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1. Mimicking bone extracellular matrix: from BMP-2-derived sequences to osteogenic-multifunctional coatings

Author: Universitat Politècnica de Catalunya. Departament de Ciència i Enginyeria de Materials, Universitat Politècnica de Catalunya. BBT - Grup de recerca en Biomaterials, Biomecànica i Enginyeria de Teixits, Tan'guk Taehakkyo, Max-Planck-Institut für medizinische Forschung, Oliver Cervelló, Lluís, Martín Gómez, Helena, Mandakhbayar, Nandin, Cavalcanti-Adam, Elisabetta Ada, Ginebra Molins, Maria Pau, Mas Moruno, Carlos, Universitat Politècnica de Catalunya. Departament de Ciència i Enginyeria de Materials, Universitat Politècnica de Catalunya. BBT - Grup de recerca en Biomaterials, Biomecànica i Enginyeria de Teixits, Tan'guk Taehakkyo, Max-Planck-Institut für medizinische Forschung, Oliver Cervelló, Lluís, Martín Gómez, Helena, Mandakhbayar, Nandin, Cavalcanti-Adam, Elisabetta Ada, Ginebra Molins, Maria Pau, and Mas Moruno, Carlos
Abstract: Cell–material interactions are regulated by mimicking bone extracellular matrix on the surface of biomaterials. In this regard, reproducing the extracellular conditions that promote integrin and growth factor (GF) signaling is a major goal to trigger bone regeneration. Thus, the use of synthetic osteogenic domains derived from bone morphogenetic protein 2 (BMP-2) is gaining increasing attention, as this strategy is devoid of the clinical risks associated with this molecule. In this work, the wrist and knuckle epitopes of BMP-2 are screened to identify peptides with potential osteogenic properties. The most active sequences (the DWIVA motif and its cyclic version) are combined with the cell adhesive RGD peptide (linear and cyclic variants), to produce tailor-made biomimetic peptides presenting the bioactive cues in a chemically and geometrically defined manner. Such multifunctional peptides are next used to functionalize titanium surfaces. Biological characterization with mesenchymal stem cells demonstrates the ability of the biointerfaces to synergistically enhance cell adhesion and osteogenic differentiation. Furthermore, in vivo studies in rat calvarial defects prove the capacity of the biomimetic coatings to improve new bone formation and reduce fibrous tissue thickness. These results highlight the potential of mimicking integrin-GF signaling with synthetic peptides, without the need for exogenous GFs., Peer Reviewed, Postprint (published version)
Published: 2022

2. An engineered biomimetic peptide regulates cell behavior by synergistic integrin and growth factor signaling

Author: Universitat Politècnica de Catalunya. Doctorat en Ciència i Enginyeria dels Materials, Universitat Politècnica de Catalunya. Departament de Ciència i Enginyeria de Materials, Universitat Politècnica de Catalunya. BBT - Biomaterials, Biomecànica i Enginyeria de Teixits, Max-Planck-Institut für medizinische Forschung, Oliver Cervelló, Lluís, Martín Gómez, Helena, Reyes, Leslie, Noureddine, Fatima, Cavalcanti-Adam, Elisabetta Ada, Ginebra Molins, Maria Pau, Mas Moruno, Carlos, Universitat Politècnica de Catalunya. Doctorat en Ciència i Enginyeria dels Materials, Universitat Politècnica de Catalunya. Departament de Ciència i Enginyeria de Materials, Universitat Politècnica de Catalunya. BBT - Biomaterials, Biomecànica i Enginyeria de Teixits, Max-Planck-Institut für medizinische Forschung, Oliver Cervelló, Lluís, Martín Gómez, Helena, Reyes, Leslie, Noureddine, Fatima, Cavalcanti-Adam, Elisabetta Ada, Ginebra Molins, Maria Pau, and Mas Moruno, Carlos
Abstract: Recreating the healing microenvironment is essential to regulate cell–material interactions and ensure the integration of biomaterials. To repair bone, such bioactivity can be achieved by mimicking its extracellular matrix (ECM) and by stimulating integrin and growth factor (GF) signaling. However, current approaches relying on the use of GFs, such as bone morphogenetic protein 2 (BMP-2), entail clinical risks. Here, a biomimetic peptide integrating the RGD cell adhesive sequence and the osteogenic DWIVA motif derived from the wrist epitope of BMP-2 is presented. The approach offers the advantage of having a spatial control over the single binding of integrins and BMP receptors. Such multifunctional platform is designed to incorporate 3,4-dihydroxyphenylalanine to bind metallic oxides with high affinity in a one step process. Functionalization of glass substrates with the engineered peptide is characterized by physicochemical methods, proving a successful surface modification. The biomimetic interfaces significantly improve the adhesion of C2C12 cells, inhibit myotube formation, and activate the BMP-dependent signaling via p38. These effects are not observed on surfaces displaying only one bioactive motif, a mixture of both motifs or soluble DWIVA. These data prove the biological potential of recreating the ECM and engaging in integrin and GF crosstalk via molecular-based mimics., Peer Reviewed, Postprint (author's final draft)
Published: 2020

3. Fachlicher Schlussbericht zu dem BMBF-Projekt 01GA0208 'Struktur und Funktion kortikaler Landkarten in genetisch veränderten Mäusen : die Rolle von Signalübertragungsbewegungen für Lernen, Gedächtnis und Regeneration'

Author: Max-Planck-Institut Für Medizinische Forschung, Heidelberg
Published: 2005
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4. 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
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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, Jacques-Philippe Colletier, 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), UCLA/DOE Institute for Genomics and Proteomics, University of California [Los Angeles] (UCLA), University of California (UC)-University of California (UC), 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), Max-Planck-Institut für Medizinische Forschung, Max-Planck-Gesellschaft, Max Planck Institute for Medical Research [Heidelberg], Linac Coherent Light Source (LCLS), SLAC National Accelerator Laboratory (SLAC), Stanford University-Stanford University, California Baptist University (CBU), Lawrence Berkeley National Laboratory [Berkeley] (LBNL), European Synchroton Radiation Facility [Grenoble] (ESRF), Laboratoire d'Ecologie Alpine (LECA ), Université Savoie Mont Blanc (USMB [Université de Savoie] [Université de Chambéry])-Centre National de la Recherche Scientifique (CNRS)-Université Grenoble Alpes (UGA), Laboratoire de Chimie - UMR5182 (LC), École normale supérieure - Lyon (ENS Lyon)-Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS), Stanford University, École normale supérieure de Lyon (ENS de Lyon)-Université Claude Bernard Lyon 1 (UCBL), ANR-17-EURE-0003,CBH-EUR-GS,CBH-EUR-GS(2017), 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: Mosquito Control, Multidisciplinary, [SDV.BBM.BS]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Structural Biology [q-bio.BM], Bacillus thuringiensis, General Physics and Astronomy, General Chemistry, [SDV.MP.BAC]Life Sciences [q-bio]/Microbiology and Parasitology/Bacteriology, General Biochemistry, Genetics and Molecular Biology, Endotoxins, Hemolysin Proteins, Bacterial Proteins, Larva, [CHIM.CRIS]Chemical Sciences/Cristallography, Animals, Nanoparticles, [CHIM]Chemical Sciences
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: 2022
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6. Mimicking Bone Extracellular Matrix: From BMP-2-Derived Sequences to Osteogenic-Multifunctional Coatings

Author: Lluís Oliver‐Cervelló, Helena Martin‐Gómez, Nandin Mandakhbayar, Young‐Woo Jo, Elisabetta Ada Cavalcanti‐Adam, Hae‐Won Kim, Maria‐Pau Ginebra, Jung‐Hwan Lee, Carlos Mas‐Moruno, Universitat Politècnica de Catalunya. Departament de Ciència i Enginyeria de Materials, Universitat Politècnica de Catalunya. BBT - Grup de recerca en Biomaterials, Biomecànica i Enginyeria de Teixits, Tan'guk Taehakkyo, and Max-Planck-Institut für medizinische Forschung
Subjects: Titanium, Integrins, Bone Regeneration, Biomedical Engineering, Pharmaceutical Science, Bone Morphogenetic Protein 2, Enginyeria biomèdica::Biomaterials [Àrees temàtiques de la UPC], Cell Differentiation, Biocompatible Materials, Rats, Extracellular Matrix, Biomaterials, Epitopes, Osteogenesis, Ossos, Animals, Bone, Peptides
Abstract: Cell–material interactions are regulated by mimicking bone extracellular matrix on the surface of biomaterials. In this regard, reproducing the extracellular conditions that promote integrin and growth factor (GF) signaling is a major goal to trigger bone regeneration. Thus, the use of synthetic osteogenic domains derived from bone morphogenetic protein 2 (BMP-2) is gaining increasing attention, as this strategy is devoid of the clinical risks associated with this molecule. In this work, the wrist and knuckle epitopes of BMP-2 are screened to identify peptides with potential osteogenic properties. The most active sequences (the DWIVA motif and its cyclic version) are combined with the cell adhesive RGD peptide (linear and cyclic variants), to produce tailor-made biomimetic peptides presenting the bioactive cues in a chemically and geometrically defined manner. Such multifunctional peptides are next used to functionalize titanium surfaces. Biological characterization with mesenchymal stem cells demonstrates the ability of the biointerfaces to synergistically enhance cell adhesion and osteogenic differentiation. Furthermore, in vivo studies in rat calvarial defects prove the capacity of the biomimetic coatings to improve new bone formation and reduce fibrous tissue thickness. These results highlight the potential of mimicking integrin-GF signaling with synthetic peptides, without the need for exogenous GFs.
Published: 2022

7. Unifying perspective of the ultrafast photodynamics of orange carotenoid proteins from synechocystis: peril of high-power excitation, existence of different S* states, and influence of tagging

Author: Stanisław Niziński, Adjéle Wilson, Lucas M. Uriarte, Cyril Ruckebusch, Elena A. Andreeva, Ilme Schlichting, Jacques-Philippe Colletier, Diana Kirilovsky, Gotard Burdzinski, 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 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), 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, Quantum Electronics Laboratory, Adam Mickiewicz University in Poznań (UAM), ANR-18-CE11-0005,DynOCP,Investigation du mécanisme de photo-activation de l'OCP par spectroscopie optique ultrarapide et cristallographie sérielle temps-résolue.(2018), Mécanismes régulateurs chez les organismes photosynthétiques (MROP), Département Biochimie, Biophysique et Biologie Structurale (B3S), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS)-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)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS), 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), and Max Planck Institute for Medical Research [Heidelberg]
Subjects: [PHYS]Physics [physics], [SDV.BBM.BS]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Structural Biology [q-bio.BM], [SDV]Life Sciences [q-bio], [CHIM]Chemical Sciences
Abstract: International audience; A substantial number of Orange Carotenoid Protein (OCP) studies have aimed to describe the evolution of singlet excited states leading to the formation of a photoactivated form, OCPR. The most recent one suggests that 3 ps-lived excited states are formed after the sub-100 fs decay of the initial S2 state. The S* state, which has the longest reported lifetime of a few to tens of picoseconds, is considered to be the precursor of the first red photoproduct P1. Here, we report the ultrafast photodynamics of the OCP from Synechocystis PCC 6803 carried out using visible-near infrared femtosecond time-resolved absorption spectroscopy as a function of the excitation pulse power and wavelength. We found that a carotenoid radical cation can form even at relatively low excitation power, obscuring the determination of photoactivation yields for P1. Moreover, the comparison of green (540 nm) and blue (470 nm) excitations revealed the existence of an hitherto uncharacterized excited state, denoted as S∼, living a few tens of picoseconds and formed only upon 470 nm excitation. Because neither the P1 quantum yield nor the photoactivation speed over hundreds of seconds vary under green and blue continuous irradiation, this S∼ species is unlikely to be involved in the photoactivation mechanism leading to OCPR. We also addressed the effect of His-tagging at the N- or C-termini on the excited-state photophysical properties. Differences in spectral signatures and lifetimes of the different excited states were observed at a variance with the usual assumption that His-tagging hardly influences protein dynamics and function. Altogether our results advocate for the careful consideration of the excitation power and His-tag position when comparing the photoactivation of different OCP variants and beg to revisit the notion that S* is the precursor of photoactivated OCPR.
Published: 2022
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8. Processing of X-ray snapshots from crystals in random orientations

