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16 results on '"Vilius Kurauskas"'

1. Ion-dependent structure, dynamics, and allosteric coupling in a non-selective cation channel

Author

Adam Lewis, Vilius Kurauskas, Marco Tonelli, and Katherine Henzler-Wildman

Subjects
Science
Abstract

NaK is a bacterial non-selective cation channel. Here, the authors use solution NMR to show that selectivity filter (SF) in NaK is dynamic, with structural differences between the Na+ and K + -bound states. The conformation of the SF is communicated to the pore-lining helices similarly as in the K + -selective channels.

Published
2021
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2. Slow conformational exchange and overall rocking motion in ubiquitin protein crystals

Author

Vilius Kurauskas, Sergei A. Izmailov, Olga N. Rogacheva, Audrey Hessel, Isabel Ayala, Joyce Woodhouse, Anastasya Shilova, Yi Xue, Tairan Yuwen, Nicolas Coquelle, Jacques-Philippe Colletier, Nikolai R. Skrynnikov, and Paul Schanda

Subjects
Science
Abstract

X-ray crystallography is the main method for protein structure determination. Here the authors combine solid-state NMR measurements and molecular dynamics simulations and show that crystal packing alters the thermodynamics and kinetics of local conformational exchange as well as overall rocking motion of protein molecules in the crystal lattice.

Published
2017
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3. Full opening of helix bundle crossing does not lead to NaK channel activation

Author

Vilius Kurauskas, Marco Tonelli, and Katherine Henzler-Wildman

Subjects
Ions, Physiology, Protein Conformation, Ion Channel Gating, Ion Channels
Abstract

A critical part of ion channel function is the ability to open and close in response to stimuli and thus conduct ions in a regulated fashion. While x-ray diffraction studies of ion channels suggested a general steric gating mechanism located at the helix bundle crossing (HBC), recent functional studies on several channels indicate that the helix bundle crossing is wide-open even in functionally nonconductive channels. Two NaK channel variants were crystallized in very different open and closed conformations, which served as important models of the HBC gating hypothesis. However, neither of these NaK variants is conductive in liposomes unless phenylalanine 92 is mutated to alanine (F92A). Here, we use NMR to probe distances at near-atomic resolution of the two NaK variants in lipid bicelles. We demonstrate that in contrast to the crystal structures, both NaK variants are in a fully open conformation, akin to Ca2+-bound MthK channel structure where the HBC is widely open. While we were not able to determine what a conductive NaK structure is like, our further inquiry into the gating mechanism suggests that the selectivity filter and pore helix are coupled to the M2 helix below and undergo changes in the structure when F92 is mutated. Overall, our data show that NaK exhibits coupling between the selectivity filter and HBC, similar to K+ channels, and has a more complex gating mechanism than previously thought, where the full opening of HBC does not lead to channel activation.

Published
2022

5. Ion-dependent structure, dynamics, and allosteric coupling in a non-selective cation channel

Author

Adam J. Lewis, Vilius Kurauskas, Katherine A. Henzler-Wildman, and Marco Tonelli

Subjects
Magnetic Resonance Spectroscopy, Potassium Channels, Direct evidence, Science, Allosteric regulation, General Physics and Astronomy, Crystal structure, Molecular Dynamics Simulation, Crystallography, X-Ray, General Biochemistry, Genetics and Molecular Biology, Sodium Channels, Article, Ion, Cyclic nucleotide-gated cation channels, Allosteric Regulation, Bacterial Proteins, Ion channel, Multidisciplinary, Bacteria, Chemistry, Sodium, General Chemistry, Potassium channel, Coupling (electronics), Crystallography, Structural change, Helix, Potassium, Permeation and transport, Solution-state NMR
Abstract

The selectivity filter (SF) determines which ions are efficiently conducted through ion channel pores. NaK is a non-selective cation channel that conducts Na+ and K+ with equal efficiency. Crystal structures of NaK suggested a rigid SF structure, but later solid-state NMR and MD simulations questioned this interpretation. Here, we use solution NMR to characterize how bound Na+ vs. K+ affects NaK SF structure and dynamics. We find that the extracellular end of the SF is flexible on the ps-ns timescale regardless of bound ion. On a slower timescale, we observe a structural change between the Na+ and K+-bound states, accompanied by increased structural heterogeneity in Na+. We also show direct evidence that the SF structure is communicated to the pore via I88 on the M2 helix. These results support a dynamic SF with multiple conformations involved in non-selective conduction. Our data also demonstrate allosteric coupling between the SF and pore-lining helices in a non-selective cation channel that is analogous to the allosteric coupling previously demonstrated for K+-selective channels, supporting the generality of this model., NaK is a bacterial non-selective cation channel. Here, the authors use solution NMR to show that selectivity filter (SF) in NaK is dynamic, with structural differences between the Na+ and K + -bound states. The conformation of the SF is communicated to the pore-lining helices similarly as in the K + -selective channels.

