Author: "Martin Weik" / Topic: chemistry - Searchworks@Jio Institute Digital Library Search Results

Your search keyword '"Martin Weik"' showing total 77 results

Start Over Author "Martin Weik" Topic chemistry

77 results on '"Martin Weik"'

1. Mutations in Tau Protein Promote Aggregation by Favoring Extended Conformations

Author: Kevin Pounot, Clara Piersson, Andrew K. Goring, Frédéric Rosu, Valérie Gabelica, Martin Weik, Songi Han, and Yann Fichou
Subjects: Chemistry, QD1-999
Published: 2023
Full Text: View/download PDF

2. Behavior of Hydrated Lipid Bilayers at Cryogenic Temperatures

Author: Jakob Meineke, Martin Weik, Giuseppe Zaccai, and Giovanna Fragneto
Subjects: flash cooling, lipids, stacked bilayers, neutrons, diffraction, hydration water, Chemistry, QD1-999
Abstract: Neutron diffraction was used to study the behavior of water present in phospholipid multilamellar stacks from 1,2-dimyristoyl-sn-glycero-3-phosphatidylcholine (DMPC) at cryogenic temperatures. Evidence was found for the existence of a highly viscous phase of water that exists between 180 and 220 K based on the observation that water can leave the intermembrane space at these low temperatures. Similar measurements are described in the literature for purple membrane (PM) samples. From a comparison with results from this natural membrane by using the same flash-cooling protocol, it is found that in the case of pure lipid samples, less water is trapped and the water flows out at lower temperatures. This suggests that the water is less hindered in its movements than in the PM case. It is shown that at least the Lβ′-phase of DMPC can be trapped likely by flash cooling; upon heating to about 260 K, it transforms to another phase that was not fully characterized.
Published: 2020
Full Text: View/download PDF

3. Rational control of off‐state heterogeneity in a photoswitchable fluorescent protein provides switching contrast enhancement

Author: Angela Mantovanelli, A. Gorel, Dominique Bourgeois, M. Hilpert, Gabriela Nass Kovacs, Mark S. Hunter, Sébastien Boutet, Ninon Zala, Kiyoshi Ueda, Jason E. Koglin, Andrew Aquila, Michel Sliwa, Ilme Schlichting, M. Stricker, Stefan Jakobs, Kensuke Tono, Martin Weik, Virgile Adam, Anne-Sophie Banneville, Lucas Martinez Uriarte, T. Domratcheva, Michel Thépaut, Mengning Liang, Oleksandr Glushonkov, Martin Byrdin, Giorgio Schirò, Marie Luise Grünbein, Robert L. Shoeman, Kyprianos Hadjidemetriou, Thomas R. M. Barends, Sergio Carbajo, Nina Eleni Christou, Thomas J. Lane, Victor Bezchastnov, Daehyun You, Tadeo Moreno Chicano, Lutz Foucar, Marco Cammarata, Nickels A. Jensen, C.M. Roome, Jacques-Philippe Colletier, Marco Kloos, Franck Fieschi, Eugenio de la Mora, Mariam El Khatib, Nicolas Coquelle, Shigeki Owada, Matthew Seaberg, R. Bruce Doak, Karol Nass, Joyce Woodhouse, 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 NanoBiophotonics [Göttingen], Max Planck Institute for Biophysical Chemistry (MPI-BPC), Max-Planck-Gesellschaft-Max-Planck-Gesellschaft, Max Planck Institute for Medical Research [Heidelberg], Max-Planck-Gesellschaft, Linac Coherent Light Source (LCLS), SLAC National Accelerator Laboratory (SLAC), Stanford University-Stanford University, Max-Planck-Institut für Medizinische Forschung, Institut de Physique de Rennes (IPR), Université de Rennes (UR)-Centre National de la Recherche Scientifique (CNRS), RIKEN SPring-8 Center [Hyogo] (RIKEN RSC), RIKEN - Institute of Physical and Chemical Research [Japon] (RIKEN), Japan Synchrotron Radiation Research Institute [Hyogo] (JASRI), Tohoku University [Sendai], Laboratoire Avancé de Spectroscopie pour les Intéractions la Réactivité et l'Environnement - UMR 8516 (LASIRE), Institut de Chimie du CNRS (INC)-Université de Lille-Centre National de la Recherche Scientifique (CNRS), Department of Chemistry, Lomonosov Moscow State University, Lomonosov Moscow State University (MSU), The XFEL experiments were carried out at BL2-EH3 of SACLA with the approval of the Japan Synchrotron Radiation Research Institute (JASRI, Proposal No. 2018 A8026, 27–29 July 2018) and at the CXI beamline at the LCLS (Proposal No. LM47 (23–27 June 2016) and LR38 (22–26 February 2018). We warmly thank the SACLA and LCLS staff for assistance. Use of the LCLS, SLAC National Accelerator Laboratory, is supported by the U.S. Department of Energy, Office of Science, Office of Basic Energy Sciences under Contract no. DE-AC02-76SF00515. Part of the sample injector used at LCLS for this research was funded by the National Institutes of Health, P41 GM103393, formerly P41RR001209. We acknowledge support from the Max Planck Society. The study was supported by travel grants from the CNRS (GoToXFEL) to MW, an ANR grant to MW, MC, MSl (BioXFEL), a PhD fellowship from Lille University to LMU and an MENESR – Univ. Grenoble Alpes fellowship to KH. This work was partially carried out at the platforms of the Grenoble Instruct-ERIC center (IBS and ISBG, UMS 3518 CNRS-CEA-UGA-EMBL) within the Grenoble Partnership for Structural Biology (PSB)., ANR-17-CE11-0047,Cryo-PALM,Microscopie super-résolution par localisation de molécules uniques à température cryogénique.(2017), ANR-10-INBS-0005,FRISBI,Infrastructure Française pour la Biologie Structurale Intégrée(2010), ANR-17-EURE-0003,CBH-EUR-GS,CBH-EUR-GS(2017), Université de Rennes 1 (UR1), Université de Rennes (UNIV-RENNES)-Université de Rennes (UNIV-RENNES)-Centre National de la Recherche Scientifique (CNRS), Laboratoire Physico-Chimie Curie [Institut Curie] (PCC), Institut Curie [Paris]-Institut de Chimie du CNRS (INC)-Sorbonne Université (SU)-Centre National de la Recherche Scientifique (CNRS), and Publica
Subjects: serial femtosecond crystallography, [SDV]Life Sciences [q-bio], RESOLFT, Green Fluorescent Proteins, nanoscopy, Atomic, quantum chemistry, Particle and Plasma Physics, Theoretical and Computational Chemistry, Microscopy, Side chain, switching contrast, Escherichia coli, Nuclear, Physical and Theoretical Chemistry, [PHYS]Physics [physics], Chemical Physics, [SDV.BBM.BS]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Structural Biology [q-bio.BM], Chemistry, photoswitchable fluorescent proteins, Resolution (electron density), Molecular, Chromophore, Fluorescence, Atomic and Molecular Physics, and Optics, Luminescent Proteins, Femtosecond, Biophysics, Generic health relevance, Biological imaging, Physical Chemistry (incl. Structural)
Abstract: Reversibly photoswitchable fluorescent proteins are essential markers for advanced biological imaging, and optimization of their photophysical properties underlies improved performance and novel applications. Here we establish a link between photoswitching contrast, a key parameter that largely dictates the achievable resolution in nanoscopy applications, and chromophore conformation in the non-fluorescent state of rsEGFP2, a widely employed label in REversible Saturable OpticaL Fluorescence Transitions (RESOLFT) microscopy. Upon illumination, the cis chromophore of rsEGFP2 isomerizes to two distinct off-state conformations, trans1 and trans2, located on either side of the V151 side chain. Reducing or enlarging the side chain at this position (V151A and V151L variants) leads to single off-state conformations that exhibit higher and lower switching contrast, respectively, compared to the rsEGFP2 parent. The combination of structural information obtained by serial femtosecond crystallography with high-level quantum chemical calculations and with spectroscopic and photophysical data determined in vitro suggests that the changes in switching contrast arise from blue- and red-shifts of the absorption bands associated to trans1 and trans2, respectively. Thus, due to elimination of trans2, the V151A variants of rsEGFP2 and its superfolding variant rsFolder2 display a more than two-fold higher switching contrast than their respective parent proteins, both in vitro and in E. coli cells. The application of the rsFolder2-V151A variant is demonstrated in RESOLFT nanoscopy. Our study rationalizes the connection between structural and photophysical chromophore properties and suggests a means to rationally improve fluorescent proteins for nanoscopy applications.
Published: 2022

4. Zinc determines dynamical properties and aggregation kinetics of human insulin

Author: Geoffrey W. Grime, Daria Noferini, Tilo Seydel, Kevin Pounot, Alessandro Longo, Martin Weik, Vito Foderà, Michaela Zamponi, Viviana Cristiglio, Elspeth F. Garman, Giorgio Schirò, Applied Physics, University of Tübingen, University of Surrey Ion Beam Centre, Istituto per lo Studio dei Materiali Nanostrutturati, Palermo, Consiglio Nazionale delle Ricerche [Roma] (CNR), Jülich Centre for Neutron Science (JCNS), Institut Laue-Langevin (ILL), ILL, Biochemistry, University of Oxford [Oxford], Institut de biologie structurale (IBS - UMR 5075), Centre National de la Recherche Scientifique (CNRS)-Université Grenoble Alpes (UGA)-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), Pharmacy, IT University of Copenhagen, University of Surrey (UNIS), National Research Council of Italy | Consiglio Nazionale delle Ricerche (CNR), University of Oxford, Centre National de la Recherche Scientifique (CNRS)-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), and IT University of Copenhagen (ITU)
Subjects: Amyloid, [SDV]Life Sciences [q-bio], medicine.medical_treatment, Biophysics, chemistry.chemical_element, Zinc, Protein aggregation, 010402 general chemistry, Fibril, 01 natural sciences, 03 medical and health sciences, 0302 clinical medicine, ddc:570, medicine, Humans, Insulin, Binding site, 030304 developmental biology, [PHYS]Physics [physics], 0303 health sciences, Amyloidosis, Biomaterial, Articles, medicine.disease, 0104 chemical sciences, Kinetics, X-Ray Absorption Spectroscopy, chemistry, 030217 neurology & neurosurgery
Abstract: Protein aggregation is a widespread process leading to deleterious consequences in the organism, with amyloid aggregates being important not only in biology but also for drug design and biomaterial production. Insulin is a protein largely used in diabetes treatment and its amyloid aggregation is at the basis of the so-called insulin-derived amyloidosis. Here we uncover the major role of zinc in both insulin dynamics and aggregation kinetics at low pH, where the formation of different amyloid superstructures (fibrils and spherulites) can be thermally induced. Amyloid aggregation is accompanied by zinc release and the suppression of water-sustained insulin dynamics, as shown by particle-induced X-ray emission and X-ray absorption spectroscopy and by neutron spectroscopy, respectively. Our study shows that zinc binding stabilizes the native form of insulin by facilitating hydration of this hydrophobic protein and suggests that introducing new binding sites for zinc can improve insulin stability and tune its aggregation propensity.Statement of SignificanceLocalized amyloidosis occurs at insulin injection sites for diabetes treatment, leading to deleterious inflammations known as insulin-derived amyloidosis. Amyloid superstructures are also promising candidates in the field of biomaterials. Here we revealed that zinc, coordinated to insulin in the native form, is released upon amyloid aggregation, when insulin forms superstructures known as fibrils and spherulites. Zinc release leads to a full suppression of functionally essential protein dynamics through a modification of the protein’s hydration properties and completely modifies insulin amyloid kinetics. The results suggest that changes in protein hydration upon zinc binding/release modifies both stability and dynamics of insulin and might then be a general strategy to control protein stability and tune protein aggregation into amorphous and ordered superstructures.
Published: 2021

5. Rivastigmine and metabolite analogues with putative Alzheimer’s disease-modifying properties in a Caenorhabditis elegans model

Author: Gawain McColl, Stephen Chan, Marie-Odile Parat, Suresh Kumar Veliyath, Zeyad D. Nassar, Xavier Brazzolotto, Israel Silman, Joel L. Sussman, Eugenio de la Mora, Satish N. Dighe, Jared A. Miles, B. Yogi Sreenivas, Martin Weik, Ross P. McGeary, Benjamin P. Ross, and Srinivas Kantham
Subjects: Drug, Rivastigmine, biology, Tertiary amine, Membrane permeability, Chemistry, Amyloid beta, media_common.quotation_subject, Metabolite, food and beverages, General Chemistry, Pharmacology, Druglikeness, Biochemistry, lcsh:Chemistry, chemistry.chemical_compound, lcsh:QD1-999, In vivo, Materials Chemistry, biology.protein, medicine, Environmental Chemistry, media_common, medicine.drug
Abstract: The development of polyphenols as drugs for Alzheimer’s disease (AD) is thwarted by their meagre brain availability due to instability and poor druglikeness. Here we describe the successful development of stable, druglike polyphenolic analogues of the current AD drug rivastigmine, that have high apparent blood-brain barrier permeabilities and multifunctional properties for AD treatment. The compounds inhibit cholinesterases and amyloid beta (Aβ) fibrillation, protect against Aβ42-induced toxicity in vitro, and demonstrate efficacy in vivo in a transgenic Caenorhabditis elegans model expressing Aβ42, with potencies similar to rivastigmine and natural polyphenols. The results suggest that a tertiary amine substituent is amenable for developing water-soluble, membrane-permeable polyphenols, and its incorporation adjacent to a hydroxy group is favourable for intramolecular hydrogen bonding that facilitates membrane permeability. Carbamylation of one hydroxy group protects the polyphenols from degradation and mostly improves their membrane permeability. These design strategies may assist in the development of polyphenol-based drugs. Polyphenols are widely studied as potential drugs for Alzheimer’s disease, but their development is limited by poor bioavailability. Here polyphenolic analogues of the Alzheimer's disease drug rivastigmine are shown to inhibit both cholinesterase and amyloid beta fibrillation in vitro and in vivo in a C. elegans model, providing a potentially general route for the development of polyphenol-based drugs.
Published: 2019

6. Mechanism and dynamics of fatty acid photodecarboxylase

Author: Sébastien Boutet, Catherine Berthomieu, Laura Antonucci, A. Gorel, Manuel Joffre, Marco Cammarata, A. Benachir, Sergio Carbajo, Solène L. Y. Moulin, Martin Weik, Michel Sliwa, Stephane Cuine, Yonghua Li-Beisson, Nicolas Coquelle, Didier Nurizzo, P. Samire, Jacques-Philippe Colletier, Alexey Aleksandrov, Robert L. Shoeman, Guillaume Gotthard, Antoine Royant, Marten H. Vos, Bo Zhuang, M. Hilpert, Adeline Bonvalet, Ilme Schlichting, Xavier Solinas, Martin Byrdin, Pascal Arnoux, Gilles Peltier, Pierre Legrand, F. Beisson, Klaus Brettel, R. Hienerwadel, Thomas R. M. Barends, R.B. Doak, Lutz Foucar, T. Domratcheva, Damien Sorigué, Marco Kloos, Stéphanie Blangy, Giorgio Schirò, Kyprianos Hadjidemetriou, Bertrand Légeret, Thomas J. Lane, Marie Luise Grünbein, Pavel Müller, Elisabeth Hartmann, Environnement, Bioénergie, Microalgues et Plantes (EBMP), Institut de Biosciences et Biotechnologies d'Aix-Marseille (ex-IBEB) (BIAM), 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)-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 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), European Synchroton Radiation Facility [Grenoble] (ESRF), Laboratoire d'Optique et Biosciences (LOB), École polytechnique (X)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Institut Laue-Langevin (ILL), Institut de Biologie Intégrative de la Cellule (I2BC), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS), SLAC National Accelerator Laboratory (SLAC), Stanford University, Institut de Physique de Rennes (IPR), Université de Rennes (UR)-Centre National de la Recherche Scientifique (CNRS), Max-Planck-Institut für Medizinische Forschung, Max-Planck-Gesellschaft, Luminy Génétique et Biophysique des Plantes (LGBP), Synchrotron SOLEIL (SSOLEIL), Centre National de la Recherche Scientifique (CNRS), Laboratoire Avancé de Spectroscopie pour les Intéractions la Réactivité et l'Environnement - UMR 8516 (LASIRE), Institut de Chimie du CNRS (INC)-Université de Lille-Centre National de la Recherche Scientifique (CNRS), Interactions Protéine Métal (IPM), Department of Chemistry, Lomonosov Moscow State University, Lomonosov Moscow State University (MSU), Microbiologie Environnementale et Moléculaire (MEM), STepLADDER (724362), European Research Council, 724362, European Research Council, SNAPsHOTs, Agence Nationale de la Recherche, Photoalkane, Agence Nationale de la Recherche, SignalBioNRJ, Agence Nationale de la Recherche, BioXFEL, Agence Nationale de la Recherche, Ministère de l’Education Nationale, de l’Enseignement Supérieur et de la Recherche, ANR-10-INBS-0005,FRISBI,Infrastructure Française pour la Biologie Structurale Intégrée(2010), ANR-18-CE11-0021,SNAPsHOTs,Dynamique structurale de l'acide gras photodécarboxylase(2018), ANR-18-CE43-0008,PHOTOALKANE,Production biosourcée d'hydrocarbures basée sur une nouvelle photoenzyme(2018), ANR-15-CE32-0004,BioXFEL,Caractérisation d'états intermédiaires de protéines fluorescentes en utilisant des lasers à électrons libres X et les spectroscopies UV-visible et infrarouge ultra-rapides(2015), European Project: 724362,STePLADDER - H2020-EU.1.1., Bioénergie et Microalgues (EBM), 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), ILL, Université de Rennes 1 (UR1), Université de Rennes (UNIV-RENNES)-Université de Rennes (UNIV-RENNES)-Centre National de la Recherche Scientifique (CNRS), Institut de Chimie du CNRS (INC)-Université de Lille-Centre National de la Recherche Scientifique (CNRS)-Centrale Lille Institut (CLIL), Vos, Marten, Infrastructure Française pour la Biologie Structurale Intégrée - - FRISBI2010 - ANR-10-INBS-0005 - INBS - VALID, APPEL À PROJETS GÉNÉRIQUE 2018 - Dynamique structurale de l'acide gras photodécarboxylase - - SNAPsHOTs2018 - ANR-18-CE11-0021 - AAPG2018 - VALID, APPEL À PROJETS GÉNÉRIQUE 2018 - Production biosourcée d'hydrocarbures basée sur une nouvelle photoenzyme - - PHOTOALKANE2018 - ANR-18-CE43-0008 - AAPG2018 - VALID, Caractérisation d'états intermédiaires de protéines fluorescentes en utilisant des lasers à électrons libres X et les spectroscopies UV-visible et infrarouge ultra-rapides - - BioXFEL2015 - ANR-15-CE32-0004 - AAPG2015 - VALID, and Solving The Pathway of LADDERane biosynthesis - STePLADDER - H2020-EU.1.1. - 724362 - INCOMING
Subjects: Models, Molecular, Light, Carboxy-Lyases, Protein Conformation, Decarboxylation, Chlorella, Reaction intermediate, Flavin group, Crystallography, X-Ray, 010402 general chemistry, Photochemistry, 01 natural sciences, [PHYS] Physics [physics], Electron Transport, 03 medical and health sciences, Catalytic Domain, Alkanes, [SDV.BBM] Life Sciences [q-bio]/Biochemistry, Molecular Biology, [SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology, Amino Acids, Alkyl, 030304 developmental biology, chemistry.chemical_classification, [PHYS]Physics [physics], Photons, 0303 health sciences, Multidisciplinary, Algal Proteins, Fatty Acids, Temperature, Fatty acid, Substrate (chemistry), Hydrogen Bonding, Carbon Dioxide, Chromophore, Electron transport chain, 0104 chemical sciences, Bicarbonates, Amino Acid Substitution, chemistry, 13. Climate action, Biocatalysis, Flavin-Adenine Dinucleotide, Mutant Proteins, Oxidation-Reduction
Abstract: Light makes light work of fatty acids Photosynthetic organisms are notable for their ability to capture light energy and use it to power biosynthesis. Some algae have gone a step beyond photosynthesis and can use light to initiate enzymatic photodecarboxylation of fatty acids, producing long-chain hydrocarbons. To understand this transformation, Sorigué et al. brought to bear an array of structural, computational, and spectroscopic techniques and fully characterized the catalytic cycle of the enzyme. These experiments are consistent with a mechanism starting with electron transfer from the fatty acid to a photoexcited oxidized flavin cofactor. Decarboxylation yields an alkyl radical, which is then reduced by back electron transfer and protonation rather than hydrogen atom transfer. The wealth of experimental data explains how algae harness light energy to produce alka(e)nes and provides an appealing model system for understanding enzyme-catalyzed photochemistry more generally. Science , this issue p. eabd5687
Published: 2021

