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1. Identification of potential aggregation hotspots on Aβ42 fibrils blocked by the anti-amyloid chaperone-like BRICHOS domain

Author

Rakesh Kumar, Tanguy Le Marchand, Laurène Adam, Raitis Bobrovs, Gefei Chen, Jēkabs Fridmanis, Nina Kronqvist, Henrik Biverstål, Kristaps Jaudzems, Jan Johansson, Guido Pintacuda, and Axel Abelein

Subjects
Science
Abstract

Abstract Protein misfolding can generate toxic intermediates, which underlies several devastating diseases, such as Alzheimer’s disease (AD). The surface of AD-associated amyloid-β peptide (Aβ) fibrils has been suggested to act as a catalyzer for self-replication and generation of potentially toxic species. Specifically tailored molecular chaperones, such as the BRICHOS protein domain, were shown to bind to amyloid fibrils and break this autocatalytic cycle. Here, we identify a site on the Aβ42 fibril surface, consisting of three C-terminal β-strands and particularly the solvent-exposed β-strand stretching from residues 26–28, which is efficiently sensed by a designed variant of Bri2 BRICHOS. Remarkably, while only a low amount of BRICHOS binds to Aβ42 fibrils, fibril-catalyzed nucleation processes are effectively prevented, suggesting that the identified site acts as a catalytic aggregation hotspot, which can specifically be blocked by BRICHOS. Hence, these findings provide an understanding how toxic nucleation events can be targeted by molecular chaperones.

Published
2024
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2. Mg2+-dependent conformational equilibria in CorA and an integrated view on transport regulation

Author

Nicolai Tidemand Johansen, Marta Bonaccorsi, Tone Bengtsen, Andreas Haahr Larsen, Frederik Grønbæk Tidemand, Martin Cramer Pedersen, Pie Huda, Jens Berndtsson, Tamim Darwish, Nageshewar Rao Yepuri, Anne Martel, Thomas Günther Pomorski, Andrea Bertarello, Mark Sansom, Mikaela Rapp, Ramon Crehuet, Tobias Schubeis, Kresten Lindorff-Larsen, Guido Pintacuda, and Lise Arleth

Subjects
cora mg2+ channel, small-angle neutron scattering, solid-state nuclear magnetic resonance, metadynamics simulation, Medicine, Science, Biology (General), QH301-705.5
Abstract

The CorA family of proteins regulates the homeostasis of divalent metal ions in many bacteria, archaea, and eukaryotic mitochondria, making it an important target in the investigation of the mechanisms of transport and its functional regulation. Although numerous structures of open and closed channels are now available for the CorA family, the mechanism of the transport regulation remains elusive. Here, we investigated the conformational distribution and associated dynamic behaviour of the pentameric Mg2+ channel CorA at room temperature using small-angle neutron scattering (SANS) in combination with molecular dynamics (MD) simulations and solid-state nuclear magnetic resonance spectroscopy (NMR). We find that neither the Mg2+-bound closed structure nor the Mg2+-free open forms are sufficient to explain the average conformation of CorA. Our data support the presence of conformational equilibria between multiple states, and we further find a variation in the behaviour of the backbone dynamics with and without Mg2+. We propose that CorA must be in a dynamic equilibrium between different non-conducting states, both symmetric and asymmetric, regardless of bound Mg2+ but that conducting states become more populated in Mg2+-free conditions. These properties are regulated by backbone dynamics and are key to understanding the functional regulation of CorA.

Published
2022
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3. Conformational dynamics in crystals reveal the molecular bases for D76N beta-2 microglobulin aggregation propensity

Author

Tanguy Le Marchand, Matteo de Rosa, Nicola Salvi, Benedetta Maria Sala, Loren B. Andreas, Emeline Barbet-Massin, Pietro Sormanni, Alberto Barbiroli, Riccardo Porcari, Cristiano Sousa Mota, Daniele de Sanctis, Martino Bolognesi, Lyndon Emsley, Vittorio Bellotti, Martin Blackledge, Carlo Camilloni, Guido Pintacuda, and Stefano Ricagno

Subjects
Science
Abstract

The aggregation prone D76N beta-2 microglobulin mutant causes systemic amyloidosis. Here the authors combine crystallography, solid-state NMR, and computational studies and show that the D76N mutation increases protein dynamics and destabilizes the outer strands, which leads to an exposure of amyloidogenic parts explaining its aggregation propensity.

Published
2018
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4. Structure of outer membrane protein G in lipid bilayers

Author

Joren S. Retel, Andrew J. Nieuwkoop, Matthias Hiller, Victoria A. Higman, Emeline Barbet-Massin, Jan Stanek, Loren B. Andreas, W. Trent Franks, Barth-Jan van Rossum, Kutti R. Vinothkumar, Lieselotte Handel, Gregorio Giuseppe de Palma, Benjamin Bardiaux, Guido Pintacuda, Lyndon Emsley, Werner Kühlbrandt, and Hartmut Oschkinat

Subjects
Science
Abstract

Porins, like OmpG, are embedded in the outer membrane of bacteria and facilitate uptake and secretion of nutrients and ions. Here the authors present a protocol for solid state NMR structure determination of proteins larger than 25 kDa and use it to structurally characterize membrane embedded OmpG.

Published
2017
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5. Electronic structure of Yb(III)[CH(SiMe3)2]3 from magnetic resonance spectroscopies

Author

Anton Ashuiev, Florian Allouche, José P. Carvalho, Kevin J. Sanders, Matthew P. Conley, Daniel Klose, Giuseppe Lapadula, Michael Wörle, Dirk Baabe, Marc D. Walter, Andrew J. Pell, Christophe Copéret, Gunnar Jeschke, Guido Pintacuda, and Richard A. Andersen

Abstract

Characterization of paramagnetic compounds, in particular regarding the detailed conformation and electronic structure, remains a challenge - still today it often relies solely on the use of X-ray crystallography, thus limiting the access to electronic structure information. This is particularly true for lanthanide elements that are often associated with peculiar structural and electronic features in relation to their partially filled f -shell. Here, we showcase the use of state-of-the-art magnetic resonance spec- troscopy (EPR and solid-state NMR) and computational approaches as well as magnetic susceptibility measurements to determine the structure of a paramagnetic Yb(III) alkyl complex, Yb(III)[CH(SiMe3)2]3, that features a notable structure according to X-ray crystallography. Each of these techniques revealed specific information about the geometry and electronic structure of the complex; taken together, they provide a detailed understanding of this paramagnetic compound. Namely, this complex displays a three-centre-two-electron Yb-γ-Me-β–Si secondary metal-ligand interaction, whose NMR spectroscopic signature was acquired for the first time for a lanthanide paramagnetic species. The electronic configuration of Yb(III)[CH(SiMe3)2]3 is demonstrated to be close to the one of the free Yb(III) ion, with the partially filled f -shell of the Yb atom having little influence on its bonding properties and with minimal delocalization of f -electron density from Yb to the directly bonded carbons.

Published
2023
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8. Backbone assignment of crystalline E. coli maltose binding protein

Author

Tobias Schubeis, Jan Stanek, Guido Pintacuda, Centre de RMN à très hauts champs de Lyon (CRMN), École normale supérieure de Lyon (ENS de 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), University of Warsaw (UW), and École normale supérieure - Lyon (ENS Lyon)-Université Claude Bernard Lyon 1 (UCBL)

Subjects
0303 health sciences, [SDV.BBM.BS]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Structural Biology [q-bio.BM], biology, Accession number (library science), Chemistry, Chemical shift, 030303 biophysics, Resonance (chemistry), [SDV.MP.BAC]Life Sciences [q-bio]/Microbiology and Parasitology/Bacteriology, Biochemistry, Maltose-Binding Proteins, NMR spectra database, 03 medical and health sciences, Crystallography, Maltose-binding protein, Microcrystalline, Molecular size, Structural Biology, biology.protein, Molecule, 030304 developmental biology
Abstract

International audience; The E.coli maltose binding protein (MBP) is a 42.5 kDa molecule widely employed in many biotechnology applications. Because of its molecular size, it has become the main model system for the development of solution NMR methods adapted to large biomolecular targets. Here, we report virtually complete (~90%) backbone resonance assignments obtained on a microcrystalline sample of MBP with 1 H-detected solidstate NMR at fast (>100 kHz) magic-angle spinning. We additionally present the detailed description of the methodology employed for the preparation of the sample and the acquisition and analysis of the NMR spectra. The chemical shifts, obtained with a single uniformly 15 N, 13 C-labelled and fully-protonated sample and about two weeks on a 800 MHz NMR spectrometer, have been deposited to the BMRB under the accession number 50089.

Published
2021
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9. Multimodal Response to Copper Binding in Superoxide Dismutase Dynamics

Author

Lyndon Emsley, Guido Pintacuda, Roberta Pierattelli, Tobias Schubeis, Marta Bonaccorsi, Hugh R W Dannatt, Michael J. Knight, Tanguy Le Marchand, Loïc Salmon, Isabella C. Felli, Centre de RMN à très hauts champs de Lyon (CRMN), 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), Université de Florence, Università degli Studi di Firenze = University of Florence [Firenze] (UNIFI), Ecole Polytechnique Fédérale de Lausanne (EPFL), École normale supérieure de Lyon (ENS de Lyon)-Université Claude Bernard Lyon 1 (UCBL), and Università degli Studi di Firenze = University of Florence (UniFI)

Subjects
Models, Molecular, Magnetic Resonance Spectroscopy, Protein Conformation, [SDV]Life Sciences [q-bio], chemistry.chemical_element, Zinc, 010402 general chemistry, 01 natural sciences, Biochemistry, Catalysis, Superoxide dismutase, Colloid and Surface Chemistry, Protein structure, Metalloproteins, Side chain, Humans, [CHIM]Chemical Sciences, Histidine, Binding site, Binding Sites, biology, Superoxide Dismutase, Chemistry, Amyotrophic Lateral Sclerosis, General Chemistry, Nuclear magnetic resonance spectroscopy, Copper, 0104 chemical sciences, Kinetics, Magnetic Fields, Biophysics, biology.protein, Protein Multimerization, Crystallization
Abstract

International audience; Copper/zinc superoxide dismutase (SOD) is a homodimeric metalloenzyme which has been extensively studied as a benchmark for structure-function relationships in proteins, in particular because of its implication in the familial form of the neurodegenerative disease amyotrophic lateral sclerosis. Here, we investigate microcrystalline preparations of two differently metallated forms of SOD, namely the fully mature functional Cu I ,Zn state and the E,Zn-SOD state in which the Cu site is empty. By using solid-state NMR with fast magic-angle spinning (MAS) at high magnetic fields (1 H Larmor frequency of 800-1000 MHz), we quantify motions spanning a dynamic range from ns to ms. We determine that metal ion uptake does not act as a rigidification element but as a switch redistributing motional processes on different timescales, with coupling of the dynamics of histidine sidechains and those of remote key backbone elements of the protein.

Published
2020
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10. Author response: Mg2+-dependent conformational equilibria in CorA and an integrated view on transport regulation

Author

Tone Bengtsen, Marta Bonaccorsi, Nicolai Tidemand Johansen, Andreas Haahr Larsen, Frederik Grønbæk Tidemand, Martin Cramer Pedersen, Pie Huda, Jens Berndtsson, Tamim Darwish, Nageshewar Rao Yepuri, Anne Martel, Thomas Günther Pomorski, Andrea Bertarello, Mark Sansom, Mikaela Rapp, Ramon Crehuet, Tobias Schubeis, Kresten Lindorff-Larsen, Guido Pintacuda, and Lise Arleth

Published
2022
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11. 1H detection and dynamic nuclear polarization–enhanced NMR of Aβ1-42 fibrils

Author

Salima Bahri, Robert Silvers, Brian Michael, Kristaps Jaudzems, Daniela Lalli, Gilles Casano, Olivier Ouari, Anne Lesage, Guido Pintacuda, Sara Linse, Robert G. Griffin, Chemistry Department [Massachusetts Institute of Technology], Massachusetts Institute of Technology (MIT), Department of Chemistry [MIT Cambridge], Francis Bitter Magnet Lab, Centre de RMN à très hauts champs de Lyon (CRMN), École normale supérieure de Lyon (ENS de 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), École normale supérieure de Lyon (ENS de Lyon), Institut de Chimie Radicalaire (ICR), Aix Marseille Université (AMU)-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS), and Department of Biochemistry & Structural Biology, Center for Molecular Protein Science

Subjects
Multidisciplinary, magic-angle spinning, [SDV.BBM.BS]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Structural Biology [q-bio.BM], Biological Sciences, [CHIM.THEO]Chemical Sciences/Theoretical and/or physical chemistry, [SDV.BBM.BP]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biophysics, dynamic nuclear polarization, Biophysics and Computational Biology, [CHIM.ANAL]Chemical Sciences/Analytical chemistry, Physical Sciences, amyloid β1-42, 1H detection
Abstract

Significance Amyloid-β (Aβ) is the subject of intense scrutiny because of its close association with Alzheimer’s disease (AD), which currently afflicts about 50 million people worldwide. The results reported in this manuscript focus on the new possibilities provided by ultrafast magic-angle spinning (MAS) 1H detection and fast-MAS dynamic nuclear polarization (DNP), which have ushered in a new era for NMR-based structural biology, but whose potential has not yet been fully exploited for the structural investigation of complex amyloid assemblies. This work demonstrates the expeditious structural analysis of amyloid fibrils, without requiring preparation of large sample amounts, and sets the stage for future studies of unlabeled AD peptides derived from tissue samples available in limited quantities., Several publications describing high-resolution structures of amyloid-β (Aβ) and other fibrils have demonstrated that magic-angle spinning (MAS) NMR spectroscopy is an ideal tool for studying amyloids at atomic resolution. Nonetheless, MAS NMR suffers from low sensitivity, requiring relatively large amounts of samples and extensive signal acquisition periods, which in turn limits the questions that can be addressed by atomic-level spectroscopic studies. Here, we show that these drawbacks are removed by utilizing two relatively recent additions to the repertoire of MAS NMR experiments—namely, 1H detection and dynamic nuclear polarization (DNP). We show resolved and sensitive two-dimensional (2D) and three-dimensional (3D) correlations obtained on 13C,15N-enriched, and fully protonated samples of M0Aβ1-42 fibrils by high-field 1H-detected NMR at 23.4 T and 18.8 T, and 13C-detected DNP MAS NMR at 18.8 T. These spectra enable nearly complete resonance assignment of the core of M0Aβ1-42 (K16-A42) using submilligram sample quantities, as well as the detection of numerous unambiguous internuclear proximities defining both the structure of the core and the arrangement of the different monomers. An estimate of the sensitivity of the two approaches indicates that the DNP experiments are currently ∼6.5 times more sensitive than 1H detection. These results suggest that 1H detection and DNP may be the spectroscopic approaches of choice for future studies of Aβ and other amyloid systems.