Published
2021
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6. Dynamics and interactions of AAC3 in DPC are not functionally relevant

Author

Christophe Chipot, Vilius Kurauskas, François Dehez, Beate Bersch, Paul Schanda, Audrey Hessel, Institut de biologie structurale (IBS - UMR 5075 ), Centre National de la Recherche Scientifique (CNRS)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA), Laboratoire de Physique et Chimie Théoriques (LPCT), Institut de Chimie du CNRS (INC)-Université de Lorraine (UL)-Centre National de la Recherche Scientifique (CNRS), Laboratoire International Associé (LIA), University of Illinois at Urbana-Champaign [Urbana], University of Illinois System-University of Illinois System-Centre National de la Recherche Scientifique (CNRS), Department of Physics [Illinois at Urbana-Champaign, USA], University of Illinois System-University of Illinois System, Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), and Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Centre National de la Recherche Scientifique (CNRS)

Subjects
0301 basic medicine, biology, Membrane transport protein, Dynamics (mechanics), Nuclear magnetic resonance spectroscopy, 010402 general chemistry, 01 natural sciences, 0104 chemical sciences, 03 medical and health sciences, chemistry.chemical_compound, Adenosine diphosphate, 030104 developmental biology, chemistry, Structural Biology, biology.protein, Biophysics, [SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology, Molecular Biology, Adenosine triphosphate, ComputingMilieux_MISCELLANEOUS
Abstract

International audience

Published
2018
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7. Dynamics and interactions of ADP/ATP transporter AAC3 in DPC detergent are not functionally relevant

Author

Vilius Kurauskas, Audrey Hessel, Paul Schanda, Beate Bersch, Christophe Chipot, François Dehez, Institut de biologie structurale ( IBS - UMR 5075 ), and Université Joseph Fourier - Grenoble 1 ( UJF ) -Commissariat à l'énergie atomique et aux énergies alternatives ( CEA ) -Centre National de la Recherche Scientifique ( CNRS ) -Université Grenoble Alpes ( UGA )

Subjects
0303 health sciences, Chemistry, Substrate (chemistry), Transporter, Nuclear magnetic resonance spectroscopy, 010402 general chemistry, 01 natural sciences, Micelle, Article, Yeast, 0104 chemical sciences, 03 medical and health sciences, Nat, Mole, Biophysics, ATP–ADP translocase, 030304 developmental biology, [ SDV.BBM.BS ] Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biomolecules [q-bio.BM]
Abstract

International audience; A recent study (S. Bruschweiler et al and J.J. Chou, Nat. Struct. Mol. Biol. 2015) used solution-state NMR spectroscopy to examine the interactions and dynamics of the yeast mitochondrial inner-membrane ADP/ATP carrier, yAAC3. The authors present NMR based interaction studies and millisecond dynamics. Their main conclusion is that yAAC3 in dodecylphosphocholine detergent micelles is functional and undergoes functionally relevant motions that depend on substrate and inhibitor. We contradict these results and show that the protein sample is partially misfolded; the dynamics are not correctly fitted and do not appear to be dependent on substrate/inhibitor. Together, our findings suggest that yAAC3 in DPC detergent is misfolded.

Published
2018

8. How Detergent Impacts Membrane Proteins: Atomic-Level Views of Mitochondrial Carriers in Dodecylphosphocholine

Author

Vilius Kurauskas, Bernhard Brutscher, Loredana Capobianco, François Dehez, Audrey Hessel, Christophe Chipot, Remy Sounier, Paola Lunetti, Paul Schanda, Peixiang Ma, Martin S. King, Katharina Weinhäupl, Vincenza Dolce, Lionel Imbert, Edmund R.S. Kunji, Beate Bersch, Kurauskas, Viliu, Audrey Hessel, †, Peixiang Ma, †, Lunetti, Paola, Katharina Weinhaupl, ‡, † Lionel Imbert, ̈, Bernhard Brutscher, †, King, † Martin S., Remy Sounier, §, ∥ Vincenza Dolce, ́, Kunji, ⊥ Edmund R. S., Capobianco, Loredana, Christophe Chipot, ‡, Francois Dehez,, ̧, Beate Bersch,, and Paul Schanda, †, Institut de biologie structurale ( IBS - UMR 5075 ), Université Joseph Fourier - Grenoble 1 ( UJF ) -Commissariat à l'énergie atomique et aux énergies alternatives ( CEA ) -Centre National de la Recherche Scientifique ( CNRS ) -Université Grenoble Alpes ( UGA ), Department of Pharmaco-Biology, University of Calabria, Medical Research Council Mitochondrial Biology Unit, University of Cambridge [UK] ( CAM ), Department of Biological and Environmental Sciences and Technologies, Università del Salento [Lecce], Structure et Réactivité des Systèmes Moléculaires Complexes ( SRSMC ), Université de Lorraine ( UL ) -Centre National de la Recherche Scientifique ( CNRS ), Institut de biologie structurale (IBS - UMR 5075 ), Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Centre National de la Recherche Scientifique (CNRS), Department of Veterinary Medicine, College of Zoology, Guizhou University, Università della Calabria [Arcavacata di Rende] (Unical), University of Cambridge [UK] (CAM), Laboratoire de Physique et Chimie Théoriques (LPCT), Institut de Chimie du CNRS (INC)-Université de Lorraine (UL)-Centre National de la Recherche Scientifique (CNRS), Laboratoire International Associé (LIA), University of Illinois at Urbana-Champaign [Urbana], University of Illinois System-University of Illinois System-Centre National de la Recherche Scientifique (CNRS), Centre National de la Recherche Scientifique (CNRS)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA), Guizhou University (GZU), Brutscher, Bernhard [0000-0001-7652-7384], Chipot, Christophe [0000-0002-9122-1698], Schanda, Paul [0000-0002-9350-7606], and Apollo - University of Cambridge Repository

