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1. Flexible-to-rigid transition is central for substrate transport in the ABC transporter BmrA from Bacillus subtilis

Author

Thomas Wiegand, Claire Chuilon, Jean-Michel Jault, Anja Böckmann, Britta Kunert, Beat H. Meier, Stéphanie Ravaud, Cédric Orelle, Denis Lacabanne, Lauriane Lecoq, Microbiologie moléculaire et biochimie structurale / Molecular Microbiology and Structural Biochemistry (MMSB), Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-Centre National de la Recherche Scientifique (CNRS), Department of Chemistry, Purdue University [West Lafayette], 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), Physical Chemistry [ETH Zürich], Department of Chemistry and Applied Biosciences [ETH Zürich] (D-CHAB), Eidgenössische Technische Hochschule - Swiss Federal Institute of Technology [Zürich] (ETH Zürich)- Eidgenössische Technische Hochschule - Swiss Federal Institute of Technology [Zürich] (ETH Zürich), France: ANR (voir liste ci-dessous) et TGIR-RMN-THC Fr3050, Suisse (Grant 200020_159707), 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
Magnetic Resonance Spectroscopy, Protein Conformation, [SDV]Life Sciences [q-bio], ATPase, Mutant, Medicine (miscellaneous), ATP-binding cassette transporter, Bacillus subtilis, 010402 general chemistry, Solid-state NMR, 01 natural sciences, Article, General Biochemistry, Genetics and Molecular Biology, Cell membrane, 03 medical and health sciences, Adenosine Triphosphate, Bacterial Proteins, [CHIM.ANAL]Chemical Sciences/Analytical chemistry, medicine, [SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology, Magnesium, lcsh:QH301-705.5, ComputingMilieux_MISCELLANEOUS, 030304 developmental biology, Adenosine Triphosphatases, 0303 health sciences, Binding Sites, Bacteria, biology, [SDV.BBM.BS]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Structural Biology [q-bio.BM], Chemistry, Hydrolysis, Cell Membrane, Substrate (chemistry), Transporter, biology.organism_classification, Drug Resistance, Multiple, 0104 chemical sciences, [SDV.BBM.BS]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biomolecules [q-bio.BM], medicine.anatomical_structure, lcsh:Biology (General), Structural biology, biology.protein, Biophysics, ATP-Binding Cassette Transporters, General Agricultural and Biological Sciences
Abstract

ATP-binding-cassette (ABC) transporters are molecular pumps that translocate molecules across the cell membrane by switching between inward-facing and outward-facing states. To obtain a detailed understanding of their mechanism remains a challenge to structural biology, as these proteins are notoriously difficult to study at the molecular level in their active, membrane-inserted form. Here we use solid-state NMR to investigate the multidrug ABC transporter BmrA reconstituted in lipids. We identify the chemical-shift differences between the inward-facing, and outward-facing state induced by ATP:Mg2+:Vi addition. Analysis of an X-loop mutant, for which we show that ATPase and transport activities are uncoupled, reveals an incomplete transition to the outward-facing state upon ATP:Mg2+:Vi addition, notably lacking the decrease in dynamics of a defined set of residues observed in wild-type BmrA. This suggests that this stiffening is required for an efficient transmission of the conformational changes to allow proper transport of substrate by the pump., Communications Biology, 2 (1), ISSN:2399-3642

Published
2019
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2. Gradient reconstitution of membrane proteins for solid-state NMR studies