7. Tracking Internal and Global Diffusive Dynamics During Protein Aggregation by High-Resolution Neutron Spectroscopy

Author: Kevin Pounot, Tilo Seydel, Hussein Chaaban, Giorgio Schirò, Martin Weik, Vito Foderà, 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), Institut Max von Laue, Department of Pharmacy, University of Copenhagen = Københavns Universitet (KU), IT University of Copenhagen, University of Copenhagen = Københavns Universitet (UCPH), IT University of Copenhagen (ITU), and ANR-17-EURE-0003,CBH-EUR-GS,CBH-EUR-GS(2017)
Subjects: Models, Molecular, Protein Conformation, Kinetics, Beta sheet, 02 engineering and technology, Protein aggregation, Diffusion, 03 medical and health sciences, Protein Aggregates, General Materials Science, Physical and Theoretical Chemistry, Spectroscopy, 030304 developmental biology, Neutrons, 0303 health sciences, [SDV.BBM.BS]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Structural Biology [q-bio.BM], Chemistry, Spectrum Analysis, 021001 nanoscience & nanotechnology, Neutron spectroscopy, Isoelectric point, Neutron backscattering, Biophysics, Muramidase, 0210 nano-technology, Superstructure (condensed matter)
Abstract: International audience; Proteins can misfold and form either amorphous or organized aggregates with different morphologies and features. Aggregates of amyloid nature are pathological hallmarks in so-called protein conformational diseases, including Alzheimer's and Parkinson's. Evidence prevails that the transient early phases of the reaction determine the aggregate morphology and toxicity. As a consequence, real-time monitoring of protein aggregation is of utmost importance. Here, we employed time-resolved neutron backscattering spectroscopy to follow center-of-mass self-diffusion and nano- to picosecond internal dynamics of lysozyme during aggregation into a specific β-sheet rich superstructure, called particulates, formed at the isoelectric point of the protein. Particulate formation is found to be a one-step process, and protein internal dynamics, to remain unchanged during the entire aggregation process. The time-resolved neutron backscattering spectroscopy approach developed here, in combination with standard kinetics assays, provides a unifying framework in which dynamics and conformational transitions can be related to the different aggregation pathways.
Published: 2020

8. Serial femtosecond crystallography on in vivo-grown crystals drives elucidation of mosquitocidal Cyt1Aa bioactivation cascade

Author: Martin Rosenthal, Maria Bacia, Maria Teresa Fernandez-Luna, Luca Signor, Guillaume Tetreau, Jayesh Arun Bafna, Enrico Gratton, Anne-Sophie Banneville, Iris D. Young, Mathias Winterhalter, Frederic Laporte, Aaron S. Brewster, Raymond G. Sierra, Manfred Burghammer, Mark S. Hunter, Sébastien Boutet, Elisabetta Boeri-Erba, Elena A. Andreeva, Ninon Zala, Jacques-Philippe Colletier, Tilman A. Grünewald, Irina Gutsche, Nicolas Coquelle, Niamh Burke, Duilio Cascio, Laurence Després, Irina Snigireva, Daphna Fenel, Alister Burt, Jean-Marie Teulon, Rabia Sadir, Nicholas K. Sauter, Jean-Luc Pellequer, Martin Weik, Brian A. Federici, Joël Beaudouin, Hyun-Woo Park, Michael R. Sawaya, Groupe Dynamique et Cinétique des processus moléculaires (IBS-DYNAMOP), 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)-Centre National de la Recherche Scientifique (CNRS)-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), Lawrence Berkeley National Laboratory [Berkeley] (LBNL), Linac Coherent Light Source (LCLS), SLAC National Accelerator Laboratory (SLAC), Stanford University-Stanford University, European Synchrotron Radiation Facility (ESRF), Department of Entomology [Riverside], University of California [Riverside] (UCR), University of California-University of California, California Baptist University (CBU), Jacobs University [Bremen], Université Grenoble Alpes [2016-2019] (UGA [2016-2019]), Institut Laue-Langevin (ILL), ILL, UCLA/DOE Institute for Genomics and Proteomics, University of California [Los Angeles] (UCLA), Laboratory for Fluorescence Dynamics [Irvine], University of California [Irvine] (UCI), AFM platform, Grenoble Instruct-ERIC center (ISBG, UMS 3518 CNRS-CEA-UGAEMBL), ANR-17-CE11-0018,X-in-vivo,Cristallisation in vivo: une nouvelle stratégie pour étudier la structure et la dynamique des protéines dans les lasers à électrons libres et les synchrotrons.(2017), ANR-18-CE11-0005,DynOCP,Investigation du mécanisme de photo-activation de l'OCP par spectroscopie optique ultrarapide et cristallographie sérielle temps-résolue.(2018), ANR-10-INBS-0005,FRISBI,Infrastructure Française pour la Biologie Structurale Intégrée(2010), ANR-17-EURE-0003,CBH-EUR-GS,CBH-EUR-GS(2017), 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), Linac Coherent Light Source (LCLS), SLAC National Accelerator Laboratory, Université Grenoble Alpes (UGA), University of California [Riverside] (UC Riverside), University of California (UC)-University of California (UC), and University of California [Irvine] (UC Irvine)
Subjects: 0301 basic medicine, Insecticides, [SDV.BIO]Life Sciences [q-bio]/Biotechnology, Protein Conformation, General Physics and Astronomy, Crystallography, X-Ray, Microscopy, Atomic Force, Atomic force microscopy, Hemolysin Proteins, Mice, 0302 clinical medicine, Sf9 Cells, Disulfides, lcsh:Science, ComputingMilieux_MISCELLANEOUS, Microscopy, Crystallography, Multidisciplinary, [SDV.BBM.BS]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Structural Biology [q-bio.BM], Nanocrystallography, Chemistry, Atomic Force, Limiting, Hydrogen-Ion Concentration, Bacillus thuringiensis israelensis, [SDV.BBM.BP]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biophysics, [SDV.BBM.BS]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biomolecules [q-bio.BM], Femtosecond, [SDV.TOX.ECO]Life Sciences [q-bio]/Toxicology/Ecotoxicology, Science, Mechanism of action, Article, General Biochemistry, Genetics and Molecular Biology, 03 medical and health sciences, Bacterial Proteins, In vivo, parasitic diseases, Animals, Humans, Bacillus thuringiensis Toxins, Cell Membrane, fungi, [SDV.BBM.BM]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Molecular biology, General Chemistry, Endotoxins, HEK293 Cells, 030104 developmental biology, Yield (chemistry), X-Ray, NIH 3T3 Cells, lcsh:Q, 030217 neurology & neurosurgery
Abstract: Cyt1Aa is the one of four crystalline protoxins produced by mosquitocidal bacterium Bacillus thuringiensis israelensis (Bti) that has been shown to delay the evolution of insect resistance in the field. Limiting our understanding of Bti efficacy and the path to improved toxicity and spectrum has been ignorance of how Cyt1Aa crystallizes in vivo and of its mechanism of toxicity. Here, we use serial femtosecond crystallography to determine the Cyt1Aa protoxin structure from sub-micron-sized crystals produced in Bti. Structures determined under various pH/redox conditions illuminate the role played by previously uncharacterized disulfide-bridge and domain-swapped interfaces from crystal formation in Bti to dissolution in the larval mosquito midgut. Biochemical, toxicological and biophysical methods enable the deconvolution of key steps in the Cyt1Aa bioactivation cascade. We additionally show that the size, shape, production yield, pH sensitivity and toxicity of Cyt1Aa crystals grown in Bti can be controlled by single atom substitution., Bacillus thuringiensis israelensis (Bti) produces the naturally-crystalline proteinaceous toxin Cyt1Aa that is toxic to mosquito larvae. Here the authors grow recombinant nanocrystals of the Cyt1Aa protoxin in vivo and use serial femtosecond crystallography to determine its structure at different redox and pH conditions and by combining their structural data with further biochemical, toxicological and biophysical analyses provide mechanistic insights into the Cyt1Aa bioactivation cascade.
Published: 2020

9. Photoswitching mechanism of a fluorescent protein revealed by time-resolved crystallography and transient absorption spectroscopy

Author: Virginia Guillon, C.M. Roome, Jacques-Philippe Colletier, Gabriela Nass Kovacs, Pauline Macheboeuf, Franck Fieschi, Marco Cammarata, Michel Thépaut, Michel Sliwa, Nicolas Coquelle, Eugenio de la Mora, Stefan Jakobs, Lucas Martinez Uriarte, Lutz Foucar, Shigeki Owada, Martin Byrdin, Robert L. Shoeman, Thomas R. M. Barends, Dominique Bourgeois, Koji Motomura, R. Bruce Doak, Kensuke Tono, Martin Weik, Virgile Adam, Martin J. Field, Makina Yabashi, Karol Nass, Ilme Schlichting, Joyce Woodhouse, Tadashi Togashi, Yasumasa Joti, Mikolaj Feliks, Cyril Ruckebusch, Giorgio Schirò, 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), Max-Planck-Institut für Medizinische Forschung, Max-Planck-Gesellschaft, Groupe Dynamique et Cinétique des processus moléculaires (IBS-DYNAMOP), 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)-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), DS/LSS Large Scale Structures group, Institut Laue-Langevin (ILL), ILL-ILL, Laboratoire Avancé de Spectroscopie pour les Intéractions la Réactivité et l'Environnement - UMR 8516 (LASIRE), Institut de Chimie du CNRS (INC)-Université de Lille-Centre National de la Recherche Scientifique (CNRS)-Centrale Lille Institut (CLIL), Department of Chemistry, University of Southern California, University of Southern California (USC), Groupe modélisation et chimie théorique (MCT ), Laboratoire de Chimie et Biologie des Métaux (LCBM - UMR 5249), Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS)-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)-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS)-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Groupe Membrane et pathogènes (IBS-MP), Department of NanoBiophotonics [Göttingen], Max Planck Institute for Biophysical Chemistry (MPI-BPC), Max-Planck-Gesellschaft-Max-Planck-Gesellschaft, Japan Synchrotron Radiation Research Institute [Hyogo] (JASRI), Groupe Pathogénie Bactérienne (IBS-PATBAC), Institute of Multidisciplinary Research for Advanced Materials, Tohoku University [Sendai], RIKEN SPring-8 Center [Hyogo] (RIKEN RSC), RIKEN - Institute of Physical and Chemical Research [Japon] (RIKEN), Institut de Physique de Rennes (IPR), Université de Rennes 1 (UR1), Université de Rennes (UNIV-RENNES)-Université de Rennes (UNIV-RENNES)-Centre National de la Recherche Scientifique (CNRS), Institut de Chimie du CNRS (INC)-Université de Lille-Centre National de la Recherche Scientifique (CNRS), and Université de Rennes (UR)-Centre National de la Recherche Scientifique (CNRS)
Subjects: 0301 basic medicine, Conformational change, Science, General Physics and Astronomy, Protonation, 010402 general chemistry, Photochemistry, 01 natural sciences, General Biochemistry, Genetics and Molecular Biology, Article, 03 medical and health sciences, Ultrafast laser spectroscopy, Spectroscopy, lcsh:Science, X-ray crystallography, [PHYS]Physics [physics], Multidisciplinary, [SDV.BBM.BS]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Structural Biology [q-bio.BM], Chemistry, Nanocrystallography, General Chemistry, Chromophore, Fluorescence, 0104 chemical sciences, 3. Good health, Time resolved crystallography, Photoexcitation, 030104 developmental biology, lcsh:Q, Structural biology
Abstract: Reversibly switchable fluorescent proteins (RSFPs) serve as markers in advanced fluorescence imaging. Photoswitching from a non-fluorescent off-state to a fluorescent on-state involves trans-to-cis chromophore isomerization and proton transfer. Whereas excited-state events on the ps timescale have been structurally characterized, conformational changes on slower timescales remain elusive. Here we describe the off-to-on photoswitching mechanism in the RSFP rsEGFP2 by using a combination of time-resolved serial crystallography at an X-ray free-electron laser and ns-resolved pump–probe UV-visible spectroscopy. Ten ns after photoexcitation, the crystal structure features a chromophore that isomerized from trans to cis but the surrounding pocket features conformational differences compared to the final on-state. Spectroscopy identifies the chromophore in this ground-state photo-intermediate as being protonated. Deprotonation then occurs on the μs timescale and correlates with a conformational change of the conserved neighbouring histidine. Together with a previous excited-state study, our data allow establishing a detailed mechanism of off-to-on photoswitching in rsEGFP2., rsEGFP2 is a reversibly photoswitchable fluorescent protein used in super-resolution light microscopy. Here the authors present the structure of an rsEGFP2 ground-state intermediate after excited state-decay that was obtained by nanosecond time-resolved serial femtosecond crystallography at an X-ray free electron laser, and time-resolved absorption spectroscopy measurements complement their structural analysis.
Published: 2020

10. Role of hydration water in the onset of protein structural dynamics

Author: Giorgio Schirò, Martin Weik, 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), and Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)
Subjects: 0303 health sciences, [SDV.BBM.BS]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Structural Biology [q-bio.BM], Chemistry, Functional protein, Protein dynamics, Dynamics (mechanics), Temperature, Proteins, Water, 010402 general chemistry, Condensed Matter Physics, 01 natural sciences, 0104 chemical sciences, 03 medical and health sciences, Water dynamics, Chemical physics, Water chemistry, General Materials Science, Mutual influence, 030304 developmental biology
Abstract: International audience; Proteins are the molecular workhorses in a living organism. Their 3D structures are animated by a multitude of equilibrium fluctuations and specific out-of-equilibrium motions that are required for proteins to be biologically active. When studied as a function of temperature, functionally relevant dynamics are observed at and above the so-called protein dynamical transition (~240 K) in hydrated, but not in dry proteins. In this review we present and discuss the main experimental and computational results that provided evidence for the dynamical transition, with a focus on the role of hydration water dynamics in sustaining functional protein dynamics. The coupling and mutual influence of hydration water dynamics and protein dynamics are discussed and the hypotheses illustrated that have been put forward to explain the physical origin of their onsets.
Published: 2019

