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1. Crystal structure of a bacterial photoactivated adenylate cyclase determined by serial femtosecond and serial synchrotron crystallography

Author: Sofia M. Kapetanaki, Nicolas Coquelle, David von Stetten, Martin Byrdin, Ronald Rios-Santacruz, Richard Bean, Johan Bielecki, Mohamed Boudjelida, Zsuzsana Fekete, Geoffrey W. Grime, Huijong Han, Caitlin Hatton, Sravya Kantamneni, Konstantin Kharitonov, Chan Kim, Marco Kloos, Faisal H. M. Koua, Iñaki de Diego Martinez, Diogo Melo, Lukas Rane, Adam Round, Ekaterina Round, Abhisakh Sarma, Robin Schubert, Joachim Schulz, Marcin Sikorski, Mohammad Vakili, Joana Valerio, Jovana Vitas, Raphael de Wijn, Agnieszka Wrona, Ninon Zala, Arwen Pearson, Katerina Dörner, Giorgio Schirò, Elspeth F. Garman, András Lukács, and Martin Weik
Subjects: bacterial adenylate cyclase, bluf domain, ac domain, serial femtosecond crystallography, xfels, synchrotrons, room-temperature x-ray crystallography, cryo-macromolecular crystallography, protein structure, light-activated enzymes, oscillatoria acuminata, Crystallography, QD901-999
Abstract: OaPAC is a recently discovered blue-light-using flavin adenosine dinucleotide (BLUF) photoactivated adenylate cyclase from the cyanobacterium Oscillatoria acuminata that uses adenosine triphosphate and translates the light signal into the production of cyclic adenosine monophosphate. Here, we report crystal structures of the enzyme in the absence of its natural substrate determined from room-temperature serial crystallography data collected at both an X-ray free-electron laser and a synchrotron, and we compare these structures with cryo-macromolecular crystallography structures obtained at a synchrotron by us and others. These results reveal slight differences in the structure of the enzyme due to data collection at different temperatures and X-ray sources. We further investigate the effect of the Y6W mutation in the BLUF domain, a mutation which results in a rearrangement of the hydrogen-bond network around the flavin and a notable rotation of the side chain of the critical Gln48 residue. These studies pave the way for picosecond–millisecond time-resolved serial crystallography experiments at X-ray free-electron lasers and synchrotrons in order to determine the early structural intermediates and correlate them with the well studied picosecond–millisecond spectroscopic intermediates.
Published: 2024
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2. Photocobilins integrate B12 and bilin photochemistry for enzyme control

Author: Shaowei Zhang, Laura N. Jeffreys, Harshwardhan Poddar, Yuqi Yu, Chuanyang Liu, Kaylee Patel, Linus O. Johannissen, Lingyun Zhu, Matthew J. Cliff, Cunyu Yan, Giorgio Schirò, Martin Weik, Michiyo Sakuma, Colin W. Levy, David Leys, Derren J. Heyes, and Nigel S. Scrutton
Subjects: Science
Abstract: Abstract Photoreceptor proteins utilise chromophores to sense light and trigger a biological response. The discovery that adenosylcobalamin (or coenzyme B12) can act as a light-sensing chromophore heralded a new field of B12-photobiology. Although microbial genome analysis indicates that photoactive B12-binding domains form part of more complex protein architectures, regulating a range of molecular–cellular functions in response to light, experimental evidence is lacking. Here we identify and characterise a sub-family of multi-centre photoreceptors, termed photocobilins, that use B12 and biliverdin (BV) to sense light across the visible spectrum. Crystal structures reveal close juxtaposition of the B12 and BV chromophores, an arrangement that facilitates optical coupling. Light-triggered conversion of the B12 affects quaternary structure, in turn leading to light-activation of associated enzyme domains. The apparent widespread nature of photocobilins implies involvement in light regulation of a wider array of biochemical processes, and thus expands the scope for B12 photobiology. Their characterisation provides inspiration for the design of broad-spectrum optogenetic tools and next generation bio-photocatalysts.
Published: 2024
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3. 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
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4. Redox driven B12-ligand switch drives CarH photoresponse

Author: Harshwardhan Poddar, Ronald Rios-Santacruz, Derren J. Heyes, Muralidharan Shanmugam, Adam Brookfield, Linus O. Johannissen, Colin W. Levy, Laura N. Jeffreys, Shaowei Zhang, Michiyo Sakuma, Jacques-Philippe Colletier, Sam Hay, Giorgio Schirò, Martin Weik, Nigel S. Scrutton, and David Leys
Subjects: Science
Abstract: Abstract CarH is a coenzyme B12-dependent photoreceptor involved in regulating carotenoid biosynthesis. How light-triggered cleavage of the B12 Co-C bond culminates in CarH tetramer dissociation to initiate transcription remains unclear. Here, a series of crystal structures of the CarH B12-binding domain after illumination suggest formation of unforeseen intermediate states prior to tetramer dissociation. Unexpectedly, in the absence of oxygen, Co-C bond cleavage is followed by reorientation of the corrin ring and a switch from a lower to upper histidine-Co ligation, corresponding to a pentacoordinate state. Under aerobic conditions, rapid flash-cooling of crystals prior to deterioration upon illumination confirm a similar B12-ligand switch occurs. Removal of the upper His-ligating residue prevents monomer formation upon illumination. Combined with detailed solution spectroscopy and computational studies, these data demonstrate the CarH photoresponse integrates B12 photo- and redox-chemistry to drive large-scale conformational changes through stepwise Co-ligation changes.
Published: 2023
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5. De novo determination of mosquitocidal Cry11Aa and Cry11Ba structures from naturally-occurring nanocrystals