Author

Lauriane Lecoq, Vlastimil Jirasko, Clément Danis, Beat H. Meier, Marie-Laure Fogeron, Vincent Chaptal, Cédric Orelle, Alons Lends, Anja Böckmann, Claire Chuilon, Pierre Falson, Britta Kunert, Jean-Michel Jault, Denis Lacabanne, Microbiologie moléculaire et biochimie structurale / Molecular Microbiology and Structural Biochemistry (MMSB), Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-Centre National de la Recherche Scientifique (CNRS), Unité de Glycobiologie Structurale et Fonctionnelle UMR 8576 (UGSF), Institut National de la Recherche Agronomique (INRA)-Université de Lille-Centre National de la Recherche Scientifique (CNRS), Physical Chemistry [ETH Zürich], Department of Chemistry and Applied Biosciences [ETH Zürich] (D-CHAB), Eidgenössische Technische Hochschule - Swiss Federal Institute of Technology [Zürich] (ETH Zürich)- Eidgenössische Technische Hochschule - Swiss Federal Institute of Technology [Zürich] (ETH Zürich), Institut de biologie et chimie des protéines [Lyon] (IBCP), Centre National de la Recherche Scientifique (CNRS)-Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon, Laboratoire d'enzymologie et biochimie structurales (LEBS), Centre National de la Recherche Scientifique (CNRS), Laboratory of Physical Chemistry [ETH Zürich] (LPC), ANR-11-LABX-0048,ECOFECT,Dynamiques eco-évolutives des maladies infectieuses(2011), ANR-11-IDEX-0007,Avenir L.S.E.,PROJET AVENIR LYON SAINT-ETIENNE(2011), Unité de Glycobiologie Structurale et Fonctionnelle - UMR 8576 (UGSF), Université de Lille-Centre National de la Recherche Scientifique (CNRS)-Institut National de la Recherche Agronomique (INRA), Eidgenössische Technische Hochschule - Swiss Federal Institute of Technology in Zürich [Zürich] (ETH Zürich)-Eidgenössische Technische Hochschule - Swiss Federal Institute of Technology in Zürich [Zürich] (ETH Zürich), and Université de Lille-Centre National de la Recherche Scientifique (CNRS)

Subjects
0301 basic medicine, [SDV]Life Sciences [q-bio], Analytical chemistry, ATP-binding cassette transporter, Solid-state NMR, Biochemistry, Mass Spectrometry, 03 medical and health sciences, Bacterial Proteins, ABC-transporter, Sample preparation, [SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology, Nuclear Magnetic Resonance, Biomolecular, Spectroscopy, ComputingMilieux_MISCELLANEOUS, Membrane proteins reconstitution, BmrA, chemistry.chemical_classification, Chromatography, Cyclodextrin, Chemistry, Membrane Proteins, Membrane Transport Proteins, Active protein, Lipids, NMR spectra database, [SDV.BBM.BP]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biophysics, 030104 developmental biology, Solid-state nuclear magnetic resonance, Membrane protein, Dialysis (biochemistry)
Abstract

Journal of Biomolecular NMR, 69 (2), ISSN:0925-2738, ISSN:1573-5001

Published
2017
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3. Sample Preparation for Membrane Protein Structural Studies by Solid-State NMR

Author

Denis, Lacabanne, Britta, Kunert, Carole, Gardiennet, Beat H, Meier, and Anja, Bo Ckmann

Subjects
Models, Molecular, Bacterial Proteins, Protein Conformation, Membrane Proteins, Nuclear Magnetic Resonance, Biomolecular, Bacillus subtilis
Abstract

Conformational studies of membrane proteins remain a challenge in the field of structural biology, and in particular the investigation of the proteins in a native-like lipid environment. Solid-state NMR presents a valuable opportunity for this, and we present here three critical steps in the solid-state NMR sample preparation, i.e., membrane reconstitution of the protein in native lipids, rotor filling, and sample quality assessment, at the example of the Bacillus subtilis ATP-binding cassette transporter BmrA.