11. Ligand pathways in neuroglobin revealed by low-temperature photodissociation and docking experiments

Author: S. Della Longa, Martin Weik, Thierry Prangé, Nathalie Colloc'h, Linda Celeste Montemiglio, Philippe Carpentier, C. Savino, Alessandro Arcovito, Cécile Exertier, Chiara Ardiccioni, Beatrice Vallone, Maurizio Brunori, P. van der Linden, G. Avella, Dominique Bourgeois, New York–Marche Structural Biology Center [Ancona, Italia] (NY-MaSBiC), Università Politecnica delle Marche [Ancona] (UNIVPM), Università cattolica del Sacro Cuore [Roma] (Unicatt), Fondazione Policlinico Universitario Agostino Gemelli, Università degli Studi dell'Aquila (UNIVAQ), European Synchrotron Radiation Facility (ESRF), Partnership for Soft Condensed Matter (PSCM), 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), Department of Biochemical Sciences 'Rossi Fanelli', Institut Pasteur, Fondation Cenci Bolognetti - Istituto Pasteur Italia, Fondazione Cenci Bolognetti, Réseau International des Instituts Pasteur (RIIP)-Réseau International des Instituts Pasteur (RIIP)-Università degli Studi di Roma 'La Sapienza' = Sapienza University [Rome], Institute of Molecular Pathology and Biology [Rome] (IPBM), Università degli Studi di Roma 'La Sapienza' = Sapienza University [Rome]-Consiglio Nazionale delle Ricerche (CNR), Biocatalyse (BIOCAT ), Laboratoire de Chimie et Biologie des Métaux (LCBM - UMR 5249), Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS)-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 de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS)-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]), Cibles Thérapeutiques et conception de médicaments (CiTCoM - UMR 8038), Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS)-Université Paris Descartes - Paris 5 (UPD5), Imagerie et Stratégies Thérapeutiques des pathologies Cérébrales et Tumorales (ISTCT), Université de Caen Normandie (UNICAEN), Normandie Université (NU)-Normandie Université (NU)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Centre National de la Recherche Scientifique (CNRS), This work received funding from the European Union's Horizon 2020 Innovative Training Network (ITN-ETN) program under the Marie Skłodowska-Curie X-probe project (Grant Agreement No. 637295 to BV)., We acknowledge the European Synchrotron Radiation Facility for provision of synchrotron radiation facilities and we would like to thank Michel Pirocchi and Jean-Luc Ferrer for assistance in using beamline BM30-A, and Hassan Behlrali and Babu Manjasetty for assistance in using beamline BM14. IBS acknowledges integration into the Interdisciplinary Research Institute of Grenoble (IRIG, CEA)., Thomas, Frank, Advanced XFEL and Synchrotron based Probes of Protein Structure and Dynamics - X-probe - - H20202015-01-01 - 2018-12-31 - 637295 - VALID, Università cattolica del Sacro Cuore = Catholic University of the Sacred Heart [Roma] (Unicatt), Fondazione Policlinico Universitario Agostino Gemelli IRCCS, Università degli Studi dell'Aquila = University of L'Aquila (UNIVAQ), European Synchroton Radiation Facility [Grenoble] (ESRF), Institut de biologie structurale (IBS - UMR 5075), Centre National de la Recherche Scientifique (CNRS)-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), Consiglio Nazionale delle Ricerche (CNR), Università degli Studi di Roma 'La Sapienza' = Sapienza University [Rome] (UNIROMA)-Consiglio Nazionale delle Ricerche (CNR), Réseau International des Instituts Pasteur (RIIP)-Réseau International des Instituts Pasteur (RIIP)-Università degli Studi di Roma 'La Sapienza' = Sapienza University [Rome] (UNIROMA), Biocatalyse (BIOCAT), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Grenoble Alpes (UGA)-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS)-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS)-Université Paris Cité (UPCité), Imagerie et Stratégies Thérapeutiques pour les Cancers et Tissus cérébraux (ISTCT), Normandie Université (NU)-Normandie Université (NU)-Centre National de la Recherche Scientifique (CNRS), the European Synchrotron Radiation Facilityry Research Institute of Grenoble (IRIG, CEA)., European Project: 637295,H2020,H2020-MSCA-ITN-2014,X-probe(2015), 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), National Research Council of Italy | Consiglio Nazionale delle Ricerche (CNR), CNR Istituto di Biologia e Patologia Molecolari [Roma] (CNR | IBPM), Institut de Chimie du CNRS (INC)-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)-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG)
Subjects: Hemeprotein, crystal microspectroscopy, [SDV.BBM.BS] Life Sciences [q-bio]/Biochemistry, Molecular Biology/Structural Biology [q-bio.BM], Biochemistry, cryo-trapping, 03 medical and health sciences, structural biology, General Materials Science, Globin, lcsh:Science, Settore BIO/10 - BIOCHIMICA, Structure determination, 030304 developmental biology, ultralow-temperature X-ray crystallography, 0303 health sciences, CO photolysis, Heme protein, [SDV.BBM.BS]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Structural Biology [q-bio.BM], Chemistry, soak-and-freeze pressurization, 030302 biochemistry & molecular biology, Photodissociation, Hexacoordinate, Oxygen transport, heme protein, neuroglobin, neuroprotection, oxygen binding, protein structure, structure determination, XANES, General Chemistry, Condensed Matter Physics, Research Papers, Neuroprotection, 3. Good health, Docking (molecular), Neuroglobin, Biophysics, Protein structure, lcsh:Q, Oxygen binding
Abstract: Ultralow-temperature X-ray crystallography, in crystallo microspectroscopy and X-ray absorption spectroscopy were used to study the carbon monoxide photodissociation intermediate in neuroglobin. Moreover, X-ray crystallography under high O2 pressure allowed the identification of a novel storage site for dioxygen in hexacoordinate ferric neuroglobin., A combined biophysical approach was applied to map gas-docking sites within murine neuroglobin (Ngb), revealing snapshots of events that might govern activity and dynamics in this unique hexacoordinate globin, which is most likely to be involved in gas-sensing in the central nervous system and for which a precise mechanism of action remains to be elucidated. The application of UV–visible microspectroscopy in crystallo, solution X-ray absorption near-edge spectroscopy and X-ray diffraction experiments at 15–40 K provided the structural characterization of an Ngb photolytic intermediate by cryo-trapping and allowed direct observation of the relocation of carbon monoxide within the distal heme pocket after photodissociation. Moreover, X-ray diffraction at 100 K under a high pressure of dioxygen, a physiological ligand of Ngb, unravelled the existence of a storage site for O2 in Ngb which coincides with Xe-III, a previously described docking site for xenon or krypton. Notably, no other secondary sites were observed under our experimental conditions.
Published: 2019

12. Remote oxidative modifications induced by oxygen free radicals modify T/R allosteric equilibrium of a hyperthermophilic lactate dehydrogenase

Author: Frédéric Halgand, Chantal Houée-Levin, Dominique Madern, Martin Weik, Laboratoire de Chimie Physique D'Orsay (LCPO), Université Paris-Sud - Paris 11 (UP11)-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS), 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), ANR-16-CE11-0011,AlloAnc,Allostérie Ancestrale(2016), European Project: Radiodommages, Institut de Chimie Physique (ICP), and Institut de Chimie du CNRS (INC)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS)
Subjects: Circular dichroism, Free Radicals, Allosteric regulation, Dehydrogenase, Oxidative phosphorylation, Structure-Activity Relationship, 03 medical and health sciences, chemistry.chemical_compound, Allosteric Regulation, Structural Biology, Catalytic Domain, Lactate dehydrogenase, [CHIM]Chemical Sciences, [SDV.BBM.BC]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biochemistry [q-bio.BM], ComputingMilieux_MISCELLANEOUS, 030304 developmental biology, chemistry.chemical_classification, 0303 health sciences, Reactive oxygen species, Binding Sites, L-Lactate Dehydrogenase, [SDV.BBM.BS]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Structural Biology [q-bio.BM], biology, 030302 biochemistry & molecular biology, Active site, [SDV.BBM.BP]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biophysics, Kinetics, Oxidative Stress, Enzyme, chemistry, biology.protein, Biophysics
Abstract: L-Lactate dehydrogenase (LDH) is a model protein allowing to shed light on the fundamental molecular mechanisms that drive the acquisition, evolution and regulation of enzyme properties. In this study, we test the hypothesis of a link between thermal stability of LDHs and their capacity against unfolding induced by reactive oxygen species (ROS) generated by γ-rays irradiation. By using circular dichroism spectroscopy, we analysed that high thermal stability of a thermophilic LDH favours strong resistance against ROS-induced unfolding, in contrast to its psychrophilic and mesophilic counterparts that are less resistant. We suggest that a protein's phenotype linking strong thermal stability and resistance against ROS damages would have been a selective evolutionary advantage. We also find that the enzymatic activity of the thermophilic LDH that is strongly resistant against ROS-unfolding is very sensitive to inactivation by irradiation. To address this counter-intuitive observation, we combined mass spectrometry analyses and enzymatic activity measurements. We demonstrate that the dramatic change on LDH activity was linked to remote chemical modifications away from the active site, that change the equilibrium between low-affinity tense (T-inactive) and high-affinity relaxed (R-active) forms. We found the T-inactive thermophilic enzyme obtained after irradiation can recover its LDH activity by addition of the allosteric effector 1, 6 fructose bis phosphate. We analyse our data within the general framework of allosteric regulation, which requires that an enzyme in solution populates a large diversity of dynamically-interchanging conformations. Our work demonstrates that the radiation-induced inactivation of an enzyme is controlled by its dynamical properties.
Published: 2020

13. Structure-Based Optimization of Nonquaternary Reactivators of Acetylcholinesterase Inhibited by Organophosphorus Nerve Agents

Author: J. De Sousa Jr., E. De la Mora, Richard C. D. Brown, Israel Silman, Gianluca Santoni, Ludovic Jean, Rachid Baati, J. A. Dias, F. Nachon, Joel L. Sussman, Martin Weik, Pierre Renard, 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), Departamento de Engenharia Geografica (DEGGE), Universidade de Lisboa (ULISBOA), Laboratory of Oceanography, Institute of Earth Science of the St. Petersburg State University, Chimie Organique et Bioorganique : Réactivité et Analyse (COBRA), Institut Normand de Chimie Moléculaire Médicinale et Macromoléculaire (INC3M), Institut de Chimie du CNRS (INC)-École Nationale Supérieure d'Ingénieurs de Caen (ENSICAEN), Normandie Université (NU)-Normandie Université (NU)-Institut national des sciences appliquées Rouen Normandie (INSA Rouen Normandie), Institut National des Sciences Appliquées (INSA)-Normandie Université (NU)-Institut National des Sciences Appliquées (INSA)-Université Le Havre Normandie (ULH), Normandie Université (NU)-Université de Rouen Normandie (UNIROUEN), Normandie Université (NU)-Centre National de la Recherche Scientifique (CNRS)-Université de Caen Normandie (UNICAEN), Normandie Université (NU)-Institut de Chimie du CNRS (INC)-École Nationale Supérieure d'Ingénieurs de Caen (ENSICAEN), Normandie Université (NU)-Centre National de la Recherche Scientifique (CNRS)-Institut de Chimie Organique Fine (IRCOF), Université de Rouen Normandie (UNIROUEN), Institut National des Sciences Appliquées (INSA)-Normandie Université (NU)-Institut National des Sciences Appliquées (INSA)-Centre National de la Recherche Scientifique (CNRS)-Centre National de la Recherche Scientifique (CNRS), Weizmann Institute of Science [Rehovot, Israël], 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), Universidade de Lisboa = University of Lisbon (ULISBOA), Saint Petersburg State University (SPBU), Institut de Chimie Organique Fine (IRCOF), Institut National des Sciences Appliquées (INSA)-Normandie Université (NU)-Institut National des Sciences Appliquées (INSA)-Centre National de la Recherche Scientifique (CNRS)-Université de Rouen Normandie (UNIROUEN), Institut National des Sciences Appliquées (INSA)-Normandie Université (NU)-Institut National des Sciences Appliquées (INSA)-Centre National de la Recherche Scientifique (CNRS)-Institut Normand de Chimie Moléculaire Médicinale et Macromoléculaire (INC3M), Université de Caen Normandie (UNICAEN), Normandie Université (NU)-Normandie Université (NU)-École Nationale Supérieure d'Ingénieurs de Caen (ENSICAEN), Normandie Université (NU)-Université Le Havre Normandie (ULH), Normandie Université (NU)-Institut national des sciences appliquées Rouen Normandie (INSA Rouen Normandie), Institut National des Sciences Appliquées (INSA)-Normandie Université (NU)-Institut National des Sciences Appliquées (INSA)-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS)-Université de Caen Normandie (UNICAEN), Normandie Université (NU)-École Nationale Supérieure d'Ingénieurs de Caen (ENSICAEN), and Normandie Université (NU)-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS)-Centre National de la Recherche Scientifique (CNRS)
Subjects: Fish Proteins, Cholinesterase Reactivators, Obidoxime Chloride, Aché, Drug Evaluation, Preclinical, 010402 general chemistry, Crystallography, X-Ray, 01 natural sciences, chemistry.chemical_compound, Structure-Activity Relationship, Organophosphorus Compounds, Catalytic Domain, Drug Discovery, medicine, [CHIM]Chemical Sciences, Structure–activity relationship, Animals, Humans, Nerve agent, chemistry.chemical_classification, biology, 010405 organic chemistry, Rational design, Active site, Oxime, Acetylcholinesterase, language.human_language, 0104 chemical sciences, 3. Good health, Molecular Docking Simulation, Enzyme, chemistry, Biophysics, language, biology.protein, Molecular Medicine, Nerve Agents, medicine.drug
Abstract: International audience; Acetylcholinesterase (AChE), a key enzyme in the central and peripheral nervous systems, is the principal target of organophosphorus nerve agents. Quaternary oximes can regenerate AChE activity by displacing the phosphyl group of the nerve agent from the active site, but they are poorly distributed in the central nervous system. A promising reactivator based on tetrahydroacridine linked to a nonquaternary oxime is also an undesired submicromolar reversible inhibitor of AChE. X-ray structures and molecular docking indicate that structural modification of the tetrahydroacridine might decrease inhibition without affecting reactivation. The chlorinated derivative was synthesized and, in line with the prediction, displayed a 10-fold decrease in inhibition but no significant decrease in reactivation efficiency. X-ray structures with the derivative rationalize this outcome. We thus show that rational design based on structural studies permits the refinement of new-generation pyridine aldoxime reactivators that may be more effective in the treatment of nerve agent intoxication.
Published: 2018

14. Photochemical Mechanism of an Atypical Algal Phytochrome

Author: Eugenio de la Mora, Michiyo Sakuma, Giorgio Schirò, Igor V. Sazanovich, Samantha J. O. Hardman, Michael Wulff, Derren J. Heyes, Joyce Woodhouse, Martin Nors Pedersen, Martin Weik, Uzma Choudry, Nigel S. Scrutton, School of Chemistry & Manchester Institute of Biotechnology, University of Manchester, Central Laser Facility, Research Complex at Harwell, Science and Technology Facilities Council ( STFC ), Institut de biologie structurale ( IBS - UMR 5075 ), Université Joseph Fourier - Grenoble 1 ( UJF ) -Commissariat à l'énergie atomique et aux énergies alternatives ( CEA ) -Centre National de la Recherche Scientifique ( CNRS ) -Université Grenoble Alpes ( UGA ), European Synchrotron Radiation Facility ( ESRF ), University of Manchester [Manchester], Science and Technology Facilities Council (STFC), 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), European Synchrotron Radiation Facility (ESRF), 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: 0301 basic medicine, Spectrophotometry, Infrared, Protein Conformation, Optogenetics, 010402 general chemistry, Photochemistry, 01 natural sciences, Biochemistry, time-resolved spectroscopy, 03 medical and health sciences, X-Ray Diffraction, Chlorophyta, Manchester Institute of Biotechnology, biophysics, Molecular Biology, phytochrome, photochemistry, Phytochrome, [SDV.BBM.BS]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Structural Biology [q-bio.BM], Chemistry, Organic Chemistry, Photoreceptor protein, Photochemical Processes, ResearchInstitutes_Networks_Beacons/manchester_institute_of_biotechnology, photoreceptor, 0104 chemical sciences, 030104 developmental biology, Molecular Medicine, Time-resolved spectroscopy, Visible spectrum, [ SDV.BBM.BS ] Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biomolecules [q-bio.BM]
Abstract: International audience; Phytochromes are bilin-containing photoreceptors that are typically sensitive to the red/far-red region of the visible spectrum. Recently, phytochromes from certain eukaryotic algae have become attractive targets for optogenetic applications because of their unique ability to respond to multiple wavelengths of light. Herein, a combination of time-resolved spectroscopy and structural approaches across picosecond to second timescales have been used to map photochemical mechanisms and structural changes in this atypical group of phytochromes. The photochemistry of an orange/far-red light-sensitive algal phytochrome from Dolihomastix tenuilepis has been investigated by using a combination of visible, IR and X-ray scattering probes. The entire photocycle, correlated with accompanying structural changes in the cofactor/protein, are reported. This study identifies a complex photocycle for this atypical phytochrome. It also highlights a need to combine outcomes from a range of biophysical approaches to unravel complex photochemical and macromolecular processes in multi-domain photoreceptor proteins that are the basis of biological light-mediated signalling.
Published: 2018

15. Potent 3‐Hydroxy‐2‐Pyridine Aldoxime Reactivators of Organophosphate‐Inhibited Cholinesterases with Predicted Blood–Brain Barrier Penetration