Published
2021
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12. Mg2+-dependent conformational equilibria in CorA: an integrated view on transport regulation

Author

Jens Berndtsson, Tamim A. Darwish, Anne L. Martel, Guido Pintacuda, Mikaela Rapp, Tobias Schubeis, Lise Arleth, Andreas Haahr Larsen, Frederik Grønbæk Tidemand, Thomas Günther Pomorski, Marta Bonaccorsi, Ramon Crehuet, Martin Cramer Pedersen, Kresten Lindorff-Larsen, Andrea Bertarello, Mark S.P. Sansom, Pie Huda, Nageshewar Rao Yepuri, Nicolai Tidemand Johansen, Tone Bengtsen, Centre de RMN à très hauts champs de Lyon (CRMN), 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), Niels Bohr Institute [Copenhagen] (NBI), Faculty of Science [Copenhagen], University of Copenhagen = Københavns Universitet (KU)-University of Copenhagen = Københavns Universitet (KU), Australian Nuclear Science and Technology Organisation [Australie] (ANSTO), École normale supérieure de Lyon (ENS de Lyon)-Université Claude Bernard Lyon 1 (UCBL), and University of Copenhagen = Københavns Universitet (UCPH)-University of Copenhagen = Københavns Universitet (UCPH)

Subjects
0303 health sciences, [SDV.BBM.BS]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Structural Biology [q-bio.BM], Chemistry, Nuclear magnetic resonance spectroscopy, Neutron scattering, 010402 general chemistry, 01 natural sciences, 0104 chemical sciences, 03 medical and health sciences, Molecular dynamics, Chemical physics, [CHIM.ANAL]Chemical Sciences/Analytical chemistry, Divalent metal ions, Dynamic equilibrium, 030304 developmental biology
Abstract

The CorA family of proteins regulates the homeostasis of divalent metal ions in many bacteria, archaea, and eukaryotic mitochondria, making it an important target in the investigation of the mechanisms of transport and its functional regulation. Although numerous structures of open and closed channels are now available for the CorA family, the mechanism of the transport regulation remains elusive. Here, we investigated the conformational distribution and associated dynamic behaviour of the pentameric Mg2+ channel CorA at room temperature using small-angle neutron scattering (SANS) in combination with molecular dynamics (MD) simulations and solid-state nuclear magnetic resonance spectroscopy (NMR). We find that neither the Mg2+-bound closed structure nor the Mg2+-free open forms are sufficient to explain the average conformation of CorA. Our data support the presence of conformational equilibria between multiple states, and we further find a variation in the behaviour of the backbone dynamics with and without Mg2+. We propose that CorA must be in a dynamic equilibrium between different non-conducting states, both symmetric and asymmetric, regardless of bound Mg2+ but that conducting states become more populated in Mg2+-free conditions. These properties are regulated by backbone dynamics and are key to understanding the functional regulation of CorA.

Published
2021

13. Proton-detected fast-magic-angle spinning NMR of paramagnetic inorganic solids

Author

David Proriol, Andrew J. Pell, Guido Pintacuda, Leonor Catita, Benjamin Burcher, Ladislav Benda, Kevin J. Sanders, Erwann Jeanneau, Anne-Agathe Quoineaud, Pierre-Alain Breuil, Arthur L. Lejeune, Jan Blahut, Centre de RMN à très hauts champs de Lyon (CRMN), 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), IFP Energies nouvelles (IFPEN), Centre de diffractométrie Henri Longchambon, Institut de Chimie de Lyon, Université de Lyon-Université de Lyon-École normale supérieure - Lyon (ENS Lyon)-Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon, Axens, École normale supérieure de Lyon (ENS de Lyon)-Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-École normale supérieure de Lyon (ENS de Lyon)-Université Claude Bernard Lyon 1 (UCBL), Stockholm University, and ANR-15-CE29-0025,MRCAT,Spectroscopies de Résonance Magnétique (RMN, RPE) pour la Catalyse de Polymérisation(2015)

Subjects
Materials science, Proton, 010405 organic chemistry, Abundance (chemistry), General Chemical Engineering, Analytical chemistry, General Chemistry, [CHIM.CATA]Chemical Sciences/Catalysis, 010402 general chemistry, 01 natural sciences, 0104 chemical sciences, 3. Good health, Catalysis, Paramagnetism, Polymerization, [CHIM.ANAL]Chemical Sciences/Analytical chemistry, Magic angle spinning
Abstract

International audience; Fast (60 kHz) magic angle spinning solid-state NMR allows very sensitive proton detection in highly paramagnetic organometallic powders. We showcase this technique with the complete assignment of 1 H and 13 C resonances in a high-spin Fe(II) polymerisation catalyst with less than 2 mg of sample at natural abundance.

Published
2021
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14. Monitoring Dynamics, Structure, and Magnetism of Switchable Metal-Organic Frameworks via 1 H-Detected MAS NMR

Author

Florian M. Wisser, Sebastian Ehrling, Arthur L. Lejeune, Guido Pintacuda, Jan Blahut, Stefan Kaskel, Irena Senkovska, Centre de RMN à très hauts champs de Lyon (CRMN), École normale supérieure de Lyon (ENS de 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), Charles University [Prague] (CU), IFP Energies nouvelles (IFPEN), Technische Universität Dresden = Dresden University of Technology (TU Dresden), Chair of Inorganic Chemistry I, Institut de recherches sur la catalyse et l'environnement de Lyon (IRCELYON), Université Claude Bernard Lyon 1 (UCBL), Universität Regensburg (UR), and École normale supérieure - Lyon (ENS Lyon)-Université Claude Bernard Lyon 1 (UCBL)

Subjects
Materials science, Resolution (mass spectrometry), Magnetism, Natural abundance, DUT-8(Ni), 010402 general chemistry, paramagnetic NMR, 01 natural sciences, Catalysis, Metal, metal–organic framework, Paramagnetism, [CHIM.ANAL]Chemical Sciences/Analytical chemistry, fast magic-angle spinning, Electronic properties, 010405 organic chemistry, Communication, General Chemistry, Atmospheric temperature range, Communications, 0104 chemical sciences, proton detection, Chemical physics, visual_art, visual_art.visual_art_medium, Metal-organic framework, Structure Elucidation
Abstract

We present a toolbox for the rapid characterisation of powdered samples of paramagnetic metal–organic frameworks at natural abundance by 1H‐detected solid‐state NMR. Very fast MAS rates at room and cryogenic temperatures and a set of tailored radiofrequency irradiation schemes help overcome the sensitivity and resolution limits often associated with the characterisation of MOF materials. We demonstrate the approach on DUT‐8(Ni), a framework containing Ni2+ paddle‐wheel units which can exist in two markedly different architectures. Resolved 1H and 13C resonances of organic linkers are detected and assigned in few hours with only 1–2 mg of sample at natural isotopic abundance, and used to rapidly extract information on structure and local internal dynamics of the assemblies, as well as to elucidate the metal electronic properties over an extended temperature range. The experiments disclose new possibilities for describing local and global structural changes and correlating them to electronic and magnetic properties of the assemblies., Paramagnetic DUT‐8(Ni) metal–organic frameworks were characterised over an extended temperature range with 1H‐detected NMR techniques under fast magic‐angle spinning. The sensitivity of the approach allows rapid access to structure and dynamics of the organic linkers as well as the electronic properties of the metal centres in these materials.

Published
2021
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15. DNP NMR of biomolecular assemblies

Author

Tatyana Polenova, Kristaps Jaudzems, Guido Pintacuda, Hartmut Oschkinat, Anne Lesage, Centre de RMN à très hauts champs de Lyon (CRMN), École normale supérieure de Lyon (ENS de 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), Department of Chemistry and Biochemistry, University of Delaware [Newark], Leibniz Forschungsinstitut für Molekulare Pharmakolgie = Leibniz Institute for Molecular Pharmacology [Berlin, Allemagne] (FMP), Leibniz Association, and École normale supérieure - Lyon (ENS Lyon)-Université Claude Bernard Lyon 1 (UCBL)

Subjects
0303 health sciences, Magnetic Resonance Spectroscopy, Materials science, Free Radicals, Molecular Structure, [SDV]Life Sciences [q-bio], 030302 biochemistry & molecular biology, Temperature, Membrane Proteins, Nanotechnology, Nuclear magnetic resonance spectroscopy, 03 medical and health sciences, Solid-state nuclear magnetic resonance, Unpaired electron, Structural Biology, Microwave irradiation, Magic angle spinning, [CHIM]Chemical Sciences, Capsid Proteins, Microwaves, Peptides, Polarization (electrochemistry), Nuclear Magnetic Resonance, Biomolecular, ComputingMilieux_MISCELLANEOUS, 030304 developmental biology
Abstract

Dynamic Nuclear Polarization (DNP) is an effective approach to alleviate the inherently low sensitivity of solid-state NMR (ssNMR) under magic angle spinning (MAS) towards large-sized multi-domain complexes and assemblies. DNP relies on a polarization transfer at cryogenic temperatures from unpaired electrons to adjacent nuclei upon continuous microwave irradiation. This is usually made possible via the addition in the sample of a polarizing agent. The first pioneering experiments on biomolecular assemblies were reported in the early 2000s on bacteriophages and membrane proteins. Since then, DNP has experienced tremendous advances, with the development of extremely efficient polarizing agents or with the introduction of new microwaves sources, suitable for NMR experiments at very high magnetic fields (currently up to 900 MHz). After a brief introduction, several experimental aspects of DNP enhanced NMR spectroscopy applied to biomolecular assemblies are discussed. Recent demonstration experiments of the method on viral capsids, the type III and IV bacterial secretion systems, ribosome and membrane proteins are then described.

Published
2019
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16. Mg

Author

Nicolai Tidemand, Johansen, Marta, Bonaccorsi, Tone, Bengtsen, Andreas Haahr, Larsen, Frederik Grønbæk, Tidemand, Martin Cramer, Pedersen, Pie, Huda, Jens, Berndtsson, Tamim, Darwish, Nageshewar Rao, Yepuri, Anne, Martel, Thomas Günther, Pomorski, Andrea, Bertarello, Mark, Sansom, Mikaela, Rapp, Ramon, Crehuet, Tobias, Schubeis, Kresten, Lindorff-Larsen, Guido, Pintacuda, and Lise, Arleth

Subjects
Models, Molecular, Magnetic Resonance Spectroscopy, Models, Chemical, Protein Conformation, Biological Transport, Magnesium, Molecular Dynamics Simulation, Cation Transport Proteins
Abstract

The CorA family of proteins regulates the homeostasis of divalent metal ions in many bacteria, archaea, and eukaryotic mitochondria, making it an important target in the investigation of the mechanisms of transport and its functional regulation. Although numerous structures of open and closed channels are now available for the CorA family, the mechanism of the transport regulation remains elusive. Here, we investigated the conformational distribution and associated dynamic behaviour of the pentameric Mg

Published
2021

17. Spectroscopic Signature and Structure of Active Sites in Ziegler-Natta Polymerization Catalysts revealed by Electron Paramagnetic Resonance

Author

Anton Ashuiev, Matthieu Humbert, David Gajan, Sebastien Norsic, Jan Blahut, Keith Searles, Daniel Klose, Anne Lesage, Guido Pintacuda, Jean Raynaud, Vincent Monteil, Christophe Copéret, and Gunnar Jeschke

Subjects
inorganic chemicals
Abstract

Despite decades of extensive studies, the atomic-scale structure of the active sites in heterogeneous Ziegler-Natta (ZN) catalysts, one of the most important processes of the chemical industry, remains elusive and a matter of debate. In the present work, the structure of “active sites” of ZN catalysts in the absence of ethylene, referred to as “dormant active sites”, is elucidated from magnetic resonance experiments, carried out on samples reacted with increasing amounts of BCl3 so as to enhance the concentration of active sites and observe clear spectroscopic signatures. Using EPR and NMR spectroscopies, in particular 2D HYSCORE experiments complemented by DFT calculations, we show that the activated ZN catalysts contain bimetallic alkyl-Ti(III),Al species whose amount is directly linked to the polymerization activity of MgCl2-supported Ziegler-Natta catalysts. This connects those spectroscopic signatures to the active species formed in the presence of ethylene, and enables us propose an ethylene polymerization mechanism on the observed bimetallic alkyl-Ti(III),Al species based on DFT computations

Published
2021
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18. Structural and molecular basis of cross-seeding barriers in amyloids

Author

Denis Martinez, Abdelmajid Noubhani, Loren B. Andreas, Guido Pintacuda, Benjamin Bardiaux, Brice Kauffmann, Asen Daskalov, Mélanie Berbon, Antoine Loquet, Joseph S. Wall, Virginie Coustou, Nadia El Mammeri, Jan Stanek, Sven J. Saupe, Birgit Habenstein, Chimie et Biologie des Membranes et des Nanoobjets (CBMN), Université de Bordeaux (UB)-École Nationale d'Ingénieurs des Travaux Agricoles - Bordeaux (ENITAB)-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS), Institut de biochimie et génétique cellulaires (IBGC), Université Bordeaux Segalen - Bordeaux 2-Centre National de la Recherche Scientifique (CNRS), Centre de Résonance Magnetique Nucleaire (CRMN), Institut de Chimie et Biochimie Moléculaires et Supramoléculaires (ICBMS), Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-École Supérieure de Chimie Physique Électronique de Lyon (CPE)-Institut National des Sciences Appliquées de Lyon (INSA Lyon), Université de Lyon-Institut National des Sciences Appliquées (INSA)-Institut National des Sciences Appliquées (INSA)-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS)-Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Institut National des Sciences Appliquées (INSA)-Institut National des Sciences Appliquées (INSA)-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS), Bioinformatique structurale - Structural Bioinformatics, Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS), Institut Européen de Chimie et Biologie (IECB), Université de Bordeaux (UB)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Brookhaven National Laboratory [Upton, NY] (BNL), UT-Battelle, LLC-Stony Brook University [SUNY] (SBU), State University of New York (SUNY)-State University of New York (SUNY)-U.S. Department of Energy [Washington] (DOE), We acknowledge financial support from the European Research Council (ERC) under the European Unions Horizon 2020 Research and Innovation Programme (ERC-2015-CoG GA 648974 to G.P. and ERC-2015-StG GA 639020 to A.L.), IdEx Bordeaux (Chaire d’Installation to B.H., ANR-10-IDEX-03-02), the Agence Nationale de la Recherche (ANR) (ANR-14-CE09-0020-01 to A.L., ANR-13-PDOC-0017-01 to B.H. and ANR-17-CE11-0035 to S.J.S), the INCEPTION project (Programme d'investissements d'avenir/ANR-16-CONV-0005) and the CNRS (IR-RMN FR3050). J.S. and L.B.A. were supported by individual Marie Sklodowska-Curie incoming fellowships (grant agreements 661799 'COMPLEX-FAST-MAS' and 624918 'MEM-MAS'). A.D. was supported by the Nouvelle Aquitaine Regional Council., We thank the Nouvelle Aquitaine Regional Council, University of Bordeaux, and the Contrat Plan Etat-Region (CPER) CampusB Bordeaux for the acquisition of the NMR equipment. This work has benefited from the Biophysical and Structural Chemistry Platform at IECB, CNRS UMS 3033, INSERM US001., ANR-10-IDEX-0003,IDEX BORDEAUX,Initiative d'excellence de l'Université de Bordeaux(2010), ANR-14-CE09-0020,NanoSSNMR,Nanostructures biologiques et synthétiques étudiées par Résonance Magnétique Nucléaire du Solide(2014), ANR-13-PDOC-0017,SUPRAMOL,Structures d'Assemblages Supramoléculaires par RMN du Solide : le Pseudopilus du Système de Sécrétion de Type II et le Tube de Queue du Bactériophage(2013), ANR-17-CE11-0035,SFAS,Structure et fonctions de motifs amyloïdes impliqués dans la transduction du signal(2017), ANR-16-CONV-0005,INCEPTION,Institut Convergences pour l'étude de l'Emergence des Pathologies au Travers des Individus et des populatiONs(2016), European Project: 648974,H2020,ERC-2014-CoG,P-MEM-NMR(2015), European Project: 639020,H2020,ERC-2014-STG,Weakinteract(2015), European Project: 661799,H2020,H2020-MSCA-IF-2014,COMPLEX-fastMAS-NMR(2016), European Project: 624918,EC:FP7:PEOPLE,FP7-PEOPLE-2013-IIF,MEM-MAS(2014), École Nationale d'Ingénieurs des Travaux Agricoles - Bordeaux (ENITAB)-Institut de Chimie du CNRS (INC)-Université de Bordeaux (UB)-Centre National de la Recherche Scientifique (CNRS), Université de Lyon-Université de Lyon-Institut National des Sciences Appliquées de Lyon (INSA Lyon), Université de Lyon-Institut National des Sciences Appliquées (INSA)-Institut National des Sciences Appliquées (INSA)-Institut de Chimie du CNRS (INC)-École Supérieure Chimie Physique Électronique de Lyon-Centre National de la Recherche Scientifique (CNRS)-Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Institut National des Sciences Appliquées (INSA)-Institut National des Sciences Appliquées (INSA)-Institut de Chimie du CNRS (INC)-École Supérieure Chimie Physique Électronique de Lyon-Centre National de la Recherche Scientifique (CNRS), Institut Pasteur [Paris]-Centre National de la Recherche Scientifique (CNRS), Centre National de la Recherche Scientifique (CNRS)-Université de Bordeaux (UB)-Institut National de la Santé et de la Recherche Médicale (INSERM), U.S. Department of Energy [Washington] (DOE)-UT-Battelle, LLC-Stony Brook University [SUNY] (SBU), and State University of New York (SUNY)-State University of New York (SUNY)