Published
2017

4. Sample Preparation for Membrane Protein Structural Studies by Solid-State NMR

Author

Carole Gardiennet, Beat H. Meier, Britta Kunert, Anja Bo¨ckmann, Denis Lacabanne, Microbiologie moléculaire et biochimie structurale / Molecular Microbiology and Structural Biochemistry (MMSB), Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-Centre National de la Recherche Scientifique (CNRS), Cristallographie, Résonance Magnétique et Modélisations (CRM2), Université de Lorraine (UL)-Centre National de la Recherche Scientifique (CNRS), Physical Chemistry [ETH Zürich], Department of Chemistry and Applied Biosciences [ETH Zürich] (D-CHAB), Eidgenössische Technische Hochschule - Swiss Federal Institute of Technology [Zürich] (ETH Zürich)- Eidgenössische Technische Hochschule - Swiss Federal Institute of Technology [Zürich] (ETH Zürich), and Lacapère, Jean-Jacques

Subjects
0301 basic medicine, [SDV.BBM.BS]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Structural Biology [q-bio.BM], biology, Chemistry, [SDV]Life Sciences [q-bio], Bacillus subtilis, 010402 general chemistry, biology.organism_classification, 01 natural sciences, 0104 chemical sciences, Sample quality, 03 medical and health sciences, 030104 developmental biology, Solid-state nuclear magnetic resonance, Membrane protein, Structural biology, Membrane reconstitution, Biophysics, [SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology, Sample preparation, ComputingMilieux_MISCELLANEOUS
Abstract

Conformational studies of membrane proteins remain a challenge in the field of structural biology, and in particular the investigation of the proteins in a native-like lipid environment. Solid-state NMR presents a valuable opportunity for this, and we present here three critical steps in the solid-state NMR sample preparation, i.e., membrane reconstitution of the protein in native lipids, rotor filling, and sample quality assessment, at the example of the Bacillus subtilis ATP-binding cassette transporter BmrA.

Published
2017
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5. Variability and conservation of structural domains in divide-and-conquer approaches

Author

Anja Böckmann, Britta Kunert, Thomas Wiegand, Riccardo Cadalbert, Laurent Terradot, Beat H. Meier, Carole Gardiennet, Denis Lacabanne, Physical Chemistry [ETH Zürich], Department of Chemistry and Applied Biosciences [ETH Zürich] (D-CHAB), Eidgenössische Technische Hochschule - Swiss Federal Institute of Technology [Zürich] (ETH Zürich)- Eidgenössische Technische Hochschule - Swiss Federal Institute of Technology [Zürich] (ETH Zürich), Cristallographie, Résonance Magnétique et Modélisations (CRM2), Université de Lorraine (UL)-Centre National de la Recherche Scientifique (CNRS), Microbiologie moléculaire et biochimie structurale / Molecular Microbiology and Structural Biochemistry (MMSB), Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-Centre National de la Recherche Scientifique (CNRS), Institut de biologie et chimie des protéines [Lyon] (IBCP), Laboratory of Physical Chemistry [ETH Zürich] (LPC), and Centre National de la Recherche Scientifique (CNRS)-Université de Lorraine (UL)

Subjects
0301 basic medicine, Divide and conquer algorithms, Models, Molecular, Magnetic Resonance Spectroscopy, Protein Conformation, [SDV]Life Sciences [q-bio], Protein domain, DnaB, Context (language use), Computational biology, Domains, Protein, Solid-state NMR, Biochemistry, Domain (software engineering), Evolution, Molecular, 03 medical and health sciences, Protein structure, Protein Domains, [CHIM]Chemical Sciences, Invariant (mathematics), Nuclear Magnetic Resonance, Biomolecular, Spectroscopy, dnaB helicase, ComputingMilieux_MISCELLANEOUS, [SDV.BBM.BS]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Structural Biology [q-bio.BM], Chemistry, Genetic Variation, Proteins, Crystallography, 030104 developmental biology, Superstructure (condensed matter), DnaB Helicases
Abstract

Journal of Biomolecular NMR, 65 (2), ISSN:0925-2738, ISSN:1573-5001

Published
2016
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6. Solid-state NMR sequential assignments of the N-terminal domain of HpDnaB helicase