Author: Kabsch, Wolfgang [Max-Planck-Institut für medizinische Forschung, Jahnstrasse 29, D-69120 Heidelberg (Germany)]
Published: 2014
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9. 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
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10. An engineered biomimetic peptide regulates cell behavior by synergistic integrin and growth factor signaling

Author: Leslie Reyes, Maria-Pau Ginebra, Carlos Mas-Moruno, Elisabetta Ada Cavalcanti-Adam, Fatima Noureddine, Lluís Oliver-Cervelló, Helena Martin-Gómez, Universitat Politècnica de Catalunya. Doctorat en Ciència i Enginyeria dels Materials, Universitat Politècnica de Catalunya. Departament de Ciència i Enginyeria de Materials, Universitat Politècnica de Catalunya. BBT - Biomaterials, Biomecànica i Enginyeria de Teixits, and Max-Planck-Institut für medizinische Forschung
Subjects: Integrins, medicine.medical_treatment, Cellular differentiation, Integrin, Biomedical Engineering, Pharmaceutical Science, Enginyeria biomèdica::Biomaterials [Àrees temàtiques de la UPC], 02 engineering and technology, Biomimètica, 010402 general chemistry, 01 natural sciences, Bone morphogenetic protein 2, Biomaterials, Extracellular matrix, Biomimetics, Cell Adhesion, medicine, Cell adhesion, biology, Chemistry, Growth factor, Cell Differentiation, 021001 nanoscience & nanotechnology, 0104 chemical sciences, Cell biology, Crosstalk (biology), biology.protein, Intercellular Signaling Peptides and Proteins, Peptides, 0210 nano-technology, C2C12
Abstract: Recreating the healing microenvironment is essential to regulate cell–material interactions and ensure the integration of biomaterials. To repair bone, such bioactivity can be achieved by mimicking its extracellular matrix (ECM) and by stimulating integrin and growth factor (GF) signaling. However, current approaches relying on the use of GFs, such as bone morphogenetic protein 2 (BMP-2), entail clinical risks. Here, a biomimetic peptide integrating the RGD cell adhesive sequence and the osteogenic DWIVA motif derived from the wrist epitope of BMP-2 is presented. The approach offers the advantage of having a spatial control over the single binding of integrins and BMP receptors. Such multifunctional platform is designed to incorporate 3,4-dihydroxyphenylalanine to bind metallic oxides with high affinity in a one step process. Functionalization of glass substrates with the engineered peptide is characterized by physicochemical methods, proving a successful surface modification. The biomimetic interfaces significantly improve the adhesion of C2C12 cells, inhibit myotube formation, and activate the BMP-dependent signaling via p38. These effects are not observed on surfaces displaying only one bioactive motif, a mixture of both motifs or soluble DWIVA. These data prove the biological potential of recreating the ECM and engaging in integrin and GF crosstalk via molecular-based mimics.
Published: 2020

11. Shock Damage Analysis in Serial Femtosecond Crystallography Data Collected at MHz X-ray Free-Electron Lasers

Author: Herbert Zimmermann, Jacques-Philippe Colletier, Michele Cascella, Marco Kloos, Mark S. Hunter, Thomas R. M. Barends, Richard Bean, Henry Kirkwood, Thomas Michelat, Robert L. Shoeman, Jolanta Sztuk-Dambietz, Johan Bielecki, Romain Letrun, A. Gorel, Guillaume Tetreau, Lutz Foucar, Claudiu A. Stan, Hans Fangohr, Marie Luise Grünbein, Elisabeth Hartmann, M. Hilpert, Ilme Schlichting, Adrian P. Mancuso, R. Bruce Doak, Max-Planck-Institut für Medizinische Forschung, Max-Planck-Gesellschaft, European XFEL GmbH (XFEL), European XFEL GmbH, 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 Institute for Medical Research [Heidelberg], SLAC National Accelerator Laboratory (SLAC), Stanford University, Department of Physics, Rutgers University [Newark], Rutgers University System (Rutgers)-Rutgers University System (Rutgers), 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, Diffraction, Free electron model, Shock wave, MESH: serial femtosecond crystallography, 030103 biophysics, serial femtosecond crystallography, Materials science, shock wave, General Chemical Engineering, law.invention, Inorganic Chemistry, 03 medical and health sciences, law, protein crystallography, lcsh:QD901-999, General Materials Science, Supersonic speed, Uncategorized, MESH: protein crystallography, [SDV.BBM.BS]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Structural Biology [q-bio.BM], X-ray, MESH: shock wave, Condensed Matter Physics, Laser, Crystallography, 030104 developmental biology, X-ray crystallography, Femtosecond, X-ray free-electron laser, lcsh:Crystallography, MESH: X-ray free-electron laser
Abstract: International audience; Serial femtosecond crystallography (SFX) data were recorded at the European X-ray free-electron laser facility (EuXFEL) with protein microcrystals delivered via a microscopic liquid jet. An XFEL beam striking such a jet may launch supersonic shock waves up the jet, compromising the oncoming sample. To investigate this efficiently, we employed a novel XFEL pulse pattern to nominally expose the sample to between zero and four shock waves before being probed. Analyzing hit rate, indexing rate, and resolution for diffraction data recorded at MHz pulse rates, we found no evidence of damage. Notably, however, this conclusion could only be drawn after careful identification and assimilation of numerous interrelated experimental factors, which we describe in detail. Failure to do so would have led to an erroneous conclusion. Femtosecond photography of the sample-carrying jet revealed critically different jet behavior from that of all homogeneous liquid jets studied to date in this manner.
Published: 2020
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12. 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
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13. Three-dimensional view of ultrafast dynamics in photoexcited bacteriorhodopsin

Author: Gabriela Nass Kovacs, Yang Yang, Lutz Foucar, Tatiana Domratcheva, A. Gorel, Ramona Schlesinger, Ilme Schlichting, Robert L. Shoeman, C.M. Roome, Stefan Haacke, Marco Kloos, Jason E. Koglin, Marie Luise Grünbein, Jacques-Philippe Colletier, M. Stricker, Sébastien Boutet, Karsten Heyne, Sergio Carbajo, Till Stensitzki, D. Ehrenberg, Alexander Batyuk, Raphael Gasper, M. Hilpert, R.B. Doak, Jochen Reinstein, Thomas R. M. Barends, Joachim Heberle, Matthew Seaberg, Max-Planck-Institut für Medizinische Forschung, Max-Planck-Gesellschaft, 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), SLAC National Accelerator Laboratory (SLAC), Stanford University, Max-Planck-Institut für Molekulare Physiologie, Institut de Physique et Chimie des Matériaux de Strasbourg (IPCMS), 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)-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), 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), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA), Université de Strasbourg (UNISTRA)-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)-Institut de Chimie du CNRS (INC)-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), 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), Max Planck Institute for Medical Research [Heidelberg], Freie Universität Berlin, Linac Coherent Light Source (LCLS), Stanford University-Stanford University, Fachbereich Physik [Freie Univeristät Berlin] | Department of Physics [Freie Univeristät Berlin], Max Planck Institute of Molecular Physiology, 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), Fachbereich Physik, Freie Universität Berlin, Fachbereich Physik [Berlin], Université de Strasbourg (UNISTRA)-Centre National de la Recherche Scientifique (CNRS)-Matériaux et nanosciences d'Alsace (FMNGE), and 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
Subjects: photon absorption, 0301 basic medicine, Science, [PHYS.PHYS.PHYS-BIO-PH]Physics [physics]/Physics [physics]/Biological Physics [physics.bio-ph], [SDV]Life Sciences [q-bio], FOS: Physical sciences, General Physics and Astronomy, 02 engineering and technology, Photochemistry, Quantum chemistry, Article, General Biochemistry, Genetics and Molecular Biology, quantum chemistry, 03 medical and health sciences, chemistry.chemical_compound, Molecule, [CHIM]Chemical Sciences, Physics - Biological Physics, Spectroscopy, lcsh:Science, Photons, Multidisciplinary, biology, [SDV.BBM.BS]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Structural Biology [q-bio.BM], bacteriorhodopsin, Nanocrystallography, Hydrogen bond, Biomolecules (q-bio.BM), Retinal, Bacteriorhodopsin, General Chemistry, 021001 nanoscience & nanotechnology, Photobiology, 3. Good health, [CHIM.THEO]Chemical Sciences/Theoretical and/or physical chemistry, 030104 developmental biology, chemistry, Quantitative Biology - Biomolecules, Biological Physics (physics.bio-ph), FOS: Biological sciences, Bacteriorhodopsins, Excited state, Picosecond, Retinaldehyde, biology.protein, lcsh:Q, 0210 nano-technology
Abstract: Bacteriorhodopsin (bR) is a light-driven proton pump. The primary photochemical event upon light absorption is isomerization of the retinal chromophore. Here we used time-resolved crystallography at an X-ray free-electron laser to follow the structural changes in multiphoton-excited bR from 250 femtoseconds to 10 picoseconds. Quantum chemistry and ultrafast spectroscopy were used to identify a sequential two-photon absorption process, leading to excitation of a tryptophan residue flanking the retinal chromophore, as a first manifestation of multiphoton effects. We resolve distinct stages in the structural dynamics of the all-trans retinal in photoexcited bR to a highly twisted 13-cis conformation. Other active site sub-picosecond rearrangements include correlated vibrational motions of the electronically excited retinal chromophore, the surrounding amino acids and water molecules as well as their hydrogen bonding network. These results show that this extended photo-active network forms an electronically and vibrationally coupled system in bR, and most likely in all retinal proteins., Bacteriorhodopsin (bR) is a light-driven proton pump. Here the authors combine time-resolved crystallography at a free-electron laser, ultrafast spectroscopy and quantum chemistry to study the structural changes following multiphoton photoexcitation of bR and find that they occur within 300 fs not only in the light-absorbing chromophore but also in the surrounding protein.
Published: 2019
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14. Mutations in GFPT1-related congenital myasthenic syndromes underlie a tubular aggregates myopathy with synaptopathy

Author: Bauché, Stéphanie, Vellieux, Geoffroy, Sternberg, Damien, Fardeau, Michel, Lacène, Emmanuelle, Romero, Norma Beatriz, Koenig, Jeanine, Fournier, Emmanuel, Daniel, Hantaï, Laforêt, Pascal, Fontaine, Bertrand, Eymard, Bruno, Chevessier, Frédéric, Stojkovic, Tanya, Nicole, Sophie, Centre de recherche en Myologie – U974 SU-INSERM, Institut National de la Santé et de la Recherche Médicale (INSERM)-Sorbonne Université (SU), Université Paris Descartes - Faculté de Médecine (UPD5 Médecine), Université Paris Descartes - Paris 5 (UPD5), CHU Pitié-Salpêtrière [AP-HP], Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Sorbonne Université (SU), Institut de Myologie, Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Association française contre les myopathies (AFM-Téléthon)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Sorbonne Université (SU)-Centre National de la Recherche Scientifique (CNRS), Centre de recherche en myologie, Université Pierre et Marie Curie - Paris 6 (UPMC)-Association française contre les myopathies (AFM-Téléthon)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Centre de Recherche de l'Institut du Cerveau et de la Moelle épinière (CRICM), Université Pierre et Marie Curie - Paris 6 (UPMC)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Centre de Référence des Maladies Neuromusculaires, Max-Planck-Institut für Medizinische Forschung, Max-Planck-Gesellschaft, Institut de Génomique Fonctionnelle (IGF), and Université Montpellier 1 (UM1)-Université Montpellier 2 - Sciences et Techniques (UM2)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)
Subjects: [SDV]Life Sciences [q-bio]
Abstract: International audience; Mutations in GFPT1 (glutamine-fructose-6-phosphate transaminase 1), a gene encoding an enzyme involved in glycosylation of ubiquitous proteins, cause a limb-girdle congenital myasthenic syndrome (LG-CMS) with tubular aggregates (TAs) characterized predominantly by affection of the proximal skeletal muscles and presence of highly organized and remodeled sarcoplasmic tubules in patients’ muscle biopsies. GFPT1 is the first and rate-limiting enzyme of the hexosamine biosynthetic pathway (HBP) involved in ubiquitous glycosylation processes (Senderek J. et al., 2011). GFPT1 is the primary genetic cause of ubiquitous CMS. Since 2011, a total of 52 patients with GFPT1 mutations have been clinically reported (Guergueltcheva V. et al., 2012; Senderek et al., 2011;Selcen D. et al., 2013; Huh S-Y. et al., 2012). Interestingly, GFPT1 is expressed in various tissues, including skeletal muscle and motor nerve, raising the intriguing question of why mutations in such a ubiquitous gene would specifically lead to synaptic and muscular disorganization. We report here the first retrospective clinical description and the molecular investigations of 11 individuals with LG-CMS linked to recessively-inherited mutations in GFPT1.In this study, we report the first long-term clinical follow-up of 11 French individuals from 9 unrelated families suffering from LG-CMS with TAs due to GFPT1 mutations, of which 9 are new. Our retrospective clinical evaluation stresses an evolution toward a myopathic weakness that occurs concomitantly to ineffectiveness of usual CMS treatments. Analysis of neuromuscular biopsies from 3 unrelated individuals demonstrates that the maintenance of neuromuscular junctions (NMJs) is dramatically impaired with loss of post-synaptic junctional folds and evidence of denervation-reinnervation processes affecting the 3 main NMJ components. Moreover, molecular analyses of the human muscle biopsies confirm glycosylation defects of proteins with reduced O-glycosylation and show reduced sialylation of transmembrane proteins in extrajunctional area. Altogether, these results pave the way for understanding the etiology of this rare neuromuscular disorder that may be considered as a “tubular aggregates myopathy with synaptopathy”
Published: 2018