Subjects
0301 basic medicine, [ SDV.BBM.BP ] Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biophysics, Letter, Saccharomyces cerevisiae Proteins, Protein Conformation, Phosphorylcholine, [SDV]Life Sciences [q-bio], Detergents, Membrane Proteins, Mitochondrial Carriers, Dodecylphosphocholine, Saccharomyces cerevisiae, Molecular Dynamics Simulation, Mitochondrial Membrane Transport Proteins, Micelle, 03 medical and health sciences, Mitochondrial membrane transport protein, Molecular dynamics, Protein structure, [CHIM]Chemical Sciences, General Materials Science, Physical and Theoretical Chemistry, Lipid bilayer, Nuclear Magnetic Resonance, Biomolecular, [ SDV.BBM ] Life Sciences [q-bio]/Biochemistry, Molecular Biology, Micelles, ComputingMilieux_MISCELLANEOUS, Thermostability, Substrate Interaction, biology, [ SDV ] Life Sciences [q-bio], Protein Stability, Chemistry, 030104 developmental biology, Membrane protein, biology.protein, Biophysics, Mitochondrial ADP, ATP Translocases
Abstract

Characterizing the structure of membrane proteins (MPs) generally requires extraction from their native environment, most commonly with detergents. Yet, the physicochemical properties of detergent micelles and lipid bilayers differ markedly and could alter the structural organization of MPs, albeit without general rules. Dodecylphosphocholine (DPC) is the most widely used detergent for MP structure determination by NMR, but the physiological relevance of several prominent structures has been questioned, though indirectly, by other biophysical techniques, e.g., functional/thermostability assay (TSA) and molecular dynamics (MD) simulations. Here, we resolve unambiguously this controversy by probing the functional relevance of three different mitochondrial carriers (MCs) in DPC at the atomic level, using an exhaustive set of solution-NMR experiments, complemented by functional/TSA and MD data. Our results provide atomic-level insight into the structure, substrate interaction and dynamics of the detergent−membrane protein complexes and demonstrates cogently that, while high-resolution NMR signals can be obtained for MCs in DPC, they systematically correspond to nonfunctional states .

Published
2018
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9. Slow conformational exchange and overall rocking motion in ubiquitin protein crystals

Author

Isabel Ayala, Anastasya Shilova, Nikolai R. Skrynnikov, Tairan Yuwen, Joyce Woodhouse, Paul Schanda, Nicolas Coquelle, Jacques-Philippe Colletier, Vilius Kurauskas, Yi Xue, Olga N. Rogacheva, Sergei A. Izmailov, Audrey Hessel, Institut de biologie structurale (IBS - UMR 5075 ), Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Centre National de la Recherche Scientifique (CNRS), Department of Chemistry, Purdue University [West Lafayette], European Synchrotron Radiation Facility (ESRF), European Research Council ERC StG-311318, Centre National de la Recherche Scientifique (CNRS)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA), Laboratory of Biomolecular NMR, and St Petersburg State University (SPbU)

Subjects
0301 basic medicine, Magnetic Resonance Spectroscopy, Materials science, Protein Conformation, Science, General Physics and Astronomy, Crystal structure, Molecular Dynamics Simulation, Crystallography, X-Ray, 010402 general chemistry, 01 natural sciences, Article, General Biochemistry, Genetics and Molecular Biology, Crystal, Motion, 03 medical and health sciences, Molecular dynamics, Nuclear magnetic resonance, Protein structure, Humans, [CHIM]Chemical Sciences, 030304 developmental biology, [PHYS]Physics [physics], 0303 health sciences, Quantitative Biology::Biomolecules, Multidisciplinary, [SDV.BBM.BS]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Structural Biology [q-bio.BM], Ubiquitin, Intermolecular force, Dynamics (mechanics), Relaxation (NMR), General Chemistry, 0104 chemical sciences, Kinetics, Microsecond, 030104 developmental biology, Chemical physics, Thermodynamics, Protein crystallization, Dispersion (chemistry), Algorithms
Abstract