Author: Martin Weik, Ludovic Jean, Guillaume Mercey, Romain Mougeot, Tamara Zorbaz, Nikolina Maček Hrvat, Nikola Maraković, Florian Nachon, Maja Katalinić, Joel L. Sussman, Eugenio de la Mora, Anissa Braïki, Zrinka Kovarik, Belén Pérez, Pierre-Yves Renard, Catherine Gomez, Julien Renou, Rachid Baati, Israel Silman, Institute for Medical Research and Occupational Health, Chimie Organique et Bioorganique : Réactivité et Analyse (COBRA), Institut Normand de Chimie Moléculaire Médicinale et Macromoléculaire (INC3M), Institut de Chimie du CNRS (INC)-École Nationale Supérieure d'Ingénieurs de Caen (ENSICAEN), Normandie Université (NU)-Normandie Université (NU)-Institut national des sciences appliquées Rouen Normandie (INSA Rouen Normandie), Institut National des Sciences Appliquées (INSA)-Normandie Université (NU)-Institut National des Sciences Appliquées (INSA)-Université Le Havre Normandie (ULH), Normandie Université (NU)-Université de Rouen Normandie (UNIROUEN), Normandie Université (NU)-Centre National de la Recherche Scientifique (CNRS)-Université de Caen Normandie (UNICAEN), Normandie Université (NU)-Institut de Chimie du CNRS (INC)-École Nationale Supérieure d'Ingénieurs de Caen (ENSICAEN), Normandie Université (NU)-Centre National de la Recherche Scientifique (CNRS)-Institut de Chimie Organique Fine (IRCOF), Université de Rouen Normandie (UNIROUEN), Institut National des Sciences Appliquées (INSA)-Normandie Université (NU)-Institut National des Sciences Appliquées (INSA)-Centre National de la Recherche Scientifique (CNRS)-Centre National de la Recherche Scientifique (CNRS), 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), Weizmann Institute of Science [Rehovot, Israël], Universitat Autònoma de Barcelona (UAB), Institut de chimie et procédés pour l'énergie, l'environnement et la santé (ICPEES), Université de Strasbourg (UNISTRA)-Matériaux et nanosciences d'Alsace (FMNGE), Institut de Chimie du CNRS (INC)-Université de Strasbourg (UNISTRA)-Université de Haute-Alsace (UHA) Mulhouse - Colmar (Université de Haute-Alsace (UHA))-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Institut de Chimie du CNRS (INC)-Université de Strasbourg (UNISTRA)-Université de Haute-Alsace (UHA) Mulhouse - Colmar (Université de Haute-Alsace (UHA))-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), Institut de Recherche Biomédicale des Armées (IRBA), Centre National de la Recherche Scientifique (CNRS)-Institut de Chimie Organique Fine (IRCOF), Institut National des Sciences Appliquées (INSA)-Normandie Université (NU)-Institut National des Sciences Appliquées (INSA)-Centre National de la Recherche Scientifique (CNRS)-Université de Rouen Normandie (UNIROUEN), Institut National des Sciences Appliquées (INSA)-Normandie Université (NU)-Institut National des Sciences Appliquées (INSA)-Centre National de la Recherche Scientifique (CNRS), 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), Weizmann Institute of Science, Laboratoire d'étude des Interactions Sol - Agrosystème - Hydrosystème (UMR LISAH), Institut de Recherche pour le Développement (IRD)-Institut National de la Recherche Agronomique (INRA)-Centre international d'études supérieures en sciences agronomiques (Montpellier SupAgro)-Institut de Recherche pour le Développement (IRD [ Madagascar])-Institut national d’études supérieures agronomiques de Montpellier (Montpellier SupAgro), UMR 5567-CNRS, University of Montpellier II, France, Université Montpellier 2 - Sciences et Techniques (UM2)-Centre National de la Recherche Scientifique (CNRS), Université de Strasbourg (UNISTRA)-Centre National de la Recherche Scientifique (CNRS)-Matériaux et nanosciences d'Alsace, Université de Strasbourg (UNISTRA)-Université de Haute-Alsace (UHA) Mulhouse - Colmar (Université de Haute-Alsace (UHA))-Institut National de la Santé et de la Recherche Médicale (INSERM)-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 la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Institut de Chimie Organique Fine (IRCOF), Institut National des Sciences Appliquées (INSA)-Normandie Université (NU)-Institut National des Sciences Appliquées (INSA)-Centre National de la Recherche Scientifique (CNRS)-Institut Normand de Chimie Moléculaire Médicinale et Macromoléculaire (INC3M), Université de Caen Normandie (UNICAEN), Normandie Université (NU)-Normandie Université (NU)-École Nationale Supérieure d'Ingénieurs de Caen (ENSICAEN), Normandie Université (NU)-Université Le Havre Normandie (ULH), Normandie Université (NU)-Institut national des sciences appliquées Rouen Normandie (INSA Rouen Normandie), Institut National des Sciences Appliquées (INSA)-Normandie Université (NU)-Institut National des Sciences Appliquées (INSA)-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS)-Université de Caen Normandie (UNICAEN), Normandie Université (NU)-École Nationale Supérieure d'Ingénieurs de Caen (ENSICAEN), Normandie Université (NU)-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS)-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), Université de Strasbourg (UNISTRA)-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS)-Matériaux et Nanosciences Grand-Est (MNGE), Université de Strasbourg (UNISTRA)-Université de Haute-Alsace (UHA) Mulhouse - Colmar (Université de Haute-Alsace (UHA))-Institut National de la Santé et de la Recherche Médicale (INSERM)-Institut de Chimie du CNRS (INC)-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 la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), and Institut de Recherche Biomédicale des Armées [Brétigny-sur-Orge] (IRBA)
Subjects: 0301 basic medicine, Sarin, Stereochemistry, Cyclosarin, Catalysis, Structure-Activity Relationship, 03 medical and health sciences, chemistry.chemical_compound, 0302 clinical medicine, Oximes, medicine, Humans, [CHIM]Chemical Sciences, HI-6, pesticide, sarin, VX, acetylcholinesterase, butyrylcholinesterase, CNS active, PAMPA, Butyrylcholinesterase, Tabun, Paraoxon, Organic Chemistry, Organophosphate, General Chemistry, Oxime, Acetylcholinesterase, Organophosphates, 030104 developmental biology, chemistry, Blood-Brain Barrier, 030217 neurology & neurosurgery, medicine.drug
Abstract: International audience; A new series of 3‐hydroxy‐2‐pyridine aldoxime compounds have been designed, synthesised and tested in vitro, in silico, and ex vivo as reactivators of human acetylcholinesterase (hAChE) and butyrylcholinesterase (hBChE) inhibited by organophosphates (OPs), for example, VX, sarin, cyclosarin, tabun, and paraoxon. The reactivation rates of three oximes (16–18) were determined to be greater than that of 2‐PAM and comparable to that of HI‐6, two pyridinium aldoximes currently used by the armies of several countries. The interactions important for a productive orientation of the oxime group within the OP‐inhibited enzyme have been clarified by molecular‐modelling studies, and by the resolution of the crystal structure of the complex of oxime 17 with Torpedo californica AChE. Blood–brain barrier penetration was predicted for oximes 15–18 based on their physicochemical properties and an in vitro brain membrane permeation assay. Among the evaluated compounds, two morpholine‐3‐hydroxypyridine aldoxime conjugates proved to be promising reactivators of OP‐inhibited cholinesterases. Moreover, efficient ex vivo reactivation of phosphylated native cholinesterases by selected oximes enabled significant hydrolysis of VX, sarin, paraoxon, and cyclosarin in whole human blood, which indicates that the oximes have scavenging potential.
Published: 2018

16. Porin self-association enables cell-to-cell contact in Providencia stuartii floating communities

Author: Jean-Marie Pagès, Guillaume Tetreau, Julie Lopes, Michel Vivaudou, Que-Tien Tran, Mathilde Lethier, Mathias Winterhalter, Jean-Michel Bolla, Hind Basbous, Martin Weik, Chady Nasrallah, Mariam El-Khatib, Jacques-Philippe Colletier, Daphna Fenel, Benoit Gallet, 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), Jacobs University [Bremen], Membranes et cibles thérapeutiques (MCT), Aix Marseille Université (AMU)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Institut de Recherche Biomédicale des Armées [Brétigny-sur-Orge] (IRBA), Institut de pharmacologie et de biologie structurale (IPBS), Université Toulouse III - Paul Sabatier (UT3), Université de Toulouse (UT)-Université de Toulouse (UT)-Centre National de la Recherche Scientifique (CNRS), We thank E. Kandiah, D. Cascio, M. R. Sawaya, I. Silman, and J. Zaccaï for critically reading the manuscript, C. Breyton and D. Levy for suggesting on-column delipidation of porins and sucrose gradient experiments, respectively, and A. Martel, A. Le Roy, A. Flayhan, A. Laganowsky, A. Davin-Regli, E. Moiseeva, M. Zhao, G. Schoehn, and T. Vernet for stimulating discussions. We are indebted to I. Snigireva for scanning electron micrographs and to J.-P. Kleman and F. Lacroix for technical support and advice during epifluorescence microscopy experiments. This work used the platforms of the Grenoble Instruct Center [Integrated Structural Biology Grenoble: Unité mixte de service 3518 CNRS–CEA–UGA–European Molecular Biology Laboratory (EMBL)], with support from the French Infrastructure for Integrated Structural Biology (Grant ANR-10-INSB-05-02) and the Grenoble Alliance for Integrated Structural Cell Biology (GRAL) (Grant ANR-10-LABX-49-01) within the Grenoble Partnership for Structural Biology. The electron microscopy facility is supported by the Rhône-Alpes Region, the Fondation de la Recherche Medicale, the Fonds Européen de Développement Régional, the CNRS, the CEA, the UGA, the EMBL, and the Groupement d’Intérêt Scientifique-Infrastrutures en Biologie, Santé et Agronomie. We are grateful to the ESRF for beam time under the long-term projects MX722, MX1464, and MX1583 (IBS beamtime allocation group). We acknowledge financial support from the CEA, the CNRS, the UGA, Agence Nationale de la Recherche Grant ANR-15-CE18-0005-02 (to J.-P.C.), GRAL Grant C7H-LXG11A20-COLLETIER (to J.-P.C.), Laboratory of Excellence 'Ion Channel Science and Therapeutics' Grant ANR-11-LABX-0015-01 (to M.V.), and Aix-Marseille University and the Service de Santé des Armées (J.-M.P.). M.E.-K. is supported by a joint CEA-GRAL doctoral fellowship (Grant C7H-LXG11A20-DYNAMOP)., ANR-10-INBS-0005,FRISBI,Infrastructure Française pour la Biologie Structurale Intégrée(2010), ANR-10-LABX-0049,GRAL,Grenoble Alliance for Integrated Structural Cell Biology(2010), ANR-11-LABX-0015,ICST,Canaux ioniques d'intérêt thérapeutique(2011), 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), Aix Marseille Université (AMU)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Institut de Recherche Biomédicale des Armées (IRBA), Centre National de la Recherche Scientifique (CNRS)-Université Toulouse III - Paul Sabatier (UT3), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées, Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Centre National de la Recherche Scientifique (CNRS), rosa, emmanuelle, Infrastructure Française pour la Biologie Structurale Intégrée - - FRISBI2010 - ANR-10-INBS-0005 - INBS - VALID, Grenoble Alliance for Integrated Structural Cell Biology - - GRAL2010 - ANR-10-LABX-0049 - LABX - VALID, and Laboratoires d'excellence - Canaux ioniques d'intérêt thérapeutique - - ICST2011 - ANR-11-LABX-0015 - LABX - VALID
Subjects: 0301 basic medicine, porins, [SDV.BBM.BS] Life Sciences [q-bio]/Biochemistry, Molecular Biology/Structural Biology [q-bio.BM], 030106 microbiology, Providencia, 03 medical and health sciences, Extracellular, Cell adhesion, Multidisciplinary, biology, [SDV.BBM.BS]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Structural Biology [q-bio.BM], Chemistry, Providencia stuartii, Biofilm, steric zippers, cell adhesion, biochemical phenomena, metabolism, and nutrition, biology.organism_classification, [SDV.MP.BAC]Life Sciences [q-bio]/Microbiology and Parasitology/Bacteriology, Transport protein, 030104 developmental biology, Porin, Biophysics, intercellular communication, bacteria, [SDV.MP.BAC] Life Sciences [q-bio]/Microbiology and Parasitology/Bacteriology, biofilms, Bacterial outer membrane
Abstract: International audience; The gram-negative pathogen Providencia stuartii forms floating communities within which adjacent cells are in apparent contact, before depositing as canonical surface-attached biofilms. Because porins are the most abundant proteins in the outer membrane of gram-negative bacteria, we hypothesized that they could be involved in cell-to-cell contact and undertook a structure-function relationship study on the two porins of P. stuartii, Omp-Pst1 and Omp-Pst2. Our crystal structures reveal that these porins can self-associate through their extracellular loops, forming dimers of tri-mers (DOTs) that could enable cell-to-cell contact within floating communities. Support for this hypothesis was obtained by studying the porin-dependent aggregation of liposomes and model cells. The observation that facing channels are open in the two porin structures suggests that DOTs could not only promote cell-to-cell contact but also contribute to intercellular communication. biofilms | porins | intercellular communication | cell adhesion | steric zippers
Published: 2018

17. Chromophore twisting in the excited state of a photoswitchable fluorescent protein captured by time-resolved serial femtosecond crystallography

Author: Martin J. Field, Jacqueline Ridard, Stefan Jakobs, Dominique Bourgeois, Giorgio Schirò, Martin Weik, Virgile Adam, C.M. Roome, Lutz Foucar, Jacques-Philippe Colletier, R. Bruce Doak, M. Hilpert, Franck Fieschi, Karol Nass, Andrew Aquila, Martin Byrdin, Thomas R. M. Barends, Thomas J. Lane, Joyce Woodhouse, Michel Sliwa, Mengning Liang, Virginia Guillon, Isabelle Demachy, Eugenio de la Mora, Bernard I. Levy, Robert L. Shoeman, Mark S. Hunter, Sébastien Boutet, Marco Cammarata, Nicolas Coquelle, Jason E. Koglin, Sergio Carbajo, Matthew Seaberg, Ilme Schlichting, Michel Thépaut, Mikolaj Feliks, Gabriela Kovacsova, Joseph Robinson, Cyril Ruckebusch, 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 Avancé de Spectroscopie pour les Intéractions la Réactivité et l'Environnement - UMR 8516 (LASIRE), Institut de Chimie du CNRS (INC)-Université de Lille-Centre National de la Recherche Scientifique (CNRS), Linac Coherent Light Source (LCLS), SLAC National Accelerator Laboratory (SLAC), Stanford University-Stanford University, Max Planck Institute for Medical Research [Heidelberg], Max-Planck-Gesellschaft, Department of NanoBiophotonics [Göttingen], Max Planck Institute for Biophysical Chemistry (MPI-BPC), Max-Planck-Gesellschaft-Max-Planck-Gesellschaft, Laboratoire de Chimie Physique D'Orsay (LCPO), Université Paris-Sud - Paris 11 (UP11)-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS), Institut de Physique de Rennes (IPR), Université de Rennes (UR)-Centre National de la Recherche Scientifique (CNRS), Max-Planck-Institut für Medizinische Forschung, ANR-15-CE32-0004,BioXFEL,Caractérisation d'états intermédiaires de protéines fluorescentes en utilisant des lasers à électrons libres X et les spectroscopies UV-visible et infrarouge ultra-rapides(2015), ANR-10-INBS-0005,FRISBI,Infrastructure Française pour la Biologie Structurale Intégrée(2010), ANR-10-LABX-0049,GRAL,Grenoble Alliance for Integrated Structural Cell Biology(2010), European Project: 317079,EC:FP7:PEOPLE,FP7-PEOPLE-2012-ITN,NANOMEM(2013), 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), Institut de Chimie du CNRS (INC)-Université de Lille-Centre National de la Recherche Scientifique (CNRS)-Centrale Lille Institut (CLIL), Université de Rennes 1 (UR1), and Université de Rennes (UNIV-RENNES)-Université de Rennes (UNIV-RENNES)-Centre National de la Recherche Scientifique (CNRS)
Subjects: 0301 basic medicine, [SDV.BBM.BS]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Structural Biology [q-bio.BM], Chemistry, General Chemical Engineering, Quantum yield, General Chemistry, Chromophore, Laser, law.invention, 03 medical and health sciences, Molecular dynamics, Crystallography, 030104 developmental biology, law, Picosecond, Excited state, Femtosecond, Physics::Chemical Physics, Ultrashort pulse
Abstract: International audience; Chromophores absorb light in photosensitive proteins and thereby initiate fundamental biological processes such as photosynthesis, vision and biofluorescence. An important goal in their understanding is the provision of detailed structural descriptions of the ultrafast photochemical events that they undergo, in particular of the excited states that connect chemistry to biological function. Here we report on the structures of two excited states in the reversibly photoswitchable fluorescent protein rsEGFP2. We populated the states through femtosecond illumination of rsEGFP2 in its non-fluorescent off state and observed their build-up (within less than one picosecond) and decay (on the several picosecond timescale). Using an X-ray free-electron laser, we performed picosecond time-resolved crystallography and show that the hydroxybenzylidene imidazolinone chromophore in one of the excited states assumes a near-canonical twisted configuration halfway between the trans and cis isomers. This is in line with excited-state quantum mechanics/molecular mechanics and classical molecular dynamics simulations. Our new understanding of the structure around the twisted chromophore enabled the design of a mutant that displays a twofold increase in its off-to-on photoswitching quantum yield.
Published: 2018

18. Neutrons describe ectoine effects on water H-bonding and hydration around a soluble protein and a cell membrane

Author: Martine Moulin, Michael Haertlein, Anne L. Martel, Susanne von Gronau, François-Xavier Gallat, Dieter Oesterhelt, Gabriel J. Cuello, Bruno Demé, Irina Bagyan, Giuseppe Zaccai, Victor M. Galván Josa, Markus Neumann, Yann Fichou, Jérôme Combet, 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), Bitop, Institut Charles Sadron (ICS), Université de Strasbourg (UNISTRA)-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS)-Matériaux et Nanosciences Grand-Est (MNGE), Université de Strasbourg (UNISTRA)-Université de Haute-Alsace (UHA) Mulhouse - Colmar (Université de Haute-Alsace (UHA))-Institut National de la Santé et de la Recherche Médicale (INSERM)-Institut de Chimie du CNRS (INC)-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 la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Réseau nanophotonique et optique, Université de Strasbourg (UNISTRA)-Université de Haute-Alsace (UHA) Mulhouse - Colmar (Université de Haute-Alsace (UHA))-Centre National de la Recherche Scientifique (CNRS)-Université de Strasbourg (UNISTRA)-Centre National de la Recherche Scientifique (CNRS), Institut Laue-Langevin (ILL), Max-Planck-Institut für Biochemie = Max Planck Institute of Biochemistry (MPIB), Max-Planck-Gesellschaft, 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 de Strasbourg (UNISTRA)-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS)-Réseau nanophotonique et optique, Centre National de la Recherche Scientifique (CNRS)-Université de Strasbourg (UNISTRA)-Université de Haute-Alsace (UHA) Mulhouse - Colmar (Université de Haute-Alsace (UHA))-Centre National de la Recherche Scientifique (CNRS)-Université de Strasbourg (UNISTRA)-Université de Haute-Alsace (UHA) Mulhouse - Colmar (Université de Haute-Alsace (UHA))-Matériaux et nanosciences d'Alsace (FMNGE), Institut de Chimie du CNRS (INC)-Université de Strasbourg (UNISTRA)-Université de Haute-Alsace (UHA) Mulhouse - Colmar (Université de Haute-Alsace (UHA))-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Université de Strasbourg (UNISTRA)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), ILL, and Max-Planck-Institut für Biochemie (MPIB)
Subjects: 0301 basic medicine, Neutron diffraction, Ectoine, Article, 03 medical and health sciences, chemistry.chemical_compound, Molecular dynamics, Bacterial Proteins, Scattering, Small Angle, Escherichia coli, ComputingMilieux_MISCELLANEOUS, Multidisciplinary, Aqueous solution, 030102 biochemistry & molecular biology, [SDV.BBM.BS]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Structural Biology [q-bio.BM], Chemistry, Cell Membrane, Amino Acids, Diamino, Water, Hydrogen Bonding, Deuterium, Small-angle neutron scattering, Neutron Diffraction, 030104 developmental biology, Membrane, Biochemistry, 13. Climate action, Osmolyte, Isotope Labeling, Biophysics, Halomonas
Abstract: Understanding adaptation to extreme environments remains a challenge of high biotechnological potential for fundamental molecular biology. The cytosol of many microorganisms, isolated from saline environments, reversibly accumulates molar concentrations of the osmolyte ectoine to counterbalance fluctuating external salt concentrations. Although they have been studied extensively by thermodynamic and spectroscopic methods, direct experimental structural data have, so far, been lacking on ectoine-water-protein interactions. In this paper, in vivo deuterium labeling, small angle neutron scattering, neutron membrane diffraction and inelastic scattering are combined with neutron liquids diffraction to characterize the extreme ectoine-containing solvent and its effects on purple membrane of H. salinarum and E. coli maltose binding protein. The data reveal that ectoine is excluded from the hydration layer at the membrane surface and does not affect membrane molecular dynamics, and prove a previous hypothesis that ectoine is excluded from a monolayer of dense hydration water around the soluble protein. Neutron liquids diffraction to atomic resolution shows how ectoine enhances the remarkable properties of H-bonds in water—properties that are essential for the proper organization, stabilization and dynamics of biological structures.
Published: 2017