15. 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
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16. Mutations in GFPT1-related congenital myasthenic syndromes are associated with synaptic morphological defects and underlie a tubular aggregates myopathy with synaptopathy

Author: Bauché, Stéphanie, Vellieux, Geoffroy, Sternberg, Damien, Fontenille, Marie-Joséphine, Brochier, Guy, Lacène, Emmanuelle, Koenig, Jeanine, Fournier, Emmanuelle, Daniel, Hantaï, Eymard, Bruno, Chevessier, Frederic, Stojkovic, Tanya, Nicole, Sophie, Centre de recherche en Myologie – U974 SU-INSERM, Institut National de la Santé et de la Recherche Médicale (INSERM)-Sorbonne Université (SU), Université Paris Descartes - Faculté de Médecine (UPD5 Médecine), Université Paris Descartes - Paris 5 (UPD5), Paris-Centre de Recherche Cardiovasculaire (PARCC (UMR_S 970/ U970)), Hôpital Européen Georges Pompidou [APHP] (HEGP), Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Hôpitaux Universitaires Paris Ouest - Hôpitaux Universitaires Île de France Ouest (HUPO)-Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Hôpitaux Universitaires Paris Ouest - Hôpitaux Universitaires Île de France Ouest (HUPO)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Paris Cité (UPCité), CHU Pitié-Salpêtrière [AP-HP], Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Sorbonne Université (SU), Institut de Myologie, Université Pierre et Marie Curie - Paris 6 (UPMC)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Association française contre les myopathies (AFM-Téléthon)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Association française contre les myopathies (AFM-Téléthon)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Sorbonne Université (SU)-Centre National de la Recherche Scientifique (CNRS), Centre de Recherche de l'Institut du Cerveau et de la Moelle épinière (CRICM), Université Pierre et Marie Curie - Paris 6 (UPMC)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Laboratoire Sciences Cognitives et Sciences Affectives - UMR 9193 (SCALab), Université de Lille-Centre National de la Recherche Scientifique (CNRS), Max-Planck-Institut für Medizinische Forschung, Max-Planck-Gesellschaft, Affections de la Myeline et des Canaux Ioniques Musculaires, Université Pierre et Marie Curie - Paris 6 (UPMC)-Institut National de la Santé et de la Recherche Médicale (INSERM), and AFM-Théléton
Subjects: GFPT1, neuromuscular junction, CMS, [SDV]Life Sciences [q-bio]
Abstract: International audience; Mutations in GFPT1 (glutamine-fructose-6-phosphate transaminase 1), a gene encoding an enzyme involved in glycosylation of ubiquitous proteins, cause a limb-girdle congenital myasthenic syndrome (LG-CMS) with tubular aggregates (TAs) characterized predominantly by affection of the proximal skeletal muscles and presence of highly organized and remodeled sarcoplasmic tubules in patients’ muscle biopsies. GFPT1 is the first and rate-limiting enzyme of the hexosamine biosynthetic pathway (HBP) involved in ubiquitous glycosylation processes (Senderek J. et al., 2011). Since 2011, a total of 52 patients with GFPT1 mutations have been clinically reported (Guergueltcheva V. et al., 2012; Senderek et al., 2011;Selcen D. et al., 2013; Huh S-Y. et al., 2012). In this study, we report the retrospective clinical description and the molecular investigations of 11 individuals from 9 unrelated families with LG-CMS linked to recessively-inherited mutations in GFPT1.Interestingly, GFPT1 is expressed in various tissues, including skeletal muscle and motor nerve, raising the intriguing question of why mutations in such a ubiquitous gene would specifically lead to synaptic and muscular disorganization.In this study, we report here the first long-term clinical follow-up of 11 French individuals suffering from LG-CMS with TAs due to GFPT1 mutations, of which 9 are new. Our retrospective clinical evaluation stresses an evolution toward a myopathic weakness that occurs concomitantly to ineffectiveness of usual CMS treatments. Analysis of neuromuscular biopsies from 3 unrelated individuals demonstrates that the maintenance of neuromuscular junctions (NMJs) is dramatically impaired with loss of post-synaptic junctional folds and evidence of denervation-reinnervation processes affecting the 3 main NMJ components. Moreover, molecular analyses of the human muscle biopsies confirm glycosylation defects of proteins with reduced O-glycosylation and show reduced sialylation of transmembrane proteins in extrajunctional area. Altogether, these results pave the way for understanding the etiology of this rare neuromuscular disorder that may be considered as a “tubular aggregates myopathy with synaptopathy”
Published: 2017

17. Sympathetic coinnervation of NMJs and its importance for synaptic homeostasis

Author: Khan, Muzamil Majid, Lustrino, Danilo, Silveira, Willian A, Wild, Franziska, Benoit, Evelyne, Molgó, Jordi, Witzemann, Veit, Lochmuller, Hanns, Kettelhut, Isis C, Navegantes, Luiz C C, Pozzan, Tullio, Rudolf, Rüdiger, Karlsruher Institut für Technologie (KIT), Department of Physiology, Faculdade de Medicina de Ribeirão Preto, University of São Paulo, 14049-900 Ribeirão Preto, Centre de recherche du CEA/DSV/iBiTec-S/SIMOPRO, Institut des Neurosciences Paris-Saclay (NeuroPSI), Université Paris-Sud - Paris 11 (UP11)-Centre National de la Recherche Scientifique (CNRS), Max-Planck-Institut für Medizinische Forschung, Max-Planck-Gesellschaft, Karlsruhe Institute of Technology (KIT), Division of regenerative Medicine, and San Raffaele Scientific Institute
Subjects: [SDV.NEU.NB]Life Sciences [q-bio]/Neurons and Cognition [q-bio.NC]/Neurobiology, [SDV.NEU]Life Sciences [q-bio]/Neurons and Cognition [q-bio.NC], ComputingMilieux_MISCELLANEOUS
Abstract: International audience
Published: 2017
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18. Identification of an Agrin Mutation that Causes Congenital Myasthenia and Affects Synapse Function

Author: Michel Fardeau, Laurent Schaeffer, Thierry Kuntzer, Isabelle Grosjean, Daniel Hantaï, A. Rouche, Annie Chaboud, Asma Ben Ammar, Nektaria Alexandri, Frédéric Chevessier, Caroline Huzé, Emmanuel Fournier, Andrea Brancaccio, K. Gaudon, Bruno Eymard, Jeanine Koenig, S. Bauche, Evelyne Goillot, Véronique Bernard, Heba-Aude Lecuyer, Pascale Richard, Markus A. Rüegg, Laboratoire de Biologie Moléculaire de la Cellule ( LBMC ), École normale supérieure - Lyon ( ENS Lyon ) -Université Claude Bernard Lyon 1 ( UCBL ), Université de Lyon-Université de Lyon-Institut National de la Santé et de la Recherche Médicale ( INSERM ) -Centre National de la Recherche Scientifique ( CNRS ), Centre de Recherche de l'Institut du Cerveau et de la Moelle épinière ( CRICM ), Université Pierre et Marie Curie - Paris 6 ( UPMC ) -Institut National de la Santé et de la Recherche Médicale ( INSERM ) -Centre National de la Recherche Scientifique ( CNRS ), Unité Fonctionnelle de Cardiogénétique et Myogénétique Moléculaire et Cellulaire, Assistance publique - Hôpitaux de Paris (AP-HP)-Centre de Génétique Moléculaire et Chromosomique du GH Pitié-Salpêtrière-CHU Pitié-Salpêtrière [APHP], Max-Planck-Institut für Medizinische Forschung, Max-Planck-Gesellschaft, Institut National de Neurologie, Université Tunis El Manar ( UTM ), Plateau d'analyse des protéines, IFR128, Biologie des Jonctions Neuromusculaires Normales et Pathologiques ( U686 ), Université Paris Descartes - Paris 5 ( UPD5 ) -Institut National de la Santé et de la Recherche Médicale ( INSERM ) -Centre National de la Recherche Scientifique ( CNRS ), Nerve-muscle unit, neurology service, Université de Lausanne ( UNIL ) -Centre Hospitalier Universitaire Vaudois [Lausanne] ( CHUV ), Centre de référence des maladies rares neuromusculaires, Assistance publique - Hôpitaux de Paris (AP-HP)-CHU Pitié-Salpêtrière [APHP], Service d'électrophysiologie, Istituto di Chimica del Riconoscimento Molecolare, Università Cattolica del Sacro Cuore, Biozentrum, University of Basel ( Unibas ), ANR-07-MRAR-0001,MRAR,Syndromes myasthéniques congénitaux : le réseau français et approches fondamentales ( 2007 ), Hantaï, Daniel, Programme Pluriannuel de Recherche sur les Maladies Rares (MRAR) - Implication de CXCL13 et CCL21 dans les mécanismes pathogéniques de la Myasthenie. - - MG chemokines2006 - ANR-06-MRAR-0001 - MRAR - VALID, Laboratoire de Biologie Moléculaire de la Cellule (LBMC), École normale supérieure - Lyon (ENS Lyon)-Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Centre de Recherche de l'Institut du Cerveau et de la Moelle épinière (CRICM), Université Pierre et Marie Curie - Paris 6 (UPMC)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-CHU Pitié-Salpêtrière [AP-HP], Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Sorbonne Université (SU)-Sorbonne Université (SU), Université de Tunis El Manar (UTM), Biologie des Jonctions Neuromusculaires Normales et Pathologiques (U686), Université Paris Descartes - Paris 5 (UPD5)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Neuroscience Paris Seine (NPS), Université Pierre et Marie Curie - Paris 6 (UPMC)-Institut de Biologie Paris Seine (IBPS), Université Pierre et Marie Curie - Paris 6 (UPMC)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Université Pierre et Marie Curie - Paris 6 (UPMC)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Université de Lausanne = University of Lausanne (UNIL)-Centre Hospitalier Universitaire Vaudois [Lausanne] (CHUV), Centre de référence des maladies rares neuromusculaires [CHU Pitié-Salpétriêre], CHU Pitié-Salpêtrière [AP-HP], Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Sorbonne Université (SU)-Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Sorbonne Université (SU), Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Sorbonne Université (SU), Università cattolica del Sacro Cuore [Milano] (Unicatt), Biozentrum [Basel, Suisse], University of Basel (Unibas), ANR-06-MRAR-0001,MG chemokines,Implication de CXCL13 et CCL21 dans les mécanismes pathogéniques de la Myasthenie.(2006), École normale supérieure de Lyon (ENS de Lyon)-Université Claude Bernard Lyon 1 (UCBL), Centre National de la Recherche Scientifique (CNRS)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Pierre et Marie Curie - Paris 6 (UPMC), Sorbonne Université (SU)-Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Sorbonne Université (SU), Neurosciences Paris Seine (NPS), Université Pierre et Marie Curie - Paris 6 (UPMC)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Institut de Biologie Paris Seine (IBPS), Université Pierre et Marie Curie - Paris 6 (UPMC)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Université Pierre et Marie Curie - Paris 6 (UPMC)-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), Centre Hospitalier Universitaire Vaudois [Lausanne] (CHUV)-Université de Lausanne (UNIL), Sorbonne Université (SU)-Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP), Sorbonne Université (SU)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Institut de Biologie Paris Seine (IBPS), Sorbonne Université (SU)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Sorbonne Université (SU)-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 du Cerveau et de la Moëlle Epinière = Brain and Spine Institute (ICM), Université Pierre et Marie Curie - Paris 6 (UPMC)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-CHU Pitié-Salpêtrière [AP-HP], Sorbonne Université-Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Sorbonne Université-Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP), Sorbonne Université-Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Sorbonne Université, Institut National de la Santé et de la Recherche Médicale (INSERM)-Sorbonne Université (SU)-Centre National de la Recherche Scientifique (CNRS)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Sorbonne Université (SU)-Centre National de la Recherche Scientifique (CNRS)-Centre National de la Recherche Scientifique (CNRS), Université Pierre et Marie Curie - Paris 6 (UPMC)-Institut National de la Santé et de la Recherche Médicale (INSERM)-CHU Pitié-Salpêtrière [AP-HP], and Sorbonne Université-Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Sorbonne Université-Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Centre National de la Recherche Scientifique (CNRS)
Subjects: Male, Biopsy, DNA Mutational Analysis, Muscle Fibers, Skeletal, Mutant, [SDV.GEN] Life Sciences [q-bio]/Genetics, 0302 clinical medicine, Mutant protein, CHRNE, Receptors, Cholinergic, Genetics(clinical), Dystroglycans, ComputingMilieux_MISCELLANEOUS, Genetics (clinical), Genetics, 0303 health sciences, Agrin, Congenital myasthenic syndrome, Recombinant Proteins, Pedigree, Cell biology, medicine.anatomical_structure, Female, [SDV.NEU]Life Sciences [q-bio]/Neurons and Cognition [q-bio.NC], Dok-7, Adult, animal structures, Mutation, Missense, Neuromuscular Junction, Biology, Article, Neuromuscular junction, Cell Line, 03 medical and health sciences, medicine, Animals, Humans, [SDV.NEU] Life Sciences [q-bio]/Neurons and Cognition [q-bio.NC], Muscle, Skeletal, 030304 developmental biology, Acetylcholine receptor, Myasthenic Syndromes, Congenital, [SDV.GEN]Life Sciences [q-bio]/Genetics, Correction, medicine.disease, Protein Structure, Tertiary, Rats, Models, Chemical, [ SDV.NEU ] Life Sciences [q-bio]/Neurons and Cognition [q-bio.NC], Synapses, 030221 ophthalmology & optometry, biology.protein, Agrin/chemistry, Agrin/genetics, Dystroglycans/metabolism, Muscle Fibers, Skeletal/cytology, Muscle Fibers, Skeletal/metabolism, Muscle, Skeletal/metabolism, Muscle, Skeletal/pathology, Myasthenic Syndromes, Congenital/genetics, Neuromuscular Junction/genetics, Neuromuscular Junction/metabolism, Receptors, Cholinergic/genetics, Receptors, Cholinergic/metabolism, Recombinant Proteins/chemistry, Recombinant Proteins/metabolism, Synapses/metabolism, [ SDV.GEN ] Life Sciences [q-bio]/Genetics, 030217 neurology & neurosurgery
Abstract: International audience; We report the case of a congenital myasthenic syndrome due to a mutation in AGRN, the gene encoding agrin, an extracellular matrix molecule released by the nerve and critical for formation of the neuromuscular junction. Gene analysis identified a homozygous missense mutation, c.5125G>C, leading to the p.Gly1709Arg variant. The muscle-biopsy specimen showed a major disorganization of the neuromuscular junction, including changes in the nerve-terminal cytoskeleton and fragmentation of the synaptic gutters. Experiments performed in nonmuscle cells or in cultured C2C12 myotubes and using recombinant mini-agrin for the mutated and the wild-type forms showed that the mutated form did not impair the activation of MuSK or change the total number of induced acetylcholine receptor aggregates. A solid-phase assay using the dystrophin glycoprotein complex showed that the mutation did not affect the binding of agrin to alpha-dystroglycan. Injection of wild-type or mutated agrin into rat soleus muscle induced the formation of nonsynaptic acetylcholine receptor clusters, but the mutant protein specifically destabilized the endogenous neuromuscular junctions. Importantly, the changes observed in rat muscle injected with mutant agrin recapitulated the pre- and post-synaptic modifications observed in the patient. These results indicate that the mutation does not interfere with the ability of agrin to induce postsynaptic structures but that it dramatically perturbs the maintenance of the neuromuscular junction.
Published: 2009
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19. Covariance mapping of two-photon double core hole states in C2H2 and C2H6 produced by an x-ray free electron laser