Proteins perform their functions in solution but their structures are most frequently studied inside crystals. Here we probe how the crystal packing alters microsecond dynamics, using solid-state NMR measurements and multi-microsecond MD simulations of different crystal forms of ubiquitin. In particular, near-rotary-resonance relaxation dispersion (NERRD) experiments probe angular backbone motion, while Bloch–McConnell relaxation dispersion data report on fluctuations of the local electronic environment. These experiments and simulations reveal that the packing of the protein can significantly alter the thermodynamics and kinetics of local conformational exchange. Moreover, we report small-amplitude reorientational motion of protein molecules in the crystal lattice with an ~3–5° amplitude on a tens-of-microseconds time scale in one of the crystals, but not in others. An intriguing possibility arises that overall motion is to some extent coupled to local dynamics. Our study highlights the importance of considering the packing when analyzing dynamics of crystalline proteins., X-ray crystallography is the main method for protein structure determination. Here the authors combine solid-state NMR measurements and molecular dynamics simulations and show that crystal packing alters the thermodynamics and kinetics of local conformational exchange as well as overall rocking motion of protein molecules in the crystal lattice.

Published
2017
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10. Methyl-Specific Isotope Labeling Strategies for NMR Studies of Membrane Proteins

Author

Remy Sounier, Vilius Kurauskas, Paul Schanda, Institut de biologie structurale (IBS - UMR 5075 ), Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Centre National de la Recherche Scientifique (CNRS), Institut de Génomique Fonctionnelle (IGF), Université de Montpellier (UM)-Université Montpellier 1 (UM1)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Montpellier 2 - Sciences et Techniques (UM2)-Centre National de la Recherche Scientifique (CNRS), European Research Council ERC-StG-311318, Schanda, Paul, Centre National de la Recherche Scientifique (CNRS)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA), and Institut National de la Santé et de la Recherche Médicale (INSERM)-Université de Montpellier (UM)-Centre National de la Recherche Scientifique (CNRS)

Subjects
0301 basic medicine, [SDV]Life Sciences [q-bio], Protein expression, Article, Protein Refolding, refolding, 03 medical and health sciences, NMR spectroscopy, detergent, Labelling, [CHIM] Chemical Sciences, Escherichia coli, [CHIM]Chemical Sciences, methyl labelling, Nuclear Magnetic Resonance, Biomolecular, deuteration, Carbon Isotopes, Isotope, Chemistry, Protein NMR Spectroscopy, Membrane Proteins, Nuclear magnetic resonance spectroscopy, Mitochondrial carrier, Deuterium, Recombinant Proteins, 3. Good health, [SDV] Life Sciences [q-bio], 030104 developmental biology, Membrane protein, Biochemistry, Isotope Labeling
Abstract

International audience; Methyl groups are very useful probes of structure, dynamics and interactions in protein NMR spectroscopy. In particular, methyl-directed experiments provide high sensitivity even in very large proteins, such as membrane proteins in a membrane-mimicking environment. In this chapter we discuss the approach for labelling methyl groups in E. coli based protein expression, as exemplified with the mitochondrial carrier GGC.

Published
2017
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11. Solid-State NMR H-N-(C)-H and H-N-C-C 3D/4D Correlation Experiments for Resonance Assignment of Large Proteins

Author

Maxime J. C. Audin, Vilius Kurauskas, Remco Sprangers, Pavel Macek, Carsten Krichel, Diego F. Gauto, Hugo Pacheco de Freitas Fraga, Charles-Adrien Arnaud, Jia-Ying Guan, Paul Schanda, Cécile Breyton, Jérôme Boisbouvier, Institut de biologie structurale (IBS - UMR 5075 ), Centre National de la Recherche Scientifique (CNRS)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA), Max Planck Institute for Developmental Biology, Max-Planck-Gesellschaft, ANR-16-CE11-0027,PerfoBac,Comment les bactériophages perforent-ils la paroi des bactéries ?(2016), European Project: 311318,EC:FP7:ERC,ERC-2012-StG_20111109,PROTDYN2FUNCTION(2013), Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), and Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Centre National de la Recherche Scientifique (CNRS)

Subjects
[SDV]Life Sciences [q-bio], 010402 general chemistry, 01 natural sciences, Article, chemistry.chemical_compound, Protein structure, Amide, Atomic model, Triple-resonance nuclear magnetic resonance spectroscopy, [CHIM]Chemical Sciences, protein quality control machinery, Physical and Theoretical Chemistry, Spectroscopy, NMR methods, Nuclear Magnetic Resonance, Biomolecular, 010405 organic chemistry, Chemistry, phage proteins, protein assemblies, Proteins, Nuclear magnetic resonance spectroscopy, Amides, Atomic and Molecular Physics, and Optics, 0104 chemical sciences, 3. Good health, Crystallography, Heteronuclear molecule, Solid-state nuclear magnetic resonance, NMR pulse sequences
Abstract