Author

Anja Böckmann, Denis Lacabanne, Thomas Wiegand, Britta Kunert, Laurent Terradot, Alexandre Bazin, Carole Gardiennet, Francesco Ravotti, Peter Güntert, Beat H. Meier, Riccardo Cadalbert, Physical Chemistry [ETH Zürich], Department of Chemistry and Applied Biosciences [ETH Zürich] (D-CHAB), Eidgenössische Technische Hochschule - Swiss Federal Institute of Technology [Zürich] (ETH Zürich)- Eidgenössische Technische Hochschule - Swiss Federal Institute of Technology [Zürich] (ETH Zürich), Cristallographie, Résonance Magnétique et Modélisations (CRM2), Université de Lorraine (UL)-Centre National de la Recherche Scientifique (CNRS), Microbiologie moléculaire et biochimie structurale / Molecular Microbiology and Structural Biochemistry (MMSB), Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-Centre National de la Recherche Scientifique (CNRS), Laboratorium für Physikalische Chemie (ETH-LPC), Eidgenössische Technische Hochschule - Swiss Federal Institute of Technology [Zürich] (ETH Zürich), Institut de biologie et chimie des protéines [Lyon] (IBCP), Institute of Biophysical Chemistry and Frankfurt Institute for Advanced Studies, Goethe-University Frankfurt am Main, and Laboratory of Physical Chemistry [ETH Zürich] (LPC)

Subjects
0301 basic medicine, Assignments, HpDnaB, Secondary chemical shifts, Solid-state NMR, ssFLYA, [SDV]Life Sciences [q-bio], Protein domain, 010402 general chemistry, 01 natural sciences, Biochemistry, Protein Structure, Secondary, 03 medical and health sciences, Nuclear magnetic resonance, Protein Domains, Structural Biology, [CHIM]Chemical Sciences, Amino Acid Sequence, Protein secondary structure, Nuclear Magnetic Resonance, Biomolecular, dnaB helicase, ComputingMilieux_MISCELLANEOUS, biology, Helicobacter pylori, [SDV.BBM.BS]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Structural Biology [q-bio.BM], Chemistry, Chemical shift, Helicase, Resonance, 0104 chemical sciences, 3. Good health, Crystallography, 030104 developmental biology, Solid-state nuclear magnetic resonance, Domain (ring theory), biology.protein, DnaB Helicases, Software
Abstract

We present solid-state NMR assignments of the N-terminal domain of the DnaB helicase from Helicobacter pylori (153 residues) in its microcrystalline form. We use a sequential resonance assignment strategy based on three-dimensional NMR experiments. The resonance assignments obtained are compared with automated resonance assignments computed with the ssFLYA algorithm. An analysis of the (13)C secondary chemical shifts determines the position of the secondary structure elements in this α-helical protein.

Published
2016
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8. Solid-state magic-angle spinning NMR of membrane proteins and protein–ligand interactions

Author

Hartmut Oschkinat, W. Trent Franks, Britta Kunert, Barth-Jan van Rossum, and Arne H. Linden

Subjects
Histology, Materials science, Solid-state, Membrane Proteins, Nanotechnology, Cell Biology, General Medicine, Ligands, Biological materials, Protein Structure, Tertiary, Pathology and Forensic Medicine, Membrane protein, Structural biology, Electron tomography, Atomic resolution, Magic angle spinning, Animals, Humans, Nuclear Magnetic Resonance, Biomolecular, Protein Binding, Protein ligand
Abstract

Structural biology is developing into a universal tool for visualizing biological processes in space and time at atomic resolution. The field has been built by established methodology like X-ray crystallography, electron microscopy and solution NMR and is now incorporating new techniques, such as small-angle X-ray scattering, electron tomography, magic-angle-spinning solid-state NMR and femtosecond X-ray protein nanocrystallography. These new techniques all seek to investigate non-crystalline, native-like biological material. Solid-state NMR is a relatively young technique that has just proven its capabilities for de novo structure determination of model proteins. Further developments promise great potential for investigations on functional biological systems such as membrane-integrated receptors and channels, and macromolecular complexes attached to cytoskeletal proteins. Here, we review the development and applications of solid-state NMR from the first proof-of-principle investigations to mature structure determination projects, including membrane proteins. We describe the development of the methodology by looking at examples in detail and provide an outlook towards future ‘big’ projects.