Author: S. Mondal, Robert Richter, James M. Glownia, M. Siano, Melanie Mucke, Maria Novella Piancastelli, P. Linusson, Li Fang, P. v d Meulen, Osamu Takahashi, K. C. Prince, Brendan Murphy, Vitali Zhaunerchyk, Christoph Bostedt, Leszek J. Frasinski, Shin-ichi Wada, Nora Berrah, J. Ullrich, Lutz Foucar, John D. Bozek, James P. Cryan, Koji Motomura, Timur Osipov, Richard J. Squibb, S. Schorb, Mats Larsson, K. Ueda, John H. D. Eland, R. D. Thomas, Raimund Feifel, Ryan Coffee, Marc Messerschmidt, P. Salen, Department of Physics and Astronomy [Uppsala], Uppsala University, AlbaNova University Center (ALBANOVA), Stockholm University, Department of Physics, Imperial College London, Department of Biochemistry [Oxford], University of Oxford [Oxford], Max-Planck-Institut für Medizinische Forschung, Max-Planck-Gesellschaft, Advanced Study Group of the Max Planck Society, Max-Planck-Institut für Kernphysik (MPIK), Physikalisch-Technische Bundesanstalt [Braunschweig] (PTB), Tohoku University [Sendai], Western Michigan University [Kalamazoo], Center for High Energy Density Science, University of Connecticut (UCONN), SLAC National Accelerator Laboratory (SLAC), Stanford University, Synchrotron SOLEIL (SSOLEIL), Centre National de la Recherche Scientifique (CNRS), NSF BioXFEL Science and Technology Center, Institute for Environmental Science and Sustainable Development, Department of Physical Science, Laboratoire de Chimie Physique - Matière et Rayonnement (LCPMR), Université Pierre et Marie Curie - Paris 6 (UPMC)-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS), Elettra Sincrotrone Trieste, and University of Oxford
Subjects: Physics, [PHYS]Physics [physics], Electron pair, 010304 chemical physics, Covariance mapping, Free-electron laser, General Physics and Astronomy, Synchrotron radiation, free electron laser, 01 natural sciences, Electron spectroscopy, Linear particle accelerator, covariance mapping, Auger, Ionization, few-photon process, 0103 physical sciences, Physical Sciences, Fysik, Physics::Accelerator Physics, double core hole, Atomic physics, 010306 general physics
Abstract: International audience; Few-photon ionization and relaxation processes in acetylene (C2H2) and ethane (C2H6) were investigated at the linac coherent light source x-ray free electron laser (FEL) at SLAC, Stanford using a highly efficient multi-particle correlation spectroscopy technique based on a magnetic bottle. The analysis method of covariance mapping has been applied and enhanced, allowing us to identify electron pairs associated with double core hole (DCH) production and competing multiple ionization processes including Auger decay sequences. The experimental technique and the analysis procedure are discussed in the light of earlier investigations of DCH studies carried out at the same FEL and at third generation synchrotron radiation sources. In particular, we demonstrate the capability of the covariance mapping technique to disentangle the formation of molecular DCH states which is barely feasible with conventional electron spectroscopy methods.
Published: 2015
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20. Constitutive Activation of the Calcium Sensor STIM1 Causes Tubular-Aggregate Myopathy

Author: Marc Bartoli, Marc Koch, Jocelyn Laporte, Johann Böhm, Frédéric Chevessier, Martin Krahn, Claudine Ebel, Jean-Michel Vallat, Jean Pouget, Michel Fardeau, Dominique Figarella-Branger, Daniel Hantaï, Karima Ghorab, Bruno Eymard, Shahram Attarian, Pascal Laforêt, André Maues De Paula, Norma B. Romero, Claire Feger, Nicolas Lévy, Catherine Koch, Bartoli, Marc, Institut de Génétique et de Biologie Moléculaire et Cellulaire (IGBMC), Université de Strasbourg (UNISTRA)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Max-Planck-Institut für Medizinische Forschung, Max-Planck-Gesellschaft, Service de pathologie et neuropathologie, Assistance Publique - Hôpitaux de Marseille (APHM)- Hôpital de la Timone [CHU - APHM] (TIMONE), Service des maladies neuromusculaires, Hôpital de la Timone [CHU - APHM] (TIMONE), Collège de France - Chaire Génétique Humaine, Collège de France (CdF (institution)), Centre de Recherche de l'Institut du Cerveau et de la Moelle épinière (CRICM), Université Pierre et Marie Curie - Paris 6 (UPMC)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), CHU Pitié-Salpêtrière [AP-HP], Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Sorbonne Université (SU), Centre de référence national neuropathies périphériques rares [CHU Limoges], CHU Limoges, Institut de Myologie, Université Pierre et Marie Curie - Paris 6 (UPMC)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Association française contre les myopathies (AFM-Téléthon)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Neuro-Oncologie [Hôpital de la Timone - APHM], Aix Marseille Université (AMU)-Assistance Publique - Hôpitaux de Marseille (APHM)- Hôpital de la Timone [CHU - APHM] (TIMONE), Centre de référence des maladies rares neuromusculaires [CHU Pitié-Salpétriêre], Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Sorbonne Université (SU)-Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Sorbonne Université (SU), Génétique Médicale et Génomique Fonctionnelle (GMGF), Aix Marseille Université (AMU)-Assistance Publique - Hôpitaux de Marseille (APHM)- Hôpital de la Timone [CHU - APHM] (TIMONE)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Chaire Génétique Humaine, Centre National de la Recherche Scientifique (CNRS)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Pierre et Marie Curie - Paris 6 (UPMC), Sorbonne Université (SU)-Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP), Centre National de la Recherche Scientifique (CNRS)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Association française contre les myopathies (AFM-Téléthon)-Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Pierre et Marie Curie - Paris 6 (UPMC), Centre de référence des maladies rares neuromusculaires, Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-CHU Pitié-Salpêtrière [AP-HP], Sorbonne Université (SU)-Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Sorbonne Université (SU), and Institut National de la Santé et de la Recherche Médicale (INSERM)-Aix Marseille Université (AMU)-Assistance Publique - Hôpitaux de Marseille (APHM)- Hôpital de la Timone [CHU - APHM] (TIMONE)-Centre National de la Recherche Scientifique (CNRS)
Subjects: Male, medicine.disease_cause, Myoblasts, Mice, 0302 clinical medicine, Myocyte, Homeostasis, Genetics(clinical), Child, Genetics (clinical), 0303 health sciences, Mutation, Muscles, STIM1, Middle Aged, Cell biology, Neoplasm Proteins, Pedigree, Phenotype, Female, medicine.symptom, Myopathies, Structural, Congenital, Adult, inorganic chemicals, medicine.medical_specialty, Adolescent, Molecular Sequence Data, Biology, Muscle disorder, Cell Line, 03 medical and health sciences, Young Adult, Internal medicine, Report, medicine, Genetics, [SDV.BBM] Life Sciences [q-bio]/Biochemistry, Molecular Biology, Animals, Humans, [SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology, Amino Acid Sequence, Stromal Interaction Molecule 1, Myopathy, 030304 developmental biology, Aged, Calcium metabolism, Base Sequence, Endoplasmic reticulum, Membrane Proteins, Endocrinology, Calcium, 030217 neurology & neurosurgery
Abstract: International audience; Tubular aggregates are regular arrays of membrane tubules accumulating in muscle with age. They are found as secondary features in several muscle disorders, including alcohol- and drug-induced myopathies, exercise-induced cramps, and inherited myasthenia, but also exist as a pure genetic form characterized by slowly progressive muscle weakness. We identified dominant STIM1 mutations as a genetic cause of tubular-aggregate myopathy (TAM). Stromal interaction molecule 1 (STIM1) is the main Ca2+ sensor in the endoplasmic reticulum, and all mutations were found in the highly conserved intraluminal Ca2+-binding EF hands. Ca2+ stores are refilled through a process called store-operated Ca2+ entry (SOCE). Upon Ca2+-store depletion, wild-type STIM1 oligomerizes and thereby triggers extracellular Ca2+ entry. In contrast, the missense mutations found in our four TAM-affected families induced constitutive STIM1 clustering, indicating that Ca2+ sensing was impaired. By monitoring the calcium response of TAM myoblasts to SOCE, we found a significantly higher basal Ca2+ level in TAM cells and a dysregulation of intracellular Ca2+ homeostasis. Because recessive STIM1 loss-of-function mutations were associated with immunodeficiency, we conclude that the tissue-specific impact of STIM1 loss or constitutive activation is different and that a tight regulation of STIM1-dependent SOCE is fundamental for normal skeletal-muscle structure and function.
Published: 2013
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21. A mouse model for congenital myasthenic syndrome due to MuSK mutations reveals defects in structure and function of neuromuscular junctions