Author: Guillaume Tetreau, Michael R. Sawaya, Elke De Zitter, Elena A. Andreeva, Anne-Sophie Banneville, Natalie A. Schibrowsky, Nicolas Coquelle, Aaron S. Brewster, Marie Luise Grünbein, Gabriela Nass Kovacs, Mark S. Hunter, Marco Kloos, Raymond G. Sierra, Giorgio Schiro, Pei Qiao, Myriam Stricker, Dennis Bideshi, Iris D. Young, Ninon Zala, Sylvain Engilberge, Alexander Gorel, Luca Signor, Jean-Marie Teulon, Mario Hilpert, Lutz Foucar, Johan Bielecki, Richard Bean, Raphael de Wijn, Tokushi Sato, Henry Kirkwood, Romain Letrun, Alexander Batyuk, Irina Snigireva, Daphna Fenel, Robin Schubert, Ethan J. Canfield, Mario M. Alba, Frédéric Laporte, Laurence Després, Maria Bacia, Amandine Roux, Christian Chapelle, François Riobé, Olivier Maury, Wai Li Ling, Sébastien Boutet, Adrian Mancuso, Irina Gutsche, Eric Girard, Thomas R. M. Barends, Jean-Luc Pellequer, Hyun-Woo Park, Arthur D. Laganowsky, Jose Rodriguez, Manfred Burghammer, Robert L. Shoeman, R. Bruce Doak, Martin Weik, Nicholas K. Sauter, Brian Federici, Duilio Cascio, Ilme Schlichting, and Jacques-Philippe Colletier
Subjects: Science
Abstract: An environmentally safe means of mosquito control is the application of Bacillus thuringiensis israelensis, which produces a cocktail of four naturally crystalline proteins exclusively toxic to mosquito. Here the authors report the atomic-resolution structures of Bti Cry11Aa and related Btj Cry11Ba solved de novo through Serial Femtosecond Crystallography on naturally-occurring nanocrystals.
Published: 2022
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6. Serial femtosecond crystallography on in vivo-grown crystals drives elucidation of mosquitocidal Cyt1Aa bioactivation cascade

Author: Guillaume Tetreau, Anne-Sophie Banneville, Elena A. Andreeva, Aaron S. Brewster, Mark S. Hunter, Raymond G. Sierra, Jean-Marie Teulon, Iris D. Young, Niamh Burke, Tilman A. Grünewald, Joël Beaudouin, Irina Snigireva, Maria Teresa Fernandez-Luna, Alister Burt, Hyun-Woo Park, Luca Signor, Jayesh A. Bafna, Rabia Sadir, Daphna Fenel, Elisabetta Boeri-Erba, Maria Bacia, Ninon Zala, Frédéric Laporte, Laurence Després, Martin Weik, Sébastien Boutet, Martin Rosenthal, Nicolas Coquelle, Manfred Burghammer, Duilio Cascio, Michael R. Sawaya, Mathias Winterhalter, Enrico Gratton, Irina Gutsche, Brian Federici, Jean-Luc Pellequer, Nicholas K. Sauter, and Jacques-Philippe Colletier
Subjects: Science
Abstract: 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
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7. Photoswitching mechanism of a fluorescent protein revealed by time-resolved crystallography and transient absorption spectroscopy

Author: Joyce Woodhouse, Gabriela Nass Kovacs, Nicolas Coquelle, Lucas M. Uriarte, Virgile Adam, Thomas R. M. Barends, Martin Byrdin, Eugenio de la Mora, R. Bruce Doak, Mikolaj Feliks, Martin Field, Franck Fieschi, Virginia Guillon, Stefan Jakobs, Yasumasa Joti, Pauline Macheboeuf, Koji Motomura, Karol Nass, Shigeki Owada, Christopher M. Roome, Cyril Ruckebusch, Giorgio Schirò, Robert L. Shoeman, Michel Thepaut, Tadashi Togashi, Kensuke Tono, Makina Yabashi, Marco Cammarata, Lutz Foucar, Dominique Bourgeois, Michel Sliwa, Jacques-Philippe Colletier, Ilme Schlichting, and Martin Weik
Subjects: Science
Abstract: 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
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8. Ligand pathways in neuroglobin revealed by low-temperature photodissociation and docking experiments

Author: Chiara Ardiccioni, Alessandro Arcovito, Stefano Della Longa, Peter van der Linden, Dominique Bourgeois, Martin Weik, Linda Celeste Montemiglio, Carmelinda Savino, Giovanna Avella, Cécile Exertier, Philippe Carpentier, Thierry Prangé, Maurizio Brunori, Nathalie Colloc'h, and Beatrice Vallone
Subjects: ultralow-temperature X-ray crystallography, XANES, heme protein, neuroglobin, oxygen binding, cryo-trapping, crystal microspectroscopy, neuroprotection, structural biology, CO photolysis, soak-and-freeze pressurization, structure determination, protein structure, Crystallography, QD901-999
Abstract: 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
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9. 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
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10. Megahertz data collection from protein microcrystals at an X-ray free-electron laser