19. Simple and efficient system for photoconverting light-sensitive proteins in serial crystallography experiments

Author: Robert L. Shoeman, Martin Weik, Joyce Woodhouse, Giorgio Schirò, Ilme Schlichting, 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), Max Planck Institute for Medical Research [Heidelberg], Max-Planck-Gesellschaft, ANR-15-CE32-0004,BioXFEL,Caractérisation d'états intermédiaires de protéines fluorescentes en utilisant des lasers à électrons libres X et les spectroscopies UV-visible et infrarouge ultra-rapides(2015), European Project: PEPS SASLELX, 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: 0301 basic medicine, Diffraction, [SDV.BBM.BS]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Structural Biology [q-bio.BM], Chemistry, Optogenetics, Laser, General Biochemistry, Genetics and Molecular Biology, law.invention, 03 medical and health sciences, Crystallography, 030104 developmental biology, law, Nano, Femtosecond, Fluorescence microscope, ComputingMilieux_MISCELLANEOUS, Macromolecule, Photosystem
Abstract: Proteins that change their structure in response to light absorption regulate many functional processes in living cells. Moreover, biotechnological approaches like optogenetics and super-resolution fluorescence microscopy recently triggered the generation of new genetically modified photosensitive proteins. Light-induced structural changes in photosensitive proteins can be studied by time-resolved serial femtosecond crystallography (SFX), an X-ray diffraction technique that allows the determination of macromolecular structures at X-ray free-electron lasers from a large number of nano- to micro-sized crystals. This article describes a simple and efficient system for converting photosensitive proteins into light-induced semi-stationary states by inline laser illumination prior to sample injection with a gas-focused liquid jet and subsequent optical pump–X-ray probe exposure. The simple setup of this device makes it suitable for integration into other liquid injectors (like electro-spinning and electro-kinetic injectors) and potentially also in high-viscosity extruders, provided that embedding microcrystals in viscous media does not alter protein photophysical properties. The functioning of the device is demonstrated with an example of a photoswitchable fluorescent protein pre-illuminated (photoactivated) for time-resolved SFX experiments. The device can be easily adapted for the conversion in time-resolved SFX experiments of other microcrystalline proteins, such as photosystems, phytochromes and rhodopsins.
Published: 2017

20. Structure and function of an insect α-carboxylesterase (αEsterase7) associated with insecticide resistance

Author: Paul D. Carr, Faisal Younus, Jian-Wei Liu, Robyn J. Russell, Chris M. Coppin, Tamara Meirelles, John G. Oakeshott, David L. Ollis, Mathilde Lethier, Martin Weik, Gunjan Pandey, Colin J. Jackson, Research School of Chemistry, Australian National University (ANU), Unit for Virus Host-Cell Interactions [Grenoble] (UVHCI), Centre National de la Recherche Scientifique (CNRS)-European Molecular Biology Laboratory [Grenoble] (EMBL)-Université Joseph Fourier - Grenoble 1 (UJF), CSIRO - Land & Water National Research Flagship, 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), Commonwealth Scientific and Industrial Research Organisation [Canberra] (CSIRO), Université Joseph Fourier - Grenoble 1 (UJF)-European Molecular Biology Laboratory [Grenoble] (EMBL)-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: 0106 biological sciences, Insecticides, Drug Resistance, MESH: Protein Structure, Secondary, MESH: Catalytic Domain, Genes, Insect, MESH: Genes, Insect, Crystallography, X-Ray, 01 natural sciences, Protein Structure, Secondary, Carboxylesterase, Substrate Specificity, chemistry.chemical_compound, Catalytic Domain, MESH: Animals, MESH: Carboxylesterase, Phosphorylation, directed evolution, MESH: Diptera, chemistry.chemical_classification, 0303 health sciences, Multidisciplinary, biology, [SDV.BBM.BS]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Structural Biology [q-bio.BM], MESH: Sheep Diseases, Organophosphate, Biological Sciences, Directed evolution, Acetylcholinesterase, Biochemistry, Lucilia cuprina, MESH: Drug Resistance, ali-esterase, MESH: Australia, Sheep Diseases, MESH: Sheep, 03 medical and health sciences, Animals, 030304 developmental biology, Enzyme substrate complex, Sheep, MESH: Phosphorylation, Diptera, Australia, Active site, protein engineering, MESH: Acetylcholinesterase, biology.organism_classification, MESH: Crystallography, X-Ray, MESH: Insecticides, 010602 entomology, Enzyme, chemistry, biology.protein, MESH: Substrate Specificity
Abstract: Insect carboxylesterases from the α Esterase gene cluster, such as αE7 (also known as E3) from the Australian sheep blowfly Lucilia cuprina ( Lc αE7), play an important physiological role in lipid metabolism and are implicated in the detoxification of organophosphate (OP) insecticides. Despite the importance of OPs to agriculture and the spread of insect-borne diseases, the molecular basis for the ability of α-carboxylesterases to confer OP resistance to insects is poorly understood. In this work, we used laboratory evolution to increase the thermal stability of Lc αE7, allowing its overexpression in Escherichia coli and structure determination. The crystal structure reveals a canonical α/β-hydrolase fold that is very similar to the primary target of OPs (acetylcholinesterase) and a unique N-terminal α-helix that serves as a membrane anchor. Soaking of Lc αE7 crystals in OPs led to the capture of a crystallographic snapshot of Lc αE7 in its phosphorylated state, which allowed comparison with acetylcholinesterase and rationalization of its ability to protect insects against the effects of OPs. Finally, inspection of the active site of Lc αE7 reveals an asymmetric and hydrophobic substrate binding cavity that is well-suited to fatty acid methyl esters, which are hydrolyzed by the enzyme with specificity constants (∼10 6 M −1 s −1 ) indicative of a natural substrate.
Published: 2013

21. Energy Landscapes of Human Acetylcholinesterase and Its Huperzine A-Inhibited Counterpart

Author: Flynn R. Hill, Lambert van Eijck, Marcus Trapp, M M Koza, Moeava Tehei, Judith Peters, Patrick Masson, Florian Nachon, Martin Weik, and Marie Trovaslet
Subjects: chemistry.chemical_classification, Chemistry, Molecular Dynamics Simulation, Acetylcholinesterase, Recombinant Proteins, Surfaces, Coatings and Films, Orders of magnitude (entropy), chemistry.chemical_compound, Alkaloids, Enzyme, Biochemistry, Catalytic Domain, Materials Chemistry, medicine, Biophysics, Humans, Thermodynamics, Cholinesterase Inhibitors, Physical and Theoretical Chemistry, Sesquiterpenes, Huperzine A, Protein Binding, medicine.drug
Abstract: Enzymes are animated by a hierarchy of motions occurring on time scales that span more than 15 orders of magnitude from femtoseconds (10(-15) s) to several minutes. As a consequence, an enzyme is characterized by a large number of conformations, so-called conformational substates that interconvert via molecular motions. The energy landscapes of these macromolecules are very complex, and many conformations are separated by only small energy barriers. Movements at this level are fast thermal atomic motions occurring on a time scale between 10(-7) and 10(-12) s, which are experimentally accessible by incoherent neutron scattering techniques. They correspond to local fluctuations within the molecule and are believed to act as coupling links for larger, conformational changes. Several questions related to this hierarchy of motions are a matter of very active research: which of the motions are involved in the biological functions of the macromolecule and are motions of different energy (and thus time) scale correlated? How does the distribution of motions change when an enzyme is inhibited? We report here on investigations of the enzyme human acetylcholinesterase, unliganded and in complex with the noncovalent inhibitor Huperzine A, by incoherent neutron scattering. Different time scales are explored to shed light on the interplay of enzyme activity, dynamics, and inhibition. Surprisingly the average molecular dynamics do not seem to be altered by the presence of the inhibitor used in this study within the considered time scales. The activation energy for the free and the inhibited form of the enzyme is moreover found to be almost identical despite changes of interactions inside the gorge, which leads to the active site of the enzyme.
Published: 2012

22. Mapping the Accessible Conformational Landscape of an Insect Carboxylesterase Using Conformational Ensemble Analysis and Kinetic Crystallography

Author: Peter D. Mabbitt, Colin J. Jackson, Martin Weik, Tamara Meirelles, Nicholas J. Fraser, Galen J. Correy, and Paul D. Carr
Subjects: 0301 basic medicine, chemistry.chemical_classification, Models, Molecular, 030102 biochemistry & molecular biology, Chemistry, Stereochemistry, Protein Conformation, Protein dynamics, Ensemble analysis, Kinetic energy, Crystallography, X-Ray, Carboxylesterase, 03 medical and health sciences, Crystallography, Kinetics, 030104 developmental biology, Enzyme, Catalytic cycle, Structural Biology, Hydrolase, Phosphorylation, Insect Proteins, Molecular Biology, Protein Binding
Abstract: Summary The proper function of enzymes often depends upon their efficient interconversion between particular conformational sub-states on a free-energy landscape. Experimentally characterizing these sub-states is challenging, which has limited our understanding of the role of protein dynamics in many enzymes. Here, we have used a combination of kinetic crystallography and detailed analysis of crystallographic protein ensembles to map the accessible conformational landscape of an insect carboxylesterase ( Lc αE7) as it traverses all steps in its catalytic cycle . Lc αE7 is of special interest because of its evolving role in organophosphate insecticide resistance. Our results reveal that a dynamically coupled network of residues extends from the substrate-binding site to a surface loop. Interestingly, the coupling of this network that is apparent in the apoenzyme appears to be reduced in the phosphorylated enzyme intermediate. Altogether, the results of this work highlight the importance of protein dynamics to enzyme function and the evolution of new activity.
Published: 2015

23. Snapshots of Enzymatic Baeyer-Villiger Catalysis

Author: Martin Weik, Marco W. Fraaije, Hanna M. Dudek, C. Martinoli, Daniel E. Torres Pazmino, Andrea Mattevi, R. Orru, and Antoine Royant
Subjects: chemistry.chemical_classification, 0303 health sciences, biology, Chemistry, Stereochemistry, Active site, Cell Biology, Flavin group, 010402 general chemistry, Photochemistry, 01 natural sciences, Biochemistry, Enzyme structure, 0104 chemical sciences, Enzyme catalysis, 03 medical and health sciences, Catalytic cycle, Oxidoreductase, Tetrahedral carbonyl addition compound, biology.protein, Molecular Biology, Mixed Function Oxygenases, 030304 developmental biology
Abstract: Baeyer-Villiger monooxygenases catalyze the oxidation of carbonylic substrates to ester or lactone products using NADPH as electron donor and molecular oxygen as oxidative reactant. Using protein engineering, kinetics, microspectrophotometry, crystallography, and intermediate analogs, we have captured several snapshots along the catalytic cycle which highlight key features in enzyme catalysis. After acting as electron donor, the enzyme-bound NADP(H) forms an H-bond with the flavin cofactor. This interaction is critical for stabilizing the oxygen-activating flavin-peroxide intermediate that results from the reaction of the reduced cofactor with oxygen. An essential active-site arginine acts as anchoring element for proper binding of the ketone substrate. Its positively charged guanidinium group can enhance the propensity of the substrate to undergo a nucleophilic attack by the flavin-peroxide intermediate. Furthermore, the arginine side chain, together with the NADP(+) ribose group, forms the niche that hosts the negatively charged Criegee intermediate that is generated upon reaction of the substrate with the flavin-peroxide. The fascinating ability of Baeyer-Villiger monooxygenases to catalyze a complex multistep catalytic reaction originates from concerted action of this Arg-NADP(H) pair and the flavin subsequently to promote flavin reduction, oxygen activation, tetrahedral intermediate formation, and product synthesis and release. The emerging picture is that these enzymes are mainly oxygen-activating and "Criegee-stabilizing" catalysts that act on any chemically suitable substrate that can diffuse into the active site, emphasizing their potential value as toolboxes for biocatalytic applications.
Published: 2011

24. Backdoor opening mechanism in acetylcholinesterase based on X-ray crystallography and molecular dynamics simulations

Author: Yechun Xu, Martin Weik, Hualiang Jiang, Israel Silman, Joel L. Sussman, Benoît Sanson, P. Therese Lang, and Jacques-Philippe Colletier
Subjects: 0303 health sciences, biology, Rational design, Active site, Crystal structure, Biochemistry, Acetylcholinesterase, law.invention, 03 medical and health sciences, Molecular dynamics, chemistry.chemical_compound, Crystallography, 0302 clinical medicine, chemistry, law, X-ray crystallography, biology.protein, Molecular Biology, 030217 neurology & neurosurgery, Torpedo, 030304 developmental biology, Backdoor
Abstract: The transient opening of a backdoor in the active-site wall of acetylcholinesterase, one of nature's most rapid enzymes, has been suggested to contribute to the efficient traffic of substrates and products. A crystal structure of Torpedo californica acetylcholinesterase in complex with the peripheral-site inhibitor aflatoxin is now presented, in which a tyrosine at the bottom of the active-site gorge rotates to create a 3.4-A wide exit channel. Molecular dynamics simulations show that the opening can be further enlarged by movement of Trp84. The crystallographic and molecular dynamics simulation data thus point to the interface between Tyr442 and Trp84 as the key element of a backdoor, whose opening permits rapid clearance of catalysis products from the active site. Furthermore, the crystal structure presented provides a novel template for rational design of inhibitors and reactivators, including anti-Alzheimer drugs and antidotes against organophosphate poisoning.
Published: 2011

25. Reaction of Cresyl Saligenin Phosphate, the Organophosphorus Agent Implicated in Aerotoxic Syndrome, with Human Cholinesterases: Mechanistic Studies Employing Kinetics, Mass Spectrometry, and X-ray Structure Analysis

Author: Patrick Masson, Florian Nachon, Oksana Lockridge, Eugénie Carletti, Jacques-Philippe Colletier, Martin Weik, Lawrence M. Schopfer, and Marie Thérèse Froment
Subjects: Models, Molecular, Aircraft, Stereochemistry, Metabolite, Crystallography, X-Ray, Toxicology, Mass spectrometry, Mass Spectrometry, Article, Adduct, chemistry.chemical_compound, Organophosphorus Compounds, Aerotoxic syndrome, Humans, Butyrylcholinesterase, Chromatography, Chemistry, Peripheral Nervous System Diseases, Tricresyl phosphate, General Medicine, Phosphate, Acetylcholinesterase, Kinetics, Air Pollution, Indoor, Neurotoxicity Syndromes, Cholinesterase Inhibitors
Abstract: Aerotoxic syndrome is assumed to be caused by exposure to tricresyl phosphate (TCP), an antiwear additive in jet engine lubricants and hydraulic fluid. CBDP (2-(ortho-cresyl)-4H-1,2,3-benzodioxaphosphoran-2-one) is the toxic metabolite of triortho-cresylphosphate, a component of TCP. Human butyrylcholinesterase (BChE; EC 3.1.1.8) and human acetylcholinesterase (AChE; EC 3.1.1.7) are irreversibly inhibited by CBDP. The bimolecular rate constants of inhibition (k(i)), determined under pseudo-first-order conditions, displayed a biphasic time course of inhibition with k(i) of 1.6 × 10(8) M(-1) min(-1) and 2.7 × 10(7) M(-1) min(-1) for E and E' forms of BChE. The inhibition constants for AChE were 1 to 2 orders of magnitude slower than those for BChE. CBDP-phosphorylated cholinesterases are nonreactivatable due to ultra fast aging. Mass spectrometry analysis showed an initial BChE adduct with an added mass of 170 Da from cresylphosphate, followed by dealkylation to a structure with an added mass of 80 Da. Mass spectrometry in (18)O-water showed that (18)O was incorporated only during the final aging step to form phospho-serine as the final aged BChE adduct. The crystal structure of CBDP-inhibited BChE confirmed that the phosphate adduct is the ultimate aging product. CBDP is the first organophosphorus agent that leads to a fully dealkylated phospho-serine BChE adduct.
Published: 2011