Subjects
0301 basic medicine, Protein Folding, Amyloid, Prions, Prion strain, Model system, Amyloidogenic Proteins, Computational biology, Biology, 010402 general chemistry, 01 natural sciences, Models, Biological, Podospora anserina, Fungal Proteins, prion, 03 medical and health sciences, Protein Aggregates, 0302 clinical medicine, Podospora, Amino Acid Sequence, Prion protein, [SDV.BBM.BC]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biochemistry [q-bio.BM], Conserved Sequence, 030304 developmental biology, cross-seeding, 0303 health sciences, Multidisciplinary, food and beverages, amyloid, Biological Sciences, biology.organism_classification, Sequence identity, 0104 chemical sciences, Protein Structure, Tertiary, [SDV.BBM.BP]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biophysics, Strain specificity, nuclear magnetic resonance, 030104 developmental biology, sequence to fold, Sequence Alignment, 030217 neurology & neurosurgery
Abstract

Neurodegenerative disorders are frequently associated with β-sheet-rich amyloid deposits. Amyloid-forming proteins can aggregate under different structural conformations known as strains, which can exhibit a prion-like behaviour and distinct patho-phenotypes. Precise molecular determinants defining strain specificity and cross-strain interactions (cross-seeding) are currently unknown. The HET-s prion protein from the fungus Podospora anserina represents a model system to study the fundamental properties of prion amyloids. Here, we report the amyloid prion structure of HELLF, a distant homolog of the model prion HET-s. We find that these two amyloids, sharing only 17% sequence identity, have nearly identical β-solenoid folds but lack cross-seeding ability in vivo, indicating that prion specificity can differ in extremely similar amyloid folds. We engineer the HELLF sequence to explore the limits of the sequence-to-fold conservation and to pinpoint determinants of cross-seeding and prion specificity. We find that amyloid fold conservation occurs even at an exceedingly low level of identity to HET-s (5%). Next, we derive a HELLF-based sequence, termed HEC, able to breach the cross-seeding barrier in vivo between HELLF and HET-s, unveiling determinants controlling cross-seeding at residue level. These findings show that virtually identical amyloid backbone structures might not be sufficient for cross-seeding and that critical side-chain positions could determine the seeding specificity of an amyloid fold. Our work redefines the conceptual boundaries of prion strain and shed new light on key molecular features concerning an important class of pathogenic agents.

Published
2021
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19. Protein structural dynamics by Magic-Angle Spinning NMR

Author

Guido Pintacuda, Tanguy Le Marchand, Marta Bonaccorsi, Centre de RMN à très hauts champs de Lyon (CRMN), 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), and École normale supérieure de Lyon (ENS de Lyon)-Université Claude Bernard Lyon 1 (UCBL)

Subjects
0303 health sciences, Materials science, Magnetic Resonance Spectroscopy, [SDV.BBM.BS]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Structural Biology [q-bio.BM], Dynamics (mechanics), Proteins, Crystallography, X-Ray, Characterization (materials science), 03 medical and health sciences, Motion, 0302 clinical medicine, Structural biology, Structural Biology, Chemical physics, [CHIM.ANAL]Chemical Sciences/Analytical chemistry, Magic angle spinning, Spectroscopy, Molecular Biology, Nuclear Magnetic Resonance, Biomolecular, 030217 neurology & neurosurgery, 030304 developmental biology
Abstract

International audience; Magic-Angle Spinning (MAS) Nuclear Magnetic Resonance (NMR) is a fastdeveloping technique, capable of complementing solution NMR, X-ray crystallography and electron microscopy for the biophysical characterization of microcrystalline, poorly crystalline or disordered protein samples, such as enzymes, biomolecular assemblies, membraneembedded systems or fibrils. Beyond structures, MAS NMR is an ideal tool for investigation of dynamics, since it is unique in its ability to distinguish static and dynamic disorder, and to characterize not only amplitudes, but also timescales of motion. Building on seminal work on model proteins, the technique is now ripe for widespread application in structural biology. This review briefly summarizes the recent evolutions in biomolecular MAS NMR and accounts for the growing number of systems where this spectroscopy has provided a description of conformational dynamics over the very last few years.

Published
2021
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20. Structural Analysis of an Antigen Chemically Coupled on Virus-Like Particles in Vaccine Formulation

Author

Kaspars Tars, Olivier Ouari, Anna Kirsteina, Guido Pintacuda, Gilles Casano, Andris Kazaks, Kristaps Jaudzems, Anne Lesage, Janis Bogans, and Tobias Schubeis

Subjects
Bioconjugation, biology, 010405 organic chemistry, Chemistry, Manufacturing process, Hemagglutinin (influenza), General Chemistry, 010402 general chemistry, 01 natural sciences, Catalysis, Virus, 0104 chemical sciences, Solid-state nuclear magnetic resonance, Virus-like particle, Antigen, biology.protein, Biophysics, Vaccines, Virus-Like Particle, Antigens, Viral, Nuclear Magnetic Resonance, Biomolecular, Alpha helix
Abstract

Structure determination of adjuvant-coupled antigens is essential for rational vaccine development but has so far been hampered by the relatively low antigen content in vaccine formulations and by their heterogeneous composition. Here we show that magic-angle spinning (MAS) solid-state NMR can be used to assess the structure of the influenza virus hemagglutinin stalk long alpha helix antigen, both in its free, unformulated form and once chemically coupled to the surface of large virus-like particles (VLPs). The sensitivity boost provided by high-field dynamic nuclear polarization (DNP) and proton detection at fast MAS rates allows to overcome the penalty associated with the antigen dilution. Comparison of the MAS NMR fingerprints between the free and VLP-coupled forms of the antigen provides structural evidence of the conservation of its native fold upon bioconjugation. This work demonstrates that high-sensitivity MAS NMR is ripe to play a major role in vaccine design, formulation studies, and manufacturing process development.

Published
2020

21. A β-barrel for oil transport through lipid membranes: Dynamic NMR structures of AlkL

Author

Tobias Schubeis, Jan Stanek, Guido Pintacuda, Wojciech Kopec, Bert L. de Groot, Tanguy Le Marchand, Csaba Daday, Loren B. Andreas, Kumar Tekwani Movellan, Tom S. Schwarzer, Kathrin Castiglione, Centre de RMN à très hauts champs de Lyon (CRMN), École normale supérieure de Lyon (ENS de 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), Max Planck Institute for Biophysical Chemistry, Biomolecular Dynamics Group, Max Planck Institute for Biophysical Chemistry, Department of NMR Based Structural Biology, University of Warsaw, Faculty of Chemistry, Technical University Munich, Institute of Biochemical Engineering, Friedrich Alexander University Erlangen Nurnberg, Chemical & Biological Engineering, Inst Bioproc Engn, and École normale supérieure - Lyon (ENS Lyon)-Université Claude Bernard Lyon 1 (UCBL)

Subjects
magic-angle spinning, Magnetic Resonance Spectroscopy, Molecular Dynamics Simulation, 010402 general chemistry, 01 natural sciences, Permeability, Protein Structure, Secondary, Membrane Lipids, 03 medical and health sciences, Protein structure, Bacterial Proteins, Magic angle spinning, membrane protein, protein structure, [SDV.BBM.BC]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biochemistry [q-bio.BM], Lipid bilayer, Nuclear Magnetic Resonance, Biomolecular, 030304 developmental biology, 0303 health sciences, Multidisciplinary, lipid bilayers, Chemistry, Protein dynamics, Membrane Proteins, Biological Sciences, Permeation, Magnetic Resonance Imaging, 0104 chemical sciences, ddc, [SDV.BBM.BP]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biophysics, Applied Physical Sciences, Biophysics and Computational Biology, Barrel, Membrane, Physical Sciences, protein dynamics, Biophysics, Bacterial outer membrane, Hydrophobic and Hydrophilic Interactions, Bacterial Outer Membrane Proteins
Abstract

Significance Here we show how AlkL, a minimalistic outer-membrane protein from oil-consuming bacteria, exploits dynamics of extracellular loops to channel substrate across the polysaccharide barrier and into the hydrophobic interior of the outer membrane. This work represents a unique example of a side-by-side atomic-level structure determination by solution NMR in detergents and by solid-state NMR in lipid bilayers, which critically demonstrates the importance of a lipid environment to investigate function. Corroborating our experimental measurements, molecular-dynamics simulations capture substrate transit via lateral openings. The capacity to unravel membrane protein function under near-native conditions, fueled by the latest method developments, opens up a new frontier for their investigation, and provides thereby for improved fundamental insights into biological processes., The protein AlkL is known to increase permeability of the outer membrane of bacteria for hydrophobic molecules, yet the mechanism of transport has not been determined. Differing crystal and NMR structures of homologous proteins resulted in a controversy regarding the degree of structure and the role of long extracellular loops. Here we solve this controversy by determining the de novo NMR structure in near-native lipid bilayers, and by accessing structural dynamics relevant to hydrophobic substrate permeation through molecular-dynamics simulations and by characteristic NMR relaxation parameters. Dynamic lateral exit sites large enough to accommodate substrates such as carvone or octane occur through restructuring of a barrel extension formed by the extracellular loops.

Published
2020
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22. Spectroscopic Signature and Structure of Active Centers in Ziegler-Natta Polymerization Catalysts revealed by Electron Paramagnetic Resonance

Author

Jan Blahut, Gunnar Jeschke, Anton Ashuiev, Vincent Monteil, Guido Pintacuda, Sébastien Norsic, Christophe Copéret, Jean Raynaud, Matthieu Humbert, Keith Searles, Anne Lesage, Daniel Klose, David Gajan, Eidgenössische Technische Hochschule - Swiss Federal Institute of Technology [Zürich] (ETH Zürich), Université Claude Bernard Lyon 1 (UCBL), Université de Lyon, Centre de RMN à très hauts champs de Lyon (CRMN), 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), and École normale supérieure de Lyon (ENS de Lyon)-Université Claude Bernard Lyon 1 (UCBL)

Subjects
inorganic chemicals, Ethylene, Keith, 020209 energy, Anton, 02 engineering and technology, Natta, sebastien, 010402 general chemistry, Photochemistry, 01 natural sciences, Catalysis, law.invention, chemistry.chemical_compound, gajan, law, david, et al. (2020): Spectroscopic Signature and Structure of Active Centers in Ziegler-Natta Polymerization Catalysts revealed by Electron Paramagnetic Resonance. ChemRxiv. Preprint, 0202 electrical engineering, electronic engineering, information engineering, [CHIM]Chemical Sciences, mathieu, Ziegler–Natta catalyst, Electron paramagnetic resonance, Bimetallic strip, biology, blahut, humber, Polyethylene, biology.organism_classification, 3. Good health, 0104 chemical sciences, jean, Searles, chemistry, Polymerization, CC BY-NC-ND 4.0 Citation information: Ashuiev, norsic
Abstract

Despite decades of extensive studies, the atomic-scale structure of the active sites in heterogeneous Ziegler-Natta (ZN) catalysts, one of the most important processes of the chemical industry, remains elusive and a matter of debate. In the present work, the structure of “active sites” of ZN catalysts in the absence of ethylene, referred to as “dormant active sites”, is elucidated from magnetic resonance experiments, carried out on samples reacted with increasing amounts of BCl3 so as to enhance the concentration of active sites and observe clear spectroscopic signatures. Using EPR and NMR spectroscopies, in particular 2D HYSCORE experiments complemented by DFT calculations, we show that the activated ZN catalysts contain bimetallic alkyl-Ti(III),Al species whose amount is directly linked to the polymerization activity of MgCl2-supported Ziegler-Natta catalysts. This connects those spectroscopic signatures to the active species formed in the presence of ethylene, and enables us propose an ethylene polymerization mechanism on the observed bimetallic alkyl-Ti(III),Al species based on DFT computations

Published
2020
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23. Spectroscopic Signature and Structure of Active Centers in Ziegler-Natta Polymerization Catalysts revealed by Electron Paramagnetic Resonance

Author

Anton Ashuiev, Matthieu Humbert, Sebastien Norsic, Jan Blahut, David Gajan, Keith Searles, Daniel Klose, Anne Lesage, Guido Pintacuda, Jean Raynaud, Christophe Copéret, and Gunnar Jeschke

Subjects
inorganic chemicals
Abstract

Despite decades of extensive studies, the atomic-scale structure of active sites in heterogeneous Ziegler-Natta (ZN) catalysts remains elusive and a matter of debate. Here, the structure of polymerization ZN catalysts is elucidated from magnetic resonance experiments carried out on samples reacted with increasing amounts of BCl3 so as to enhance the concentration of active sites and observe clear spectroscopic signatures. Notably, we show that EPR and NMR spectroscopy of the activated ZN catalysts enables to observe paramagnetic species whose amount increases in conjunction with the catalytic activity. The joint application of 2D HYSCORE experiments and DFT calculations reveals the presence of bimetallic alkyl-Ti(III),Al complexes that are assigned to the catalytic centers of MgCl2-supported Ziegler-Natta catalysts

Published
2020
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24. Resonance assignment of the outer membrane protein AlkL in lipid bilayers by proton-detected solid-state NMR

Author

Loren B. Andreas, Tom S. Schwarzer, Guido Pintacuda, Tobias Schubeis, Kathrin Castiglione, Tanguy Le Marchand, Jan Stanek, Kumar Tekwani Movellan, Centre de Résonance Magnetique Nucleaire (CRMN), Institut de Chimie et Biochimie Moléculaires et Supramoléculaires (ICBMS), Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-Institut National des Sciences Appliquées de Lyon (INSA Lyon), Université de Lyon-Institut National des Sciences Appliquées (INSA)-Institut National des Sciences Appliquées (INSA)-École Supérieure Chimie Physique Électronique de Lyon-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS)-Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Institut National des Sciences Appliquées (INSA)-Institut National des Sciences Appliquées (INSA)-École Supérieure Chimie Physique Électronique de Lyon-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS), Inst Biochem Engn, Technische Universität Munchen - Université Technique de Munich [Munich, Allemagne] (TUM), Max Planck Institute for Biophysical Chemistry (MPI-BPC), and Max-Planck-Gesellschaft

Subjects
0303 health sciences, Nitrogen Isotopes, Chemistry, Pseudomonas putida, Proton Magnetic Resonance Spectroscopy, 030303 biophysics, Lipid Bilayers, Membrane Proteins, Nuclear magnetic resonance spectroscopy, Biochemistry, Alkane transport, Small molecule, Protein Structure, Secondary, [SDV.BBM.BP]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biophysics, 03 medical and health sciences, Beta barrel, Membrane protein, Bacterial Proteins, Structural Biology, Biophysics, Amino Acid Sequence, Bacterial outer membrane, Lipid bilayer, Protein secondary structure, 030304 developmental biology
Abstract

International audience; Most commonly small outer membrane proteins, possessing between 8 and 12 β-strands, are not involved in transport but fulfill diverse functions such as cell adhesion or binding of ligands. An intriguing exception are the 8-stranded β-barrel proteins of the OmpW family, which are implicated in the transport of small molecules. A representative example is AlkL from Pseudomonas putida GPoI, which functions as a passive importer of hydrophobic molecules. This role is of high interest with respect to both fundamental biological understanding and industrial applications in biocatalysis, since this protein is frequently utilized in biotransformation of alkanes. While the transport function of AlkL is generally accepted, a controversy in the transport mechanism still exists. In order to address this, we are pursuing a structural study of recombinantly produced AlkL reconstituted in lipid bilayers using solid-state NMR spectroscopy. In this manuscript we present 1 H, 13 C and 15 N chemical shift assignments obtained via a suite of 3D experiments employing high magnetic fields (1 GHz and 800 MHz) and the latest magic-angle spinning (MAS) approaches at fast (60-111) kHz rates. We additionally analyze the secondary structure prediction in comparison with those of published structures of homologous proteins.