Author: Marie Luise Grünbein, Johan Bielecki, Alexander Gorel, Miriam Stricker, Richard Bean, Marco Cammarata, Katerina Dörner, Lars Fröhlich, Elisabeth Hartmann, Steffen Hauf, Mario Hilpert, Yoonhee Kim, Marco Kloos, Romain Letrun, Marc Messerschmidt, Grant Mills, Gabriela Nass Kovacs, Marco Ramilli, Christopher M. Roome, Tokushi Sato, Matthias Scholz, Michel Sliwa, Jolanta Sztuk-Dambietz, Martin Weik, Britta Weinhausen, Nasser Al-Qudami, Djelloul Boukhelef, Sandor Brockhauser, Wajid Ehsan, Moritz Emons, Sergey Esenov, Hans Fangohr, Alexander Kaukher, Thomas Kluyver, Max Lederer, Luis Maia, Maurizio Manetti, Thomas Michelat, Astrid Münnich, Florent Pallas, Guido Palmer, Gianpietro Previtali, Natascha Raab, Alessandro Silenzi, Janusz Szuba, Sandhya Venkatesan, Krzysztof Wrona, Jun Zhu, R. Bruce Doak, Robert L. Shoeman, Lutz Foucar, Jacques-Philippe Colletier, Adrian P. Mancuso, Thomas R. M. Barends, Claudiu A. Stan, and Ilme Schlichting
Subjects: Science
Abstract: The European X-ray free-electron laser (EuXFEL) in Hamburg is the first megahertz (MHz) repetition rate XFEL. Here the authors use lysozyme crystals and microcrystals from jack bean proteins and demonstrate that damage-free high quality data can be collected at a MHz repetition rate.
Published: 2018
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11. Radiation damage to biological samples: still a pertinent issue

Author: Martin Weik and Elspeth F. Garman
Subjects: Nuclear and High Energy Physics, single-wavelength anomalous dispersion, Materials science, X-ray footprinting, Synchrotron radiation, Electron, law.invention, macromolecular crystallography, law, Radiation damage, serial crystallography, Instrumentation, Radiation, electron microscopy, Scattering, X-ray imaging, dose, SAXS, Synchrotron, Computational physics, Beamline, radiation damage, XFEL simulations, Absorbed dose, Radiolysis, room-temperature crystallography
Abstract: An understanding of radiation damage effects suffered by biological samples during structural analysis using both X-rays and electrons is pivotal to obtain reliable molecular models of imaged molecules. This special issue on radiation damage contains six papers reporting analyses of damage from a range of biophysical imaging techniques., An understanding of radiation damage effects suffered by biological samples during structural analysis using both X-rays and electrons is pivotal to obtain reliable molecular models of imaged molecules. This special issue on radiation damage contains six papers reporting analyses of damage from a range of biophysical imaging techniques. For X-ray diffraction, an in-depth study of multi-crystal small-wedge data collection single-wavelength anomalous dispersion phasing protocols is presented, concluding that an absorbed dose of 5 MGy per crystal was optimal to allow reliable phasing. For small-angle X-ray scattering, experiments are reported that evaluate the efficacy of three radical scavengers using a protein designed to give a clear signature of damage in the form of a large conformational change upon the breakage of a disulfide bond. The use of X-rays to induce OH radicals from the radiolysis of water for X-ray footprinting are covered in two papers. In the first, new developments and the data collection pipeline at the NSLS-II high-throughput dedicated synchrotron beamline are described, and, in the second, the X-ray induced changes in three different proteins under aerobic and low-oxygen conditions are investigated and correlated with the absorbed dose. Studies in XFEL science are represented by a report on simulations of ultrafast dynamics in protic ionic liquids, and, lastly, a broad coverage of possible methods for dose efficiency improvement in modalities using electrons is presented. These papers, as well as a brief synopsis of some other relevant literature published since the last Journal of Synchrotron Radiation Special Issue on Radiation Damage in 2019, are summarized below.
Published: 2021

12. 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

13. 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

14. Structural information about the $trans-to-cis$ isomerization mechanism of the photoswitchable fluorescent protein rsEGFP2 revealed by multiscale infrared transient absorption

Author: Lucas M. Uriarte, Raffaele Vitale, Stanisław Niziński, Kyprianos Hadjidemetriou, Ninon Zala, Andras Lukacs, Gregory M. Greetham, Igor V. Sazanovich, Martin Weik, Cyril Ruckebusch, Stephen R. Meech, Michel Sliwa, 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), 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 Biophysics, Medical School, Central Laser Facility (CLF), STFC Rutherford Appleton Laboratory (RAL), Science and Technology Facilities Council (STFC)-Science and Technology Facilities Council (STFC), University of East Anglia [Norwich] (UEA), and 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)
Subjects: MESH: Protein Conformation, [SDV.BBM.BS]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Structural Biology [q-bio.BM], MESH: Spectrophotometry, Infrared, MESH: Isomerism, General Materials Science, MESH: Luminescent Proteins, Physical and Theoretical Chemistry, MESH: Benzylidene Compounds, MESH: Imidazoles
Abstract: International audience; RsEGFP2 is a reversibly photoswitchable fluorescent protein used in super-resolved optical microscopies, which can be toggled between a fluorescent On state and a nonfluorescent Off state. Previous time-resolved ultraviolet-visible spectroscopic studies have shown that the Off-to-On photoactivation extends over the femto- to millisecond time scale and involves two picosecond lifetime excited states and four ground state intermediates, reflecting a trans-to-cis excited state isomerization, a millisecond deprotonation, and protein structural reorganizations. Femto- to millisecond time-resolved multiple-probe infrared spectroscopy (TRMPS-IR) can reveal structural aspects of intermediate species. Here we apply TRMPS-IR to rsEGFP2 and implement a Savitzky-Golay derivative analysis to correct for baseline drift. The results reveal that a subpicosecond twisted excited state precursor controls the trans-to-cis isomerization and the chromophore reaches its final position in the protein pocket within 100 ps. A new step with a time constant of 42 ns is reported and assigned to structural relaxation of the protein that occurs prior to the deprotonation of the chromophore on the millisecond time scale.
Published: 2022