26. Similarities and differences in radiation damage at 100 Kversus160 K in a crystal of thermolysin

Author: Martin Weik and Douglas H. Juers
Subjects: Nuclear and High Energy Physics, Radiation, Chemistry, Stereochemistry, Radical, Static disorder, Solvent, Crystal, Crystallography, Residue (chemistry), Thermolysin, Radiation damage, Instrumentation, Macromolecule
Abstract: The temperature-dependence of radiation damage in macromolecular X-ray crystallography is currently much debated. Most protein crystallographic studies are based on data collected at 100 K. Data collection at temperatures below 100 K has been proposed to reduce radiation damage and above 100 K to be useful for kinetic crystallography that is aimed at the generation and trapping of protein intermediate states. Here the global and specific synchrotron-radiation sensitivity of crystalline thermolysin at 100 and 160 K are compared. Both types of damage are higher at 160 K than at 100 K. At 160 K more residue types are affected (Lys, Asp, Gln, Pro, Thr, Met, Asn) than at 100 K (Met, Asp, Glu, Lys). The X-ray-induced relative atomic B-factor increase is shown to correlate with the proximity of the atom to the nearest solvent channel at 160 K. Two models may explain the observed correlation: either an increase in static disorder or an increased attack of hydroxyl radicals from the solvent area of the crystal.
Published: 2011

27. Long Route or Shortcut? A Molecular Dynamics Study of Traffic of Thiocholine within the Active-Site Gorge of Acetylcholinesterase

Author: Yechun Xu, Guangrong Qin, Martin Weik, Hualiang Jiang, Israel Silman, Jacques-Philippe Colletier, and Joel L. Sussman
Subjects: Anions, Time Factors, Stereochemistry, Phenylalanine, Static Electricity, Biophysics, Molecular Dynamics Simulation, Torpedo, 010402 general chemistry, 01 natural sciences, law.invention, 03 medical and health sciences, chemistry.chemical_compound, Molecular dynamics, law, Catalytic Domain, medicine, Animals, Pliability, 030304 developmental biology, Thiocholine, 0303 health sciences, biology, Protein, Tryptophan, Active site, Substrate (chemistry), Biological Transport, Acetylcholinesterase, 0104 chemical sciences, chemistry, Biocatalysis, biology.protein, Cholinergic, Mutant Proteins, Acetylcholine, medicine.drug
Abstract: The principal role of acetylcholinesterase is termination of nerve impulse transmission at cholinergic synapses, by rapid hydrolysis of the neurotransmitter acetylcholine to acetate and choline. Its active site is buried at the bottom of a deep and narrow gorge, at the rim of which is found a second anionic site, the peripheral anionic site. The fact that the active site is so deeply buried has raised cogent questions as to how rapid traffic of substrate and products occurs in such a confined environment. Various theoretical and experimental approaches have been used to solve this problem. Here, multiple conventional molecular dynamics simulations have been performed to investigate the clearance of the product, thiocholine, from the active-site gorge of acetylcholinesterase. Our results indicate that thiocholine is released from the peripheral anionic site via random pathways, while three exit routes appear to be favored for its release from the active site, namely, along the axis of the active-site gorge, and through putative back- and side-doors. The back-door pathway is that via which thiocholine exits most frequently. Our results are in good agreement with kinetic and kinetic-crystallography studies. We propose the use of multiple molecular dynamics simulations as a fast yet accurate complementary tool in structural studies of enzymatic trafficking.
Published: 2010

28. Front Cover: Photochemical Mechanism of an Atypical Algal Phytochrome (ChemBioChem 10/2018)

Author: Samantha J. O. Hardman, Giorgio Schirò, Michael Wulff, Eugenio de la Mora, Uzma Choudry, Nigel S. Scrutton, Martin Nors Pedersen, Igor V. Sazanovich, Martin Weik, Joyce Woodhouse, Michiyo Sakuma, and Derren J. Heyes
Subjects: Front cover, Phytochrome, Chemistry, Organic Chemistry, Molecular Medicine, Time-resolved spectroscopy, Photochemistry, Molecular Biology, Biochemistry, Mechanism (sociology)
Published: 2018

29. Temperature-dependent macromolecular X-ray crystallography

Author: Jacques-Philippe Colletier and Martin Weik
Subjects: Models, Molecular, Macromolecular Substances, viruses, macromolecular substances, Crystallography, X-Ray, Structural Biology, Radiation damage, Intermediate state, temperature-dependent macromolecular crystallography, Chemistry, Macromolecular crystallography, Temperature, Proteins, General Medicine, Atmospheric temperature range, Research Papers, diagnosis, eye diseases, Protein Structure, Tertiary, Kinetics, Crystallography, Chemical physics, X-ray crystallography, Solvents, sense organs, Macromolecule
Abstract: The dynamical behaviour of crystalline macromolecules and their surrounding solvent as a function of cryo-temperature is reviewed., X-ray crystallography provides structural details of biological macromolecules. Whereas routine data are collected close to 100 K in order to mitigate radiation damage, more exotic temperature-controlled experiments in a broader temperature range from 15 K to room temperature can provide both dynamical and structural insights. Here, the dynamical behaviour of crystalline macromolecules and their surrounding solvent as a function of cryo-temperature is reviewed. Experimental strategies of kinetic crystallography are discussed that have allowed the generation and trapping of macromolecular intermediate states by combining reaction initiation in the crystalline state with appropriate temperature profiles. A particular focus is on recruiting X-ray-induced changes for reaction initiation, thus unveiling useful aspects of radiation damage, which otherwise has to be minimized in macromolecular crystallography.
Published: 2010

30. Crystallographic Snapshots of Nonaged and Aged Conjugates of Soman with Acetylcholinesterase, and of a Ternary Complex of the Aged Conjugate with Pralidoxime

Author: Florian Nachon, Patrick Masson, Marie-Thérèse Froment, Israel Silman, Benoît Sanson, Martin Weik, Jacques-Philippe Colletier, Lilly Toker, Yaacov Ashani, Harry M. Greenblatt, and Joel L. Sussman
Subjects: Models, Molecular, Pralidoxime, Stereochemistry, Soman, Crystallography, X-Ray, Torpedo, 010402 general chemistry, 01 natural sciences, 03 medical and health sciences, chemistry.chemical_compound, Catalytic Domain, Drug Discovery, medicine, Animals, Humans, Ternary complex, 030304 developmental biology, Cholinesterase, 0303 health sciences, Pralidoxime Compounds, biology, Organophosphate, Water, Oxime, Acetylcholinesterase, 0104 chemical sciences, Enzyme Activation, Kinetics, chemistry, Dealkylation, biology.protein, Molecular Medicine, Cholinesterase Inhibitors, Conjugate, medicine.drug
Abstract: Organophosphate compounds (OP) are potent inhibitors of acetylcholinesterases (AChEs) and can cause lethal poisoning in humans. Inhibition of AChEs by the OP soman involves phosphonylation of the catalytic serine, and subsequent dealkylation produces a form known as the "aged" enzyme. The nonaged form can be reactivated to a certain extent by nucleophiles, such as pralidoxime (2-PAM), whereas aged forms of OP-inhibited AChEs are totally resistant to reactivation. Here, we solved the X-ray crystal structures of AChE from Torpedo californica (TcAChE) conjugated with soman before and after aging. The absolute configuration of the soman stereoisomer adduct in the nonaged conjugate is P(S)C(R). A structural reorientation of the catalytic His440 side chain was observed during the aging process. Furthermore, the crystal structure of the ternary complex of the aged conjugate with 2-PAM revealed that the orientation of the oxime function does not permit nucleophilic attack on the phosphorus atom, thus providing a plausible explanation for its failure to reactivate the aged soman/AChE conjugate. Together, these three crystal structures provide an experimental basis for the design of new reactivators.
Published: 2009

31. Kinetic insight into the mechanism of cholinesterasterase inhibition by aflatoxin B1 to develop biosensors

Author: Jure Stojan, Jean-Louis Marty, Benoît Sanson, Martin Weik, Tamara Hansmann, and Didier Fournier
Subjects: Aflatoxin B1, Stereochemistry, Biomedical Engineering, Biophysics, Biosensing Techniques, Sensitivity and Specificity, Enzyme activator, chemistry.chemical_compound, Electrochemistry, Computer Simulation, Electrodes, chemistry.chemical_classification, biology, Reproducibility of Results, Active site, Substrate (chemistry), Equipment Design, General Medicine, biology.organism_classification, Binding constant, Acetylcholinesterase, Electric eel, Enzyme assay, Enzyme Activation, Equipment Failure Analysis, Kinetics, Enzyme, Models, Chemical, chemistry, biology.protein, Computer-Aided Design, Cholinesterase Inhibitors, Biotechnology
Abstract: In this paper, the inhibition effect of aflatoxin B1 on different species of cholinesterases was investigated to unravel action mechanism. The inhibition curves of several cholinesterase mutants (obtained by spectrophotometric measurements of enzyme activity, pS curves) were analyzed. They showed that this toxin reversibly inhibits cholinesterases by binding to a peripheral site located at the entrance of the active site gorge without entering inside the site. Electric eel enzyme revealed the highest inhibition extent with a binding constant estimated to 0.35 microM. This binding prevents the entrance of substrate en route to the catalytic site and also decreases chemical steps of the reaction at the catalytic site: acetylation is reduced to the half and deacetylation is reduced to the third. Electric eel acetylcholinesterase was used to settle an amperometric biosensor. The best detection was obtained by using 0.3 mU enzyme on the electrode and 0.5mM ATCh in the solution. The limit of detection was 3 microM corresponding to 20% inhibition.
Published: 2009

32. Direct Correlation between Molecular Dynamics and Enzymatic Stability: A Comparative Neutron Scattering Study of Native Human Butyrylcholinesterase and its 'Aged' Soman Conjugate

Author: Frank Gabel, Patrick Masson, Giuseppe Zaccai, Bhupendra P. Doctor, Martin Weik, Ashima Saxena, Israel Silman, and Marie-Thérèse Froment
Subjects: Protein Denaturation, Protein Conformation, Entropy, Soman, Enthalpy, Neutron diffraction, Biophysics, Analytical chemistry, Neutron scattering, Molecular dynamics, Differential scanning calorimetry, Enzyme Stability, Humans, Neutron, Denaturation (biochemistry), Quantitative Biology::Biomolecules, Calorimetry, Differential Scanning, Chemistry, Spectrum Analysis, Protein, Temperature, Neutron spectroscopy, Neutron Diffraction, Models, Chemical, Butyrylcholinesterase, Thermodynamics, Algorithms
Abstract: An incoherent elastic neutron scattering study of the molecular dynamics of native human butyrylcholinesterase and its “aged” soman-inhibited conjugate revealed a significant change in molecular flexibility on an angstrom-nanosecond scale as a function of temperature. The results were related to the stability of each state as established previously by differential scanning calorimetry. A striking relationship was found between the denaturation behavior and the molecular flexibility of the native and inhibited enzymes as a function of temperature. This was reflected in a quantitative correlation between the atomic mean-square displacements on an angstrom-nanosecond scale determined by neutron spectroscopy and the calorimetric specific heat. By the application of a simple two-state model that describes the transition from a folded to a denatured state, the denaturation temperatures of the native and the inhibited enzyme were correctly extracted from the atomic mean-square displacements. Furthermore, the transition entropy and enthalpy extracted from the model fit of the neutron data were, within the experimental accuracy, compatible with the values determined by differential scanning calorimetry.
Published: 2009

33. Colouring cryo-cooled crystals: online microspectrophotometry

Author: Robin L. Owen, James W. Murray, Sean McSweeney, Elspeth F. Garman, Martin Weik, Raimond B. G. Ravelli, John McGeehan, and Florent Cipriani
Subjects: Nuclear and High Energy Physics, Absorption spectroscopy, Radical, Analytical chemistry, Color, Solvated electron, Biochemistry, Redox, Online Systems, Sensitivity and Specificity, macromolecular crystallography, X-Ray Diffraction, Radiation damage, Absorption (electromagnetic radiation), Radiation Damage, Instrumentation, Radiation, Chemistry, Reproducibility of Results, Equipment Design, Cold Temperature, Equipment Failure Analysis, X-ray crystallography, Computer-Aided Design, Spectrophotometry, Ultraviolet, online microspectrophotometry, Crystallization, Macromolecule
Abstract: A portable and readily aligned online microspectrophotometer that can be easily installed on macromolecular crystallography beamlines is described. It allows measurement of the spectral characteristics of macromolecular crystals prior, during, and after the X-ray diffraction experiment., X-rays can produce a high concentration of radicals within cryo-cooled macromolecular crystals. Some radicals have large extinction coefficients in the visible (VIS) range of the electromagnetic spectrum, and can be observed optically and spectrally. An online microspectrophotometer with high temporal resolution has been constructed that is capable of measuring UV/VIS absorption spectra (200–1100 nm) during X-ray data collection. The typical X-ray-induced blue colour that is characteristic of a wide range of cryo-conditions has been identified as trapped solvated electrons. Disulphide-containing proteins are shown to form disulphide radicals at millimolar concentrations, with absorption maxima around 400 nm. The solvated electrons and the disulphide radicals seem to have a lifetime in the range of seconds up to minutes at 100 K. The temperature dependence of the kinetics of X-ray-induced radical formation is different for the solvated electrons compared with the disulphide radicals. The online microspectrophotometer provides a technique complementary to X-ray diffraction for analysing and characterizing intermediates and redox states of proteins and enzymes.
Published: 2009

34. Flexibility of Aromatic Residues in the Active-Site Gorge of Acetylcholinesterase: X-ray versus Molecular Dynamics

Author: Yechun Xu, Hualiang Jiang, Israel Silman, Joel L. Sussman, John Moult, Martin Weik, and Jacques-Philippe Colletier
Subjects: Models, Molecular, Protein Conformation, Stereochemistry, Biophysics, Crystal structure, Crystallography, X-Ray, Torpedo, law.invention, Amino Acids, Aromatic, Molecular dynamics, Protein structure, law, Side chain, Animals, Computer Simulation, Binding site, Binding Sites, biology, Chemistry, Proteins, Active site, Ligand (biochemistry), Crystallography, Acetylcholinesterase, biology.protein
Abstract: The high aromatic content of the deep and narrow active-site gorge of acetylcholinesterase (AChE) is a remarkable feature of this enzyme. Here, we analyze conformational flexibility of the side chains of the 14 conserved aromatic residues in the active-site gorge of Torpedo californica AChE based on the 47 three-dimensional crystal structures available for the native enzyme, and for its complexes and conjugates, and on a 20-ns molecular dynamics (MD) trajectory of the native enzyme. The degree of flexibility of these 14 aromatic side chains is diverse. Although the side-chain conformations of F330 and W279 are both very flexible, the side-chain conformations of F120, W233, W432, Y70, Y121, F288, F290 and F331 appear to be fixed. Residues located on, or adjacent to, the Ω-loop (C67–C94), namely W84, Y130, Y442, and Y334, display different flexibilities in the MD simulations and in the crystal structures. An important outcome of our study is that the majority of the side-chain conformations observed in the 47 Torpedo californica AChE crystal structures are faithfully reproduced by the MD simulation on the native enzyme. Thus, the protein can assume these conformations even in the absence of the ligand that permitted their experimental detection. These observations are pertinent to structure-based drug design.
Published: 2008
Full Text: View/download PDF

35. Shoot-and-Trap: Use of specific x-ray damage to study structural protein dynamics by temperature-controlled cryo-crystallography

Author: Dominique Bourgeois, Martin Weik, Israel Silman, Benoît Sanson, Joel L. Sussman, Jacques-Philippe Colletier, and Didier Fournier
Subjects: Models, Molecular, Binding Sites, Radiochemistry, Multidisciplinary, biology, Chemistry, Temperature, Energy landscape, Active site, Substrate (chemistry), Biological Sciences, Crystallography, X-Ray, Torpedo, Acetylcholine, Protein Structure, Tertiary, Substrate Specificity, Crystallography, Complementary experiments, Protein structure, Hydrolase, Acetylcholinesterase, biology.protein, Animals, Binding site, Macromolecule
Abstract: Although x-ray crystallography is the most widely used method for macromolecular structure determination, it does not provide dynamical information, and either experimental tricks or complementary experiments must be used to overcome the inherently static nature of crystallographic structures. Here we used specific x-ray damage during temperature-controlled crystallographic experiments at a third-generation synchrotron source to trigger and monitor (Shoot-and-Trap) structural changes putatively involved in an enzymatic reaction. In particular, a nonhydrolyzable substrate analogue of acetylcholinesterase, the “off-switch” at cholinergic synapses, was radiocleaved within the buried enzymatic active site. Subsequent product clearance, observed at 150 K but not at 100 K, indicated exit from the active site possibly via a “backdoor.” The simple strategy described here is, in principle, applicable to any enzyme whose structure in complex with a substrate analogue is available and, therefore, could serve as a standard procedure in kinetic crystallography studies.
Published: 2008

36. Dynamical Heterogeneity of Specific Amino Acids in Bacteriorhodopsin

Author: Gerald R. Kneller, Dieter Oesterhelt, M. Johnson, Sergei Grudinin, Giuseppe Zaccai, Martin Weik, Kathleen Wood, Brigitte Kessler, Institut Laue-Langevin (ILL), Max-Planck-Institute of Biochemistry [Martinsried, Germany], Laboratoire de Biophysique Moléculaire (LBM), 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)-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)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA), Forschungszentrum Jülich GmbH | Centre de recherche de Juliers, Helmholtz-Gemeinschaft = Helmholtz Association, Centre de biophysique moléculaire (CBM), Université d'Orléans (UO)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS), Synchrotron SOLEIL (SSOLEIL), Centre National de la Recherche Scientifique (CNRS), Université d'Orléans (UO), and Université d'Orléans (UO)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Institut de Chimie du CNRS (INC)
Subjects: Halobacterium salinarum, Models, Molecular, ANGSTROM RESOLUTION, [SDV.BC]Life Sciences [q-bio]/Cellular Biology, 010402 general chemistry, 01 natural sciences, Protein Structure, Secondary, 03 medical and health sciences, Molecular dynamics, Protein structure, WATER-MOLECULES, PROGRAM PACKAGE, Structural Biology, Scattering, Radiation, Computer Simulation, Dynamical heterogeneity, Amino Acids, PURPLE MEMBRANE, Molecular Biology, 030304 developmental biology, Neutrons, chemistry.chemical_classification, 0303 health sciences, biology, Chemistry, Spectrum Analysis, Protein dynamics, Temperature, Water, Bacteriorhodopsin, BIOLOGICAL-MEMBRANES, Deuterium, PROTEIN DYNAMICS, biology.organism_classification, 0104 chemical sciences, Amino acid, Crystallography, NEUTRON-SCATTERING, MOLECULAR-DYNAMICS, Bacteriorhodopsins, Isotope Labeling, THERMAL MOTIONS, SCATTERING ORIENTED ANALYSIS, Biophysics, biology.protein, Isoleucine
Abstract: International audience; Components of biological macromolecules, complexes and membranes are animated by motions occurring over a wide range of time and length scales, the synergy of which is at the basis of biological activity. Understanding biological function thus requires a detailed analysis of the underlying dynamical heterogeneity. Neutron scattering, using specific isotope labeling, and molecular dynamics simulations were combined in order to study the dynamics of specific amino acid types in bacteriorhodopsin within the purple membrane (PM) of Halobacterium salinarum. Motions of leucine, isoleucine and tyrosine residues on the pico- to nanosecond time scale were examined separately as a function of temperature from 20 to 300 K. The dynamics of the three residue types displayed different temperature dependence: isoleucine residues have larger displacements compared to the global PM above 120 K; leucine residues have displacements similar to that of PM in the entire temperature range studied; and tyrosine residues have displacements smaller than that of the average membrane in an intermediate temperature range. Experimental features were mostly well reproduced by molecular dynamics simulations performed at five temperatures, which allowed the dynamical characterisation of the amino acids under study as a function of local environment. The resulting dynamical map of bacteriorhodopsin revealed that movements of a specific residue are determined by both its environment and its residue type.
Published: 2008