Published
2020
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25. Automated Backbone NMR Resonance Assignment of Large Proteins Using Redundant Linking from a Single Simultaneous Acquisition

Author

Loren B. Andreas, Peter Güntert, Guido Pintacuda, Tobias Schubeis, Jan Stanek, Piotr Paluch, Centre de RMN à très hauts champs de Lyon (CRMN), École normale supérieure de Lyon (ENS de 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), Centre of Molecular and Macromolecular Studies, Polish Academy of Sciences, Institute of Biophysical Chemistry and Frankfurt Institute for Advanced Studies, Goethe-University Frankfurt am Main, Max Planck Institute for Biophysical Chemistry (MPI-BPC), Max-Planck-Gesellschaft, and École normale supérieure - Lyon (ENS Lyon)-Université Claude Bernard Lyon 1 (UCBL)

Subjects
Chemistry, Escherichia coli Proteins, Proton Magnetic Resonance Spectroscopy, Resonance, General Chemistry, 010402 general chemistry, 01 natural sciences, Biochemistry, Maltose-Binding Proteins, Catalysis, 0104 chemical sciences, [SDV.BBM.BP]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biophysics, Colloid and Surface Chemistry, [CHIM.ANAL]Chemical Sciences/Analytical chemistry, Escherichia coli, [SDV.BBM.BC]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biochemistry [q-bio.BM], Biological system, Nuclear Magnetic Resonance, Biomolecular, Spinning, Protein size
Abstract

International audience; Thanks to magic-angle spinning (MAS) probes with frequencies of 60-100 kHz, the benefit of high sensitivity 1 H detection can now be broadly realized in biomolecular solid-state NMR for the analysis of microcrystalline, sedimented, or lipid-embedded preparations. Nonetheless, performing the assignment of all resonances remains a rate-limiting step in protein structural studies, and even the latest optimized protocols fail to perform this step when the protein size exceeds ~20 kDa. Here we leverage the benefits of fast (100 kHz) MAS and high (800 MHz) magnetic fields to design an approach that lifts this limitation. Through the creation, conservation and acquisition of independent magnetization pathways within a single triple-resonance MAS NMR experiment, a single self-consistent dataset can be acquired, providing enhanced sensitivity, reduced vulnerability to machine or sample instabilities, and highly redundant linking that supports fully-automated peak picking and resonance assignment. The method, dubbed RAVASSA (Redundant Assignment Via A Single Simultaneous Acquisition), is demonstrated with the assignment of the largest protein to date in the solid state, the 42.5 kDa maltose binding protein, using a single fully protonated microcrystalline sample and one week of spectrometer time.

Published
2020
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26. Broadband MAS NMR spectroscopy in the low-power limit

Author

Sebastian Wegner, Clare P. Grey, Kevin J. Sanders, Guido Pintacuda, Andrew J. Pell, Biological Solid-State NMR Methods - Méthodes de RMN à l'état solide en biologie, Institut des Sciences Analytiques (ISA), Institut de Chimie du CNRS (INC)-Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-Centre National de la Recherche Scientifique (CNRS)-Institut de Chimie du CNRS (INC)-Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-Centre National de la Recherche Scientifique (CNRS), Department of Materials and Environmental Chemistry - Arrhenius Laboratory, Bruker BioSpin GmbH, D-76287 Rheinstetten, Germany, affiliation inconnue, University of Cambridge - Chemistry Department, This work was financially supported by the People Program of the European Union’s FP7 (FP7-PEOPLE-2012-ITN No. 317127 'pNMR'), the European Research Council (ERC) under the European Union’s Horizon 2020 research and innovation program (Grant No. 648974 'P-MEM-NMR'), and the Agence Nationale de la Recherche (Grant No. ANR-15-CE29-0025-01). KJS would like to thank Prof. Philip J. Grandinetti for fruitful discussions about adiabatic pulses., European Project: 317127,EC:FP7:PEOPLE,FP7-PEOPLE-2012-ITN,PNMR(2013), European Project: 648974,H2020,ERC-2014-CoG,P-MEM-NMR(2015), 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)-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), and University of Cambridge [UK] (CAM)

Subjects
Physics, Sideband, Ultrafast MAS, General Physics and Astronomy, Biasing, 02 engineering and technology, 010402 general chemistry, 021001 nanoscience & nanotechnology, 01 natural sciences, 0104 chemical sciences, Paramagnetic NMR, broadband NMR, Paramagnetism, [CHIM.ANAL]Chemical Sciences/Analytical chemistry, Broadband, Li-ion battery, Waveform, Physical and Theoretical Chemistry, Atomic physics, 0210 nano-technology, Adiabatic process, Anisotropy, Adiabatic pulses, Spinning
Abstract

International audience; We investigate the performance of broadband adiabatic inversion pulses in the high-power (short high-powered adi-abatic pulse, SHAP) and low-power (single-sideband-selective adiabatic pulse, S 3 AP) RF regimes on a spin system subjected to large anisotropic interactions. We show by combined experimental results and spin dynamics simulations that when the magic-angle spinning rate exceeds 100 kHz S 3 APs begin outperforming SHAPs. This is especially true for low-gamma nuclei, such as 6 Li in paramagnetic Li-ion battery materials. Finally, we show how S 3 APs can be improved by combining multiple waveforms sweeping over multiple sidebands simultaneously, in order to produce inverted sideband profiles free from intensity biasing.

Published
2018
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27. Paramagnetic Cobalt(II) Complexes with Cyclam Derivatives: Toward 19 F MRI Contrast Agents

Author

Guido Pintacuda, Petr Hermann, Ladislav Benda, Jan Kotek, Jan Blahut, Centre de RMN à très hauts champs de Lyon (CRMN), 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), Charles University [Prague] (CU), and École normale supérieure de Lyon (ENS de Lyon)-Université Claude Bernard Lyon 1 (UCBL)

Subjects
chemistry.chemical_element, [CHIM.INOR]Chemical Sciences/Inorganic chemistry, 010402 general chemistry, paramagnetic NMR, 01 natural sciences, cyclam derivatives, Inorganic Chemistry, chemistry.chemical_compound, Paramagnetism, Nuclear magnetic resonance, spin density delocalization, Cyclam, medicine, [CHIM.COOR]Chemical Sciences/Coordination chemistry, Physical and Theoretical Chemistry, high-spin cobalt complexes, Ligand molecule, medicine.diagnostic_test, 010405 organic chemistry, kinetic inertness, Magnetic resonance imaging, Contrast (music), 0104 chemical sciences, NMR spectra database, [CHIM.THEO]Chemical Sciences/Theoretical and/or physical chemistry, chemistry, hyperfine shift calculations, NMR relaxation, 19 F MRI contrast agents, macrocyclic complexes, Cobalt, phosphonate complexes
Abstract

International audience; In order to develop novel, more efficient and/or selective contrast agents for magnetic resonance imaging (MRI), different modi operandi are explored as alternatives to water-relaxation enhancement. In this work, cobalt(II/III) complexes of bis(N-trifluoroethy)cyclam derivatives with two acetate or two phosphonate pendant arms, H2te2f2a and H4te2f2p, were prepared and investigated. X-ray diffraction structures confirmed octahedral coordination with a very stable trans-III cyclam conformation and with fluorine atoms located about 5.3 Å from the metal center. The Co(II) complexes are kinetically inert, decomposing slowly even in 1 M aqueous HCl at 80 °C. The Co(II) complexes exhibited well-resolved paramagnetically-shifted NMR spectra. 2 These were interpreted with the help of quantum chemistry calculations. The 13 C NMR shifts of the trans-[Co II (te2f2p)] complex were successfully assigned based on spin density delocalization within the ligand molecule. The obtained spin density also helps to describe d-metal-induced NMR relaxation properties of 19 F nuclei, including the contribution of a Fermi contact relaxation mechanism. The paramagnetic complexes show convenient relaxation properties to be used as 19 F MRI contrast agents.

Published
2020
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28. Impact of Magnetic Field Strength on Resolution and Sensitivity of Proton Resonances in Biological Solids

Author

Daniela Lalli, Zdenek Tosner, Bernd Reif, Kai Xue, Guido Pintacuda, Riddhiman Sarkar, Benita Koch, Helmholtz-Zentrum München (HZM), Centre de RMN à très hauts champs de Lyon (CRMN), 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), Technische Universität München [München] (TUM), Charles University [Prague] (CU), Technische Universität Munchen - Université Technique de Munich [Munich, Allemagne] (TUM), Helmholtz Zentrum München = German Research Center for Environmental Health, and École normale supérieure de Lyon (ENS de Lyon)-Université Claude Bernard Lyon 1 (UCBL)

Subjects
Coupling, Yield (engineering), Materials science, Proton, [SDV.BBM.BS]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Structural Biology [q-bio.BM], Resolution (electron density), 02 engineering and technology, 010402 general chemistry, 021001 nanoscience & nanotechnology, 01 natural sciences, Molecular physics, 0104 chemical sciences, Surfaces, Coatings and Films, Electronic, Optical and Magnetic Materials, Magnetic field, NMR spectra database, [CHIM.THEO]Chemical Sciences/Theoretical and/or physical chemistry, General Energy, Quality (physics), Magic angle spinning, Condensed Matter::Strongly Correlated Electrons, Physical and Theoretical Chemistry, 0210 nano-technology
Abstract

International audience; Sensitivity and resolution together determine the quality of NMR spectra in biological solids. Higher magic angle spinning frequencies yield a more efficient suppression of the coupling network and enable atomic-level investigations of protonated protein samples. On the other hand, truncation effects induced by higher magnetic fields have an impact on the achievable sensitivity and resolution. In this work, we address the question of how the proton dipolar coupling network affects the magnetic field strength-dependent gains in sensitivity and resolution. We find that-beyond the canonical B0 3/2 dependence-an additional factor of 2 in sensitivity can be achieved for residues embedded in the core of the protein, when the static magnetic field induces a transition from the strong-to the weak-coupling limit. The experiments are carried out using a selectively methylprotonated (13 CH3) α-spectrin SH3 sample, at magnetic field strengths of 11.75 T (1 H Larmor frequency of 500 MHz) and 23.5 T (1 H Larmor frequency of 1 GHz).

Published
2020
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29. MAS dependent sensitivity of different isotopomers in selectively methyl protonated protein samples in solid state NMR

Author

Kai Xue, Daniela Lalli, Benita Koch, Carina Motz, Zdenek Tosner, Guido Pintacuda, Bernd Reif, Riddhiman Sarkar, Harbin Engineering University (HRBEU), Institut des sciences et d'ing:nierie chimiques (ISIC), Ecole Polytechnique Fédérale de Lausanne (EPFL), Centre de RMN à très hauts champs de Lyon (CRMN), 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), Department of Chemistry [Munich], Technische Universität Munchen - Université Technique de Munich [Munich, Allemagne] (TUM), and École normale supérieure de Lyon (ENS de Lyon)-Université Claude Bernard Lyon 1 (UCBL)

Subjects
0301 basic medicine, Proton, Analytical chemistry, Protonation, 010402 general chemistry, 01 natural sciences, Biochemistry, Methylation, Sensitivity and Specificity, Isotopomers, Matrix (chemical analysis), src Homology Domains, 03 medical and health sciences, [CHIM.ANAL]Chemical Sciences/Analytical chemistry, Nuclear Magnetic Resonance, Biomolecular, ComputingMilieux_MISCELLANEOUS, Spectroscopy, Spectrometer, Chemistry, Resolution (electron density), Spectrin, Deuterium, 0104 chemical sciences, Solid State Nmr, Magic Angle Spinning (mas), Selective Deuteration, Ch3 Labelling, Methyl Isotopomers, Microcrystalline Proteins, [CHIM.THEO]Chemical Sciences/Theoretical and/or physical chemistry, NMR spectra database, 030104 developmental biology, Solid-state nuclear magnetic resonance, Protons
Abstract

Sensitivity and resolution together determine the quality of NMR spectra in biological solids. For high-resolution structure determination with solid-state NMR, proton-detection emerged as an attractive strategy in the last few years. Recent progress in probe technology has extended the range of available MAS frequencies up to above 100 kHz, enabling the detection of resolved resonances from sidechain protons, which are important reporters of structure. Here we characterise the interplay between MAS frequency in the newly available range of 70-110 kHz and proton content on the spectral quality obtainable on a 1 GHz spectrometer for methyl resonances. Variable degrees of proton densities are tested on microcrystalline samples of the alpha-spectrin SH3 domain with selectively protonated methyl isotopomers (CH3, CH2D, CHD2) in a perdeuterated matrix. The experimental results are supported by simulations that allow the prediction of the sensitivity outside this experimental frequency window. Our results facilitate the selection of the appropriate labelling scheme at a given MAS rotation frequency.

Published
2019
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30. A Straightforward Access to Stable, 16 Valence-electron Phosphine-Stabilized Fe

Author

Benjamin, Burcher, Kevin J, Sanders, Ladislav, Benda, Guido, Pintacuda, Erwann, Jeanneau, Andreas A, Danopoulos, Pierre, Braunstein, Hélène, Olivier-Bourbigou, and Pierre-Alain R, Breuil

Subjects
Article
Abstract

The use of the dialkene divinyltetramethyldisiloxane (dvtms) allows easy access to the reactive 16 valence-electron complexes [Fe(0)(L-L)(dvtms)], (L-L) = dppe (1,2-bis(diphenylphosphino)ethane), (1), dppp (1,2-bis(diisopropylphosphino)propane), (2), pyNMeP((i)Pr)(2) (N-(diisopropylphosphino)-N-methylpyridin-2-amine), (4), dipe (1,2-bis(diisopropylphosphino)ethane), (5), and [Fe(0)(L)(2)(dvtms)], L = PMe(3), (3), by a mild reductive route using AlEt(2)(OEt) as reducing agent. In contrast, by the same methodology, the 18 valence-electron complexes [Fe(0)(L-L)(2)(ethylene)], (L-L) = dppm (1,2-bis(diphenylphosphino)methane), 6, (L-L) = dppa (1,2-bis(diphenylphosphino)amine) 7 or (L-L)=dppe, 8, were obtained, which do not contain dvtms. In addition, a combined DFT and solid-state paramagnetic NMR methodology is introduced for the structure determination of 5. A comparative study of the reactivity of 1,2,4-6 and 8 with 3-hexyne highlights emerging mechanistic implications for C-C coupling reactions using these complexes as catalysts.