Author: Sönke Bartling, Jeanine Koenig, Daniel Hantaï, Frédéric Chevessier, Emmanuelle Girard, Veit Witzemann, Jordi Molgó, Max-Planck-Institut für Medizinische Forschung, Max-Planck-Gesellschaft, Biologie des Jonctions Neuromusculaires Normales et Pathologiques (U686), Université Paris Descartes - Paris 5 (UPD5)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Laboratoire de neurobiologie cellulaire et moléculaire (NBCM), Centre National de la Recherche Scientifique (CNRS), Institut de Neurobiologie Alfred Fessard (INAF), Abteilung medizinische Physik in der Radiologie, Deutsches Krebsforschungzentrum, Physiopathologie et thérapie du muscle strié, Université Pierre et Marie Curie - Paris 6 (UPMC)-IFR14-Institut National de la Santé et de la Recherche Médicale (INSERM), Université Bordeaux Segalen - Bordeaux 2, Centre de référence des maladies neuromusculaires Paris-Est, Hôpital de la Salpêtrière AP-HP, CHU Pitié-Salpêtrière [AP-HP], Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Sorbonne Université (SU), Biologie des Jonctions Neuromusculaires Normales et Pathologiques ( U686 ), Université Paris Descartes - Paris 5 ( UPD5 ) -Institut National de la Santé et de la Recherche Médicale ( INSERM ) -Centre National de la Recherche Scientifique ( CNRS ), Laboratoire de neurobiologie cellulaire et moléculaire ( NBCM ), Centre National de la Recherche Scientifique ( CNRS ), Institut de Neurobiologie Alfred Fessard ( INAF ), and Université Pierre et Marie Curie - Paris 6 ( UPMC ) -IFR14-Institut National de la Santé et de la Recherche Médicale ( INSERM )
Subjects: Male, Refractory Period, Electrophysiological, MESH: Muscle Contraction, MESH : Muscle Weakness, medicine.disease_cause, Synaptic Transmission, MESH: Motor Endplate, Mice, MESH : Locomotion, 0302 clinical medicine, Missense mutation, MESH : Female, Receptors, Cholinergic, MESH: Animals, Genetics (clinical), MESH : Synaptic Transmission, 0303 health sciences, Mutation, Muscle Weakness, MESH: Muscle Weakness, General Medicine, Congenital myasthenic syndrome, Null allele, medicine.anatomical_structure, Female, MESH: Receptor Protein-Tyrosine Kinases, [SDV.NEU]Life Sciences [q-bio]/Neurons and Cognition [q-bio.NC], medicine.symptom, MESH: Refractory Period, Electrophysiological, Locomotion, Muscle Contraction, Muscle contraction, medicine.medical_specialty, MESH: Axons, MESH: Myasthenic Syndromes, Congenital, MESH : Male, Diaphragm, MESH : Axons, Mutation, Missense, Neuromuscular Junction, MESH: Locomotion, MESH: Microscopy, Electron, Biology, MESH : Myasthenic Syndromes, Congenital, Motor Endplate, Neuromuscular junction, 03 medical and health sciences, Internal medicine, MESH : Mice, Genetics, medicine, MESH: Synaptic Transmission, Animals, Humans, Kyphosis, Molecular Biology, MESH: Mice, 030304 developmental biology, Myasthenic Syndromes, Congenital, MESH: Mutation, Missense, MESH : Kyphosis, MESH: Receptors, Cholinergic, MESH: Humans, MESH : Humans, MESH : Motor Endplate, Receptor Protein-Tyrosine Kinases, Muscle weakness, MESH : Receptor Protein-Tyrosine Kinases, MESH : Receptors, Cholinergic, medicine.disease, MESH : Disease Models, Animal, MESH : Microscopy, Electron, Axons, Myasthenia gravis, MESH: Male, Disease Models, Animal, Microscopy, Electron, MESH : Refractory Period, Electrophysiological, Endocrinology, MESH: Diaphragm, [ SDV.NEU ] Life Sciences [q-bio]/Neurons and Cognition [q-bio.NC], MESH : Muscle Contraction, MESH : Neuromuscular Junction, MESH : Animals, MESH: Neuromuscular Junction, MESH : Diaphragm, MESH: Disease Models, Animal, MESH: Kyphosis, MESH: Female, MESH : Mutation, Missense, 030217 neurology & neurosurgery
Abstract: International audience; In the muscle-specific tyrosine kinase receptor gene MUSK, a heteroallelic missense and a null mutation were identified in a patient suffering from a congenital myasthenic syndrome (CMS). We generated one mouse line carrying the homozygous missense mutation V789M in musk (musk(V789M/V789M) mice) and a second hemizygous line, resembling the patient genotype, with the V789M mutation on one allele and an allele lacking the kinase domain (musk(V789M/-) mice). We report here that musk(V789M/V789M) mice present no obvious abnormal phenotype regarding weight, muscle function and viability. In contrast, adult musk(V789M/-) mice suffer from severe muscle weakness, exhibit shrinkage of pelvic and scapular regions and hunchback. Musk(V789M/-) diaphragm develops less force upon direct or nerve-induced stimulation. A profound tetanic fade is observed following nerve-evoked muscle contraction, and fatigue resistance is severely impaired upon a train of tetanic nerve stimulations. Electrophysiological measurements indicate that fatigable muscle weakness is due to impaired neurotransmission as observed in a patient suffering from a CMS. The diaphragm of adult musk(V789M/-) mice exhibits pronounced changes in endplate architecture, distribution and innervation pattern. Thus, the missense mutation V789M in MuSK acts as a hypomorphic mutation and leads to insufficiency in MuSK function in musk(V789M/-) mutants. These mutant mice represent valuable models for elucidating the roles of MuSK for synapse formation, maturation and maintenance as well as for studying the pathophysiology of a CMS due to MuSK mutations.
Published: 2008
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22. A Fluoro Analogue of the Menadione Derivative 6-[2‘-(3‘-Methyl)-1‘,4‘-naphthoquinolyl]hexanoic Acid Is a Suicide Substrate of Glutathione Reductase. Crystal Structure of the Alkylated Human Enzyme†

Author: Hervé Vezin, R. Heiner Schirmer, Sebastian Kühner, Elisabeth Davioud-Charvet, Bruce A. Palfey, Holger Bauer, Susan J. Little, Vanessa Yardley, Andreas Winzer, Karin Fritz-Wolf, Universität Heidelberg [Heidelberg] = Heidelberg University, Max-Planck-Institut für Medizinische Forschung, Max-Planck-Gesellschaft, London School of Hygiene and Tropical Medicine (LSHTM), Laboratoire de Chimie Organique et Macromoleculaire (UMR CNRS 8009), Université de Lille, Sciences et Technologies-Ecole Nationale Supérieure de Chimie de Lille (ENSCL), University of Michigan [Ann Arbor], University of Michigan System, Centre National de la Recherche Scientifique (CNRS), Universität Heidelberg [Heidelberg], and Ecole Nationale Supérieure de Chimie de Lille (ENSCL)-Université de Lille, Sciences et Technologies
Subjects: Models, Molecular, Stereochemistry, Protein Conformation, Glutathione reductase, Plasmodium falciparum, Peptides and proteins, 010402 general chemistry, 01 natural sciences, Biochemistry, Catalysis, Antioxidants, chemistry.chemical_compound, Colloid and Surface Chemistry, Menadione, Genetics, Animals, Humans, Redox reactions, Caproates, Hexanoic acid, chemistry.chemical_classification, Binding Sites, Molecular Structure, 010405 organic chemistry, Inhibitors, Substrate (chemistry), Vitamin K 3, General Chemistry, Glutathione, Quinone methide, 3. Good health, 0104 chemical sciences, [CHIM.THEO]Chemical Sciences/Theoretical and/or physical chemistry, Enzyme, Glutathione Reductase, chemistry, Uncompetitive inhibitor, Naphthoquinones
Abstract: International audience; Glutathione reductase is an important housekeeping enzyme for redox homeostasis both in human cells and in the causative agent of tropical malaria, Plasmodium falciparum. Glutathione reductase inhibitors were shown to have anticancer and antimalarial activity per se and to contribute to the reversal of drug resistance. The development of menadione chemistry has led to the selection of 6-[2′-(3′-methyl)-1′,4′-naphthoquinolyl] hexanoic acid, called M5, as a potent reversible and uncompetitive inhibitor of both human and P. falciparum glutathione reductases. Here we describe the synthesis and kinetic characterization of a fluoromethyl-M 5 analogue that acts as a mechanism-based inhibitor of both enzymes. In the course of enzymatic catalysis, the suicide substrate is activated by one- or two-electron reduction, and then a highly reactive quinone methide is generated upon elimination of the fluorine. Accordingly the human enzyme was found to be irreversibly inactivated with a kinact value of 0.4 ± 0.2 min-1. The crystal structure of the alkylated enzyme was solved at 1.7 Å resolution. It showed the inhibitor to bind covalently to the active site Cys58 and to interact noncovalently with His467′, Arg347, Arg37, and Tyr114. On the basis of the crystal structure of the inactivated human enzyme and stopped-flow kinetic studies with two- and four-electron- reduced forms of the unreacted P. falciparum enzyme, a mechanism is proposed which explains naphthoquinone reduction at the flavin of glutathione reductase.
Published: 2006
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23. Proton-decoupled deuterium NMR study of an asymmetric liquid crystal dimer having two nematic phases.

Author: Chamignon C, Lelli M, Emsley JW, Luckhurst GR, and Zimmermann H
Abstract: Proton-decoupled deuterium NMR spectra were obtained for an asymmetric liquid crystal dimer 1-(4-cyanobiphenyl-4'-yloxy)-6-(4-cyanobiphenyl-4'-yl)hexane (CB6OCB) containing a single -CD_{2}- group. The sample has two nematic liquid crystal phases: a twist-bend nematic, N_{TB}, at the lowest temperature followed by a uniaxial nematic, N_{U}, on increasing the temperature. Proton decoupling reduces the linewidths of the peaks in the deuterium spectrum from kHz to ∼100Hz, enabling quadrupolar splittings, Δν, to be obtained with enhanced precision as well as the dipolar coupling between deuterium nuclei within the CD_{2} group, hence enhancing the information content.
Published: 2023
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24. Bioactive Nanogels Mimicking the Antithrombogenic Nitric Oxide-Release Function of the Endothelium.

Author: Hosseinnejad A, Ludwig N, Mersmann S, Winnerbach P, Bleilevens C, Rossaint R, Rossaint J, and Singh S
Subjects: Humans, Nanogels, Cyclic GMP metabolism, Blood Platelets metabolism, Endothelium metabolism, Nitric Oxide metabolism, Thrombosis
Abstract: Nitric oxide (NO) plays a significant role in controlling the physiology and pathophysiology of the body, including the endothelial antiplatelet function and therefore, antithrombogenic property of the blood vessels. This property of NO can be exploited to prevent thrombus formation on artificial surfaces like extracorporeal membrane oxygenators, which when come into contact with blood lead to protein adsorption and thereby platelet activation causing thrombus formation. However, NO is extremely reactive and has a very short biological half-life in blood, so only endogenous generation of NO from the blood contacting material can result into a stable and kinetically controllable local delivery of NO. In this regards, highly hydrophilic bioactive nanogels are presented which can endogenously generate NO in blood plasma from endogenous NO-donors thereby maintaining a physiological NO flux. It is shown that NO releasing nanogels could initiate cGMP-dependent protein kinase signaling followed by phosphorylation of vasodilator-stimulated phosphoprotein in platelets. This prevents platelet activation and aggregation even in presence of highly potent platelet activators like thrombin, adenosine 5'-diphosphate, and U46619 (thromboxane A2 mimetic)., (© 2023 The Authors. Small published by Wiley-VCH GmbH.)
Published: 2023
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25. Insight into the structural dynamics of light sensitive proteins from time-resolved crystallography and quantum chemical calculations.