15. A guide to time-resolved structural analysis of light-activated proteins

Author: Harshwardhan Poddar, David Leys, Derren J. Heyes, Nigel S. Scrutton, Martin Weik, Giorgio Schirò, University of Manchester [Manchester], 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)), and Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Grenoble Alpes (UGA)
Subjects: 0301 basic medicine, Models, Molecular, Materials science, Time Factors, Light, Protein Conformation, Crystallography, X-Ray, time-resolved, Biochemistry, law.invention, 03 medical and health sciences, 0302 clinical medicine, Protein structure, X-Ray Diffraction, law, Manchester Institute of Biotechnology, TR-SFX, Molecular Biology, photoenzymes, [SDV.BBM.BS]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Structural Biology [q-bio.BM], Scattering, Small-angle X-ray scattering, Protein dynamics, Lasers, Proteins, photoreceptors, Cell Biology, SAXS, Chromophore, Laser, ResearchInstitutes_Networks_Beacons/manchester_institute_of_biotechnology, 030104 developmental biology, Structural change, light-activated proteins, WAXS, 030220 oncology & carcinogenesis, Femtosecond, protein dynamics, Biophysics, sense organs
Abstract: International audience; Dynamical changes in protein structures are essential for protein function and occur over femtoseconds to seconds timescales. X-ray free electron lasers have facilitated investigations of structural dynamics in proteins with unprecedented temporal and spatial resolution. Light-activated proteins are attractive targets for time-resolved structural studies, as the reaction chemistry and associated protein structural changes can be triggered by short laser pulses. Proteins with different light-absorbing centres have evolved to detect light and harness photon energy to bring about downstream chemical and biological output responses. Following light absorption, rapid chemical/small-scale structural changes are typically localised around the chromophore. These localised changes are followed by larger structural changes propagated throughout the photoreceptor/photocatalyst that enables the desired chemical and/or biological output response. Time-resolved serial femtosecond crystallography (SFX) and solution scattering techniques enable direct visualisation of early chemical change in light-activated proteins on timescales previously inaccessible, whereas scattering gives access to slower timescales associated with more global structural change. Here, we review how advances in time-resolved SFX and solution scattering techniques have uncovered mechanisms of photochemistry and its coupling to output responses. We also provide a prospective on how these time-resolved structural approaches might impact on other photoreceptors/photoenzymes that have not yet been studied by these methods.
Published: 2022

16. De novo determination of mosquitocidal Cry11Aa and Cry11Ba structures from naturally-occurring nanocrystals

Author: Guillaume Tetreau, Michael R. Sawaya, Elke De Zitter, Elena A. Andreeva, Anne-Sophie Banneville, Natalie Schibrowsky, Nicolas Coquelle, Aaron S. Brewster, Marie Luise Grünbein, Gabriela Nass Kovacs, Mark S. Hunter, Marco Kloos, Raymond G. Sierra, Giorgio Schiro, Pei Qiao, Myriam Stricker, Dennis Bideshi, Iris D. Young, Ninon Zala, Sylvain Engilberge, Alexander Gorel, Luca Signor, Jean-Marie Teulon, Mario Hilpert, Lutz Foucar, Johan Bielecki, Richard Bean, Raphael de Wijn, Tokushi Sato, Henry Kirkwood, Romain Letrun, Alexander Batyuk, Irina Snigireva, Daphna Fenel, Robin Schubert, Ethan J. Canfield, Mario M. Alba, Frédéric Laporte, Laurence Després, Maria Bacia, Amandine Roux, Christian Chapelle, François Riobé, Olivier Maury, Wai Li Ling, Sébastien Boutet, Adrian Mancuso, Irina Gutsche, Eric Girard, Thomas R. M. Barends, Jean-Luc Pellequer, Hyun-Woo Park, Arthur D. Laganowsky, Jose Rodriguez, Manfred Burghammer, Robert L. Shoeman, R. Bruce Doak, Martin Weik, Nicholas K. Sauter, Brian Federici, Duilio Cascio, Ilme Schlichting, and Jacques-Philippe Colletier
Abstract: Cry11Aa and Cry11Ba are the two most potent toxins produced by mosquitocidal Bacillus thuringiensis subsp. israelensis and jegathesan, respectively. The toxins naturally crystallize within the host; however, the crystals are too small for structure determination at synchrotron sources. Therefore, we applied serial femtosecond crystallography at X-ray free electron lasers to in vivo-grown nanocrystals of these toxins. The structure of Cry11Aa was determined de novo using the single-wavelength anomalous dispersion method, which in turn enabled the determination of the Cry11Ba structure by molecular replacement. The two structures reveal a new pattern for in vivo crystallization of Cry toxins, whereby each of their three domains packs with a symmetrically identical domain, and a cleavable crystal packing motif is located within the protoxin rather than at the termini. The diversity of in vivo crystallization patterns suggests explanations for their varied levels of toxicity and rational approaches to improve these toxins for mosquito control.
Published: 2021

17. Insight into the structural dynamics of light sensitive proteins from time-resolved crystallography and quantum chemical calculations

Author: Martin Weik and Tatiana Domratcheva
Subjects: Structural Biology, Molecular Biology
Abstract: The structural dynamics underlying molecular mechanisms of light-sensitive proteins can be studied by a variety of experimental and computational biophysical techniques. Here we review recent progress in combining time-resolved crystallography at X-ray free electron lasers and quantum chemical calculations to study structural changes in photoenzymes, photosynthetic proteins, photoreceptors, and photoswitchable fluorescent proteins following photoexcitation.
Published: 2022

