonal antiserum raised against caspartin was used to localize this protein in the shell by immunogold. The immunolocalization demonstrates that caspartin is distributed within the prisms and makes a continuous film at the interface between the prisms and the surrounding insoluble sheets. Our finding emphasizes the prominent role of aspartic acid-rich proteins for the building of calcitic prisms among molluscs.

Published
2005

47. Factorizing selectivity determinants of inhibitor binding toward aldose and aldehyde reductases: structural and thermodynamic properties of the aldose reductase mutant Leu300Pro-fidarestat complex

Author

Holger Steuber, Andrzej Joachimiak, Alberto Podjarny, Ossama El-Kabbani, Mitsuru Oka, Isabelle Hazemann, Gerhard Klebe, Roland P.-T. Chung, Alexandra Cousido-Siah, Tatiana V. Petrova, Andre Mitschler, Institut de génétique et biologie moléculaire et cellulaire (IGBMC), Centre National de la Recherche Scientifique (CNRS)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Louis Pasteur - Strasbourg I, and Université Louis Pasteur - Strasbourg I-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)

Subjects
Models, Molecular, MESH: Mutation, Protein Conformation, Stereochemistry, Calorimetry, MESH: Holoenzymes, Imidazolidines, Fidarestat, MESH: Aldehyde Reductase, MESH: Protein Conformation, Aldehyde Reductase, Oxidoreductase, Drug Discovery, Humans, MESH: Protein Binding, Binding site, MESH: Calorimetry, MESH: Crystallization, chemistry.chemical_classification, Aldose reductase, Binding Sites, MESH: Humans, biology, Hydrogen bond, [SDV.BBM.BM]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Molecular biology, MESH: Imidazolidines, Aldose, chemistry, MESH: Binding Sites, Enzyme inhibitor, Mutation, biology.protein, Thermodynamics, Molecular Medicine, MESH: Thermodynamics, Crystallization, Holoenzymes, MESH: Models, Molecular, Protein Binding
Abstract

International audience; Structure of the Leu300Pro mutant of human aldose reductase (ALR2) in complex with the inhibitor fidarestat is determined. Comparison with the hALR2-fidarestat complex and the porcine aldehyde reductase (ALR1)-fidarestat complex indicates that the hydrogen bond between the Leu300 amino group of the wild-type and the exocyclic amide group of the inhibitor is the key determinant for the specificity of fidarestat for ALR2 over ALR1. Thermodynamic data also suggest an enthalpic contribution as the predominant difference in the binding energy between the aldose reductase mutant and the wild-type. An additional selectivity-determining feature is the difference in the interaction between the inhibitor and the side chain of Trp219, ordered in the present structure but disordered (corresponding Trp220) in the ALR1-fidarestat complex. Thus, the hydrogen bond ( approximately 7 kJ/mol) corresponds to a 23-fold difference in inhibitor potency while the differences in the interactions between Trp219(ALR2) and fidarestat and between Trp220(ALR1) and fidarestat can account for an additional 10-fold difference in potency.

Published
2005

48. Functional characterization and purification of a Saccharomyces cerevisiae ADP/ATP carrier-iso 1 cytochrome c fusion protein

Author

Gérard Brandolin, Cécile Dahout-Gonzalez, Emmanuel Philippe Dassa, Anne-Christine Dianoux, Membranes bactériennes, Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS), Physiopathologie, conséquences fonctionnelles et neuroprotection des atteintes du cerveau en développement, Université Paris Diderot - Paris 7 (UPD7)-IFR2-Institut National de la Santé et de la Recherche Médicale (INSERM), Génétique et biochimie des microorganismes (GBM), Biochimie et biophysique des systèmes intégrés (BBSI), Université Joseph Fourier - Grenoble 1 (UJF)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Centre National de la Recherche Scientifique (CNRS), Institut Laue-Langevin (ILL), Institut Pasteur [Paris]-Centre National de la Recherche Scientifique (CNRS), Centre National de la Recherche Scientifique (CNRS)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Joseph Fourier - Grenoble 1 (UJF), and ILL