Published
2012
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9. Mapping Putative Contact Sites Between Subunits in a Bacterial ATP-binding Cassette (ABC) Transporter by Synthetic Peptide Libraries

Author

Britta Kunert, Heidi Landmesser, Susanne Berendt, Erwin Schneider, Viola Eckey, Rudolf Volkmer, Michael Portwich, and Bettina Blüschke

Subjects
Salmonella typhimurium, Monosaccharide Transport Proteins, Protein subunit, Molecular Sequence Data, Protein Array Analysis, ATP-binding cassette transporter, Peptide, Biology, Protein Structure, Secondary, Protein–protein interaction, Bacterial Proteins, Peptide Library, Structural Biology, Protein Interaction Mapping, Point Mutation, Amino Acid Sequence, Binding site, Maltose, Molecular Biology, Maltose transport, chemistry.chemical_classification, Periplasmic space, Protein Subunits, Biochemistry, chemistry, Biotinylation, ATP-Binding Cassette Transporters, Peptides, Sequence Alignment, Protein Binding
Abstract

The maltose ATP-binding cassette transporter of Salmonella typhimurium is composed of the soluble periplasmic receptor, MalE, and a membrane-associated complex comprising one copy each of the pore-forming hydrophobic subunits, MalF and MalG, and of a homodimer of the ATP-hydrolyzing subunit, MalK. During the transport process the subunits are thought to undergo conformational changes that might transiently alter molecular contacts between MalFG and MalK2. In order to map sites of subunit–subunit interactions we have used a comprehensive peptide mapping approach comprising large-scale microsynthesis of labelled probes and array techniques. In particular, we screened the binding of (i) MalFG-derived soluble biotinylated peptides to immobilized MalK, and (ii) radiolabelled MalK to MalFG-derived cellulose membrane-bound peptides. The first approach identified seven peptides (10mers) each of MalF and MalG that specifically bound to MalK. The peptides were localized to TMDs 3 and 6, periplasmic loop P4 and cytoplasmic loops C2 and C3 of MalF, while MalG-derived peptides localized to the N terminus, TMDs 4–6, periplasmic loop P1 and cytoplasmic loop C2. Peptides from C3 and C2, respectively, of MalF and MalG partially encompass the conserved EAA-motif, known to be crucial for interaction with MalK. These results were basically confirmed by screening MalFG-derived peptide arrays consisting of 16mers or 31mers with radiolabelled MalK. This approach also allowed us to perform complete substitutional analyses of peptides in question. The results led to the construction of MalFG variants that were subsequently analyzed for functional consequences in vivo. Growth experiments revealed that most of the mutations had no phenotype, suggesting that the mutated residues themselves are not critical but part of a discontinuous binding site. However, two novel mutations affecting residues from the EAA motifs of MalF (Ile417Glu) and MalG (Phe203Gln/Asn), respectively, displayed severe growth defects, indicating their functional importance. Together, these experimental outcomes identify specific molecular contacts made between MalK and MalFG that extend beyond the well-characterized EAA motif.

Published
2007
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10. Solid-state NMR chemical-shift perturbations indicate domain reorientation of the DnaG primase in the primosome of Helicobacter pylori