Published
2019

31. Straightforward Access to Stable, 16-Valence-Electron Phosphine-Stabilized Fe0 Olefin Complexes and Their Reactivity

Author

Benjamin Burcher, Helene Olivier-Bourbigou, Pierre Braunstein, Kevin J. Sanders, Erwann Jeanneau, Andreas A. Danopoulos, Guido Pintacuda, Ladislav Benda, and Pierre-Alain Breuil

Subjects
Olefin fiber, Ethylene, 010405 organic chemistry, Reducing agent, Organic Chemistry, 010402 general chemistry, 01 natural sciences, Medicinal chemistry, Coupling reaction, 0104 chemical sciences, 3. Good health, Catalysis, Inorganic Chemistry, chemistry.chemical_compound, chemistry, Organic chemistry, Amine gas treating, Reactivity (chemistry), Physical and Theoretical Chemistry, Phosphine
Abstract

The use of the dialkene divinyltetramethyldisiloxane (dvtms) allows easy access to the reactive 16-valence-electron complexes [Fe0(L-L)(dvtms)] (L-L = dppe (1,2-bis(diphenylphosphino)ethane; 1), dppp (1,2-bis(diphenylphosphino)propane; 2), pyNMeP(iPr)2 (N-(diisopropylphosphino)-N-methylpyridin-2-amine; 4), dipe (1,2-bis(diisopropylphosphino)ethane; 5)) and [Fe0(L)2(dvtms)] (L = PMe3; 3) by a mild reductive route using AlEt2(OEt) as reducing agent. In contrast, by the same methodology, the 18-valence-electron complexes [Fe0(L-L)2(ethylene)] (L-L = dppm (1,2-bis(diphenylphosphino)methane; 6), dppa (1,2-bis(diphenylphosphino)amine; 7), dppe (8)) were obtained, which do not contain dvtms. In addition, a combined DFT and solid-state paramagnetic NMR methodology is introduced for the structure determination of 5. A comparative study of the reactivity of 1, 2, 4–6, and 8 with 3-hexyne highlights emerging mechanistic implications for C–C coupling reactions using these complexes as catalysts.

Published
2017
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33. Target highlights from the first post-PSI CASP experiment (CASP12, May-August 2016)

Author

Shabir Najmudin, Johan C. Hill, Guido Pintacuda, Kinlin L. Chao, John Moult, Arnaud Baslé, T.H. Nguyen, Kaspars Tars, Harry J. Gilbert, Krzysztof Fidelis, Christopher S. Hayes, Marco Nardini, Reinhard Albrecht, Mark J. van Raaij, Valentina Nardone, Roman I. Koning, Celia W. Goulding, Pedro Bule, A.K. Singh, Nicole Zitzmann, Andrzej Joachimiak, Osnat Herzberg, Leila Lo Leggio, Carlos M. G. A. Fontes, Eric J. Sundberg, Pietro Roversi, Didier Ndeh, Andriy Kryshtafovych, Amir Shimon, Gert Wieland Kohring, Folmer Fredslund, Alessandro T. Caputo, Ana Luísa Carvalho, Marco Mangiagalli, Karolina Michalska, Sandra Postel, Marcus D. Hartmann, Torsten Schwede, Ron Diskin, Genome Center [UC Davis], University of California [Davis] (UC Davis), University of California-University of California, Abteilung Membranbiochemie [Martinsried], Max-Planck-Institut für Biochemie (MPIB), Max-Planck-Gesellschaft-Max-Planck-Gesellschaft, Institute for Cell and Molecular Biosciences, Newcastle University [Newcastle], Interdisciplinary Centre of Research in Animal Health, Faculdade de Medicina Veterinária, Pólo Universitário do Alto da Ajuda, Universidade de Lisboa, Oxford Glycobiol Inst, Dept Biochem, University of Oxford [Oxford], Unidade de Ciencias Biomoleculares Aplicadas (UCIBIO), Requimte, Departamento de Química (DQ), Faculdade de Ciências e Tecnologia = School of Science & Technology (FCT NOVA), Universidade Nova de Lisboa = NOVA University Lisbon (NOVA)-Universidade Nova de Lisboa = NOVA University Lisbon (NOVA)-Faculdade de Ciências e Tecnologia = School of Science & Technology (FCT NOVA), Universidade Nova de Lisboa = NOVA University Lisbon (NOVA)-Universidade Nova de Lisboa = NOVA University Lisbon (NOVA)-Universidade do Porto-Departamento de Química (DQ), Universidade Nova de Lisboa = NOVA University Lisbon (NOVA)-Universidade Nova de Lisboa = NOVA University Lisbon (NOVA)-Universidade do Porto, Dept Mol Cellular & Dev Biol, University of California [Santa Barbara] (UCSB), Structural Biology Center, Biosciences Division, Universität des Saarlandes [Saarbrücken], Department of Chemistry, University of Copenhagenn, Department of Cell Biology and Molecular Genetics, University of Maryland [College Park], University of Maryland System-University of Maryland System, Department of Biomolecular Sciences and Biotechnology, University of Milano, Biological Solid-State NMR Methods - Méthodes de RMN à l'état solide en biologie, Institut des Sciences Analytiques (ISA), Institut de Chimie du CNRS (INC)-Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-Centre National de la Recherche Scientifique (CNRS)-Institut de Chimie du CNRS (INC)-Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-Centre National de la Recherche Scientifique (CNRS), Biomedical Research & Study Centre, Department of Biochemistry [Oxford], Biozentrum [Basel, Suisse], University of Basel (Unibas), Kryshtafovych, A, Albrecht, R, Baslé, A, Bule, P, Caputo, A, Carvalho, A, Chao, K, Diskin, R, Fidelis, K, Fontes, C, Fredslund, F, Gilbert, H, Goulding, C, Hartmann, M, Hayes, C, Herzberg, O, Hill, J, Joachimiak, A, Kohring, G, Koning, R, Lo Leggio, L, Mangiagalli, M, Michalska, K, Moult, J, Najmudin, S, Nardini, M, Nardone, V, Ndeh, D, Nguyen, T, Pintacuda, G, Postel, S, Van Raaij, M, Roversi, P, Shimon, A, Singh, A, Sundberg, E, Tars, K, Zitzmann, N, and Schwede, T

Subjects
0301 basic medicine, Models, Molecular, Protein Folding, Protein Conformation, Bioinformatics, FIS/07 - FISICA APPLICATA (A BENI CULTURALI, AMBIENTALI, BIOLOGIA E MEDICINA), Computational biology, computer.software_genre, Crystallography, X-Ray, Biochemistry, Article, Mathematical Sciences, 03 medical and health sciences, Structural Biology, Models, Information and Computing Sciences, [CHIM]Chemical Sciences, Animals, Humans, [SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology, CASP, Molecular Biology, X-ray crystallography, Physics, Crystallography, Bacteria, Proteins, Computational Biology, Molecular, Protein structure prediction, Biological Sciences, BIO/10 - BIOCHIMICA, NMR, protein structure prediction, 030104 developmental biology, Biological significance, X-Ray, Data mining, computer, Software
Abstract

International audience; The functional and biological significance of the selected CASP12 targets are described by the authors of the structures. The crystallographers discuss the most interesting structural features of the target proteins and assess whether these features were correctly reproduced in the predictions submitted to the CASP12 experiment.

Published
2018
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34. 1 H magic-angle spinning NMR evolves as a powerful new tool for membrane proteins

Author

Loren B. Andreas, Tobias Schubeis, Guido Pintacuda, Tanguy Le Marchand, Biological Solid-State NMR Methods - Méthodes de RMN à l'état solide en biologie, Institut des Sciences Analytiques (ISA), Institut de Chimie du CNRS (INC)-Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-Centre National de la Recherche Scientifique (CNRS)-Institut de Chimie du CNRS (INC)-Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-Centre National de la Recherche Scientifique (CNRS), Max Planck Institute for Biophysical Chemistry (MPI-BPC), Max-Planck-Gesellschaft, Financial support from the CNRS (IR-RMN FR3050) and from the European Research Council (ERC) under the European Union’s Horizon 2020 research and innovation programme (ERC-2015-CoG GA n°648974) are gratefully acknowledged., European Project: 648974,H2020,ERC-2014-CoG,P-MEM-NMR(2015), 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)-Université Claude Bernard Lyon 1 (UCBL), and Université de Lyon-Université de Lyon-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS)

Subjects
0301 basic medicine, Nuclear and High Energy Physics, Materials science, Field (physics), Resolution (electron density), Biophysics, 010402 general chemistry, Condensed Matter Physics, 01 natural sciences, Biochemistry, 0104 chemical sciences, Characterization (materials science), 03 medical and health sciences, Dipole, 030104 developmental biology, Membrane protein, [CHIM.ANAL]Chemical Sciences/Analytical chemistry, Magic angle spinning, Sensitivity (control systems), Biological system, Spinning
Abstract

International audience; Building on a decade of continuous advances of the community, the recent development of very fast (60 kHz and above) magic-angle spinning (MAS) probes has revolutionised the field of solid-state NMR. This new spinning regime reduces the 1 H-1 H dipolar couplings, so that direct detection of the larger magnetic moment available from 1 H is now possible at high resolution, not only in deuterated molecules but also in fully-protonated substrates. Such capabilities allow rapid " fingerprinting " of samples with a tenfold reduction of the required sample amounts with respect to conventional approaches, and permit extensive, robust and expeditious assignment of small-to-medium sized proteins (up to ca. 300 residues), and the determination of inter-nuclear proximities, relative orientations of secondary structural elements, protein-cofactor interactions, local and global dynamics. Fast MAS and 1 H detection techniques have nowadays been shown to be applicable to membrane-bound systems. This paper reviews the strategies underlying this recent leap forward in sensitivity and resolution, describing its potential for the detailed characterization of membrane proteins. Graphical TOC

Published
2018
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35. Rapid access to RNA resonances by proton-detected solid-state NMR at >100 kHz MAS

Author

Alexander Marchanka, Teresa Carlomagno, Guido Pintacuda, Jan Stanek, Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7, 38124 Braunschweig, Germany., Centre for Biomolecular Drug Research (BMWZ) and Institute of Organic Chemistry, Leibniz Universität Hannover [Hannover] (LUH), Biological Solid-State NMR Methods - Méthodes de RMN à l'état solide en biologie, Institut des Sciences Analytiques (ISA), Institut de Chimie du CNRS (INC)-Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-Centre National de la Recherche Scientifique (CNRS)-Institut de Chimie du CNRS (INC)-Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-Centre National de la Recherche Scientifique (CNRS), and Helmholtz Centre for Infection Research

Subjects
RNA Resonance, Dewey Decimal Classification::500 | Naturwissenschaften::540 | Chemie, Materials science, Proton, Nuclear Magnetic Resonance, 010402 general chemistry, 01 natural sciences, Catalysis, Nuclear magnetic resonance, [CHIM.ANAL]Chemical Sciences/Analytical chemistry, Materials Chemistry, Magic angle spinning, Rapid access, Spectroscopy, ComputingMilieux_MISCELLANEOUS, Spin-½, 010405 organic chemistry, Metals and Alloys, RNA, General Chemistry, 0104 chemical sciences, Surfaces, Coatings and Films, Electronic, Optical and Magnetic Materials, Solid-state nuclear magnetic resonance, ddc:540, Ceramics and Composites
Abstract

Fast (>100 kHz) magic angle spinning solid-state NMR allows combining high-sensitive proton detection with the absence of an intrinsic molecular weight limit. Using this technique we observe for the first time narrow 1H RNA resonances and assign nucleotide spin systems with only 200 μg of uniformly 13C,15N-labelled RNA.

Published
2018
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36. 3D structure determination of amyloid fibrils using solid-state NMR spectroscopy

Author

Mélanie Berbon, Benjamin Bardiaux, Antoine Loquet, Jan Stanek, Birgit Habenstein, Nadia El Mammeri, Guido Pintacuda, Chimie et Biologie des Membranes et des Nanoobjets (CBMN), Université de Bordeaux (UB)-École Nationale d'Ingénieurs des Travaux Agricoles - Bordeaux (ENITAB)-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS), Biological Solid-State NMR Methods - Méthodes de RMN à l'état solide en biologie, Institut des Sciences Analytiques (ISA), 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)-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), Bioinformatique structurale - Structural Bioinformatics, Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS), ANR-10-IDEX-0003,IDEX BORDEAUX,Initiative d'excellence de l'Université de Bordeaux(2010), ANR-14-CE09-0020,NanoSSNMR,Nanostructures biologiques et synthétiques étudiées par Résonance Magnétique Nucléaire du Solide(2014), ANR-13-PDOC-0017,SUPRAMOL,Structures d'Assemblages Supramoléculaires par RMN du Solide : le Pseudopilus du Système de Sécrétion de Type II et le Tube de Queue du Bactériophage(2013), European Project: 639020,H2020,ERC-2014-STG,Weakinteract(2015), European Project: 648974,H2020,ERC-2014-CoG,P-MEM-NMR(2015), European Project: 661799,H2020,H2020-MSCA-IF-2014,COMPLEX-fastMAS-NMR(2016), École Nationale d'Ingénieurs des Travaux Agricoles - Bordeaux (ENITAB)-Institut de Chimie du CNRS (INC)-Université de Bordeaux (UB)-Centre National de la Recherche Scientifique (CNRS), Institut de Chimie du CNRS (INC)-Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-Centre National de la Recherche Scientifique (CNRS)-Institut de Chimie du CNRS (INC)-Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-Centre National de la Recherche Scientifique (CNRS), Institut Pasteur [Paris]-Centre National de la Recherche Scientifique (CNRS), Université Sciences et Technologies - Bordeaux 1-École Nationale d'Ingénieurs des Travaux Agricoles - Bordeaux (ENITAB)-Centre National de la Recherche Scientifique (CNRS), Chimie et Biologie des Membranes et des Nanoobjets ( CBMN ), Université Sciences et Technologies - Bordeaux 1-École Nationale d'Ingénieurs des Travaux Agricoles - Bordeaux ( ENITAB ) -Centre National de la Recherche Scientifique ( CNRS ), Institut des Sciences Analytiques ( ISA ), École normale supérieure - Lyon ( ENS Lyon ) -Université Claude Bernard Lyon 1 ( UCBL ), Université de Lyon-Université de Lyon-Centre National de la Recherche Scientifique ( CNRS ), Bioinformatique structurale, Institut Pasteur [Paris]-Centre National de la Recherche Scientifique ( CNRS ), ANR-10-IDEX-0003-02/10-IDEX-0003,IDEX BORDEAUX,IdEx Bordeaux ( 2010 ), ANR-14-CE09-0020,NanoSSNMR,Nanostructures biologiques et synthétiques étudiées par Résonance Magnétique Nucléaire du Solide ( 2014 ), ANR-13-PDOC-0017,SUPRAMOL,Structures d'Assemblages Supramoléculaires par RMN du Solide : le Pseudopilus du Système de Sécrétion de Type II et le Tube de Queue du Bactériophage ( 2013 ), European Project : 639020,H2020,ERC-2014-STG,Weakinteract ( 2015 ), European Project : 648974,H2020,ERC-2014-CoG,P-MEM-NMR ( 2015 ), and European Project : 661799,H2020,H2020-MSCA-IF-2014,COMPLEX-fastMAS-NMR ( 2016 )

Subjects
0301 basic medicine, [ SDV.BBM.BP ] Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biophysics, Amyloid, Protein Conformation, Prions, Supramolecular chemistry, Protein aggregation, 010402 general chemistry, 01 natural sciences, Solid-state NMR, General Biochemistry, Genetics and Molecular Biology, 03 medical and health sciences, [ SDV.BBM.BC ] Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biomolecules [q-bio.BM], mental disorders, Humans, [SDV.BBM.BC]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biochemistry [q-bio.BM], Spectroscopy, Molecular Biology, Nuclear Magnetic Resonance, Biomolecular, Amyloid fibrils, Proton detection, Chemistry, Isotopic labelling, Nuclear magnetic resonance spectroscopy, [CHIM.MATE]Chemical Sciences/Material chemistry, Amyloid fibril, 0104 chemical sciences, Structure calculation, [SDV.BBM.BP]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biophysics, [CHIM.THEO]Chemical Sciences/Theoretical and/or physical chemistry, 030104 developmental biology, Solid-state nuclear magnetic resonance, Structural biology, Isotope Labeling, [ CHIM.THEO ] Chemical Sciences/Theoretical and/or physical chemistry, Biophysics
Abstract

International audience; The amyloid fold is structurally characterized by a typical cross-β architecture, which is under debate to represent an energy-favourable folding state that many globular or natively unfolded proteins can adopt. Being initially solely associated with amyloid fibrils observed in the propagation of several neurodegenerative disorders, the discovery of non-pathological (or " functional ") amyloids in many native biological processes has recently further intensified the general interest invested in those cross-β supramolecular assemblies. The insoluble and non-crystalline nature of amyloid fibrils and their usually inhomogeneous appearance on the mesoscopic level pose a challenge to biophysical techniques aiming at an atomic-level structural characterization. Solid-state NMR spectroscopy (SSNMR) has granted breakthroughs in structural investigations on amyloid fibrils ranging from the assessment of the impact of polymorphism in disease development to the 3D atomic structure determination of amyloid fibrils. First landmark studies towards the characterization of atomic structures and interactions involving functional amyloids have provided new impulses in the understanding of the role of the amyloid fold in native biological functions. Over the last decade many strategies have been developed in protein isotope labelling, NMR resonance assignment, distance restraint determination and 3D structure calculation of amyloid fibrils based on SSNMR approaches. We will here discuss the emerging concepts and state-of-the-art methods related to the assessment of amyloid structures and interactions involving amyloid entities by SSNMR.