Author: Weik M and Domratcheva T
Subjects: Crystallography, X-Ray, X-Rays, Proteins chemistry, Lasers
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., (Copyright © 2022 Elsevier Ltd. All rights reserved.)
Published: 2022
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26. A dark intermediate in the fluorogenic reaction between tetrazine fluorophores and trans -cyclooctene.

Author: Hild F, Werther P, Yserentant K, Wombacher R, and Herten DP
Abstract: Fluorogenic labeling via bioorthogonal tetrazine chemistry has proven to be highly successful in fluorescence microscopy of living cells. To date, trans -cyclooctene (TCO) and bicyclonyne have been found to be the most useful substrates for live-cell labeling owing to their fast labeling kinetics, high biocompatibility, and bioorthogonality. Recent kinetic studies of fluorogenic click reactions with TCO derivatives showed a transient fluorogenic effect but could not explain the reaction sequence and the contributions of different intermediates. More recently, fluorescence quenching by potential intermediates has been investigated, suggesting their occurrence in the reaction sequence. However, in situ studies of the click reaction that directly relate these observations to the known reaction sequence are still missing. In this study, we developed a single-molecule fluorescence detection framework to investigate fluorogenic click reactions. In combination with data from ultra-performance liquid chromatography-tandem mass spectrometry, this explains the transient intensity increase by relating fluorescent intermediates to the known reaction sequence of TCO with fluorogenic tetrazine dyes. More specifically, we confirm that the reaction of TCO with tetrazine rapidly forms a fluorescent 4,5-dihydropyridazine species that slowly tautomerizes to a weakly fluorescent 1,4-dihydropyridazine, explaining the observed drop in fluorescence intensity. On a much slower timescale of hours/days, the fluorescence intensity may be recovered by oxidation of the intermediate to a pyridazine. Our findings are of importance for quantitative applications in fluorescence microscopy and spectroscopy as the achieved peak intensity with TCO depends on the specific experimental settings. They clearly indicate the requirement for more robust benchmarking of click reactions with tetrazine dyes and the need for alternative dienophiles with fast reaction kinetics and stable fluorescence emission to further applications in advanced fluorescence microscopy., Competing Interests: The authors declare no competing interests., (© 2022 The Authors.)
Published: 2022
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27. Rational Control of Off-State Heterogeneity in a Photoswitchable Fluorescent Protein Provides Switching Contrast Enhancement.

Author: Adam V, Hadjidemetriou K, Jensen N, Shoeman RL, Woodhouse J, Aquila A, Banneville AS, Barends TRM, Bezchastnov V, Boutet S, Byrdin M, Cammarata M, Carbajo S, Eleni Christou N, Coquelle N, De la Mora E, El Khatib M, Moreno Chicano T, Bruce Doak R, Fieschi F, Foucar L, Glushonkov O, Gorel A, Grünbein ML, Hilpert M, Hunter M, Kloos M, Koglin JE, Lane TJ, Liang M, Mantovanelli A, Nass K, Nass Kovacs G, Owada S, Roome CM, Schirò G, Seaberg M, Stricker M, Thépaut M, Tono K, Ueda K, Uriarte LM, You D, Zala N, Domratcheva T, Jakobs S, Sliwa M, Schlichting I, Colletier JP, Bourgeois D, and Weik M
Subjects: Green Fluorescent Proteins, Luminescent Proteins chemistry, Escherichia coli metabolism, Microscopy
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, one of the key parameters that dictate 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., (© 2022 The Authors. ChemPhysChem published by Wiley-VCH GmbH.)
Published: 2022
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28. Structure-function-dynamics relationships in the peculiar Planktothrix PCC7805 OCP1: Impact of his-tagging and carotenoid type.

Author: Wilson A, Andreeva EA, Niziński S, Talbot L, Hartmann E, Schlichting I, Burdzinski G, Sliwa M, Kirilovsky D, and Colletier JP
Subjects: Bacterial Proteins metabolism, Carotenoids metabolism, Fluorescence, Cyanobacteria metabolism, Planktothrix
Abstract: The orange carotenoid protein (OCP) is a photoactive protein involved in cyanobacterial photoprotection. Here, we report on the functional, spectral and structural characteristics of the peculiar Planktothrix PCC7805 OCP (Plankto-OCP). We show that this OCP variant is characterized by higher photoactivation and recovery rates, and a stronger energy-quenching activity, compared to other OCP studied thus far. We characterize the effect of the functionalizing carotenoid and of his-tagging on these reactions, and identify the time scales on which these modifications affect photoactivation. The presence of a his-tag at the C-terminus has a large influence on photoactivation, thermal recovery and PBS-fluorescence quenching, and likewise for the nature of the carotenoid that additionally affects the yield and characteristics of excited states and the ns-s dynamics of photoactivated OCP. By solving the structures of Plankto-OCP in the ECN- and CAN-functionalized states, each in two closely-related crystal forms, we further unveil the molecular breathing motions that animate Plankto-OCP at the monomer and dimer levels. We finally discuss the structural changes that could explain the peculiar properties of Plankto-OCP., (Copyright © 2022. Published by Elsevier B.V.)
Published: 2022
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29. Time-resolved serial femtosecond crystallography on fatty-acid photodecarboxylase: lessons learned.

Author: Hadjidemetriou K, Coquelle N, Barends TRM, De Zitter E, Schlichting I, Colletier JP, and Weik M
Subjects: Crystallography, Crystallography, X-Ray, Light, Spectrum Analysis, Fatty Acids, Lasers
Abstract: Upon absorption of a blue-light photon, fatty-acid photodecarboxylase catalyzes the decarboxylation of free fatty acids to form hydrocarbons (for example alkanes or alkenes). The major components of the catalytic mechanism have recently been elucidated by combining static and time-resolved serial femtosecond crystallography (TR-SFX), time-resolved vibrational and electronic spectroscopies, quantum-chemical calculations and site-directed mutagenesis [Sorigué et al. (2021), Science, 372, eabd5687]. The TR-SFX experiments, which were carried out at four different picosecond to microsecond pump-probe delays, yielded input for the calculation of Fourier difference maps that demonstrated light-induced decarboxylation. Here, some of the difficulties encountered during the experiment as well as during data processing are highlighted, in particular regarding space-group assignment, a pump-laser power titration is described and data analysis is extended by structure-factor extrapolation of the TR-SFX data. Structure refinement against extrapolated structure factors reveals a reorientation of the generated hydrocarbon and the formation of a photoproduct close to Cys432 and Arg451. Identification of its chemical nature, CO 2 or bicarbonate, was not possible because of the limited data quality, which was assigned to specificities of the crystalline system. Further TR-SFX experiments on a different crystal form are required to identify the photoproducts and their movements during the catalytic cycle., (open access.)
Published: 2022
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30. Is orange carotenoid protein photoactivation a single-photon process?

Author: Niziński S, Schlichting I, Colletier JP, Kirilovsky D, Burdzinski G, and Sliwa M
Abstract: In all published photoactivation mechanisms of orange carotenoid protein (OCP), absorption of a single photon by the orange dark state starts a cascade of red-shifted OCP ground-state intermediates that subsequently decay within hundreds of milliseconds, resulting in the formation of the final red form OCP R , which is the biologically active form that plays a key role in cyanobacteria photoprotection. A major challenge in deducing the photoactivation mechanism is to create a uniform description explaining both single-pulse excitation experiments, involving single-photon absorption, and continuous light irradiation experiments, where the red-shifted OCP intermediate species may undergo re-excitation. We thus investigated photoactivation of Synechocystis OCP using stationary irradiation light with a biologically relevant photon flux density coupled with nanosecond laser pulse excitation. The kinetics of photoactivation upon continuous and nanosecond pulse irradiation light show that the OCP R formation quantum yield increases with photon flux density; thus, a simple single-photon model cannot describe the data recorded for OCP in vitro . The results strongly suggest a consecutive absorption of two photons involving a red intermediate with ≈100 millisecond lifetime. This intermediate is required in the photoactivation mechanism and formation of the red active form OCP R ., Competing Interests: The authors declare no competing interests., (© 2022 The Authors.)
Published: 2022
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31. Oligomerization processes limit photoactivation and recovery of the orange carotenoid protein.

Author: Andreeva EA, Niziński S, Wilson A, Levantino M, De Zitter E, Munro R, Muzzopappa F, Thureau A, Zala N, Burdzinski G, Sliwa M, Kirilovsky D, Schirò G, and Colletier JP
Subjects: Carotenoids metabolism, Bacterial Proteins metabolism, Cyanobacteria
Abstract: The orange carotenoid protein (OCP) is a photoactive protein involved in cyanobacterial photoprotection by quenching of the excess of light-harvested energy. The photoactivation mechanism remains elusive, in part due to absence of data pertaining to the timescales over which protein structural changes take place. It also remains unclear whether or not oligomerization of the dark-adapted and light-adapted OCP could play a role in the regulation of its energy-quenching activity. Here, we probed photoinduced structural changes in OCP by a combination of static and time-resolved X-ray scattering and steady-state and transient optical spectroscopy in the visible range. Our results suggest that oligomerization partakes in regulation of the OCP photocycle, with different oligomers slowing down the overall thermal recovery of the dark-adapted state of OCP. They furthermore reveal that upon non-photoproductive excitation a numbed state forms, which remains in a non-photoexcitable structural state for at least ≈0.5 μs after absorption of a first photon., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2022 Biophysical Society. Published by Elsevier Inc. All rights reserved.)
Published: 2022
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32. De novo determination of mosquitocidal Cry11Aa and Cry11Ba structures from naturally-occurring nanocrystals.

Author: Tetreau G, Sawaya MR, De Zitter E, Andreeva EA, Banneville AS, Schibrowsky NA, Coquelle N, Brewster AS, Grünbein ML, Kovacs GN, Hunter MS, Kloos M, Sierra RG, Schiro G, Qiao P, Stricker M, Bideshi D, Young ID, Zala N, Engilberge S, Gorel A, Signor L, Teulon JM, Hilpert M, Foucar L, Bielecki J, Bean R, de Wijn R, Sato T, Kirkwood H, Letrun R, Batyuk A, Snigireva I, Fenel D, Schubert R, Canfield EJ, Alba MM, Laporte F, Després L, Bacia M, Roux A, Chapelle C, Riobé F, Maury O, Ling WL, Boutet S, Mancuso A, Gutsche I, Girard E, Barends TRM, Pellequer JL, Park HW, Laganowsky AD, Rodriguez J, Burghammer M, Shoeman RL, Doak RB, Weik M, Sauter NK, Federici B, Cascio D, Schlichting I, and Colletier JP
Subjects: Animals, Bacterial Proteins toxicity, Endotoxins, Hemolysin Proteins toxicity, Larva, Mosquito Control, Bacillus thuringiensis, Nanoparticles
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., (© 2022. The Author(s).)
Published: 2022
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33. Xtrapol8 enables automatic elucidation of low-occupancy intermediate-states in crystallographic studies.

Author: De Zitter E, Coquelle N, Oeser P, Barends TRM, and Colletier JP
Subjects: Bayes Theorem, Kinetics, Ligands, Crystallography methods
Abstract: Unstable states studied in kinetic, time-resolved and ligand-based crystallography are often characterized by a low occupancy, which hinders structure determination by conventional methods. To automatically extract structural information pertaining to these states, we developed Xtrapol8, a program which (i) applies various flavors of Bayesian-statistics weighting to generate the most informative Fourier difference maps; (ii) determines the occupancy of the intermediate states by use of methods hitherto not available; (iii) calculates extrapolated structure factors using the various proposed formalisms while handling the issue of negative structure factor amplitudes, and (iv) refines the corresponding structures in real and reciprocal-space. The use of Xtrapol8 could accelerate data processing in kinetic and time-resolved crystallographic studies, and as well foster the identification of drug-targetable states in ligand-based crystallography., (© 2022. The Author(s).)
Published: 2022
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34. A facile method for grafting functional hydrogel films on PTFE, PVDF, and TPX polymers.