18. X-ray radiation damage to biological samples: recent progress

Author: Martin Weik and Elspeth F. Garman
Subjects: 0303 health sciences, Nuclear and High Energy Physics, Radiation, X-Rays, 030303 biophysics, Bremsstrahlung, X-ray, Synchrotron radiation, DNA, Particle detector, Computational physics, 03 medical and health sciences, X-Ray Diffraction, Scattering, Small Angle, X-ray crystallography, Cadmium Compounds, Radiation damage, Irradiation, Tellurium, Small-angle scattering, Crystallization, Instrumentation, Synchrotrons, DNA Damage, 030304 developmental biology
Abstract: With the continuing development of beamlines for macromolecular crystallography (MX) over the last few years providing ever higher X-ray flux densities, it has become even more important to be aware of the effects of radiation damage on the resulting structures. Nine papers in this issue cover a range of aspects related to the physics and chemistry of the manifestations of this damage, as observed in both MX and small-angle X-ray scattering (SAXS) on crystals, solutions and tissue samples. The reports include measurements of the heating caused by X-ray irradiation in ruby microcrystals, low-dose experiments examining damage rates as a function of incident X-ray energy up to 30 keV on a metallo-enzyme using a CdTe detector of high quantum efficiency as well as a theoretical analysis of the gains predicted in diffraction efficiency using these detectors, a SAXS examination of low-dose radiation exposure effects on the dissociation of a protein complex related to human health, theoretical calculations describing radiation chemistry pathways which aim to explain the specific structural damage widely observed in proteins, investigation of radiation-induced damage effects in a DNA crystal, a case study on a metallo-enzyme where structural movements thought to be mechanism related might actually be radiation-damage-induced changes, and finally a review describing what X-ray radiation-induced cysteine modifications can teach us about protein dynamics and catalysis. These papers, along with some other relevant literature published since the last Journal of Synchrotron Radiation Radiation Damage special issue in 2017, are briefly summarized below.
Published: 2019

19. 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

20. 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

21. High-resolution structure and reaction cycle of fatty acid photodecarboxylase: anatomy of a crime scene

Author: Damien Sorigué, Kyprianos Hadjidemetriou, Stéphanie Blangy, Guillaume Gotthard, Pierre Legrand, Didier Nurizzo, Antoine Royant, Catherine Berthomieu, Martin Weik, Tatiana Domratcheva, Klaus Brettel, Martin H. Vos, Ilme Schlichting, Pavel Müller, Fred Beisson, and Pascal Arnoux
Subjects: Inorganic Chemistry, Structural Biology, General Materials Science, Physical and Theoretical Chemistry, Condensed Matter Physics, Biochemistry
Abstract: Fatty Acid Photodecarboxylase (FAP) is a recently discovered photoenzyme that catalyzes the conversion of fatty acids into alkane and CO2 under light, with potential importance in green chemistry applications [1]. Its mechanism was still not fully understood and partly relied on a low-resolution crystal structure obtained from crystals with a twinning default [1]. Here, we present high-resolution crystal structures of FAP obtained in the dark and after light illumination at cryogenic temperatures (Figure 1). Combined with structural, computational, and spectroscopic techniques we are now able to provide a detailed reaction cycle of FAP. The reaction mechanism starts with an 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. Along with flavin reoxidation by the alkyl radical intermediate, a major fraction of the cleaved CO2 unexpectedly transforms in 100 ns, most likely into bicarbonate. This is orders of magnitude faster than in solution, which indicates a catalytic step. FT-IR, structural and functional studies on variants centered on two conserved active site residues (R451 and C432) showed that R451 is essential for substrate stabilization and proton transfer. Altogether this study provides a detailed characterization of this unique enzyme and reveals a striking and unanticipated mechanistic complexity [2].
Published: 2021

22. 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

23. 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

24. Radiation damage and dose limits in serial synchrotron crystallography at cryo- and room temperatures

Author: James M. Holton, Charles S. Bury, Ian Carmichael, Jacques-Philippe Colletier, Nicolas Coquelle, Eugenio de la Mora, Martin Rosenthal, Elspeth F. Garman, Manfred Burghammer, Martin Weik, 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 Laue-Langevin (ILL), 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), European Synchrotron Radiation Facility (ESRF), University of California (UC), Notre Dame Radiation Laboratory, University of Notre Dame [Indiana] (UND), University of Oxford, 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), Department of Biochemistry, Glycobiology Institute, University of Oxford, Oxford, Institut de Science des Matériaux de Mulhouse (IS2M), Centre National de la Recherche Scientifique (CNRS)-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)-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), Department of Biochemistry and Biophysics [San Francisco], and University of California
Subjects: Diffraction, Materials science, [SDV]Life Sciences [q-bio], 030303 biophysics, Crystallography, X-Ray, law.invention, 03 medical and health sciences, law, Energy absorbing, Radiation damage, [CHIM]Chemical Sciences, serial crystallography, 030304 developmental biology, 0303 health sciences, Multidisciplinary, [SDV.BBM.BS]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Structural Biology [q-bio.BM], Resolution (electron density), Temperature, Biological Sciences, Synchrotron, X-ray radiation damage, Crystallography, Biophysics and Computational Biology, PNAS Plus, room temperature synchrotron data collection, Muramidase, Dose rate, Crystallization, Beam (structure), Synchrotrons, Macromolecule
Abstract: Significance Macromolecular X-ray crystallography (MX) is the most prolific structure determination method in structural biology but is limited by radiation damage. To reduce damage progression, MX is usually carried out at cryogenic temperatures, sometimes blocking functionally important conformational heterogeneity. Lacking this shortcoming, room temperature MX has gained momentum with the recent advent of serial crystallography, whereby distribution of the X-ray dose over thousands of crystals mitigates damage. Here, an approach to serial crystallography is presented allowing visualization of specific damage to amino acids at room temperature and determination of a dose limit above which structural information from electron density maps decreases due to radiation damage. This limit provides important guidance for the growing number of synchrotron room temperature MX experiments., Radiation damage limits the accuracy of macromolecular structures in X-ray crystallography. Cryogenic (cryo-) cooling reduces the global radiation damage rate and, therefore, became the method of choice over the past decades. The recent advent of serial crystallography, which spreads the absorbed energy over many crystals, thereby reducing damage, has rendered room temperature (RT) data collection more practical and also extendable to microcrystals, both enabling and requiring the study of specific and global radiation damage at RT. Here, we performed sequential serial raster-scanning crystallography using a microfocused synchrotron beam that allowed for the collection of two series of 40 and 90 full datasets at 2- and 1.9-Å resolution at a dose rate of 40.3 MGy/s on hen egg white lysozyme (HEWL) crystals at RT and cryotemperature, respectively. The diffraction intensity halved its initial value at average doses (D1/2) of 0.57 and 15.3 MGy at RT and 100 K, respectively. Specific radiation damage at RT was observed at disulfide bonds but not at acidic residues, increasing and then apparently reversing, a peculiar behavior that can be modeled by accounting for differential diffraction intensity decay due to the nonuniform illumination by the X-ray beam. Specific damage to disulfide bonds is evident early on at RT and proceeds at a fivefold higher rate than global damage. The decay modeling suggests it is advisable not to exceed a dose of 0.38 MGy per dataset in static and time-resolved synchrotron crystallography experiments at RT. This rough yardstick might change for proteins other than HEWL and at resolutions other than 2 Å.
Published: 2020