37. Dynamics of hydration water in deuterated purple membranes explored by neutron scattering

Author: Brigitte Kessler, Giuseppe Zaccai, Martin Weik, Frank Gabel, Kathleen Wood, Dieter Oesterhelt, and Marie Plazanet
Subjects: Halobacterium salinarum, Dynamical transition, Time Factors, Movement, Neutron diffraction, Biophysics, Analytical chemistry, Neutron scattering, Hydration water dynamics, Neutron spectroscopy, Kinetic isotope effect, Lamellar structure, Original Paper, Chemistry, Dynamics (mechanics), Temperature, Membrane Proteins, Water, General Medicine, Deuterium, Purple membrane, Neutron Diffraction, Crystallography, Membrane
Abstract: The function and dynamics of proteins depend on their direct environment, and much evidence has pointed to a strong coupling between water and protein motions. Recently however, neutron scattering measurements on deuterated and natural-abundance purple membrane (PM), hydrated in H2O and D2O, respectively, revealed that membrane and water motions on the ns–ps time scale are not directly coupled below 260 K (Wood et al. in Proc Natl Acad Sci USA 104:18049–18054, 2007). In the initial study, samples with a high level of hydration were measured. Here, we have measured the dynamics of PM and water separately, at a low-hydration level corresponding to the first layer of hydration water only. As in the case of the higher hydration samples previously studied, the dynamics of PM and water display different temperature dependencies, with a transition in the hydration water at 200 K not triggering a transition in the membrane at the same temperature. Furthermore, neutron diffraction experiments were carried out to monitor the lamellar spacing of a flash-cooled deuterated PM stack hydrated in H2O as a function of temperature. At 200 K, a sudden decrease in lamellar spacing indicated the onset of long-range translational water diffusion in the second hydration layer as has already been observed on flash-cooled natural-abundance PM stacks hydrated in D2O (Weik et al. in J Mol Biol 275:632–634, 2005), excluding thus a notable isotope effect. Our results reinforce the notion that membrane-protein dynamics may be less strongly coupled to hydration water motions than the dynamics of soluble proteins.
Published: 2008

38. Coupling of protein and hydration-water dynamics in biological membranes

Author: Martin Weik, Marie Plazanet, Giuseppe Zaccai, Brigitte Kessler, Douglas J. Tobias, Frank Gabel, Dieter Oesterhelt, and Kathleen Wood
Subjects: Multidisciplinary, Chemistry, Cell Membrane, Membrane Proteins, Water, Biological membrane, Context (language use), Biological Sciences, Neutron scattering, Crystallography, Molecular dynamics, Membrane, Membrane protein, Deuterium, Chemical physics, Deuterium Oxide, Protein Binding, Macromolecule
Abstract: The dynamical coupling between proteins and their hydration water is important for the understanding of macromolecular function in a cellular context. In the case of membrane proteins, the environment is heterogeneous, composed of lipids and hydration water, and the dynamical coupling might be more complex than in the case of the extensively studied soluble proteins. Here, we examine the dynamical coupling between a biological membrane, the purple membrane (PM), and its hydration water by a combination of elastic incoherent neutron scattering, specific deuteration, and molecular dynamics simulations. Examining completely deuterated PM, hydrated in H 2 O, allowed the direct experimental exploration of water dynamics. The study of natural abundance PM in D 2 O focused on membrane dynamics. The temperature-dependence of atomic mean-square displacements shows inflections at 120 K and 260 K for the membrane and at 200 K and 260 K for the hydration water. Because transition temperatures are different for PM and hydration water, we conclude that ps–ns hydration water dynamics are not directly coupled to membrane motions on the same time scale at temperatures
Published: 2007

39. Activity, Stability and Structural Studies of Lactate Dehydrogenases Adapted to Extreme Thermal Environments

Author: Frédéric M. D. Vellieux, Dominique Madern, Nicolas Coquelle, Emanuela Fioravanti, and Martin Weik
Subjects: Models, Molecular, Hot Temperature, Protein Conformation, Surface Properties, Molecular Sequence Data, Crystallography, X-Ray, Protein Structure, Secondary, chemistry.chemical_compound, Structural Biology, Oxidoreductase, Lactate dehydrogenase, Enzyme Stability, Amino Acid Sequence, Psychrophile, Molecular Biology, chemistry.chemical_classification, Binding Sites, Gram-Negative Anaerobic Bacteria, L-Lactate Dehydrogenase, Sequence Homology, Amino Acid, biology, Hydrogen Bonding, Deinococcus radiodurans, Thermus thermophilus, biology.organism_classification, Adaptation, Physiological, Amino acid, Enzyme, chemistry, Biochemistry, Biophysics, Dimerization, Mesophile
Abstract: Lactate dehydrogenase (LDH) catalyzes the conversion of pyruvate to lactate with concomitant oxidation of NADH during the last step in anaerobic glycolysis. In the present study, we present a comparative biochemical and structural analysis of various LDHs adapted to function over a large temperature range. The enzymes were from Champsocephalus gunnari (an Antarctic fish), Deinococcus radiodurans (a mesophilic bacterium) and Thermus thermophilus (a hyperthermophilic bacterium). The thermodynamic activation parameters of these LDHs indicated that temperature adaptation from hot to cold conditions was due to a decrease in the activation enthalpy and an increase in activation entropy. The crystal structures of these LDHs have been solved. Pairwise comparisons at the structural level, between hyperthermophilic versus mesophilic LDHs and mesophilic versus psychrophilic LDHs, have revealed that temperature adaptation is due to a few amino acid substitutions that are localized in critical regions of the enzyme. These substitutions, each having accumulating effects, play a role in either the conformational stability or the local flexibility or in both. Going from hot- to cold-adapted LDHs, the various substitutions have decreased the number of ion pairs, reduced the size of ionic networks, created unfavorable interactions involving charged residues and induced strong local disorder. The analysis of the LDHs adapted to extreme temperatures shed light on how evolutionary processes shift the subtle balance between overall stability and flexibility of an enzyme.
Published: 2007

40. Use of a 'caged' analogue to study the traffic of choline within acetylcholinesterase by kinetic crystallography

Author: Giuseppe Zaccai, Patrick Masson, Dominique Bourgeois, Antoine Royant, Martin Weik, Alexandre Specht, Florian Nachon, Joel L. Sussman, Maurice Goeldner, Jacques-Philippe Colletier, Israel Silman, and Benoît Sanson
Subjects: Models, Molecular, Protein Conformation, Ultraviolet Rays, Stereochemistry, Crystallography, X-Ray, Torpedo, Cleavage (embryo), Arsenicals, Catalysis, Choline, law.invention, chemistry.chemical_compound, Protein structure, Structural Biology, law, Catalytic Domain, Hydrolase, Animals, Binding site, Binding Sites, Photolysis, biology, Chemistry, Lasers, Temperature, Active site, General Medicine, Acetylcholinesterase, Protein Structure, Tertiary, Kinetics, Crystallography, biology.protein, Cholinergic, Spectrophotometry, Ultraviolet, Crystallization, Protein Binding
Abstract: Acetylcholinesterase plays a crucial role in nerve-impulse transmission at cholinergic synapses. The apparent paradox that it displays high turnover despite its active site being buried raises cogent questions as to how the traffic of substrates and products to and from the active site can occur so rapidly in such circumstances. Here, a kinetic crystallography strategy aimed at structurally addressing the issue of product traffic in acetylcholinesterase is presented, in which UV-laser-induced cleavage of a photolabile precursor of the enzymatic product analogue arsenocholine, 'caged' arsenocholine, is performed in a temperature-controlled X-ray crystallography regime. The 'caged' arsenocholine was shown to bind at both the active and peripheral sites of acetylcholinesterase. UV irradiation of a complex with acetylcholinesterase during a brief temperature excursion from 100 K to room temperature is most likely to have resulted in a decrease in occupancy by the caged compound. Microspectrophotometric experiments showed that the caged compound had indeed been photocleaved. It is proposed that a fraction of the arsenocholine molecules released within the crystal had been expelled from both the active and the peripheral sites. Partial q-weighted difference refinement revealed a relative movement of the two domains in acetylcholinesterase after photolysis and the room-temperature excursion, resulting in an increase in the active-site gorge volume of 30% and 35% in monomers A and B of the asymmetric unit, respectively. Moreover, an alternative route to the active-site gorge of the enzyme appeared to open. This structural characterization of acetylcholinesterase 'at work' is consistent with the idea that choline exits from the enzyme after catalysis either via the gorge or via an alternative 'backdoor' trajectory.
Published: 2007

41. Mechanisms of cholinesterase inhibition by inorganic mercury

Author: Jure Stojan, Félix Carvalho, Manuela F. Frasco, Didier Fournier, Martin Weik, Jacques-Philippe Colletier, and Lúcia Guilhermino
Subjects: chemistry.chemical_classification, biology, chemistry.chemical_element, Cell Biology, Biochemistry, Acetylcholinesterase, law.invention, Mercury (element), chemistry.chemical_compound, Enzyme, chemistry, law, Hydrolase, Electrophorus, biology.protein, Molecular Biology, Torpedo, Butyrylcholinesterase, Cholinesterase
Abstract: The poorly known mechanism of inhibition of cholinesterases by inorganic mercury (HgCl2) has been studied with a view to using these enzymes as biomarkers or as biological components of biosensors to survey polluted areas. The inhibition of a variety of cholinesterases by HgCl2 was investigated by kinetic studies, X-ray crystallography, and dynamic light scattering. Our results show that when a free sensitive sulfhydryl group is present in the enzyme, as in Torpedo californica acetylcholinesterase, inhibition is irreversible and follows pseudo-first-order kinetics that are completed within 1 h in the micromolar range. When the free sulfhydryl group is not sensitive to mercury (Drosophila melanogaster acetylcholinesterase and human butyrylcholinesterase) or is otherwise absent (Electrophorus electricus acetylcholinesterase), then inhibition occurs in the millimolar range. Inhibition follows a slow binding model, with successive binding of two mercury ions to the enzyme surface. Binding of mercury ions has several consequences: reversible inhibition, enzyme denaturation, and protein aggregation, protecting the enzyme from denaturation. Mercury-induced inactivation of cholinesterases is thus a rather complex process. Our results indicate that among the various cholinesterases that we have studied, only Torpedo californica acetylcholinesterase is suitable for mercury detection using biosensors, and that a careful study of cholinesterase inhibition in a species is a prerequisite before using it as a biomarker to survey mercury in the environment.
Published: 2007

42. Translational diffusion of hydration water correlates with functional motions in folded and intrinsically disordered proteins

Author: Jacques-Philippe Colletier, Matthias Heyden, Francois Xavier Gallat, Joachim Wuttke, Michael Härtlein, Douglas J. Tobias, Giorgio Schirò, Kathleen Wood, Martine Moulin, Martin Weik, Frank Gabel, Alessandro Paciaroni, Andrea Orecchini, Yann Fichou, 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), Institut Laue-Langevin (ILL), ILL, Australian Nuclear Science and Technology Organisation (ANSTO), Australian Nuclear Science and Technology Organisation, Unité de recherche Géosciences Marines (Ifremer) (GM), Institut Français de Recherche pour l'Exploitation de la Mer (IFREMER), Dipartimento di Fisica e Geologia [Perugia], Università degli Studi di Perugia (UNIPG), Department of Chemistry [Irvine], University of California [Irvine] (UCI), University of California-University of California, ANR-11-BSV5-0027,Bieau,Combining experimental and computational methods to study the impact of biomolecular hydration water on protein dynamics: application to intrinsically disordered proteins and solvent-free protein-polymer hybrids(2011), European Project: HPRI-2001-50065, 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), Università degli Studi di Perugia = University of Perugia (UNIPG), University of California [Irvine] (UC Irvine), and University of California (UC)-University of California (UC)
Subjects: Protein Folding, Protein Conformation, Diffusion, Neutron diffraction, General Physics and Astronomy, tau Proteins, 02 engineering and technology, Molecular Dynamics Simulation, Neutron scattering, 010402 general chemistry, Intrinsically disordered proteins, 01 natural sciences, Maltose-Binding Proteins, Article, General Biochemistry, Genetics and Molecular Biology, Enzyme catalysis, Motion, Molecular dynamics, Protein structure, Humans, Scattering, Radiation, Computer Simulation, Neutrons, Binding Sites, Multidisciplinary, [SDV.BBM.BS]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Structural Biology [q-bio.BM], Chemistry, Temperature, Proteins, Water, General Chemistry, 021001 nanoscience & nanotechnology, 0104 chemical sciences, Intrinsically Disordered Proteins, Neutron Diffraction, Biochemistry, Chemical physics, Protein folding, ddc:500, 0210 nano-technology
Abstract: Hydration water is the natural matrix of biological macromolecules and is essential for their activity in cells. The coupling between water and protein dynamics has been intensively studied, yet it remains controversial. Here we combine protein perdeuteration, neutron scattering and molecular dynamics simulations to explore the nature of hydration water motions at temperatures between 200 and 300 K, across the so-called protein dynamical transition, in the intrinsically disordered human protein tau and the globular maltose binding protein. Quasi-elastic broadening is fitted with a model of translating, rotating and immobile water molecules. In both experiment and simulation, the translational component markedly increases at the protein dynamical transition (around 240 K), regardless of whether the protein is intrinsically disordered or folded. Thus, we generalize the notion that the translational diffusion of water molecules on a protein surface promotes the large-amplitude motions of proteins that are required for their biological activity., Hydration water plasticizes protein structures and is essential for their biological functions, such as enzymatic catalysis. Here, the authors use neutron scattering and molecular dynamics simulations to study hydration water at the dynamical transition of folded and disordered proteins.
Published: 2015

43. Effects of Soman Inhibition and of Structural Differences on Cholinesterase Molecular Dynamics: A Neutron Scattering Study

Author: Israel Silman, Didier Fournier, Frank Gabel, Bhupendra P. Doctor, L. Brochier, Frédérique Renault, Patrick Masson, Giuseppe Zaccai, Martin Weik, and Ashima Saxena
Subjects: Protein Denaturation, Protein Folding, Circular dichroism, Glycosylation, Time Factors, Protein Conformation, Ultraviolet Rays, Entropy, Soman, Normal Distribution, Biophysics, Biophysical Phenomena, Catalysis, Substrate Specificity, chemistry.chemical_compound, Molecular dynamics, Protein structure, Animals, Cholinesterases, Humans, Scattering, Radiation, Denaturation (biochemistry), Enzyme Inhibitors, Phosphorylation, Protein Structure, Quaternary, Butyrylcholinesterase, Neutrons, Binding Sites, Models, Statistical, Circular Dichroism, Temperature, Water, Proteins, Crystallography, Drosophila melanogaster, chemistry, Acetylcholinesterase, Thermodynamics, Protein quaternary structure, Protein folding, Cholinesterase Inhibitors, Dimerization, Hydrogen
Abstract: Incoherent elastic neutron scattering experiments on members of the cholinesterase family were carried out to investigate how molecular dynamics is affected by covalent inhibitor binding and by differences in primary and quaternary structure. Tetrameric native and soman-inhibited human butyrylcholinesterase (HuBChE) as well as native dimeric Drosophila melanogaster acetylcholinesterase (DmAChE) hydrated protein powders were examined. Atomic mean-square displacements (MSDs) were found to be identical for native HuBChE and for DmAChE in the whole temperature range examined, leading to the conclusion that differences in activity and substrate specificity are not reflected by a global modification of subnanosecond molecular dynamics. MSDs of native and soman-inhibited HuBChE were identical below the thermal denaturation temperature of the native enzyme, indicating a common mean free-energy surface. Denaturation of the native enzyme is reflected by a relative increase of MSDs consistent with entropic stabilization of the unfolded state. The results suggest that the stabilization of HuBChE phosphorylated by soman is due to an increase in free energy of the unfolded state due to a decrease in entropy.
Published: 2005

44. The Influence of Solvent Composition on Global Dynamics of Human Butyrylcholinesterase Powders: A Neutron-Scattering Study