Author

Anja Böckmann, Britta Kunert, Alexandre Bazin, Denis Lacabanne, Laurent Terradot, Irina Gutsche, Riccardo Cadalbert, Thomas Wiegand, Carole Gardiennet, Beat H. Meier, Cristallographie, Résonance Magnétique et Modélisations (CRM2), Université de Lorraine (UL)-Centre National de la Recherche Scientifique (CNRS), Microbiologie moléculaire et biochimie structurale / Molecular Microbiology and Structural Biochemistry (MMSB), Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-Centre National de la Recherche Scientifique (CNRS), Physical Chemistry [ETH Zürich], Department of Chemistry and Applied Biosciences [ETH Zürich] (D-CHAB), Eidgenössische Technische Hochschule - Swiss Federal Institute of Technology [Zürich] (ETH Zürich)- Eidgenössische Technische Hochschule - Swiss Federal Institute of Technology [Zürich] (ETH Zürich), 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), TGIR-RMN-THC FR3050, ANR-12-BS08-0013,XLproteinSSNMR,Etudes structurales du 75 kDa prion Sup35 : Vers la RMN du solide des protéines extra-larges(2012), 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, Protein complexes, DnaB, Bazin, DnaG, 01 natural sciences, Biochemistry, Solid-state NMR, Primosome, Sediments, law.invention, law, MESH: Nuclear Magnetic Resonance, Biomolecular, Crystallization, Spectroscopy, Irina, [SDV.BBM.BS]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Structural Biology [q-bio.BM], Chemistry, Wiegand, Riccardo, 3. Good health, Solid-state nuclear magnetic resonance, MESH: DNA Primase, Denis, MESH: Protein Domains, Primase, Böckmann, Anja, Carole, Protein domain, Terradot, Laurent, DNA Primase, 010402 general chemistry, Domain (mathematical analysis), 03 medical and health sciences, Protein Domains, Alexandre, Beat H, Nuclear Magnetic Resonance, Biomolecular, dnaB helicase, Gutsche, Helicobacter pylori, Gardiennet, Cadalbert, 0104 chemical sciences, Meier, Crystallography, Thomas, 030104 developmental biology, Lacabanne, Biophysics, MESH: Helicobacter pylori, Kunert, Britta
Abstract

International audience; We here investigate the interactions between the DnaB helicase and the C-terminal domain of the corresponding DnaG primase of Helicobacter pylori using solid-state NMR. The difficult crystallization of this 387 kDa complex, where the two proteins interact in a six to three ratio, is circumvented by simple co-sedimentation of the two proteins directly into the MAS-NMR rotor. While the amount of information that can be extracted from such a large protein is still limited, we can assign a number of amino-acid residues experiencing significant chemical-shift perturbations upon helicase-primase complex formation. The location of these residues is used as a guide to model the interaction interface between the two proteins in the complex. Chemical-shift perturbations also reveal changes at the interaction interfaces of the hexameric HpDnaB assembly on HpDnaG binding. A structural model of the complex that explains the experimental findings is obtained.

Published
2015
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11. Efficient and stable reconstitution of the ABC transporter BmrA for solid-state NMR studies

Author

Anja Böckmann, Britta Kunert, Denis Lacabanne, Beat H. Meier, François Penin, Carole Gardiennet, Pierre Falson, Daniel Calles-Garcia, Jean-Michel Jault, 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 biologie et chimie des protéines [Lyon] (IBCP), Université Claude Bernard Lyon 1 (UCBL), 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), Université de Lyon-Université de Lyon-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS), Centre National de la Recherche Scientifique (CNRS)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), and Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)

Subjects
[SDV]Life Sciences [q-bio], ATP-binding cassette transporter, Bacillus subtilis, Biochemistry, Genetics and Molecular Biology (miscellaneous), Biochemistry, ABC-transporter, Sample preparation, Molecular Biosciences, Original Research Article, isotope labeling, lcsh:QH301-705.5, Molecular Biology, Protein secondary structure, biology, Chemistry, B. subtilis lipids, Transporter, biology.organism_classification, 3. Good health, Membrane, BmrA, lcsh:Biology (General), Solid-state nuclear magnetic resonance, Membrane protein, Solid-state NMR, Isotope labeling, Biophysics, solid-state NMR
Abstract