25. 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

26. 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

27. 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

28. MHz data collection of a microcrystalline mixture of different jack bean proteins

Author: Gabriela Nass Kovacs, Djelloul Boukhelef, Alexander Kaukher, Sandhya Venkatesan, Astrid Münnich, Michel Sliwa, Nasser Al-Qudami, Jolanta Sztuk-Dambietz, Sergey Esenov, Lutz Foucar, A. Gorel, Krzysztof Wrona, Janusz Szuba, Maurizio Manetti, Thomas Kluyver, Marco Cammarata, Jun Zhu, Johan Bielecki, Matthias Scholz, Marc Messerschmidt, Richard Bean, Gianpietro Previtali, Thomas Michelat, Sandor Brockhauser, Natascha Raab, M. Hilpert, Thomas R. M. Barends, Alessandro Silenzi, Katerina Dörner, Moritz Emons, Yoonhee Kim, Grant Mills, C.M. Roome, Luis Maia, Max Lederer, Jacques-Philippe Colletier, Wajid Ehsan, Steffen Hauf, Britta Weinhausen, Marco Ramilli, Marco Kloos, Elisabeth Hartmann, Romain Letrun, Claudiu A. Stan, Florent Pallas, Marie Luise Grünbein, R. Bruce Doak, Martin Weik, Lars Fröhlich, M. Stricker, Robert L. Shoeman, Guido Palmer, Tokushi Sato, Hans Fangohr, Ilme Schlichting, Adrian P. Mancuso, European XFEL GmbH (XFEL), European XFEL GmbH, Max Planck Institute for Medical Research [Heidelberg], Max-Planck-Gesellschaft, Institut de Physique de Rennes (IPR), Université de Rennes (UR)-Centre National de la Recherche Scientifique (CNRS), Elettra Sincrotrone Trieste, National Science Foundation [Arlington] (NSF), Alma Mater Studiorum Università di Bologna [Bologna] (UNIBO), KEK (High energy accelerator research organization), TOPTICA Photonics AG, 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), 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), European Molecular Biology Laboratory [Grenoble] (EMBL), Institut de Microélectronique, Electromagnétisme et Photonique (IMEP), Université Joseph Fourier - Grenoble 1 (UJF)-Institut National Polytechnique de Grenoble (INPG)-Centre National de la Recherche Scientifique (CNRS), NINGBO UNIVERSITY, Ningbo University (NBU), Department of Biomolecular Mechanisms, Max-Planck-Gesellschaft-Max-Planck-Gesellschaft, Rutgers University [Newark], Rutgers University System (Rutgers), Deutsches Elektronen-Synchrotron, ANR-17-CE11-0018-01, Agence Nationale de la Recherche, European XFEL, ANR Grant, European XFEL - travel funding provided by the International Synchrotron Access Program (ISAP) managed by the Australian Synchrotron, part of ANSTO, and funded by the Australian Government, ANR Grant (BioXFEL) CEA, CNRS, Universit&amp, x00E9, Grenoble Alpes, startup funds from Rutgers University Newark, 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), 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), 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 Centre National de la Recherche Scientifique (CNRS)-Institut National Polytechnique de Grenoble (INPG)-Université Joseph Fourier - Grenoble 1 (UJF)
Subjects: Statistics and Probability, Data Descriptor, 010504 meteorology & atmospheric sciences, Computer science, Library and Information Sciences, Crystallography, X-Ray, 01 natural sciences, Education, law.invention, 03 medical and health sciences, Optics, law, Concanavalin A, [CHIM]Chemical Sciences, lcsh:Science, ComputingMilieux_MISCELLANEOUS, 030304 developmental biology, 0105 earth and related environmental sciences, Plant Proteins, [PHYS]Physics [physics], 0303 health sciences, Data collection, business.industry, Nanocrystallography, Detector, Proteins, Frame rate, Laser, Sample (graphics), Urease, Computer Science Applications, Data set, Microcrystalline, Femtosecond, Enzyme mechanisms, lcsh:Q, ddc:500, Statistics, Probability and Uncertainty, business, Crystallization, Information Systems
Abstract: Scientific data 6(1), 18 (2019). doi:10.1038/s41597-019-0010-0, We provide a detailed description of a serial femtosecond crystallography (SFX) dataset collected at the European X-ray free-electron laser facility (EuXFEL). The EuXFEL is the first high repetition rate XFEL delivering MHz X-ray pulse trains at 10 Hz. The short spacing (, Published by Nature Publ. Group, London
Published: 2019