Author: Ashima Saxena, Giuseppe Zaccai, L. Brochier, Didier Fournier, Martin Weik, Frédérique Renault, Frank Gabel, Israel Silman, Bhupendra P. Doctor, and Patrick Masson
Subjects: Sodium, Biophysics, Analytical chemistry, Salt (chemistry), chemistry.chemical_element, Buffers, Neutron scattering, Biophysical Phenomena, Ion, Molecular dynamics, Humans, Scattering, Radiation, Deuterium Oxide, Ions, Neutrons, chemistry.chemical_classification, Models, Statistical, Chemistry, Temperature, Proteins, Water, Atmospheric temperature range, Solvent, Freeze Drying, Butyrylcholinesterase, Intramolecular force, Solvents, Salts, Protons
Abstract: A major result of incoherent elastic neutron-scattering experiments on protein powders is the strong dependence of the intramolecular dynamics on the sample environment. We performed a series of incoherent elastic neutron-scattering experiments on lyophilized human butyrylcholinesterase (HuBChE) powders under different conditions (solvent composition and hydration degree) in the temperature range from 20 to 285 K to elucidate the effect of the environment on the enzyme atomic mean-square displacements. Comparing D(2)O- with H(2)O-hydrated samples, we were able to investigate protein as well as hydration water molecular dynamics. HuBChE lyophilized from three distinct buffers showed completely different atomic mean-square displacements at temperatures above approximately 200 K: a salt-free sample and a sample containing Tris-HCl showed identical small-amplitude motions. A third sample, containing sodium phosphate, displayed highly reduced mean-square displacements at ambient temperature with respect to the other two samples. Below 200 K, all samples displayed similar mean-square displacements. We draw the conclusion that the reduction of intramolecular protein mean-square displacements on an Angstrom-nanosecond scale by the solvent depends not only on the presence of salt ions but also on their type.
Published: 2004

45. Temperature Derivative Fluorescence Spectroscopy as a Tool to Study Dynamical Changes in Protein Crystals

Author: Dominique Bourgeois, Antoine Royant, Martin Weik, and X. Vernede
Subjects: Glycerol, Time Factors, Fluorophore, Biophysics, Crystallography, X-Ray, Torpedo, Protein Structure, Secondary, Fluorescence spectroscopy, Crystal, chemistry.chemical_compound, Animals, Humans, chemistry.chemical_classification, Binding Sites, Chemistry, Biomolecule, Temperature, Proteins, Water, Energy landscape, Fluorescence, Solvent, Kinetics, Crystallography, Spectrometry, Fluorescence, Spectrophotometry, Chemical physics, Butyrylcholinesterase, Acetylcholinesterase, Fluorescein, Muramidase, Glass, Protein crystallization
Abstract: Motions through the energy landscape of proteins lead to biological function. At temperatures below a dynamical transition (150–250K), some of these motions are arrested and the activity of some proteins ceases. Here, we introduce the technique of temperature-derivative fluorescence microspectrophotometry to investigate the dynamical behavior of single protein crystals. The observation of glass transitions in thin films of water/glycerol mixtures allowed us to demonstrate the potential of the technique. Then, protein crystals were investigated, after soaking the samples in a small amount of fluorescein. If the fluorophore resides within the crystal channels, temperature-dependent changes in solvent dynamics can be monitored. Alternatively, if the fluorophore binds to the protein, local dynamical transitions within the biomolecule can be probed directly. A clear dynamical transition was observed at 175K in the active site of crystalline human butyrylcholinesterase. The results suggest that the dynamics of crystalline proteins is strongly dependent on solvent composition and confinement in the crystal channels. Beyond applications in the field of kinetic crystallography, the highly sensitive temperature-derivative fluorescence microspectrophotometry technique opens the way to many studies on the dynamics of biological nanosamples.
Published: 2004
Full Text: View/download PDF

46. Evidence for the formation of disulfide radicals in protein crystals upon X-ray irradiation

Author: Maria L. Raves, Piet Gros, Sean McSweeney, Jacqueline Bergès, Chantal Houée-Levin, Israel Silman, Joel L. Sussman, Martin Weik, and Raimond B. G. Ravelli
Subjects: Models, Molecular, Nuclear and High Energy Physics, Radiation, Free Radicals, Absorption spectroscopy, Protein Conformation, Chemistry, Radical, Proteins, Dose-Response Relationship, Radiation, Electrons, law.invention, Crystal, Crystallography, Protein structure, X-Ray Diffraction, law, Humans, Disulfides, Irradiation, Crystallization, Protein crystallization, Instrumentation, Single crystal
Abstract: Irradiation of proteins with intense X-ray radiation produced by third-generation synchrotron sources generates specific structural and chemical alterations, including breakage of disulfide bonds and decarboxylation. In this paper, disulfide bond lengths in irradiated crystals of the enzyme Torpedo californica acetylcholinesterase are examined based on quantum simulations and on experimental data published previously. The experimental data suggest that one disulfide bond elongates by approximately 0.7 A upon X-ray irradiation as seen in a series of nine data sets collected on a single crystal. Simulation of the same bond suggests elongation by a similar value if a disulfide-radical anion is formed by trapping an electron. The absorption spectrum of a crystal irradiated under similar conditions shows a peak at approximately 400 nm, which in aqueous solution has been attributed to disulfide radicals. The results suggest that the formation of disulfide radicals in protein crystals owing to X-ray irradiation can be observed experimentally, both by structural means and by absorption spectroscopy.
Published: 2002

47. Low-dose X-ray radiation induces structural alterations in proteins

Author: Ivan S. Erofeev, Dieter Willbold, Ivan Gushchin, Georg Büldt, Valentin Gordeliy, Andrii Ishchenko, Ekaterina Round, Valentin Borshchevskiy, Alexey Mishin, Martin Weik, Moscow Institute of Physics and Technology [Moscow] (MIPT), Institute of Complex Systems (ICS), Forschungszentrum Jülich GmbH | Centre de recherche de Juliers, Helmholtz-Gemeinschaft = Helmholtz Association-Helmholtz-Gemeinschaft = Helmholtz Association, 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), Institute of Crystallography [Aachen], Rheinisch-Westfälische Technische Hochschule Aachen (RWTH), Heinrich Heine Universität Düsseldorf = Heinrich Heine University [Düsseldorf], Helmholtz-Gemeinschaft = Helmholtz Association, 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), Rheinisch-Westfälische Technische Hochschule Aachen University (RWTH), and Thomas, Frank
Subjects: Models, Molecular, biology, Fourier Analysis, [SDV.BBM.BS]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Structural Biology [q-bio.BM], [SDV.BBM.BS] Life Sciences [q-bio]/Biochemistry, Molecular Biology/Structural Biology [q-bio.BM], Chemistry, Protein Conformation, X-Rays, X-ray, Active site, Proteins, Bacteriorhodopsin, Dose-Response Relationship, Radiation, General Medicine, Radiation, Crystallography, X-Ray, Transport protein, Crystallography, Protein structure, Membrane protein, Structural Biology, Bacteriorhodopsins, Catalytic Domain, biology.protein, Radiation damage
Abstract: X-ray-radiation-induced alterations to protein structures are still a severe problem in macromolecular crystallography. One way to avoid the influence of radiation damage is to reduce the X-ray dose absorbed by the crystal during data collection. However, here it is demonstrated using the example of the membrane protein bacteriorhodopsin (bR) that even a low dose of less than 0.06 MGy may induce structural alterations in proteins. This dose is about 500 times smaller than the experimental dose limit which should ideally not be exceeded per data set (i.e.30 MGy) and 20 times smaller than previously detected specific radiation damage at the bR active site. To date, it is the lowest dose at which radiation modification of a protein structure has been described. Complementary use was made of high-resolution X-ray crystallography and online microspectrophotometry to quantitatively study low-dose X-ray-induced changes. It is shown that structural changes of the protein correlate with the spectroscopically observed formation of the so-called bR orange species. Evidence is provided for structural modifications taking place at the protein active site that should be taken into account in crystallographic studies which aim to elucidate the molecular mechanisms of bR function.
Published: 2014

48. 300-Fold increase in production of the Zn2+-dependent dechlorinase TrzN in soluble form via apoenzyme stabilization

Author: Thomas S. Peat, Colin J. Jackson, Alexey Aleksandrov, Joanna Ubels, Robyn J. Russell, John G. Oakeshott, Colin Scott, Martin Weik, Paul D. Carr, Christopher W. Coppin, Matthew Wilding, Michael Paks, Martin J. Field, Janet Newman, Elena Sugrue, Institut Jacques Monod (IJM (UMR_7592)), Université Paris Diderot - Paris 7 (UPD7)-Centre National de la Recherche Scientifique (CNRS), Institute of Mathematical, Physics and Computer Sciences (IMAPCS), Aberystwyth University, Universite de Californie, 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), Udall Center for Studies in Public Policy, University of Arizona, 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), Thomas, Frank, 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: Models, Molecular, [SDV.BBM.BS] Life Sciences [q-bio]/Biochemistry, Molecular Biology/Structural Biology [q-bio.BM], Hydrolases, Protein Conformation, Mutation, Missense, medicine.disease_cause, Crystallography, X-Ray, Applied Microbiology and Biotechnology, Catalysis, 03 medical and health sciences, chemistry.chemical_compound, Hydrolysis, Protein structure, Apoenzymes, Arthrobacter, Hydrolase, Enzyme Stability, medicine, Escherichia coli, Computer Simulation, Enzymology and Protein Engineering, 030304 developmental biology, Triazine, chemistry.chemical_classification, 0303 health sciences, Ecology, biology, [SDV.BBM.BS]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Structural Biology [q-bio.BM], 030306 microbiology, Chemistry, Herbicides, Triazines, biology.organism_classification, Recombinant Proteins, [SDV.BBM.BS]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biomolecules [q-bio.BM], Kinetics, Enzyme, Biochemistry, Solubility, Mutant Proteins, Food Science, Biotechnology
Abstract: Microbial metalloenzymes constitute a large library of biocatalysts, a number of which have already been shown to catalyze the breakdown of toxic chemicals or industrially relevant chemical transformations. However, while there is considerable interest in harnessing these catalysts for biotechnology, for many of the enzymes, their large-scale production in active, soluble form in recombinant systems is a significant barrier to their use. In this work, we demonstrate that as few as three mutations can result in a 300-fold increase in the expression of soluble TrzN, an enzyme from Arthrobacter aurescens with environmental applications that catalyzes the hydrolysis of triazine herbicides, in Escherichia coli . Using a combination of X-ray crystallography, kinetic analysis, and computational simulation, we show that the majority of the improvement in expression is due to stabilization of the apoenzyme rather than the metal ion-bound holoenzyme. This provides a structural and mechanistic explanation for the observation that many compensatory mutations can increase levels of soluble-protein production without increasing the stability of the final, active form of the enzyme. This study provides a molecular understanding of the importance of the stability of metal ion free states to the accumulation of soluble protein and shows that differences between apoenzyme and holoenzyme structures can result in mutations affecting the stability of either state differently.
Published: 2014

49. Reaction site-driven regioselective synthesis of AChE inhibitors

Author: Martin Weik, Olga A. Syzgantseva, Ludovic Jean, Laurent Joubert, Vincent Tognetti, Pierre-Yves Renard, Florian Nachon, Gianluca Santoni, Anthony Romieu, Emilia Oueis, Cyrille Sabot, Cyril Ronco, Chimie Organique et Bioorganique : Réactivité et Analyse (COBRA), Institut de Chimie Organique Fine (IRCOF), Université de Rouen Normandie (UNIROUEN), Normandie Université (NU)-Normandie Université (NU)-Institut national des sciences appliquées Rouen Normandie (INSA Rouen Normandie), Institut National des Sciences Appliquées (INSA)-Normandie Université (NU)-Institut National des Sciences Appliquées (INSA)-Centre National de la Recherche Scientifique (CNRS)-Université de Rouen Normandie (UNIROUEN), Institut National des Sciences Appliquées (INSA)-Normandie Université (NU)-Institut National des Sciences Appliquées (INSA)-Centre National de la Recherche Scientifique (CNRS)-Institut Normand de Chimie Moléculaire Médicinale et Macromoléculaire (INC3M), Université de Caen Normandie (UNICAEN), Normandie Université (NU)-Normandie Université (NU)-École Nationale Supérieure d'Ingénieurs de Caen (ENSICAEN), Normandie Université (NU)-Université Le Havre Normandie (ULH), Normandie Université (NU)-Université de Rouen Normandie (UNIROUEN), Normandie Université (NU)-Institut national des sciences appliquées Rouen Normandie (INSA Rouen Normandie), Institut National des Sciences Appliquées (INSA)-Normandie Université (NU)-Institut National des Sciences Appliquées (INSA)-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS)-Université de Caen Normandie (UNICAEN), Normandie Université (NU)-École Nationale Supérieure d'Ingénieurs de Caen (ENSICAEN), Normandie Université (NU)-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS)-Centre National de la Recherche Scientifique (CNRS), Centre d'études prospectives et d'informations internationales (CEPII), Laboratoire d'Electrochimie et de Chimie Analytique (LECA), Université Pierre et Marie Curie - Paris 6 (UPMC)-Ecole Nationale Supérieure de Chimie de Paris - Chimie ParisTech-PSL (ENSCP), Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-Centre National de la Recherche Scientifique (CNRS), 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 de Recherche Biomédicale des Armées [Brétigny-sur-Orge] (IRBA), Institut Normand de Chimie Moléculaire Médicinale et Macromoléculaire (INC3M), Institut de Chimie du CNRS (INC)-École Nationale Supérieure d'Ingénieurs de Caen (ENSICAEN), Institut National des Sciences Appliquées (INSA)-Normandie Université (NU)-Institut National des Sciences Appliquées (INSA)-Université Le Havre Normandie (ULH), Normandie Université (NU)-Centre National de la Recherche Scientifique (CNRS)-Université de Caen Normandie (UNICAEN), Normandie Université (NU)-Institut de Chimie du CNRS (INC)-École Nationale Supérieure d'Ingénieurs de Caen (ENSICAEN), Normandie Université (NU)-Centre National de la Recherche Scientifique (CNRS)-Institut de Chimie Organique Fine (IRCOF), Institut National des Sciences Appliquées (INSA)-Normandie Université (NU)-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), 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 Institut de Recherche Biomédicale des Armées (IRBA)
Subjects: Models, Molecular, Denticity, Stereochemistry, Ligands, Biochemistry, chemistry.chemical_compound, Humans, [CHIM]Chemical Sciences, Physical and Theoretical Chemistry, ComputingMilieux_MISCELLANEOUS, chemistry.chemical_classification, Molecular Structure, Chemistry, Organic Chemistry, Regioselectivity, Stereoisomerism, Combinatorial chemistry, Acetylcholinesterase, Cycloaddition, Recombinant Proteins, Enzyme, Biological target, Cyclization, Click chemistry, Aminoquinolines, Click Chemistry, Cholinesterase Inhibitors, Selectivity
Abstract: The enzyme-directed synthesis is an emerging fragment-based lead discovery approach in which the biological target is able to assemble its own multidentate ligands from a pool of building blocks. Here, we report for the first time the use of the human acetylcholinesterase (AChE) as an enzyme for the design and synthesis of new potent heterodimeric huprine-based inhibitors. Both the specific click chemistry site within the protein and the regioselectivity of the Huisgen cycloaddition observed suggest promising alternatives in the design of efficient mono- and dimeric ligands of AChE. Finally, a detailed computational modelling of the click reaction was conducted to further understand the origin of this TGS selectivity.
Published: 2014

50. Correlation of the dynamics of native human acetylcholinesterase and its inhibited huperzine A counterpart from sub-picoseconds to nanoseconds

Author: Judith Peters, Florian Nachon, Patrick Masson, Marcus Trapp, Moeava Tehei, M M Koza, Martin Weik, Marie Trovaslet, N Martinez, Thomas, Frank, Institute of human genetics, Medical University Graz, Biocomplexité des écosystèmes coralliens de l'Indo-Pacifique (CoReUS2), 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), Institut Laue-Langevin (ILL), ILL, 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: MESH: Enzyme Activation, [SDV.BBM.BS] Life Sciences [q-bio]/Biochemistry, Molecular Biology/Structural Biology [q-bio.BM], Statistics as Topic, Biomedical Engineering, Biophysics, Thermal fluctuations, Bioengineering, Neutron scattering, Molecular Dynamics Simulation, Biochemistry, Inelastic neutron scattering, Catalysis, Biomaterials, Molecular dynamics, Alkaloids, MESH: Alkaloids, Humans, MESH: Protein Binding, MESH: Molecular Dynamics Simulation, Research Articles, MESH: Statistics as Topic, Binding Sites, MESH: Humans, MESH: Kinetics, [SDV.BBM.BS]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Structural Biology [q-bio.BM], Chemistry, Relaxation (NMR), MESH: Models, Chemical, Nanosecond, MESH: Acetylcholinesterase, MESH: Catalysis, Enzyme Activation, Crystallography, Kinetics, Models, Chemical, MESH: Binding Sites, Chemical physics, Picosecond, Density of states, Acetylcholinesterase, MESH: Sesquiterpenes, Cholinesterase Inhibitors, MESH: Cholinesterase Inhibitors, Sesquiterpenes, Biotechnology, Protein Binding
Abstract: It is a long debated question whether catalytic activities of enzymes, which lie on the millisecond timescale, are possibly already reflected in variations in atomic thermal fluctuations on the pico- to nanosecond timescale. To shed light on this puzzle, the enzyme human acetylcholinesterase in its wild-type form and complexed with the inhibitor huperzine A were investigated by various neutron scattering techniques and molecular dynamics simulations. Previous results on elastic neutron scattering at various timescales and simulations suggest that dynamical processes are not affected on average by the presence of the ligand within the considered time ranges between 10 ps and 1 ns. In the work presented here, the focus was laid on quasi-elastic (QENS) and inelastic neutron scattering (INS). These techniques give access to different kinds of individual diffusive motions and to the density of states of collective motions at the sub-picoseconds timescale. Hence, they permit going beyond the first approach of looking at mean square displacements. For both samples, the autocorrelation function was well described by a stretched-exponential function indicating a linkage between the timescales of fast and slow functional relaxation dynamics. The findings of the QENS and INS investigation are discussed in relation to the results of our earlier elastic incoherent neutron scattering and molecular dynamics simulations.
Published: 2014

Catalog

Books, media, physical & digital resources

See catalog results

Searchworks

Select search scope, currently: Articles Catalog books, media & more in Jio Institute collections Articles journal articles & other e-resources

Search

Search Constraints

Refine your results

Search Limiters

Topic

Publication Year Range

Language

Publication Type

Journal

Database

Publisher

77 results on '"Martin Weik"'

Search Results

Catalog

Select search scope, currently: Articles

Catalog

books, media & more in Jio Institute collections

Articles

journal articles & other e-resources