International audience; Solid-state NMR can provide insight into protein structure and dynamics at the atomic level without inherent protein size limitations. However, a major hurdle to studying large proteins by solid-state NMR spectroscopy is related to spectral complexity and resonance overlap, which increase with molecular weight and severely hamper the assignment process. Here we show the use of two sets of experiments that expand the tool kit of 1H-detected assignment approaches, and which correlate a given amide pair either to the two adjacent CO-CA pairs (4D hCOCANH/hCOCAcoNH), or to the amide 1H of the neighboring residue (3D HcocaNH/HcacoNH, which can be extended to up to 5D). The experiments are based on efficient coherence transfers between backbone atoms using INEPT transfers between carbons and cross-polarization for heteronuclear transfers. We exemplify the usefulness of these experiments with applications to assemblies of deuterated, fully amide-protonated proteins from ca. 20 to 60 kDa monomer, at MAS frequencies from ca. 40 to 55 kHz. These experiments will also be applicable to protonated proteins at higher MAS frequencies. We report the resonance assignment of a domain within the 50.4 kDa bacteriophage T5 tube protein pb6, and compare these to solution-state NMR assignments of the isolated domain in solution. This comparison reveals contacts of this domain to the core of the polymeric tail tube assembly. Solid‐state NMR H‐N‐(C)‐H and H‐N‐C‐C 3D/4D correlation experiments for resonance assignment of large proteins. Available from: https://www.researchgate.net/publication/319020315_Solid-state_NMR_H-N-C-H_and_H-N-C-C_3D4D_correlation_experiments_for_resonance_assignment_of_large_proteins.

Published
2017
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12. Protein conformational dynamics studied by 15N and 1H R1ρ relaxation dispersion: application to wild-type and G53A ubiquitin crystals

Author

Diego F. Gauto, Vilius Kurauskas, Audrey Hessel, Paul Schanda, Rasmus Linser, Petra Rovó, Institut de biologie structurale (IBS - UMR 5075 ), Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Centre National de la Recherche Scientifique (CNRS), Department Chemie, Physikalische Chemie, Ludwig-Maximilians-Universität München (LMU), Centre National de la Recherche Scientifique (CNRS)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), and Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)

Subjects
0301 basic medicine, Models, Molecular, Nuclear and High Energy Physics, Protein Conformation, Field strength, Protein dynamics, Fast MAS, 010402 general chemistry, Crystallography, X-Ray, 01 natural sciences, Solid-state NMR, Article, 03 medical and health sciences, β-turn, Protein structure, Dispersion (optics), Instrumentation, Nuclear Magnetic Resonance, Biomolecular, Proton detection, Radiation, [SDV.BBM.BS]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Structural Biology [q-bio.BM], Chemistry, Ubiquitin, General Chemistry, Nuclear magnetic resonance spectroscopy, 0104 chemical sciences, Proton relaxation, Microsecond, Crystallography, 030104 developmental biology, Solid-state nuclear magnetic resonance, Chemical physics, Mutation, Relaxation (physics), Mutant Proteins, Spin relaxation
Abstract

International audience; Solid-state NMR spectroscopy can provide site-resolved information about protein dynamics over many time scales. Here we combine protein deuteration, fast magic-angle spinning (~45-60kHz) and proton detection to study dynamics of ubiquitin in microcrystals, and in particular a mutant in a region that undergoes microsecond motions in a β-turn region in the wild-type protein. We use (15)N R1ρ relaxation measurements as a function of the radio-frequency (RF) field strength, i.e. relaxation dispersion, to probe how the G53A mutation alters these dynamics. We report a population-inversion of conformational states: the conformation that in the wild-type protein is populated only sparsely becomes the predominant state. We furthermore explore the potential to use amide-(1)H R1ρ relaxation to obtain insight into dynamics. We show that while quantitative interpretation of (1)H relaxation remains beyond reach under the experimental conditions, due to coherent contributions to decay, one may extract qualitative information about flexibility.