29. Evolution of Protein Quaternary Structure in Response to Selective Pressure for Increased Thermostability

Author: Martin Weik, James M. Murphy, Chris W. Coppin, Jian-Wei Liu, Peter D. Mabbitt, John G. Oakeshott, Mathilde Lethier, Galen J. Correy, Matthew A. Perugini, Nicholas J. Fraser, and Colin J. Jackson
Subjects: 0301 basic medicine, medicine.disease_cause, 03 medical and health sciences, Protein structure, Structural Biology, Hydrolase, medicine, Animals, Amino Acid Sequence, Protein Structure, Quaternary, Molecular Biology, Thermostability, Mutation, Sheep, 030102 biochemistry & molecular biology, biology, Australia, Proteins, biology.organism_classification, Directed evolution, Biological Evolution, 030104 developmental biology, Biochemistry, Thermotoga maritima, Protein quaternary structure, Protein Multimerization, Protein stabilization, Sequence Alignment
Abstract: Oligomerization has been suggested to be an important mechanism for increasing or maintaining the thermostability of proteins. Although it is evident that protein-protein contacts can result in substantial stabilization in many extant proteins, evidence for evolutionary selection for oligomerization is largely indirect and little is understood of the early steps in the evolution of oligomers. A laboratory-directed evolution experiment that selected for increased thermostability in the αE7 carboxylesterase from the Australian sheep blowfly, Lucilia cuprina, resulted in a thermostable variant, LcαE7-4a, that displayed increased levels of dimeric and tetrameric quaternary structure. A trade-off between activity and thermostability was made during the evolution of thermostability, with the higher-order oligomeric species displaying the greatest thermostability and lowest catalytic activity. Analysis of monomeric and dimeric LcαE7-4a crystal structures revealed that only one of the oligomerization-inducing mutations was located at a potential protein-protein interface. This work demonstrates that by imposing a selective pressure demanding greater thermostability, mutations can lead to increased oligomerization and stabilization, providing support for the hypothesis that oligomerization is a viable evolutionary strategy for protein stabilization.
Published: 2016

30. 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

31. Novel multitarget-directed ligands targeting acetylcholinesterase and σ

Author: Julien, Lalut, Gianluca, Santoni, Delphine, Karila, Cédric, Lecoutey, Audrey, Davis, Florian, Nachon, Israel, Silman, Joel, Sussman, Martin, Weik, Tangui, Maurice, Patrick, Dallemagne, and Christophe, Rochais
Subjects: Aniline Compounds, Indoles, Crystallography, X-Ray, Ligands, Torpedo, Serotonin 5-HT4 Receptor Agonists, Piperidines, Alzheimer Disease, Drug Design, Acetylcholinesterase, Animals, Humans, Cholinesterase Inhibitors, Receptors, Serotonin, 5-HT4
Abstract: Pleiotropic intervention may be a requirement for effective limitation of the progression of multifactorial diseases such as Alzheimer's Disease. One approach to such intervention is to design a single chemical entity capable of acting on two or more targets of interest, which are accordingly known as Multi-Target Directed Ligands (MTDLs). We recently described donecopride, the first MTDL able to simultaneously inhibit acetylcholinesterase and act as an agonist of the 5-HT
Published: 2018

32. 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

33. 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

34. Porin self-association enables cell-to-cell contact in

Author: Mariam, El-Khatib, Chady, Nasrallah, Julie, Lopes, Que-Tien, Tran, Guillaume, Tetreau, Hind, Basbous, Daphna, Fenel, Benoit, Gallet, Mathilde, Lethier, Jean-Michel, Bolla, Jean-Marie, Pagès, Michel, Vivaudou, Martin, Weik, Mathias, Winterhalter, and Jacques-Philippe, Colletier
Subjects: PNAS Plus, Biofilms, bacteria, Porins, Providencia, Crystallography, X-Ray, Dimerization, Bacterial Outer Membrane Proteins
Abstract: We report that porins, the main gateway for hydrophilic nutrients and ions into bacterial cells, assemble face-to-face in crystals, yielding dimers of trimers (DOTs). Further investigation suggests that DOTs could support cell-to-cell contact in the floating communities formed by Providencia stuartii, and could enable direct communication between adjacent cells. Self-matching steric zipper interactions are observed at the dimer interface, suggesting a means by which DOT formation could be restricted to cells of the same clonal origin.
Published: 2018