Author: Fischer T, Tenbusch J, Möller M, and Singh S
Subjects: Amines, Fluorocarbon Polymers, Methylgalactosides, Polytetrafluoroethylene chemistry, Polyvinyls chemistry, Surface Properties, Hydrogels, Polymers chemistry
Abstract: The use of polymeric materials in biomedical applications requires a judicious control of surface properties as they are directly related to cellular interactions and biocompatibility. The most desired chemical surface properties include hydrophilicity and the presence of functional groups for surface modification. In this work, we describe a method to graft a highly stable, ultra-thin, amine-functional hydrogel layer onto highly inert surfaces of poly(tetrafluoroethylene) (PTFE), poly(vinylidene fluoride) (PVDF), and poly(4-methyl-1-pentene) (PMP or TPX). Covalent grafting is realized with hydrophilic poly(vinylamine- co -acetamide)s by C-H insertion crosslinking (CHic) chemistry initiated by UV light. These polyvinylamides carry tetrafluorophenyl azide groups as photo or thermo activated binding sites and contain further free amine groups, which can be used to bind peptides such as biological ligands, polysaccharides, or other hydrogel layers. The covalently bound surface layers resist intensive Soxhlet extraction confirming the stability of the coating. Fluorescent staining verified the accessibility of free primary amine groups, which can be used for the functionalization of the surface with bioactive molecules. The coating demonstrates hydrophobic wetting behavior when conditioned in air and hydrophilic wetting behavior when conditioned in water showing the presence of loosely crosslinked polymer chains that can re-orient. We believe that the reported application of CHic for the surface modification of fluorinated polymers like PTFE and PVDF as well as TPX can form the basis for advanced biocompatible and biofunctional surface engineering.
Published: 2022
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35. Unifying Perspective of the Ultrafast Photodynamics of Orange Carotenoid Proteins from Synechocystis : Peril of High-Power Excitation, Existence of Different S* States, and Influence of Tagging.

Author: Niziński S, Wilson A, Uriarte LM, Ruckebusch C, Andreeva EA, Schlichting I, Colletier JP, Kirilovsky D, Burdzinski G, and Sliwa M
Abstract: A substantial number of Orange Carotenoid Protein (OCP) studies have aimed to describe the evolution of singlet excited states leading to the formation of a photoactivated form, OCP R . The most recent one suggests that 3 ps-lived excited states are formed after the sub-100 fs decay of the initial S 2 state. The S* state, which has the longest reported lifetime of a few to tens of picoseconds, is considered to be the precursor of the first red photoproduct P 1 . Here, we report the ultrafast photodynamics of the OCP from Synechocystis PCC 6803 carried out using visible-near infrared femtosecond time-resolved absorption spectroscopy as a function of the excitation pulse power and wavelength. We found that a carotenoid radical cation can form even at relatively low excitation power, obscuring the determination of photoactivation yields for P 1 . Moreover, the comparison of green (540 nm) and blue (470 nm) excitations revealed the existence of an hitherto uncharacterized excited state, denoted as S ∼ , living a few tens of picoseconds and formed only upon 470 nm excitation. Because neither the P 1 quantum yield nor the photoactivation speed over hundreds of seconds vary under green and blue continuous irradiation, this S ∼ species is unlikely to be involved in the photoactivation mechanism leading to OCP R . We also addressed the effect of His-tagging at the N- or C-termini on the excited-state photophysical properties. Differences in spectral signatures and lifetimes of the different excited states were observed at a variance with the usual assumption that His-tagging hardly influences protein dynamics and function. Altogether our results advocate for the careful consideration of the excitation power and His-tag position when comparing the photoactivation of different OCP variants and beg to revisit the notion that S* is the precursor of photoactivated OCP R ., Competing Interests: The authors declare no competing financial interest., (© 2022 The Authors. Published by American Chemical Society.)
Published: 2022
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36. Sizes of pure and doped helium droplets from single shot x-ray imaging.

Author: Tanyag RMP, Bacellar C, Pang W, Bernando C, Gomez LF, Jones CF, Ferguson KR, Kwok J, Anielski D, Belkacem A, Boll R, Bozek J, Carron S, Chen G, Delmas T, Englert L, Epp SW, Erk B, Foucar L, Hartmann R, Hexemer A, Huth M, Leone SR, Ma JH, Marchesini S, Neumark DM, Poon BK, Prell J, Rolles D, Rudek B, Rudenko A, Seifrid M, Swiggers M, Ullrich J, Weise F, Zwart P, Bostedt C, Gessner O, and Vilesov AF
Abstract: Advancements in x-ray free-electron lasers on producing ultrashort, ultrabright, and coherent x-ray pulses enable single-shot imaging of fragile nanostructures, such as superfluid helium droplets. This imaging technique gives unique access to the sizes and shapes of individual droplets. In the past, such droplet characteristics have only been indirectly inferred by ensemble averaging techniques. Here, we report on the size distributions of both pure and doped droplets collected from single-shot x-ray imaging and produced from the free-jet expansion of helium through a 5 μm diameter nozzle at 20 bars and nozzle temperatures ranging from 4.2 to 9 K. This work extends the measurement of large helium nanodroplets containing 10 9 -10 11 atoms, which are shown to follow an exponential size distribution. Additionally, we demonstrate that the size distributions of the doped droplets follow those of the pure droplets at the same stagnation condition but with smaller average sizes.
Published: 2022
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37. Endogenous Nitric Oxide-Releasing Microgel Coating Prevents Clot Formation on Oxygenator Fibers Exposed to In Vitro Blood Flow.

Author: Winnersbach P, Hosseinnejad A, Breuer T, Fechter T, Jakob F, Schwaneberg U, Rossaint R, Bleilevens C, and Singh S
Abstract: Background: Clot formation on foreign surfaces of extracorporeal membrane oxygenation systems is a frequent event. Herein, we show an approach that mimics the enzymatic process of endogenous nitric oxide (NO) release on the oxygenator membrane via a biomimetic, non-fouling microgel coating to spatiotemporally inhibit the platelet (PLT) activation and improve antithrombotic properties. This study aims to evaluate the potential of this biomimetic coating towards NO-mediated PLT inhibition and thereby the reduction of clot formation under flow conditions., Methods: Microgel-coated (NOrel) or bare (Control) poly(4-methyl pentene) (PMP) fibers were inserted into a test channel and exposed to a short-term continuous flow of human blood. The analysis included high-resolution PLT count, pooled PLT activation via β-Thromboglobulin (β-TG) and the visualization of remnants and clots on the fibers using scanning electron microscopy (SEM)., Results: In the Control group, PLT count was significantly decreased, and β-TG concentration was significantly elevated in comparison to the NOrel group. Macroscopic and microscopic visualization showed dense layers of stable clots on the bare PMP fibers, in contrast to minimal deposition of fibrin networks on the coated fibers., Conclusion: Endogenously NO-releasing microgel coating inhibits the PLT activation and reduces the clot formation on PMP fibers under dynamic flow.
Published: 2022
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38. DNase I functional microgels for neutrophil extracellular trap disruption.

Author: Hosseinnejad A, Ludwig N, Wienkamp AK, Rimal R, Bleilevens C, Rossaint R, Rossaint J, and Singh S
Subjects: Deoxyribonuclease I, Humans, Neutrophils, Extracellular Traps, Microgels, Thrombosis
Abstract: Neutrophil extracellular traps (NETs) are web-like chromatin structures produced and liberated by neutrophils under inflammatory conditions which also promote the activation of the coagulation cascade and thrombus formation. The formation of NETs is quite prominent when blood comes in contact with artificial surfaces like extracorporeal circuits, oxygenator membranes, or intravascular grafts. DNase I as a factor of the host defense system, digests the DNA backbone of NETs, which points out its treatment potential for NET-mediated thrombosis. However, the low serum stability of DNase I restricts its clinical/therapeutic applications. To improve the bioavailability of the enzyme, DNase I was conjugated to the microgels (DNase I MG) synthesized from highly hydrophilic N -(2-hydroxypropyl) methacrylamide (HPMA) and zwitterionic carboxybetaine methacrylamide (CBMAA). The enzyme was successfully conjugated to the microgels without any alternation to its secondary structure. The K m value representing the enzymatic activity of the conjugated DNase I was calculated to be 0.063 μM demonstrating a high enzyme-substrate affinity. The DNase I MGs were protein repellant and were able to digest NETs more efficiently compared to free DNase in a biological media, remarkably even after long-term exposure to the stimulated neutrophils continuously releasing NETs. Overall, the conjugation of DNase I to a non-fouling microgel provides a novel biohybrid platform that can be exploited as non-thrombogenic active microgel-based coatings for blood-contacting surfaces to reduce the NET-mediated inflammation and microthrombi formation.
Published: 2021
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39. Enzyme mimetic microgel coating for endogenous nitric oxide mediated inhibition of platelet activation.

Author: Hosseinnejad A, Fischer T, Jain P, Bleilevens C, Jakob F, Schwaneberg U, Rossaint R, and Singh S
Subjects: Adsorption, Humans, Nitric Oxide, Platelet Activation, Polymerization, Microgels
Abstract: Nitric oxide (NO) continuously generated by healthy endothelium prevents platelet activation and maintains vascular homeostasis. However, when artificial surfaces, like of extracorporeal membrane oxygenator comes in contact with blood, protein adsorption and thereby platelet activation takes place, which eventually leads to thrombus formation. To overcome this, we present an antifouling microgel coating mimicking the function of enzyme glutathione peroxidase to endogenously generate NO in the blood plasma from endogenous NO-donors and maintain a physiological NO flux. Microgels are synthesized by copolymerization of highly hydrophilic N-(2-hydroxypropyl)methacrylamide (HPMA) and glycidyl methacrylate (GMA) with diselenide crosslinks. For immobilization of the microgels on hydrophobic poly(4-methylpentene) (TPX) membranes bioengineered amphiphilic anchor peptides with free thiols are used. The anchor peptide attaches to the TPX membranes by hydrophobic interactions while the free thiols are presented for crosslinking with the microgels. The hydrophilic nature of the microgel coating prevents protein adsorption while the reversible diselenide bridges make the microgels responsive to the reducing environment and lead to the formation of reactive selenols/selenolates. The generated selenols/selenolates provide an efficient and sustained NO-release from endogenous S-nitrosothiols (RSNOs) mimicking the enzymatic function of glutathione peroxidase. On exposure to the whole blood, the microgel coating inhibited platelet activation and prolonged the blood clotting time., Competing Interests: Declaration of Competing Interest R.R. has received honoraria for lectures and consultancy from CSL Behring (Germany), Bayer HealthCare (Germany), Boehringer Ingelheim (Germany), and Novo Nordisk (Germany). The other authors have no conflicts of interest to declare., (Copyright © 2021 Elsevier Inc. All rights reserved.)
Published: 2021
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40. Hyperpolarization of cis- 15 N 2 -Azobenzene by Parahydrogen at Ultralow Magnetic Fields*.

Author: Sheberstov KF, Kozinenko VP, Kiryutin AS, Vieth HM, Zimmermann H, Ivanov KL, and Yurkovskaya AV
Abstract: The development of nuclear spins hyperpolarization, and the search for molecules that can be efficiently hyperpolarized is an active area in nuclear magnetic resonance. In this work we present a detailed study of SABRE SHEATH (signal amplification by reversible exchange in shield enabled alignment transfer to heteronuclei) experiments on 15 N 2 -azobenzene. In SABRE SHEATH experiments the nuclear spins of the target are hyperpolarized through transfer of spin polarization from parahydrogen at ultralow fields during a reversible chemical process. Azobenzene exists in two isomers, trans and cis. We show that all nuclear spins in cis-azobenzene can be efficiently hyperpolarized by SABRE at suitable magnetic fields. Enhancement factors (relative to 9.4 T) reach up to 3000 for 15 N spins and up to 30 for the 1 H spins. We compare two approaches to observe either hyperpolarized magnetization of 15 N/ 1 H spins, or hyperpolarized singlet order of the 15 N spin pair. The results presented here will be useful for further experiments in which hyperpolarized cis- 15 N 2 -azobenzene is switched by light to trans- 15 N 2 -azobenzene for storing the produced hyperpolarization in the long-lived spin state of the 15 N pair of trans- 15 N 2 -azobenzene., (© 2021 The Authors. ChemPhysChem published by Wiley-VCH GmbH.)
Published: 2021
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41. Mechanism and dynamics of fatty acid photodecarboxylase.