Published
2017
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13. A ring-shaped conduit connects the mother cell and forespore during sporulation in Bacillus subtilis

Author

Emmanuelle Neumann, Paul Schanda, Xavier Henry, Yann Fichou, Vilius Kurauskas, Laure Bellard, Guy Schoehn, Christopher D. A. Rodrigues, David Z. Rudner, Cécile Morlot, Department of Microbiology and Immunobiology, Harvard Medical School [Boston] (HMS), Institut de biologie structurale (IBS - UMR 5075 ), Centre National de la Recherche Scientifique (CNRS)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA), Institute for Physical Chemistry II, Ruhr-Universität Bochum [Bochum], NIH [GM086466], Agence Nationale de la Recherche (ANR) [ANR-11-BSV8-005-01 PILIPATH], French Infrastructure for Integrated Structural Biology Initiative (FRISBI) Grant [ANR-10-INSB-05-02], Grenoble Alliance for Integrated Structural Cell Biology (GRAL) Grant [ANR-10-LABX-49-01], Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Centre National de la Recherche Scientifique (CNRS), Rodrigues, Christopher D. A., Henry, Xavier, and Morlot, Cécile

Subjects
0301 basic medicine, Models, Molecular, Magnetic Resonance Spectroscopy, sporulation, Operon, Protein domain, EscJ/PrgK/FliF, SigG, Bacillus subtilis, Biology, Type three secretion system, Microbiology, 03 medical and health sciences, Imaging, Three-Dimensional, Bacterial Proteins, Protein Domains, Secretion, Computer Simulation, Amino Acid Sequence, Peptide sequence, Spores, Bacterial, SpoIIIAG, type III secretion system, espace intercellulaire, Multidisciplinary, Sequence Homology, Amino Acid, [SDV.BBM.BS]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Structural Biology [q-bio.BM], Cryoelectron Microscopy, Biological Sciences, biology.organism_classification, Cell biology, 030104 developmental biology, Mutation, structure des protéines, Intermembrane space, Sporulation in Bacillus subtilis
Abstract

International audience; During spore formation in Bacillus subtilis a transenvelope complex is assembled across the double membrane that separates the mother cell and forespore. This complex (called the "A-Q complex") is required to maintain forespore development and is composed of proteins with remote homology to components of type II, III, and IV secretion systems found in Gram-negative bacteria. Here, we show that one of these proteins, SpoIIIAG, which has remote homology to ring-forming proteins found in type III secretion systems, assembles into an oligomeric ring in the periplasmic-like space between the two membranes. Three-dimensional reconstruction of images generated by cryo-electron microscopy indicates that the SpoIIIAG ring has a cup-and-saucer architecture with a 6-nm central pore. Structural modeling of SpoIIIAG generated a 24-member ring with dimensions similar to those of the EM-derived saucer. Point mutations in the predicted oligomeric interface disrupted ring formation in vitro and impaired forespore gene expression and efficient spore formation in vivo. Taken together, our data provide strong support for the model in which the A-Q transenvelope complex contains a conduit that connects the mother cell and forespore. We propose that a set of stacked rings spans the intermembrane space, as has been found for type III secretion systems.

Published
2016
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14. Cross-Correlated Relaxation of Dipolar Coupling and Chemical-Shift Anisotropy in Magic-Angle Spinning R 1ρ NMR Measurements: Application to Protein Backbone Dynamics Measurements

Author

Paul Schanda, Isabel Ayala, Emmanuelle Weber, Dominique Marion, Vilius Kurauskas, Audrey Hessel, Institut de biologie structurale (IBS - UMR 5075 ), Centre National de la Recherche Scientifique (CNRS)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA), ERC Stg ProtDyn2Function 2012-311318, European Project: 311318,EC:FP7:ERC,ERC-2012-StG_20111109,PROTDYN2FUNCTION(2013), Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), and Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Centre National de la Recherche Scientifique (CNRS)

Subjects
[PHYS.PHYS.PHYS-BIO-PH]Physics [physics]/Physics [physics]/Biological Physics [physics.bio-ph], Molecular Dynamics Simulation, 010402 general chemistry, 01 natural sciences, Molecular physics, Article, Spin–spin relaxation, numerical spin simulations, Proton spin crisis, Materials Chemistry, Magic angle spinning, rotating frame relaxation, Physical and Theoretical Chemistry, spin-diffusion, Anisotropy, Nuclear Magnetic Resonance, Biomolecular, J-doublet, Condensed matter physics, [SDV.BBM.BS]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Structural Biology [q-bio.BM], 010405 organic chemistry, Chemistry, Spin–lattice relaxation, Proteins, 0104 chemical sciences, Surfaces, Coatings and Films, Spin diffusion, Relaxation (physics), protein, Magnetic dipole–dipole interaction
Abstract

International audience; Transverse relaxation rate measurements in MAS solid-state NMR provide information about molecular motions occurring on nanoseconds-to-milliseconds (ns-ms) time scales. The measurement of heteronuclear (13C , 15N) relaxation rate constants in the presence of a spin-lock radio-frequency field (R1ρ relaxation) provides access to such motions, and an increasing number of studies involving R1ρ relaxation in proteins has been reported. However, two factors that influence the observed relaxation rate constants have so far been neglected, namely (i) the role of CSA/dipolar cross-correlated relaxation (CCR), and (ii) the impact of fast proton spin flips (i.e. proton spin diffusion and relaxation). We show that CSA/D CCR in R1ρ experiments is measurable, and that this cross-correlated relaxation rate constant depends on ns-ms motions, and can thus itself provide insight into dynamics. We find that proton spin-diffusion attenuates this cross-correlated relaxation, due to its decoupling effect on the doublet components. For measurements of dynamics, the use of R1ρ rate constants has practical advantages over the use of CCR rates, and the present manuscript reveals factors that have so far been disregarded and which are important for accurate measurements and interpretation.