Published
2018
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37. Probing Membrane Protein Insertion into Lipid Bilayers by Solid‐State NMR

Author

Tom S. Schwarzer, Eszter E. Najbauer, Guido Pintacuda, Loren B. Andreas, Stefan Becker, Kathrin Castiglione, Kumar Tekwani Movellan, Tobias Schubeis, Karin Giller, Max Planck Institute for Biophysical Chemistry (MPI-BPC), Max-Planck-Gesellschaft, Biological Solid-State NMR Methods - Méthodes de RMN à l'état solide en biologie, Institut des Sciences Analytiques (ISA), Institut de Chimie du CNRS (INC)-Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-Centre National de la Recherche Scientifique (CNRS)-Institut de Chimie du CNRS (INC)-Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-Centre National de la Recherche Scientifique (CNRS), Friedrich-Alexander Universität Erlangen-Nürnberg (FAU), and ISA - Centre de RMN à très hauts champs

Subjects
0301 basic medicine, Voltage-dependent anion channel, biology, [SDV.BBM.BS]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Structural Biology [q-bio.BM], Chemistry, 010402 general chemistry, biology.organism_classification, 01 natural sciences, Atomic and Molecular Physics, and Optics, Pseudomonas putida, 0104 chemical sciences, [CHIM.THEO]Chemical Sciences/Theoretical and/or physical chemistry, 03 medical and health sciences, 030104 developmental biology, Membrane, Solid-state nuclear magnetic resonance, Membrane protein, Magic angle spinning, Spin diffusion, Biophysics, biology.protein, Physical and Theoretical Chemistry, Lipid bilayer
Abstract

International audience; Determination of the environment surrounding a protein is often key to understanding its function and can also be used to infer the structural properties of the protein. By using proton-detected solid-state NMR, we show that reduced spin diffusion within the protein under conditions of fast magic-angle spinning, high magnetic field, and sample deuteration allows the efficient measurement of site-specific exposure to mobile water and lipids. We demonstrate this site specificity on two membrane proteins, the human voltage dependent anion channel, and the alkane transporter AlkL from Pseudomonas putida. Transfer from lipids is observed selectively in the membrane spanning region, and an average lipid-protein transfer rate of 6 s−1 was determined for residues protected from exchange. Transfer within the protein, as tracked in the 15N-1H 2D plane, was estimated from initial rates and found to be in a similar range of about 8 to 15 s−1 for several resolved residues, explaining the site specificity

Published
2018
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38. Lipid bilayer-bound conformation of an integral membrane beta barrel protein by multidimensional MAS NMR

Author

Yongchao Su, Robert Silvers, Guido Pintacuda, Robert G. Griffin, Lyndon Emsley, Lindsay Clark, Loren B. Andreas, Matthew T. Eddy, Gerhard Wagner, Massachusetts Institute of Technology. Department of Chemistry, Francis Bitter Magnet Laboratory (Massachusetts Institute of Technology), Griffin, Robert Guy, Eddy, Matthew Thomas, Su, Yongchao, Silvers, Robert, Andreas, Loren, Clark, Lindsay, Department of Chemistry [MIT Cambridge], Massachusetts Institute of Technology (MIT), Francis Bitter Magnet Lab, Biological Solid-State NMR Methods - Méthodes de RMN à l'état solide en biologie, Institut des Sciences Analytiques (ISA), Institut de Chimie du CNRS (INC)-Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-Centre National de la Recherche Scientifique (CNRS)-Institut de Chimie du CNRS (INC)-Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-Centre National de la Recherche Scientifique (CNRS), Dept Biol Chem & Mol Pharmacol, Harvard University [Cambridge], Solid-State NMR Methods for Materials - Méthodes de RMN à l'état solide pour les matériaux, and NIH Grants EB001960 and EB002026.

Subjects
Models, Molecular, Protein Folding, Lipid Bilayers, 010402 general chemistry, 01 natural sciences, Biochemistry, Protein Structure, Secondary, Article, CHIMERIC POTASSIUM CHANNEL, 03 medical and health sciences, Protein structure, [CHIM.ANAL]Chemical Sciences/Analytical chemistry, Magic angle spinning, Membrane fluidity, Humans, NICOTINIC ACETYLCHOLINE-RECEPTOR, Lipid bilayer, Nuclear Magnetic Resonance, Biomolecular, Integral membrane protein, Micelles, Spectroscopy, 030304 developmental biology, 0303 health sciences, VDAC, Chemistry, Voltage-Dependent Anion Channel 1, PHOSPHOLAMBAN PENTAMER, ANION-SELECTIVE CHANNEL, MITOCHONDRIAL OUTER-MEMBRANE, Magnetic Resonance Imaging, TRANSMEMBRANE DOMAIN, Protein Structure, Tertiary, SOLID-STATE NMR, 2D lipid crystals, 3. Good health, 0104 chemical sciences, Crystallography, ANGLE-SPINNING NMR, MAS, Beta barrel, Solid-state nuclear magnetic resonance, Membrane protein, BACTERIORHODOPSIN PHOTOCYCLE, Recoupling, lipids (amino acids, peptides, and proteins), ROTATIONAL RESONANCE
Abstract

The human voltage dependent anion channel 1 (VDAC) is a 32 kDa β-barrel integral membrane protein that controls the transport of ions across the outer mitochondrial membrane. Despite the determination of VDAC solution and diffraction structures, a structural basis for the mechanism of its function is not yet fully understood. Biophysical studies suggest VDAC requires a lipid bilayer to achieve full function, motivating the need for atomic resolution structural information of VDAC in a membrane environment. Here we report an essential step toward that goal: extensive assignments of backbone and side chain resonances for VDAC in DMPC lipid bilayers via magic angle spinning nuclear magnetic resonance (MAS NMR). VDAC reconstituted into DMPC lipid bilayers spontaneously forms two-dimensional lipid crystals, showing remarkable spectral resolution (0.5–0.3 ppm for [superscript 13]C line widths and, National Institutes of Health (U.S.) (Grant EB001960), National Institutes of Health (U.S.) (Grant EB002026)

Published
2015
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39. Structure of outer membrane protein G in lipid bilayers

Author

Emeline Barbet-Massin, Gregorio Giuseppe de Palma, Barth-Jan van Rossum, Andrew J. Nieuwkoop, W. Trent Franks, Werner Kühlbrandt, Benjamin Bardiaux, Loren B. Andreas, Lyndon Emsley, Victoria A. Higman, Hartmut Oschkinat, Lieselotte Handel, Guido Pintacuda, Jan Stanek, Kutti R. Vinothkumar, J. Retel, Matthias Hiller, Leibniz Forschungsinstitut für Molekulare Pharmakolgie = Leibniz Institute for Molecular Pharmacology [Berlin, Allemagne] (FMP), Leibniz Association, Biological Solid-State NMR Methods - Méthodes de RMN à l'état solide en biologie, Institut des Sciences Analytiques (ISA), 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)-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), Max-Planck-Institut für Biophysik - Max Planck Institute of Biophysics (MPIBP), Max-Planck-Gesellschaft, Bioinformatique structurale - Structural Bioinformatics, Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS), Institut des Sciences et Ingénierie Chimiques (ISIC), Ecole Polytechnique Fédérale de Lausanne (EPFL), This work was supported from a Joint Research Activity in the 7th Framework program of the EC (BioNMR No. 261863), the EU-project iNext (infrastructure for NMR, EM, and X-rays for Translational Research, GA 653706), and the Deutsche Forschungsgemeinschaft (SFB 740 and OS106/9). A.J.N. was supported by fellowships from the Fulbright Program and the Alexander von Humboldt Foundation., European Project, European Project: 653706,H2020,H2020-INFRAIA-2014-2015,iNEXT(2015), Institut de Chimie du CNRS (INC)-Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-Centre National de la Recherche Scientifique (CNRS)-Institut de Chimie du CNRS (INC)-Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-Centre National de la Recherche Scientifique (CNRS), and Institut Pasteur [Paris]-Centre National de la Recherche Scientifique (CNRS)

Subjects
Models, Molecular, 0301 basic medicine, Magnetic Resonance Spectroscopy, Science, Lipid Bilayers, Porins, General Physics and Astronomy, Crystallography, X-Ray, 010402 general chemistry, 01 natural sciences, Protein Structure, Secondary, Article, General Biochemistry, Genetics and Molecular Biology, 03 medical and health sciences, [CHIM.ANAL]Chemical Sciences/Analytical chemistry, Escherichia coli, Lipid bilayer, lcsh:Science, Multidisciplinary, Chemistry, Escherichia coli Proteins, General Chemistry, Nuclear magnetic resonance spectroscopy, 0104 chemical sciences, 030104 developmental biology, Membrane, Membrane protein, Helix, ddc:540, Biophysics, Protein folding, lcsh:Q, Bacterial outer membrane, Bacterial Outer Membrane Proteins, Outer membrane protein G
Abstract

β-barrel proteins mediate nutrient uptake in bacteria and serve vital functions in cell signaling and adhesion. For the 14-strand outer membrane protein G of Escherichia coli, opening and closing is pH-dependent. Different roles of the extracellular loops in this process were proposed, and X-ray and solution NMR studies were divergent. Here, we report the structure of outer membrane protein G investigated in bilayers of E. coli lipid extracts by magic-angle-spinning NMR. In total, 1847 inter-residue 1H–1H and 13C–13C distance restraints, 256 torsion angles, but no hydrogen bond restraints are used to calculate the structure. The length of β-strands is found to vary beyond the membrane boundary, with strands 6–8 being the longest and the extracellular loops 3 and 4 well ordered. The site of barrel closure at strands 1 and 14 is more disordered than most remaining strands, with the flexibility decreasing toward loops 3 and 4. Loop 4 presents a well-defined helix., Porins, like OmpG, are embedded in the outer membrane of bacteria and facilitate uptake and secretion of nutrients and ions. Here the authors present a protocol for solid state NMR structure determination of proteins larger than 25 kDa and use it to structurally characterize membrane embedded OmpG.

Published
2017
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40. Degree of Biomimicry of Artificial Spider Silk Spinning Assessed by NMR Spectroscopy

Author

Qiupin Jia, Loren B. Andreas, Kristaps Jaudzems, Martins Otikovs, Kerstin Nordling, Guido Pintacuda, Qing Meng, Jan Johansson, Marlene Andersson, Anna Rising, Latvian Institute of Organic Synthesis, Swedish University of Agricultural Sciences (SLU), Institute of Biological Sciences and Biotechnology, Donghua University [Shanghai], Center for Alzheimer Research, Karolinska Institutet [Stockholm], Biological Solid-State NMR Methods - Méthodes de RMN à l'état solide en biologie, Institut des Sciences Analytiques (ISA), Institut de Chimie du CNRS (INC)-Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-Centre National de la Recherche Scientifique (CNRS)-Institut de Chimie du CNRS (INC)-Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-Centre National de la Recherche Scientifique (CNRS), We acknowledge financial support from the EC FP7 grant no. 316149 'InnovaBalt', the Swedish Research Council, Center for Innovative Medicine (CIMED) at Karolinska Institutet, and Stockholm City Council., European Project: 316149,EC:FP7:REGPOT,FP7-REGPOT-2012-2013-1,INNOVABALT(2013), Latvian Institute of Organic Synthesis [Riga, Lituania], 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)-Université Claude Bernard Lyon 1 (UCBL), and Université de Lyon-Université de Lyon-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS)

Subjects
0301 basic medicine, Materials science, Polymer science, fungi, Silk, Spiders, General Medicine, General Chemistry, Nuclear magnetic resonance spectroscopy, Protein Structure, Secondary, Catalysis, Arthropod Proteins, 03 medical and health sciences, 030104 developmental biology, SILK, Solid-state nuclear magnetic resonance, Protein Domains, Biomimetic Materials, [CHIM.ANAL]Chemical Sciences/Analytical chemistry, Animals, Spider silk, Fiber, Biomimetics, Nuclear Magnetic Resonance, Biomolecular, Spinning
Abstract

International audience; Biomimetic spinning of artificial spider silk requires that the terminal domains of designed minispidroins undergo specific structural changes in concert with the β-sheet conversion of the repetitive region. Herein, we combine solution and solid-state NMR methods to probe domain-specific structural changes in the NT2RepCT minispidroin, which allows us to assess the degree of biomimicry of artificial silk spinning. In addition, we show that the structural effects of post-spinning procedures can be examined. By studying the impact of NT2RepCT fiber drying, we observed a reversible beta-to-alpha conversion. We think that this approach will be useful for guiding the optimization of artificial spider silk fibers.

Published
2017
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41. Expanding the horizons for structural analysis of fully protonated protein assemblies by NMR spectroscopy at MAS frequencies above 100 kHz

Author

Tatyana Polenova, Jodi Kraus, Jan Stanek, Werner E. Maas, Caitlin M. Quinn, Guangjin Hou, Loren B. Andreas, Anne Lesage, Jochem Struppe, Xingyu Lu, Angela M. Gronenborn, Manman Lu, Mingzhang Wang, Guido Pintacuda, Daniela Lalli, Bruker BioSpin Corporation, University of Pittsburgh School of Medicine, Pennsylvania Commonwealth System of Higher Education (PCSHE), Department of Chemistry and Biochemistry, University of Delaware [Newark], Biological Solid-State NMR Methods - Méthodes de RMN à l'état solide en biologie, Institut des Sciences Analytiques (ISA), Institut de Chimie du CNRS (INC)-Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-Centre National de la Recherche Scientifique (CNRS)-Institut de Chimie du CNRS (INC)-Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-Centre National de la Recherche Scientifique (CNRS), Solid-State NMR Methods for Materials - Méthodes de RMN à l'état solide pour les matériaux, Department of Structural Biology, Pennsylvania Commonwealth System of Higher Education (PCSHE)-Pennsylvania Commonwealth System of Higher Education (PCSHE), and This work was supported by the National Institutes of Health (NIH Grant P50 GM082251), we acknowledge the support of the National Science Foundation (NSF Grant CHE0959496) for the acquisition of the 850 MHz NMR spectrometer, and the National Institutes of Health (NIH Grant P30GM110758) for the support of core instrumentation infrastructure at the University of Delaware.