Subjects
Mitochondrion, Atractyloside, MESH: Histidine, Adenosine Triphosphate, MESH: Saccharomyces cerevisiae Proteins, MESH: Adenosine Triphosphate, Cloning, Molecular, MESH: Crystallization, 0303 health sciences, Chromatography, biology, MESH: Kinetics, MESH: Escherichia coli, Cytochrome c, 030302 biochemistry & molecular biology, MESH: Molecular Probes, Cytochromes c, MESH: Cytochromes c, MESH: Saccharomyces cerevisiae, Adenosine Diphosphate, Biochemistry, MESH: Heme, MESH: Genetic Engineering, ATP–ADP translocase, MESH: Mitochondrial ADP, ATP Translocases, Crystallization, Genetic Engineering, Biotechnology, Saccharomyces cerevisiae Proteins, Recombinant Fusion Proteins, Saccharomyces cerevisiae, Heme, MESH: Chromatography, 03 medical and health sciences, Affinity chromatography, Escherichia coli, MESH: Recombinant Fusion Proteins, Histidine, MESH: Cloning, Molecular, [SDV.BBM.BC]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biochemistry [q-bio.BM], 030304 developmental biology, ATP transport, MESH: Adenosine Diphosphate, biology.organism_classification, Fusion protein, Yeast, Kinetics, MESH: Atractyloside, Molecular Probes, biology.protein, Mitochondrial ADP, ATP Translocases
Abstract

International audience; A recombinant fusion protein combining the mitochondrial ADP/ATP carrier (Anc2p) and the iso-1-cytochrome c (Cyc1p), both from Saccharomyces cerevisiae, has been genetically elaborated with the aim of increasing the polar surface area of the carrier to facilitate its crystallization. The gene encoding the his-tagged fusion protein was expressed in yeast under the control of the regulatory sequences of ScANC2. The chimeric carrier, Anc2-Cyc1(His6)p, was able to restore growth on a non-fermentable carbon source of a yeast strain devoid of functional ADP/ATP carrier, which demonstrated its transport activity. The kinetic exchange properties of Anc2-Cyc1(His6)p and the wild type his-tagged carrier Anc2(His6)p were very similar. However, Anc2-Cyc1(His6)p restored cell growth less efficiently than Anc2(His6)p which correlates with the lower amount found in mitochondria. Purification of Anc2-Cyc1(His6)p in complex with carboxyatractyloside (CATR), a high affinity inhibitor of ADP/ATP transport, was achieved by combining ion-exchange chromatography and ion-metal affinity chromatography in the presence of LAPAO, an aminoxide detergent. As characterized by absorption in the visible range, heme was found to be present in isolated Anc2-Cyc1(His6)p, giving the protein a red color. Large-scale purification of Anc2-Cyc1(His6)p-CATR complex opens up novel possibilities for the use of crystallographic approaches to the yeast ADP/ATP carrier.

Published
2005

49. Translational operator of mRNA on the ribosome: how repressor proteins exclude ribosome binding

Author

Bernard Ehresmann, Gulnara Yusupova, B. Rees, Mathias Springer, Pascale Romby, Lasse Jenner, Marat Yusupov, Dino Moras, Chantal Ehresmann, Clemens Schulze-Briese, Institut de génétique et biologie moléculaire et cellulaire (IGBMC), Université Louis Pasteur - Strasbourg I-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Architecture et réactivité de l'ARN (ARN), and Université Louis Pasteur - Strasbourg I-Centre National de la Recherche Scientifique (CNRS)