Published
2016
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15. Sensitive proton-detected solid-state NMR spectroscopy of large proteins with selective CH3 labelling: application to the 50S ribosome subunit

Author

Paul Schanda, Pavel Macek, Vilius Kurauskas, Diego F. Gauto, Elodie Crublet, Rime Kerfah, Jérôme Boisbouvier, Institut de biologie structurale (IBS - UMR 5075 ), Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Centre National de la Recherche Scientifique (CNRS), European Research Council ERC-StG-2012-311318, European Research Council ERC-StG-2010-260887, Centre National de la Recherche Scientifique (CNRS)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), and Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)

Subjects
0301 basic medicine, Protein subunit, 010402 general chemistry, 01 natural sciences, Ribosome, Catalysis, Article, 03 medical and health sciences, [CHIM.ANAL]Chemical Sciences/Analytical chemistry, Labelling, Materials Chemistry, Spectroscopy, 50S, biology, [SDV.BBM.BS]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Structural Biology [q-bio.BM], Chemistry, Metals and Alloys, General Chemistry, Nuclear magnetic resonance spectroscopy, Thermus thermophilus, biology.organism_classification, 0104 chemical sciences, Surfaces, Coatings and Films, Electronic, Optical and Magnetic Materials, Crystallography, 030104 developmental biology, Solid-state nuclear magnetic resonance, Ceramics and Composites, Biophysics
Abstract

International audience; Solid-state NMR spectroscopy allows the characterization of structure, interactions and dynamics of insoluble and/or very large proteins. Sensitivity and resolution are often major challenges for obtaining atomic-resolution information, in particular for very large protein complexes. Here we show that the use of deuterated, specifically CH3-labelled proteins result in significant sensitivity gains compared to previously employed CHD2 labelling, while line widths only marginally increase. We apply this labelling strategy to a 468 kDa-large dodecameric aminopeptidase, TET2, and the 1.6 MDa-large 50S ribosome subunit of Thermus thermophilus.

Published
2016
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16. Comprehensive Fragment Screening of the SARS‐CoV‐2 Proteome Explores Novel Chemical Space for Drug Development

Author

Hannes Berg, Maria A. Wirtz Martin, Nadide Altincekic, Islam Alshamleh, Jasleen Kaur Bains, Julius Blechar, Betül Ceylan, Vanessa de Jesus, Karthikeyan Dhamotharan, Christin Fuks, Santosh L. Gande, Bruno Hargittay, Katharina F. Hohmann, Marie T. Hutchison, Sophie Marianne Korn, Robin Krishnathas, Felicitas Kutz, Verena Linhard, Tobias Matzel, Nathalie Meiser, Anna Niesteruk, Dennis J. Pyper, Linda Schulte, Sven Trucks, Kamal Azzaoui, Marcel J. J. Blommers, Yojana Gadiya, Reagon Karki, Andrea Zaliani, Philip Gribbon, Marcius da Silva Almeida, Cristiane Dinis Anobom, Anna L. Bula, Matthias Bütikofer, Ícaro Putinhon Caruso, Isabella Caterina Felli, Andrea T. Da Poian, Gisele Cardoso de Amorim, Nikolaos K. Fourkiotis, Angelo Gallo, Dhiman Ghosh, Francisco Gomes‐Neto, Oksana Gorbatyuk, Bing Hao, Vilius Kurauskas, Lauriane Lecoq, Yunfeng Li, Nathane Cunha Mebus‐Antunes, Miguel Mompeán, Thais Cristtina Neves‐Martins, Martí Ninot‐Pedrosa, Anderson S. Pinheiro, Letizia Pontoriero, Yulia Pustovalova, Roland Riek, Angus J. Robertson, Marie Jose Abi Saad, Miguel Á. Treviño, Aikaterini C. Tsika, Fabio C. L. Almeida, Ad Bax, Katherine Henzler‐Wildman, Jeffrey C. Hoch, Kristaps Jaudzems, Douglas V. Laurents, Julien Orts, Roberta Pierattelli, Georgios A. Spyroulias, Elke Duchardt‐Ferner, Jan Ferner, Boris Fürtig, Martin Hengesbach, Frank Löhr, Nusrat Qureshi, Christian Richter, Krishna Saxena, Andreas Schlundt, Sridhar Sreeramulu, Anna Wacker, Julia E. Weigand, Julia Wirmer‐Bartoschek, Jens Wöhnert, Harald Schwalbe, State of Hesse, German Research Foundation, European Commission, Ministero dell'Istruzione, dell'Università e della Ricerca, Agence Nationale de la Recherche (France), Centre National de la Recherche Scientifique (France), National Institutes of Health (US), National Science Foundation (US), Latvian Council of Science, Berg, Hannes, Wirtz Martin, Maria A., Altincekic, Nadide, Alshamleh, Islam, Dhamotharan, Karthikeyan, Marianne Korn, Sophie, Schulte, Linda, da Silva Almeida, Marcius, Caterina Felli, Isabella, Fourkiotis, Nikolaos K., Gallo, Angelo, Ninot-Pedrosa, Martí, Pontoriero, Letizia, Treviño, Miguel A., Tsika, Aikaterini C., Almeida, Fabio C.L., Bax, Ad, Henzler-Wildman, Katherine, Hoch, Jeffrey C., Jaudzems, Kristaps, Laurents, D.V., Ferner, Jan, Hengesbach, Martin, Löhr, Frank, Qureshi, Nusrat, Richter, Christian, Schlundt, Andreas, Weigand, Julia E., Wirmer-Bartoschek, Julia, Schwalbe, Harald, and Publica