Subjects
0301 basic medicine, Nuclear and High Energy Physics, Proton, Analytical chemistry, Protonation, 010402 general chemistry, 01 natural sciences, Article, 03 medical and health sciences, Nuclear magnetic resonance, [CHIM.ANAL]Chemical Sciences/Analytical chemistry, [SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology, Protein assemblies, Instrumentation, Nuclear Magnetic Resonance, Biomolecular, Radiation, Chemistry, Resolution (electron density), Resonance, Proteins, General Chemistry, Nuclear magnetic resonance spectroscopy, HIV-1 capsid protein, 0104 chemical sciences, Dipole, 030104 developmental biology, Heteronuclear molecule, Proton-detected MAS NMR correlations, HIV-1, MAS NMR, Capsid Proteins, Protons, Two-dimensional nuclear magnetic resonance spectroscopy, Oligopeptides
Abstract

International audience; The recent breakthroughs in NMR probe technologies resulted in the development of MAS NMR probes with rotation frequencies exceeding 100 kHz. Herein, we explore dramatic increases in sensitivity and resolution observed at MAS frequencies of 110-111 kHz in a novel 0.7 mm HCND probe that enable structural analysis of fully protonated biological systems. Proton- detected 2D and 3D correlation spectroscopy under such conditions requires only 0.1-0.5 mg of sample and a fraction of time compared to conventional 13C-detected experiments. We discuss the performance of several proton- and heteronuclear- (13C-,15N-) based correlation experiments in terms of sensitivity and resolution, using a model microcrystalline fMLF tripeptide. We demonstrate the applications of ultrafast MAS to a large, fully protonated protein assembly of the 231-residue HIV-1 CA capsid protein. Resonance assignments of protons and heteronuclei, as well as 1H-15N dipolar and 1HN CSA tensors are readily obtained from the high sensitivity and resolution proton-detected 3D experiments. The approach demonstrated here is expected to enable the determination of atomic-resolution structures of large protein assemblies, inaccessible by current methodologies.

Published
2017
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42. Paramagnetic Properties of a Crystalline Iron-Sulfur Protein by Magic-Angle Spinning NMR Spectroscopy

Author

Giacomo Parigi, Andrea Bertarello, Lyndon Emsley, Carmelo Fuccio, Enrico Ravera, Marco Fragai, Claudio Luchinat, Guido Pintacuda, Tobias Schubeis, Biological Solid-State NMR Methods - Méthodes de RMN à l'état solide en biologie, Institut des Sciences Analytiques (ISA), 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)-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), Giotto Biotech SRL, Magnet Resonance Ctr CERM & Interunivers Consoriu, Dept Chem Ugo Schiff, Università degli Studi di Firenze = University of Florence (UniFI), Institut des Sciences et Ingénierie Chimiques (ISIC), This research was supported by the CNRS (IR-RMN FR3050), the Fondazione Cassa di Risparmio di Firenze, the MIUR PRIN 2012SK7ASN, the People Programme of the European Union's FP7 (FP7-PEOPLE-2012-ITN no. 317127 'pNMR'), the European Research Council (ERC) under the European Union's Horizon 2020 research and innovation programme (grant no. 648974 'P-MEM-NMR'), the European Commission projects BioMedBridges No. 284209, iNEXT No. 653706, and Instruct, part of the European Strategy Forum on Research Infrastructures (ESFRI). Specifically, we thank the EU ESFRI Instruct Core Centre CERM, Italy. E.R. holds a FIRC triennial fellowship 'Gino Mazzega and Guglielmina Lucatello' (17941)., European Project: 317127,EC:FP7:PEOPLE,FP7-PEOPLE-2012-ITN,PNMR(2013), European Project: 653706,H2020,H2020-INFRAIA-2014-2015,iNEXT(2015), European Project: 648974,H2020,ERC-2014-CoG,P-MEM-NMR(2015), Institut de Chimie du CNRS (INC)-Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-Centre National de la Recherche Scientifique (CNRS)-Institut de Chimie du CNRS (INC)-Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-Centre National de la Recherche Scientifique (CNRS), and Università degli Studi di Firenze = University of Florence [Firenze] (UNIFI)

Subjects
Iron-Sulfur Proteins, chemistry.chemical_classification, 010405 organic chemistry, Chemistry, Nuclear magnetic resonance spectroscopy, 010402 general chemistry, 01 natural sciences, 0104 chemical sciences, Inorganic Chemistry, Crystallography, Paramagnetism, [CHIM.ANAL]Chemical Sciences/Analytical chemistry, Magic angle spinning, Cluster (physics), Metalloprotein, Irradiation, Physical and Theoretical Chemistry, Crystallization, Nuclear Magnetic Resonance, Biomolecular, Spinning, Cysteine
Abstract

International audience; We present the first solid-state NMR study of an iron sulfur protein. The combined use of very fast (60 kHz) magic-angle spinning and tailored radiofrequency irradiation schemes allows the detection and the assignment of most of the H-1 and C-13 resonances of the oxidized high-potential iron sulfur protein I from Ectothiorhodospira halophila (EhHiPIP I), including those in residues coordinating the Fe4S4 cluster. For these residues, contact shifts as large as 100 and 400 ppm for H-1 and C-13 resonances, respectively, were observed, which represent the most shifted solid-state NMR signals ever measured in metalloproteins. Interestingly, by targeting EhHiPIP I in a crystalline environment, we were able to capture distinct paramagnetic signatures from the two conformations present in the asymmetric unit. The magnetic properties of the system were verified by following the temperature dependence of the contact-shifted cysteine resonances.

Published
2017
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43. Low-power broadband solid-state MAS NMR of

Author

Andrew J, Pell, Kevin J, Sanders, Sebastian, Wegner, Guido, Pintacuda, and Clare P, Grey

Subjects
ARTICLES
Abstract

We propose two broadband pulse schemes for 14N solid-state magic-angle-spinning (MAS) nuclear magnetic resonance (NMR) that achieves (i) complete population inversion and (ii) efficient excitation of the double-quantum spectrum using low-power single-sideband-selective pulses. We give a comprehensive theoretical description of both schemes using a common framework that is based on the jolting-frame formalism of Caravatti et al. [J. Magn. Reson. 55, 88 (1983)]. This formalism is used to determine for the first time that we can obtain complete population inversion of 14N under low-power conditions, which we do here using single-sideband-selective adiabatic pulses. It is then used to predict that double-quantum coherences can be excited using low-power single-sideband-selective pulses. We then proceed to design a new experimental scheme for double-quantum excitation. The final double-quantum excitation pulse scheme is easily incorporated into other NMR experiments, as demonstrated here for double quantum–single quantum 14N correlation spectroscopy, and 1H–14N dipolar heteronuclear multiple-quantum correlation experiments. These pulses and irradiation schemes are evaluated numerically using simulations on single crystals and full powders, as well as experimentally on ammonium oxalate ((NH4)2C2O4) at moderate MAS and glycine at ultra-fast MAS. The performance of these new NMR methods is found to be very high, with population inversion efficiencies of 100% and double-quantum excitation efficiencies of 30%–50%, which are hitherto unprecedented for the low radiofrequency field amplitudes, up to the spinning frequency, that are used here.

Published
2017

44. Low-power broadband solid-state MAS NMR of 14 N

Author

Clare P. Grey, Kevin J. Sanders, Sebastian Wegner, Andrew J. Pell, Guido Pintacuda, Department of Chemistry, University of Cambridge [UK] (CAM), Biological Solid-State NMR Methods - Méthodes de RMN à l'état solide en biologie, Institut des Sciences Analytiques (ISA), 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)-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), Bruker BioSpin GmbH, D-76287 Rheinstetten, Germany, affiliation inconnue, A.J.P. and C.P.G. were supported by the Assistant Secretary for Energy Efficiency and Renewable Energy, Office of Vehicle Technologies of the US Department of Energy under Contract No. DE-AC02-05CH11231, under the Batteries for Advanced Transportation Technologies (BATT) Program Subcontract No. 7057154. G.P. acknowledges support from the European Research Council under the European Union’s Horizon 2020 Research and Innovation Programme (ERC consolidator Grant No. 648974 'P-MEM-NMR')., European Project: 648974,H2020,ERC-2014-CoG,P-MEM-NMR(2015), Institut de Chimie du CNRS (INC)-Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-Centre National de la Recherche Scientifique (CNRS)-Institut de Chimie du CNRS (INC)-Université Claude Bernard Lyon 1 (UCBL), and Université de Lyon-Université de Lyon-Centre National de la Recherche Scientifique (CNRS)

Subjects
010304 chemical physics, Chemistry, Analytical chemistry, General Physics and Astronomy, 010402 general chemistry, Population inversion, 01 natural sciences, 0104 chemical sciences, Dipole, Amplitude, Heteronuclear molecule, [CHIM.ANAL]Chemical Sciences/Analytical chemistry, Excited state, 0103 physical sciences, Physical and Theoretical Chemistry, Atomic physics, Adiabatic process, Two-dimensional nuclear magnetic resonance spectroscopy, Excitation
Abstract

We acknowledge Dr. Dominique Massiot and Dr. Michael Deschamps (Université d’Orléans), and Professor Philip J. Grandinetti (Ohio State University) for many useful discussions about various aspects of broadband NMR sequences, adiabaticity, and the jolting frame. A.J.P. also thanks Professor Malcolm H. Levitt (University of Southampton) for his invaluable support with SpinDynamica and an interesting discussion regarding some subtle properties of the dynamics of spin-one nuclear spins.; International audience; We propose two broadband pulse schemes for 14N solid-state magic-angle-spinning (MAS) nuclear magnetic resonance (NMR) that achieves (i) complete population inversion and (ii) efficient excitation of the double-quantum spectrum using low-power single-sideband-selective pulses. We give a comprehensive theoretical description of both schemes using a common framework that is based on the jolting-frame formalism of Caravatti et al. [J. Magn. Reson. 55, 88 (1983)]. This formalism is used to determine for the first time that we can obtain complete population inversion of 14N under low-power conditions, which we do here using single-sideband-selective adiabatic pulses. It is then used to predict that double-quantum coherences can be excited using low-power single-sideband-selective pulses. We then proceed to design a new experimental scheme for double-quantum excitation. The final double-quantum excitation pulse scheme is easily incorporated into other NMR experiments, as demonstrated here for double quantum–single quantum 14N correlation spectroscopy, and 1H–14N dipolar heteronuclear multiple-quantum correlation experiments. These pulses and irradiation schemes are evaluated numerically using simulations on single crystals and full powders, as well as experimentally on ammonium oxalate ((NH4)2C2O4) at moderate MAS and glycine at ultra-fast MAS. The performance of these new NMR methods is found to be very high, with population inversion efficiencies of 100% and double-quantum excitation efficiencies of 30%–50%, which are hitherto unprecedented for the low radiofrequency field amplitudes, up to the spinning frequency, that are used here.

Published
2017
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45. Correlating Local Compositions and Structures with the Macroscopic Optical Properties of Ce3+-Doped CaSc2O4, an Efficient Green-Emitting Phosphor

Author

Ram Seshadri, Géraldine Dantelle, Jakoah Brgoch, Andrew J. Pell, Adam Jaffe, Nathan C. George, Clayton Cozzan, Guido Pintacuda, Bradley F. Chmelka, Anna Llobet, Mat Res Lab, University of California [Santa Barbara] (UCSB), University of California-University of California, Dept Chem Engn, Mat Dept, Dept Chem, University of Houston, Biological Solid-State NMR Methods - Méthodes de RMN à l'état solide en biologie, Institut des Sciences Analytiques (ISA), Institut de Chimie du CNRS (INC)-Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-Centre National de la Recherche Scientifique (CNRS)-Institut de Chimie du CNRS (INC)-Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-Centre National de la Recherche Scientifique (CNRS), Surface and Aqueous Geochemistry Group [Stanford], Advanced Light Source [LBNL Berkeley] (ALS), Lawrence Berkeley National Laboratory [Berkeley] (LBNL)-Lawrence Berkeley National Laboratory [Berkeley] (LBNL)-Stanford Synchrotron Radiation Lightsource (SSRL SLAC), SLAC National Accelerator Laboratory (SLAC), Stanford University-Stanford University-SLAC National Accelerator Laboratory (SLAC), Stanford University-Stanford University, Optique et Matériaux (OPTIMA ), Institut Néel (NEEL), Institut polytechnique de Grenoble - Grenoble Institute of Technology (Grenoble INP )-Centre National de la Recherche Scientifique (CNRS)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut polytechnique de Grenoble - Grenoble Institute of Technology (Grenoble INP )-Centre National de la Recherche Scientifique (CNRS)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019]), Lujan Center for Neutron Scattering, LANL, Los Alamos National Laboratory (LANL), The work at the ENS-Lyon was supported by the Initial Training Network on Paramagnetic NMR funded by the European Union's Seventh Framework Program for research, technological development, and demonstration (FP7-PEOPLE-2012-ITN contract n. 317127 'pNMR'). N.C.G. acknowledges support from the ConvEne IGERT Program of the U.S. National Science Foundation (NSF-DGE 0801627). A.J.P. and G.P. were supported by the LABEX iMUST (ANR-10-LABX-0064) of the Universite de Lyon, within the program 'Investissements d'Avenir' (ANR-11-IDEX-0007) operated by the Agence Nationale de la Recherche (ANR). The research made extensive use of the Central Facilities of the UCSB Materials Research Laboratory, supported by the MRSEC Program of the NSF under Award DMR-1121053, which is a member of the NSF-funded Materials Research Facilities Network (www.mrfn.org). Use of the Advanced Photon Source at Argonne National Laboratory was supported by the U.S. Department of Energy, Office of Science, Office of Basic Energy Sciences, under Contract No. DE-AC02-06CH11357. This work benefited from the use of the HIPD beamline at the Lujan Center at Los Alamos National Laboratory, funded by the DOE Office of Basic Energy Sciences, LANL is operated by Los Alamos National Security LLC under DE-AC52-06NA25396. The solid-state NMR measurements at 23.5 T were conducted at the Centre Europeen de Resonance Magnetique Nucleaire a Tres Hauts Champs in Lyon, France. Solid-state 43Ca NMR measurements were conducted at the U.S. National High Magnetic Field Laboratory, Tallahassee, FL, supported by the U.S. NSF. B.F.C. thanks the ENS-Lyon for support as a 'Professeur Invite' in 2013., ANR-11-IDEX-0007,Avenir L.S.E.,PROJET AVENIR LYON SAINT-ETIENNE(2011), European Project: 317127,EC:FP7:PEOPLE,FP7-PEOPLE-2012-ITN,PNMR(2013), University of California [Santa Barbara] (UC Santa Barbara), University of California (UC)-University of California (UC), 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)-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 Synchrotron Radiation Lightsource (SSRL SLAC), Stanford University-Stanford University-Advanced Light Source [LBNL Berkeley] (ALS), Lawrence Berkeley National Laboratory [Berkeley] (LBNL)-Lawrence Berkeley National Laboratory [Berkeley] (LBNL), and Optique et Matériaux (NEEL - OPTIMA)

Subjects
Materials science, Photoluminescence, Scattering, General Chemical Engineering, Neutron diffraction, Analytical chemistry, Phosphor, 02 engineering and technology, General Chemistry, [CHIM.MATE]Chemical Sciences/Material chemistry, 010402 general chemistry, 021001 nanoscience & nanotechnology, 01 natural sciences, Synchrotron, 0104 chemical sciences, law.invention, [CHIM.THEO]Chemical Sciences/Theoretical and/or physical chemistry, law, [CHIM.ANAL]Chemical Sciences/Analytical chemistry, Materials Chemistry, Density functional theory, Quantum efficiency, 0210 nano-technology, Electron paramagnetic resonance
Abstract

The authors thank J. Siewenie for assistance with collection of the neutron powder diffraction data and Dr. Z. Gan for assistance with the 43Ca NMR measurements.; International audience; Calcium scandate (CaSc2O4) substituted with small amounts (

Published
2017
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46. Local and Global Dynamics in Klebsiella pneumoniae Outer Membrane Protein a in Lipid Bilayers Probed at Atomic Resolution