35. 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

36. 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

37. 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

38. Megahertz Data Collection from Protein Microcrystals at an X-Ray Free-Electron Laser

Author: Florent Pallas, Alexander Kaukher, Martin Weik, Richard Bean, Thomas Michelat, Nasser Al-Qudami, Natascha Raab, Guido Palmer, Yoonhee Kim, Grant Mills, M. Hilpert, Gianpietro Previtali, M. Stricker, Lars Fröhlich, Claudiu A. Stan, Tokushi Sato, Maurizio Manetti, Sergey Esenov, Gabriela Nass Kovacs, Thomas R. M. Barends, Katerina Dörner, Matthias Scholz, Marie Luise Grünbein, Jolanta Sztuk-Dambietz, Elisabeth Hartmann, Steffen Hauf, Robert L. Shoeman, A. Gorel, Britta Weinhausen, Janusz Szuba, Thomas Kluyver, Alessandro Silenzi, Astrid Münnich, Michel Sliwa, Djelloul Boukhelef, Marc Messerschmidt, Krzysztof Wrona, Sandhya Venkatesan, Moritz Emons, C.M. Roome, Jacques-Philippe Colletier, Wajid Ehsan, Marco Kloos, Marco Cammarata, Jun Zhu, Lutz Foucar, Luis Maia, Max Lederer, Hans Fangohr, Marco Ramilli, Johan Bielecki, Ilme Schlichting, Sandor Brockhauser, Adrian P. Mancuso, Romain Letrun, R. Bruce Doak, Max Planck Institute for Medical Research [Heidelberg], Max-Planck-Gesellschaft, European XFEL GmbH (XFEL), European XFEL GmbH, 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), Deutsches Elektronensynchrotron DESY, D-22607 Hamburg, Germany, 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), 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), Rutgers University [Newark], Rutgers University System (Rutgers), Université de Rennes (UR)-Centre National de la Recherche Scientifique (CNRS), Institut de Chimie du CNRS (INC)-Université de Lille-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), and 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)
Subjects: 0301 basic medicine, Shock wave, Materials science, Science, General Physics and Astronomy, 02 engineering and technology, General Biochemistry, Genetics and Molecular Biology, Article, law.invention, 03 medical and health sciences, Optics, law, lcsh:Science, [PHYS]Physics [physics], Multidisciplinary, [SDV.BBM.BS]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Structural Biology [q-bio.BM], Femtosecond pulse, business.industry, Free-electron laser, X-ray, General Chemistry, 021001 nanoscience & nanotechnology, Laser, Pulse (physics), 030104 developmental biology, Femtosecond, lcsh:Q, ddc:500, 0210 nano-technology, business, Beam (structure)
Abstract: Nature Communications 9(1), 3487 (2018). doi:10.1038/s41467-018-05953-4, X-ray free-electron lasers (XFELs) enable novel experiments because of their high peak brilliance and femtosecond pulse duration. However, non-superconducting XFELs offer repetition rates of only 10–120 Hz, placing significant demands on beam time and sample consumption. We describe serial femtosecond crystallography experiments performed at the European XFEL, the first MHz repetition rate XFEL, delivering 1.128 MHz X-ray pulse trains at 10 Hz. Given the short spacing between pulses, damage caused by shock waves launched by one XFEL pulse on sample probed by subsequent pulses is a concern. To investigate this issue, we collected data from lysozyme microcrystals, exposed to a ~15 μm XFEL beam. Under these conditions, data quality is independent of whether the first or subsequent pulses of the train were used for data collection. We also analyzed a mixture of microcrystals of jack bean proteins, from which the structure of native, magnesium-containing concanavalin A was determined., Published by Springer Nature, London
Published: 2018

39. Radiation Damage in Macromolecular Crystallography

Author: Elspeth F. Garman and Martin Weik
Subjects: 0301 basic medicine, 030103 biophysics, Nuclear and High Energy Physics, Materials science, Free Radicals, Macromolecular Substances, Protein Conformation, Electrons, Nanotechnology, Crystallography, X-Ray, Radiation Dosage, law.invention, 03 medical and health sciences, law, Fourth generation, Radiation damage, Lasers, X-Rays, Macromolecular crystallography, Proteins, Free Radical Scavengers, Limiting, Engineering physics, Synchrotron, Third generation, Atomic and Molecular Physics, and Optics, 030104 developmental biology, Crystallization, Synchrotrons
Abstract: Interest in radiation damage to macromolecules during structural experiments has a long history dating back to 1962, when the first room-temperature study of the phenomenon was carried out on crystals of myoglobin [1], and this interest has not abated over the last few years, since there remains a need to understand both the parameters that affect radiation damage progression and also the artifacts produced by it. Although there is now a growing body of literature pertaining to this topic, clear and foolproof methods for experimenters to routinely minimize damage have yet to emerge. Additionally, radiation damage is also a concern and limiting problem in other methods used in structural biology, such as electron microscopy [2] and SAXS [3, 4]. However, the recently available free electron lasers (FELs) have presented the possibility and promise that samples will give “diffraction before destruction”; is this indeed the “cure” for the challenges of radiation damage?
Published: 2015

40. 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

41. 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

42. Radiation damage to macromolecules: kill or cure?

Author: Elspeth F. Garman, Martin Weik, Laboratory of Molecular Biophysics, University of Oxford [Oxford], 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), University of Oxford, 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), and Thomas, Frank
Subjects: Models, Molecular, Diffraction, Nuclear and High Energy Physics, Radiation, Materials science, [SDV.BBM.BS]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Structural Biology [q-bio.BM], Macromolecular Substances, [SDV.BBM.BS] Life Sciences [q-bio]/Biochemistry, Molecular Biology/Structural Biology [q-bio.BM], business.industry, Macromolecular crystallography, A protein, Context (language use), Crystallography, X-Ray, Sensitivity and Specificity, Optics, X-Ray Diffraction, Scattering, Small Angle, Radiation damage, Humans, Computer Simulation, Amino acid residue, Radiation Injuries, business, Instrumentation
Abstract: Radiation damage induced by X-ray beams during macromolecular diffraction experiments remains an issue of concern in structural biology. While advances in our understanding of this phenomenon, driven in part by a series of workshops in this area, undoubtedly have been and are still being made, there are still questions to be answered. Eight papers in this volume give a flavour of ongoing investigations, addressing various issues. These range over: a proposed new metric derived from atomicB-factors for identifying potentially damaged amino acid residues, a study of the relative damage susceptibility of protein and DNA in a DNA/protein complex, a report of an indication of specific radiation damage to a protein determined from data collected using an X-ray free-electron laser (FEL), an account of the challenges in FEL raw diffraction data analysis, an exploration of the possibilities of using radiation damage induced phasing to solve structures using FELs, simulations of radiation damage as a function of FEL temporal pulse profiles, results on the influence of radiation damage during scanning X-ray diffraction measurements and, lastly, consideration of strategies for minimizing radiation damage during SAXS experiments. In this short introduction, these contributions are briefly placed in the context of other current work on radiation damage in the field.
Published: 2017

43. 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

44. 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

45. 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

46. 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

47. 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

48. 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

49. 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

50. 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

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