Subjects
Proteome, SARS-CoV-2, Protein, COVID19-NMR, General Medicine, General Chemistry, Ligands, NMR Spectroscopy, Catalysis, COVID-19 Drug Treatment, Fragment Screening, Drug Design, Drug Discovery, Humans, COVID19 * drug discovery * fragment screening * NMR spectroscopy * SARS-CoV-2
Abstract

12 pags., 4 figs., 3 tabs., SARS-CoV-2 (SCoV2) and its variants of concern pose serious challenges to the public health. The variants increased challenges to vaccines, thus necessitating for development of new intervention strategies including anti-virals. Within the international Covid19-NMR consortium, we have identified binders targeting the RNA genome of SCoV2. We established protocols for the production and NMR characterization of more than 80 % of all SCoV2 proteins. Here, we performed an NMR screening using a fragment library for binding to 25 SCoV2 proteins and identified hits also against previously unexplored SCoV2 proteins. Computational mapping was used to predict binding sites and identify functional moieties (chemotypes) of the ligands occupying these pockets. Striking consensus was observed between NMR-detected binding sites of the main protease and the computational procedure. Our investigation provides novel structural and chemical space for structure-based drug design against the SCoV2 proteome., Work at BMRZ is supported by the state of Hesse. Work in Covid19-NMR was supported by the Goethe Corona Funds, by the IWBEFRE-program 20007375 of state of Hesse, the DFG through CRC902: “Molecular Principles of RNA-based regulation.” and through infrastructure funds (project numbers: 277478796, 277479031, 392682309, 452632086, 70653611) and by European Union’s Horizon 2020 research and innovation program iNEXT-discovery under grant agreement No 871037. BY-COVID receives funding from the European Union’s Horizon Europe Research and Innovation Programme under grant agreement number 101046203. “INSPIRED” (MIS 5002550) project, implemented under the Action “Reinforcement of the Research and Innovation Infrastructure,” funded by the Operational Program “Competitiveness, Entrepreneurship and Innovation” (NSRF 2014–2020) and co-financed by Greece and the EU (European Regional Development Fund) and the FP7 REGPOT CT-2011-285950—“SEE-DRUG” project (purchase of UPAT’s 700 MHz NMR equipment). The support of the CERM/CIRMMP center of Instruct-ERIC is gratefully acknowledged. This work has been funded in part by a grant of the Italian Ministry of University and Research (FISR2020IP_02112, ID-COVID) and by Fondazione CR Firenze. A.S. is supported by the Deutsche Forschungsgemeinschaft [SFB902/B16, SCHL2062/2-1] and the Johanna Quandt Young Academy at Goethe [2019/AS01]. M.H. and C.F. thank SFB902 and the Stiftung Polytechnische Gesellschaft for the Scholarship. L.L. work was supported by the French National Research Agency (ANR, NMR-SCoV2-ORF8), the Fondation de la Recherche Médicale (FRM, NMR-SCoV2-ORF8), FINOVI and the IR-RMN-THC Fr3050 CNRS. Work at UConn Health was supported by grants from the US National Institutes of Health (R01 GM135592 to B.H., P41 GM111135 and R01 GM123249 to J.C.H.) and the US National Science Foundation (DBI 2030601 to J.C.H.). Latvian Council of Science Grant No. VPP-COVID-2020/1-0014. National Science Foundation EAGER MCB-2031269. This work was supported by the grant Krebsliga KFS-4903-08-2019 and SNF-311030_192646 to J.O. P.G. (ITMP) The EOSC Future project is co-funded by the European Union Horizon Programme call INFRAEOSC-03-2020—Grant Agreement Number 101017536. Open Access funding enabled and organized by Projekt DEAL

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