Author

Olivier Saurel, Pascal Demange, Iordan Iordanov, Guillaume Nars, Tanguy Le Marchand, Loren B. Andreas, Guido Pintacuda, Alain Milon, Institut de pharmacologie et de biologie structurale (IPBS), Centre National de la Recherche Scientifique (CNRS)-Université Toulouse III - Paul Sabatier (UT3), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées, Dept Med Biochem, Semmelweis University [Budapest], Biological Solid-State NMR Methods - Méthodes de RMN à l'état solide en biologie, Institut des Sciences Analytiques (ISA), Institut de Chimie du CNRS (INC)-Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-Centre National de la Recherche Scientifique (CNRS)-Institut de Chimie du CNRS (INC)-Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-Centre National de la Recherche Scientifique (CNRS), PICT - Genotoul platform of Toulouse, CNRS, Universite de Toulouse-UPS, European structural funds, the Midi-Pyrenees region, CNRS (IR-RMN FR3050) and from the European Research Council (ERC) under the European Union's Horizon 2020 research and innovation programme (ERC-2015-CoG GA No. 648974). II was funded by an EU Initial Training network (FP7/2007-2013, Grant Agreement No. 211800). L.B.A. was supported by an MC incoming fellowships (REA Grant Agreement No. 624918 'MEM-MAS')., Université Toulouse III - Paul Sabatier (UT3), and Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Centre National de la Recherche Scientifique (CNRS)

Subjects
0301 basic medicine, [SDV]Life Sciences [q-bio], SPIN-LATTICE-RELAXATION, Lipid Bilayers, 010402 general chemistry, 01 natural sciences, Biochemistry, Catalysis, Mass Spectrometry, 03 medical and health sciences, Colloid and Surface Chemistry, [CHIM.ANAL]Chemical Sciences/Analytical chemistry, Lipid bilayer, Nuclear Magnetic Resonance, Biomolecular, ComputingMilieux_MISCELLANEOUS, Liposome, RESONANCE ASSIGNMENT, Chemistry, Relaxation (NMR), Spin–lattice relaxation, CONFORMATIONAL DYNAMICS, CRYSTALLINE PROTEIN, General Chemistry, HETERONUCLEAR DIPOLAR COUPLINGS, Recombinant Proteins, TRANSMEMBRANE DOMAIN, 0104 chemical sciences, SOLID-STATE NMR, Transmembrane domain, Crystallography, Klebsiella pneumoniae, 030104 developmental biology, Membrane, Solid-state nuclear magnetic resonance, Membrane protein, FULLY PROTONATED PROTEINS, Proteolysis, Biophysics, MAS NMR, Thermodynamics, BACKBONE DYNAMICS, Bacterial Outer Membrane Proteins
Abstract

We thank Dr. Lewandowski for providing a Matlab code for simulating 15N R1 values via the website http://www.ens-lyon.fr/crmn/crmn/index.html.; International audience; The role of membrane proteins in cellular mechanism strongly depends on their dynamics, and solid-state magic-angle spinning (MAS) nuclear magnetic resonance (NMR) is a unique method to exhaustively characterize motions of proteins in a lipid environment. Herein, we make use of advances in H-1-detected MAS NMR to describe the dynamics of the membrane domain of the Outer membrane protein A of Klebsiella pneumoniae (KpOmpA). By measuring H-1-N-15 dipolar coupling as well as N-15 R-1 and R-1 rho relaxation rates at fast (60 kHz) MAS and high magnetic field (1 GHz), we were able to describe the motions of the residues of the beta-barrel as a collective rocking of low amplitude and of hundreds of nanoseconds time scale. Residual local motions at the edges of the strands, underscored by enhanced (NT)-N-15 R-1 rho relaxation rates, report on the mobility of the connected loops. In agreement with MAS NMR data, proteolysis experiments performed on the full length KpOmpA as well as on its membrane domain, reconstituted in liposomes or in detergent micelles, revealed in all cases the existence of a unique trypsin cleavage site within the membrane domain (out of 16 potential Lys and Arg sites). This site is located in the extracellular loop L3, showing that it is highly accessible to protein-protein interactions. KpOmpA is involved in cell-cell recognition, for adhesion and immune response mechanisms. The L3 region may therefore play a key role in pathogenicity.

Published
2017
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47. Selective 1H–1H Distance Restraints in Fully Protonated Proteins by Very Fast Magic-Angle Spinning Solid-State NMR

Author

Vipin Agarwal, Jan Stanek, Kathrin Castiglione, Mukul G. Jain, Perunthiruthy K. Madhu, Tom S. Schwarzer, Tobias Schubeis, Loren B. Andreas, Chandrakala Gowda, Guido Pintacuda, Daniela Lalli, Satya Prakash, TIFR Centre for Interdisciplinary Sciences [Hyderabad] (TCIS), Tata Institute for Fundamental Research (TIFR), Biological Solid-State NMR Methods - Méthodes de RMN à l'état solide en biologie, Institut des Sciences Analytiques (ISA), Institut de Chimie du CNRS (INC)-Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-Centre National de la Recherche Scientifique (CNRS)-Institut de Chimie du CNRS (INC)-Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-Centre National de la Recherche Scientifique (CNRS), Inst Biochem Engn, Technische Universität Munchen - Université Technique de Munich [Munich, Allemagne] (TUM), Max Planck Institute for Biophysical Chemistry (MPI-BPC), Max-Planck-Gesellschaft, Department of Chemical Sciences [TIFR] (DCS), We acknowledge financial support from CNRS (IR-RMN FR3050, TGIR-RMN00874) and from the European Research Council (ERC) under the European Union's Horizon 2020 research and innovation programme (ERC-2015-CoG GA No. 648974). J.S. is supported by a MC incoming fellowship (REA Grant Agreement No. 661799 'COMPLEX-FAST- MAS'). T.S. and K.C. acknowledge funding from German Federal Ministry of Education and Research (BMBF Grant No. 031A178)., European Project: 648974,H2020,ERC-2014-CoG,P-MEM-NMR(2015), 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)-Université Claude Bernard Lyon 1 (UCBL), and Université de Lyon-Université de Lyon-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS)

Subjects
Analytical chemistry, Protonation, 010402 general chemistry, 01 natural sciences, Spectral line, Magnetization, [CHIM.ANAL]Chemical Sciences/Analytical chemistry, Magic angle spinning, General Materials Science, Physical and Theoretical Chemistry, [SDV.BBM.BC]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biochemistry [q-bio.BM], Spinning, 010405 organic chemistry, Chemistry, AlkL, Resolution (electron density), GB1, structure determination, NMR, 0104 chemical sciences, structural characterization, [CHIM.THEO]Chemical Sciences/Theoretical and/or physical chemistry, Crystallography, Membrane, MAS, Solid-state nuclear magnetic resonance, protein, beta-barrel membrane protein
Abstract

We acknowledge Prof Matthias Ernst, ETH, Zurich, for useful discussions and Dr Rajalakshmi and Dr Kaustubh Mote for proofreading the manuscript.; International audience; Very fast magic-angle spinning (MAS > 80 kHz) NMR combined with high-field magnets has enabled the acquisition of proton-detected spectra in fully protonated solid samples with sufficient resolution and sensitivity. One of the primary challenges in structure determination of protein is observing long-range H-1-H-1 contacts. Here we use band-selective spin-lock pulses to obtain selective H-1-H-1 contacts (e.g., H-N-H-N) on the order of 5-6 angstrom in fully protonated proteins at 111 kHz MAS. This approach is a major advancement in structural characterization of proteins given that magnetization can be selectively transferred between protons that are 5-6 angstrom apart despite the presence of other protons at shorter distance. The observed contacts are similar to those previously observed only in perdeuterated proteins with selective protonation. Simulations and experiments show the proposed method has performance that is superior to that of the currently used methods. The method is demonstrated on GB1 and a beta-barrel membrane protein, AlkL.

Published
2017
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48. Dynamic Nuclear Polarization Enhanced MAS NMR Spectroscopy for Structural Analysis of HIV‑1 Protein Assemblies

Author

Andrea Bertarello, Marcella Orwick-Rydmark, Hartmut Oschkinat, Marc A. Caporini, Melanie Rosay, In-Ja L. Byeon, Rupal Gupta, Guangjin Hou, Trent W. Franks, Anne Lesage, Jochem Struppe, Christopher L. Suiter, Werner E. Maas, Manman Lu, Guido Pintacuda, Tatyana Polenova, Angela M. Gronenborn, Jinwoo Ahn, Department of Chemistry and Biochemistry, University of Delaware [Newark], University of Pittsburgh School of Medicine, Pennsylvania Commonwealth System of Higher Education (PCSHE), Bruker BioSpin Corporation, Department of Structural Biology, Pennsylvania Commonwealth System of Higher Education (PCSHE)-Pennsylvania Commonwealth System of Higher Education (PCSHE), Leibniz Forschungsinstitut für Molekulare Pharmakolgie = Leibniz Institute for Molecular Pharmacology [Berlin, Allemagne] (FMP), Leibniz Association, Biological Solid-State NMR Methods - Méthodes de RMN à l'état solide en biologie, Institut des Sciences Analytiques (ISA), 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)-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), and Solid-State NMR Methods for Materials - Méthodes de RMN à l'état solide pour les matériaux

Subjects
0301 basic medicine, Magnetic Resonance Spectroscopy, Protein Conformation, Intermolecular correlations, Peptide, 010402 general chemistry, Cleavage (embryo), 01 natural sciences, Article, Viral Proteins, 03 medical and health sciences, Capsid, Protein structure, Dynamic nuclear polarization, Sensitivity enhancements, [CHIM.ANAL]Chemical Sciences/Analytical chemistry, Materials Chemistry, Molecule, Physical and Theoretical Chemistry, Conformational isomerism, Structure determination, Nuclear magnetic resonance spectroscopy, chemistry.chemical_classification, Chemistry, Tubular steel structures Cryogenic temperatures, Intermolecular force, Relaxation (NMR), Proteins, 0104 chemical sciences, 3. Good health, Surfaces, Coatings and Films, Temperature distribution, Crystallography, 030104 developmental biology, Proteolytic cleavage, Temperature dependence, Magnetic field strengths, HIV-1, Amino acids, Peptides, Molecular structure
Abstract

International audience; Mature infectious HIV-1 virions contain conical capsids composed of CA protein, generated by the proteolytic cleavage cascade of the Gag polyprotein, termed maturation. The mechanism of capsid core formation through the maturation process remains poorly understood. We present DNP-enhanced MAS NMR studies of tubular assemblies of CA and Gag CA-SP1 maturation intermediate and report 20-64-fold sensitivity enhancements due to DNP at 14.1 T. These sensitivity enhancements enabled direct observation of spacer peptide 1 (SP1) resonances in CA-SP1 by dipolar-based correlation experiments, unequivocally indicating that the SP1 peptide is unstructured in assembled CA-SP1 at cryogenic temperatures, corroborating our earlier results. Furthermore, the dependence of DNP enhancements and spectral resolution on magnetic field strength (9.4-18.8 T) and temperature (109-180 K) was investigated. Our results suggest that DNP-based measurements could potentially provide residue-specific dynamics information by allowing for the extraction of the temperature dependence of the anisotropic tensorial or relaxation parameters. With DNP, we were able to detect multiple well-resolved isoleucine side-chain conformers; unique intermolecular correlations across two CA molecules; and functionally relevant conformationally disordered states such as the 14-residue SP1 peptide, none of which are visible at ambient temperatures. The detection of isolated conformers and intermolecular correlations can provide crucial constraints for structure determination of these assemblies. Overall, our results establish DNP-based MAS NMR spectroscopy as an excellent tool for the characterization of HIV-1 assemblies.

Published
2017
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49. Frontispiz: Zuordnung der Rückgrat- und Seitenketten-Protonen in vollständig protonierten Proteinen durch Festkörper-NMR-Spektroskopie: Mikrokristalle, Sedimente und Amyloidfibrillen

Author

Jan Stanek, Loren B. Andreas, Kristaps Jaudzems, Diane Cala, Daniela Lalli, Andrea Bertarello, Tobias Schubeis, Inara Akopjana, Svetlana Kotelovica, Kaspars Tars, Andrea Pica, Serena Leone, Delia Picone, Zhi-Qiang Xu, Nicholas E. Dixon, Denis Martinez, Mélanie Berbon, Nadia El Mammeri, Abdelmajid Noubhani, Sven Saupe, Birgit Habenstein, Antoine Loquet, and Guido Pintacuda

Subjects
General Medicine
Published
2016
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50. Structural Characterization of the EtOH-TiCl 4 -MgCl 2 Ziegler-Natta Precatalyst

Author

Sébastien Norsic, Christophe Copéret, Andrew J. Pell, Guido Pintacuda, Vincent Monteil, Anne Lesage, Philippe Sautet, David Gajan, Kevin J. Sanders, V. d'Anna, Laboratoire de Chimie - UMR5182 (LC), Centre National de la Recherche Scientifique (CNRS)-Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-École normale supérieure - Lyon (ENS Lyon)-Institut de Chimie du CNRS (INC), Laboratoire de Chimie, Catalyse, Polymères et Procédés, R 5265 (C2P2), Centre National de la Recherche Scientifique (CNRS)-École supérieure de Chimie Physique Electronique de Lyon (CPE)-Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-Institut de Chimie du CNRS (INC), Solid-State NMR Methods for Materials - Méthodes de RMN à l'état solide pour les matériaux, Institut des Sciences Analytiques (ISA), 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)-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), Biological Solid-State NMR Methods - Méthodes de RMN à l'état solide en biologie, Department of Chemistry and Applied Biosciences [ETH Zürich], Eidgenössische Technische Hochschule - Swiss Federal Institute of Technology [Zürich] (ETH Zürich), V.D. acknowledges the Swiss National Science Foundation (Early Post-Doc mobility, no. P2GEP2_155679)., École normale supérieure de Lyon (ENS de Lyon)-Université Claude Bernard Lyon 1 (UCBL), and Université de Lyon-Université de Lyon-École Supérieure de Chimie Physique Électronique de Lyon (CPE)-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS)

Subjects
biology, 010405 organic chemistry, Chemistry, Nuclear magnetic resonance spectroscopy, [CHIM.MATE]Chemical Sciences/Material chemistry, Natta, Polyethylene, 010402 general chemistry, biology.organism_classification, 01 natural sciences, 0104 chemical sciences, Surfaces, Coatings and Films, Electronic, Optical and Magnetic Materials, Catalysis, Characterization (materials science), NMR spectra database, chemistry.chemical_compound, General Energy, Polymerization, [CHIM.ANAL]Chemical Sciences/Analytical chemistry, Polymer chemistry, Organic chemistry, Lewis acids and bases, Physical and Theoretical Chemistry
Abstract

The authors thank the PSMN, CINES, and IDRIS for the computational resources.; International audience; The Ziegler-Natta polymerization is one major example of application of catalysis in industry. Since the first discovery of Ziegler and Natta, several modifications of the catalyst have been developed, in order to improve its performance. Nowadays, a typical Ziegler-Natta catalyst for polyethylene synthesis consists of a precatalyst, composed of TiCl4 supported on MgCl2 in the presence of a Lewis base, activated by organoaluminum. The atomic-scale characterization of the Ziegler-Natta catalyst is crucial for further improvement of the catalyst. Here, the precatalyst TiCl4-MgCl2 with EtOH as internal. Lewis base is characterized combining solid-state NMR spectroscopy and periodic density functional theory calculations. From total energy and NMR spectra, eight surface species were proposed showing EtO- ligands on the Ti and EtOH/EtO- on the surface Mg species, These species lead to a complete interpretation of the NMR two-dimensional spectra. Hence a detailed molecular scale description of the precatalyst was obtained.

Published
2016
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