Author: Sorigué D, Hadjidemetriou K, Blangy S, Gotthard G, Bonvalet A, Coquelle N, Samire P, Aleksandrov A, Antonucci L, Benachir A, Boutet S, Byrdin M, Cammarata M, Carbajo S, Cuiné S, Doak RB, Foucar L, Gorel A, Grünbein M, Hartmann E, Hienerwadel R, Hilpert M, Kloos M, Lane TJ, Légeret B, Legrand P, Li-Beisson Y, Moulin SLY, Nurizzo D, Peltier G, Schirò G, Shoeman RL, Sliwa M, Solinas X, Zhuang B, Barends TRM, Colletier JP, Joffre M, Royant A, Berthomieu C, Weik M, Domratcheva T, Brettel K, Vos MH, Schlichting I, Arnoux P, Müller P, and Beisson F
Subjects: Algal Proteins chemistry, Algal Proteins metabolism, Alkanes metabolism, Amino Acid Substitution, Amino Acids metabolism, Bicarbonates metabolism, Biocatalysis, Carbon Dioxide metabolism, Catalytic Domain, Crystallography, X-Ray, Decarboxylation, Electron Transport, Flavin-Adenine Dinucleotide chemistry, Hydrogen Bonding, Light, Models, Molecular, Mutant Proteins chemistry, Mutant Proteins metabolism, Oxidation-Reduction, Photons, Protein Conformation, Temperature, Carboxy-Lyases chemistry, Carboxy-Lyases metabolism, Chlorella enzymology, Fatty Acids metabolism
Abstract: Fatty acid photodecarboxylase (FAP) is a photoenzyme with potential green chemistry applications. By combining static, time-resolved, and cryotrapping spectroscopy and crystallography as well as computation, we characterized Chlorella variabilis FAP reaction intermediates on time scales from subpicoseconds to milliseconds. High-resolution crystal structures from synchrotron and free electron laser x-ray sources highlighted an unusual bent shape of the oxidized flavin chromophore. We demonstrate that decarboxylation occurs directly upon reduction of the excited flavin by the fatty acid substrate. Along with flavin reoxidation by the alkyl radical intermediate, a major fraction of the cleaved carbon dioxide unexpectedly transformed in 100 nanoseconds, most likely into bicarbonate. This reaction is orders of magnitude faster than in solution. Two strictly conserved residues, R451 and C432, are essential for substrate stabilization and functional charge transfer., (Copyright © 2021 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)
Published: 2021
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42. Illumination guidelines for ultrafast pump-probe experiments by serial femtosecond crystallography.

Author: Grünbein ML, Stricker M, Nass Kovacs G, Kloos M, Doak RB, Shoeman RL, Reinstein J, Lecler S, Haacke S, and Schlichting I
Subjects: Electromagnetic Radiation, Lasers, Light, Scattering, Radiation, Crystallography, X-Ray methods, Photons
Abstract: Time-resolved crystallography with X-ray free-electron lasers enables structural characterization of light-induced reactions on ultrafast timescales. To be biologically and chemically relevant, such studies must be carried out in an appropriate photoexcitation regime to avoid multiphoton artifacts, a common issue in recent studies. We describe numerical and experimental approaches to determine how many photons are needed for single-photon excitation in microcrystals, taking into account losses by scattering.
Published: 2020
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43. Structural dynamics in proteins induced by and probed with X-ray free-electron laser pulses.

Author: Nass K, Gorel A, Abdullah MM, V Martin A, Kloos M, Marinelli A, Aquila A, Barends TRM, Decker FJ, Bruce Doak R, Foucar L, Hartmann E, Hilpert M, Hunter MS, Jurek Z, Koglin JE, Kozlov A, Lutman AA, Kovacs GN, Roome CM, Shoeman RL, Santra R, Quiney HM, Ziaja B, Boutet S, and Schlichting I
Subjects: Disulfides chemistry, Sulfur chemistry, X-Rays, Bacterial Proteins chemistry, Electrons, Lasers
Abstract: X-ray free-electron lasers (XFELs) enable crystallographic structure determination beyond the limitations imposed upon synchrotron measurements by radiation damage. The need for very short XFEL pulses is relieved through gating of Bragg diffraction by loss of crystalline order as damage progresses, but not if ionization events are spatially non-uniform due to underlying elemental distributions, as in biological samples. Indeed, correlated movements of iron and sulfur ions were observed in XFEL-irradiated ferredoxin microcrystals using unusually long pulses of 80 fs. Here, we report a femtosecond time-resolved X-ray pump/X-ray probe experiment on protein nanocrystals. We observe changes in the protein backbone and aromatic residues as well as disulfide bridges. Simulations show that the latter's correlated structural dynamics are much slower than expected for the predicted high atomic charge states due to significant impact of ion caging and plasma electron screening. This indicates that dense-environment effects can strongly affect local radiation damage-induced structural dynamics.
Published: 2020
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44. Author Correction: Generating and sustaining long-lived spin states in 15 N, 15 N'-azobenzene.

Author: Sheberstov KF, Vieth HM, Zimmermann H, Rodin BA, Ivanov KL, Kiryutin AS, and Yurkovskaya AV
Abstract: An amendment to this paper has been published and can be accessed via a link at the top of the paper.
Published: 2020
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45. Photoswitching mechanism of a fluorescent protein revealed by time-resolved crystallography and transient absorption spectroscopy.

Author: Woodhouse J, Nass Kovacs G, Coquelle N, Uriarte LM, Adam V, Barends TRM, Byrdin M, de la Mora E, Bruce Doak R, Feliks M, Field M, Fieschi F, Guillon V, Jakobs S, Joti Y, Macheboeuf P, Motomura K, Nass K, Owada S, Roome CM, Ruckebusch C, Schirò G, Shoeman RL, Thepaut M, Togashi T, Tono K, Yabashi M, Cammarata M, Foucar L, Bourgeois D, Sliwa M, Colletier JP, Schlichting I, and Weik M
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.
Published: 2020
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46. Generating and sustaining long-lived spin states in 15 N, 15 N'-azobenzene.

Author: Sheberstov KF, Vieth HM, Zimmermann H, Rodin BA, Ivanov KL, Kiryutin AS, and Yurkovskaya AV
Abstract: Long-Lived spin States (LLSs) hold a great promise for sustaining non-thermal spin order and investigating various slow processes by Nuclear Magnetic Resonance (NMR) spectroscopy. Of special interest for such application are molecules containing nearly equivalent magnetic nuclei, which possess LLSs even at high magnetic fields. In this work, we report an LLS in trans- 15 N, 15 N'-azobenzene. The singlet state of the 15 N spin pair exhibits a long-lived character. We solve the challenging problem of generating and detecting this LLS and further increase the LLS population by converting the much higher magnetization of protons into the 15 N singlet spin order. As far as the longevity of this spin order is concerned, various schemes have been tested for sustaining the LLS. Lifetimes of 17 minutes have been achieved at 16.4 T, a value about 250 times longer than the longitudinal relaxation time of 15 N in this magnetic field. We believe that such extended relaxation times, along with the photochromic properties of azobenzene, which changes conformation upon light irradiation and can be hyperpolarized by using parahydrogen, are promising for designing new experiments with photo-switchable long-lived hyperpolarization.
Published: 2019
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47. Three-dimensional view of ultrafast dynamics in photoexcited bacteriorhodopsin.

Author: Nass Kovacs G, Colletier JP, Grünbein ML, Yang Y, Stensitzki T, Batyuk A, Carbajo S, Doak RB, Ehrenberg D, Foucar L, Gasper R, Gorel A, Hilpert M, Kloos M, Koglin JE, Reinstein J, Roome CM, Schlesinger R, Seaberg M, Shoeman RL, Stricker M, Boutet S, Haacke S, Heberle J, Heyne K, Domratcheva T, Barends TRM, and Schlichting I
Subjects: Crystallography, Isomerism, Light, Photons, Protein Conformation, Spectrum Analysis, Water chemistry, Bacteriorhodopsins chemistry, Halobacterium salinarum metabolism, Retinaldehyde chemistry
Abstract: Bacteriorhodopsin (bR) is a light-driven proton pump. The primary photochemical event upon light absorption is isomerization of the retinal chromophore. Here we used time-resolved crystallography at an X-ray free-electron laser to follow the structural changes in multiphoton-excited bR from 250 femtoseconds to 10 picoseconds. Quantum chemistry and ultrafast spectroscopy were used to identify a sequential two-photon absorption process, leading to excitation of a tryptophan residue flanking the retinal chromophore, as a first manifestation of multiphoton effects. We resolve distinct stages in the structural dynamics of the all-trans retinal in photoexcited bR to a highly twisted 13-cis conformation. Other active site sub-picosecond rearrangements include correlated vibrational motions of the electronically excited retinal chromophore, the surrounding amino acids and water molecules as well as their hydrogen bonding network. These results show that this extended photo-active network forms an electronically and vibrationally coupled system in bR, and most likely in all retinal proteins.
Published: 2019
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48. A versatile liquid-jet setup for the European XFEL.

Author: Schulz J, Bielecki J, Doak RB, Dörner K, Graceffa R, Shoeman RL, Sikorski M, Thute P, Westphal D, and Mancuso AP
Abstract: The SPB/SFX instrument of the European XFEL provides unique possibilities for high-throughput serial femtosecond crystallography. This publication presents the liquid-jet sample delivery setup of this instrument. The setup is compatible with state-of-the-art gas dynamic virtual nozzle systems as well as high-viscosity extruders and provides space and flexibility for other liquid injection devices and future upgrades. The liquid jets are confined in a differentially pumped catcher assembly and can be replaced within a couple of minutes through a load-lock. A two-microscope imaging system allows visual control of the jets from two perspectives., (open access.)
Published: 2019
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49. Constant-adiabaticity radiofrequency pulses for generating long-lived singlet spin states in NMR.

Author: Rodin BA, Sheberstov KF, Kiryutin AS, Hill-Cousins JT, Brown LJ, Brown RCD, Jamain B, Zimmermann H, Sagdeev RZ, Yurkovskaya AV, and Ivanov KL
Abstract: A method is implemented to perform "fast" adiabatic variation of the spin Hamiltonian by imposing the constant adiabaticity condition. The method is applied to improve the performance of singlet-state Nuclear Magnetic Resonance (NMR) experiments, specifically, for efficient generation and readout of the singlet spin order in coupled spin pairs by applying adiabatically ramped RF-fields. Test experiments have been performed on a specially designed molecule having two strongly coupled 13 C spins and on selectively isotopically labelled glycerol having two pairs of coupled protons. Optimized RF-ramps show improved performance in comparison, for example, to linear ramps. We expect that the methods described here are useful not only for singlet-state NMR experiments but also for other experiments in magnetic resonance, which utilize adiabatic variation of the spin Hamiltonian.
Published: 2019
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50. Free-electron laser data for multiple-particle fluctuation scattering analysis.

Author: Pande K, Donatelli JJ, Malmerberg E, Foucar L, Poon BK, Sutter M, Botha S, Basu S, Bruce Doak R, Dörner K, Epp SW, Englert L, Fromme R, Hartmann E, Hartmann R, Hauser G, Hattne J, Hosseinizadeh A, Kassemeyer S, Lomb L, Montero SFC, Menzel A, Rolles D, Rudenko A, Seibert MM, Sierra RG, Schwander P, Ourmazd A, Fromme P, Sauter NK, Bogan M, Bozek J, Bostedt C, Schlichting I, Kerfeld CA, and Zwart PH
Subjects: Phycodnaviridae, Scattering, Small Angle, X-Ray Diffraction
Abstract: Fluctuation X-ray scattering (FXS) is an emerging experimental technique in which solution scattering data are collected using X-ray exposures below rotational diffusion times, resulting in angularly anisotropic X-ray snapshots that provide several orders of magnitude more information than traditional solution scattering data. Such experiments can be performed using the ultrashort X-ray pulses provided by a free-electron laser source, allowing one to collect a large number of diffraction patterns in a relatively short time. Here, we describe a test data set for FXS, obtained at the Linac Coherent Light Source, consisting of close to 100 000 multi-particle diffraction patterns originating from approximately 50 to 200 Paramecium Bursaria Chlorella virus particles per snapshot. In addition to the raw data, a selection of high-quality pre-processed diffraction patterns and a reference SAXS profile are provided.
Published: 2018
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