We present solid-state NMR sample preparation and first 2D spectra of the Bacillus subtilis ATP-binding cassette (ABC) transporter BmrA, a membrane protein involved in multidrug resistance. The homodimeric 130-kDa protein is a challenge for structural characterization due to its membrane-bound nature, size, inherent flexibility and insolubility. We show that reconstitution of this protein in lipids from Bacillus subtilis at a lipid-protein ratio of 0.5 w/w allows for optimal protein insertion in lipid membranes with respect to two central NMR requirements, high signal-to-noise in the spectra and sample stability over a time period of months. The obtained spectra point to a well-folded protein and a highly homogenous preparation, as witnessed by the narrow resonance lines and the signal dispersion typical for the expected secondary structure distribution of BmrA. This opens the way for studies of the different conformational states of the transporter in the export cycle, as well as on interactions with substrates, via chemical-shift fingerprints and sequential resonance assignments. ISSN:2296-889X

Published
2014
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12. Quadruple-resonance magic-angle spinning NMR spectroscopy of deuterated solid proteins

Author

Andrew J. Nieuwkoop, Niels Chr. Nielsen, Frank Engelke, Hartmut Oschkinat, Anja Voreck, Sebastian Wegner, Priyanga Bandara, Britta Kunert, and Ümit Akbey

Subjects
Deuterium NMR, Carbon-13 NMR satellite, Chemistry, Protein Conformation, General Chemistry, Fluorine-19 NMR, Nuclear magnetic resonance spectroscopy, General Medicine, Deuterium, Catalysis, Geobacillus stearothermophilus, Crystallography, Structural biology, Bacterial Proteins, Magic angle spinning, Transverse relaxation-optimized spectroscopy, Lipid bilayer, Nuclear Magnetic Resonance, Biomolecular
Abstract

(1)H-detected magic-angle spinning NMR experiments facilitate structural biology of solid proteins, which requires using deuterated proteins. However, often amide protons cannot be back-exchanged sufficiently, because of a possible lack of solvent exposure. For such systems, using (2)H excitation instead of (1)H excitation can be beneficial because of the larger abundance and shorter longitudinal relaxation time, T1, of deuterium. A new structure determination approach, "quadruple-resonance NMR spectroscopy", is presented which relies on an efficient (2)H-excitation and (2)H-(13)C cross-polarization (CP) step, combined with (1)H detection. We show that by using (2)H-excited experiments better sensitivity is possible on an SH3 sample recrystallized from 30 % H2O. For a membrane protein, the ABC transporter ArtMP in native lipid bilayers, different sets of signals can be observed from different initial polarization pathways, which can be evaluated further to extract structural properties.

Published
2013
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13. A sedimented sample of a 59 kDa dodecameric helicase yields high-resolution solid-state NMR spectra

Author

Laurent Terradot, Andreas Hunkeler, Anne K. Schütz, Carole Gardiennet, Beat H. Meier, Anja Böckmann, Britta Kunert, Institut de biologie et chimie des protéines [Lyon] (IBCP), Université Claude Bernard Lyon 1 (UCBL), and Université de Lyon-Université de Lyon-Centre National de la Recherche Scientifique (CNRS)

Subjects
[SDV]Life Sciences [q-bio], Analytical chemistry, 010402 general chemistry, 01 natural sciences, Catalysis, Spectral line, 03 medical and health sciences, Preparative Centrifugation, MESH: Nuclear Magnetic Resonance, Biomolecular, Centrifugation, Sample preparation, Nuclear Magnetic Resonance, Biomolecular, dnaB helicase, 030304 developmental biology, MESH: DnaB Helicases, 0303 health sciences, biology, Chemistry, Helicase, General Chemistry, 0104 chemical sciences, Crystallography, Solid-state nuclear magnetic resonance, Yield (chemistry), biology.protein, DnaB Helicases
Abstract

International audience; Crystal clear: Preparing solid-state NMR samples that yield high-resolution spectra displaying high sensitivity is time-consuming and complicated. A sample of the 59 kDa protein DnaB, prepared simply by preparative centrifugation, provides spectra that are as good as the ones from carefully grown microcrystals.

Published
2012
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