Subjects
Models, Molecular, MESH: Thermus thermophilus, Crystallography, X-Ray, RNA, Ribosomal, 16S, 30S, Ribosome profiling, Base Pairing, MESH: Bacterial Proteins, MESH: Crystallization, MESH: Threonine-tRNA Ligase, 0303 health sciences, Multidisciplinary, Fourier Analysis, 030302 biochemistry & molecular biology, Translation (biology), MESH: Ribosomal Proteins, Cell biology, MESH: RNA, Ribosomal, 16S, RNA, Bacterial, MESH: Nucleic Acid Conformation, MESH: Repressor Proteins, MESH: Protein Biosynthesis, T arm, Crystallization, Eukaryotic Ribosome, MESH: RNA, Bacterial, MESH: Models, Molecular, Ribosomal Proteins, RNA, Transfer, Met, MESH: Base Pairing, Regulatory Sequences, Ribonucleic Acid, Biology, 03 medical and health sciences, Bacterial Proteins, Threonine-tRNA Ligase, RNA, Messenger, 030304 developmental biology, MESH: RNA, Messenger, Binding Sites, Thermus thermophilus, MESH: RNA, Transfer, Met, MESH: Regulatory Sequences, Ribonucleic Acid, [SDV.BBM.BM]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Molecular biology, MESH: Crystallography, X-Ray, Molecular biology, Ribosomal binding site, Repressor Proteins, A-site, Internal ribosome entry site, MESH: Binding Sites, Protein Biosynthesis, Nucleic Acid Conformation, Ribosomes, MESH: Ribosomes, MESH: Fourier Analysis
Abstract

The ribosome of Thermus thermophilus was cocrystallized with initiator transfer RNA (tRNA) and a structured messenger RNA (mRNA) carrying a translational operator. The path of the mRNA was defined at 5.5 angstroms resolution by comparing it with either the crystal structure of the same ribosomal complex lacking mRNA or with an unstructured mRNA. A precise ribosomal environment positions the operator stem-loop structure perpendicular to the surface of the ribosome on the platform of the 30 S subunit. The binding of the operator and of the initiator tRNA occurs on the ribosome with an unoccupied tRNA exit site, which is expected for an initiation complex. The positioning of the regulatory domain of the operator relative to the ribosome elucidates the molecular mechanism by which the bound repressor switches off translation. Our data suggest a general way in which mRNA control elements must be placed on the ribosome to perform their regulatory task.

Published
2005

50. Structural genomics of eukaryotic targets at a laboratory scale

Author

David Rosé, Raymond Ripp, Didier Busso, Jean-Claude Thierry, Pierre Poussin-Courmontagne, Dino Moras, Alain Litt, Institut de génétique et biologie moléculaire et cellulaire (IGBMC), Centre National de la Recherche Scientifique (CNRS)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Louis Pasteur - Strasbourg I, and Université Louis Pasteur - Strasbourg I-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)

Subjects
Proteomics, 030303 biophysics, Nanotechnology, Genomics, Biology, Laboratory scale, Biochemistry, Protein expression, Structural genomics, 03 medical and health sciences, Human health, MESH: Gene Expression Profiling, Structural Biology, Strategic investment, Genetics, MESH: Proteins, 030304 developmental biology, MESH: Crystallization, 0303 health sciences, Gene Expression Profiling, MESH: Proteomics, Proteins, [SDV.BBM.BM]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Molecular biology, General Medicine, Data science, Structural biology, MESH: Laboratories, Crystallization, Laboratories, Protein Structure Initiative
Abstract

International audience; Structural genomics programs are distributed worldwide and funded by large institutions such as the NIH in United-States, the RIKEN in Japan or the European Commission through the SPINE network in Europe. Such initiatives, essentially managed by large consortia, led to technology and method developments at the different steps required to produce biological samples compatible with structural studies. Besides specific applications, method developments resulted mainly upon miniaturization and parallelization. The challenge that academic laboratories faces to pursue structural genomics programs is to produce, at a higher rate, protein samples. The Structural Biology and Genomics Department (IGBMC - Illkirch - France) is implicated in a structural genomics program of high eukaryotes whose goal is solving crystal structures of proteins and their complexes (including large complexes) related to human health and biotechnology. To achieve such a challenging goal, the Department has established a medium-throughput pipeline for producing protein samples suitable for structural biology studies. Here, we describe the setting up of our initiative from cloning to crystallization and we demonstrate that structural genomics may be manageable by academic laboratories by strategic investments in robotic and by adapting classical bench protocols and new developments, in particular in the field of protein expression, to parallelization.

Published
2005

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