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1. Molecular recognition of Escherichia coli R1-type core lipooligosaccharide by DC-SIGN

Author: Ferran Nieto-Fabregat, Angela Marseglia, Michel Thépaut, Jean-Philippe Kleman, Massilia Abbas, Aline Le Roy, Christine Ebel, Meriem Maalej, Jean-Pierre Simorre, Cedric Laguri, Antonio Molinaro, Alba Silipo, Franck Fieschi, and Roberta Marchetti
Subjects: Microbiology, Structural biology, Science
Abstract: Summary: Due to their ability to recognize carbohydrate structures, lectins emerged as potential receptors for bacterial lipopolysaccharides (LPS). Despite growing interest in investigating the association between host receptor lectins and exogenous glycan ligands, the molecular mechanisms underlying bacterial recognition by human lectins are still not fully understood. We contributed to fill this gap by unveiling the molecular basis of the interaction between the lipooligosaccharide of Escherichia coli and the dendritic cell-specific intracellular adhesion molecules (ICAM)-3 grabbing non-integrin (DC-SIGN). Specifically, a combination of different techniques, including fluorescence microscopy, surface plasmon resonance, NMR spectroscopy, and computational studies, demonstrated that DC-SIGN binds to the purified deacylated R1 lipooligosaccharide mainly through the recognition of its outer core pentasaccharide, which acts as a crosslinker between two different tetrameric units of DC-SIGN. Our results contribute to a better understanding of DC-SIGN-LPS interaction and may support the development of pharmacological and immunostimulatory strategies for bacterial infections, prevention, and therapy.
Published: 2024
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2. A multilaboratory comparison of calibration accuracy and the performance of external references in analytical ultracentrifugation.

Author: Huaying Zhao, Rodolfo Ghirlando, Carlos Alfonso, Fumio Arisaka, Ilan Attali, David L Bain, Marina M Bakhtina, Donald F Becker, Gregory J Bedwell, Ahmet Bekdemir, Tabot M D Besong, Catherine Birck, Chad A Brautigam, William Brennerman, Olwyn Byron, Agnieszka Bzowska, Jonathan B Chaires, Catherine T Chaton, Helmut Cölfen, Keith D Connaghan, Kimberly A Crowley, Ute Curth, Tina Daviter, William L Dean, Ana I Díez, Christine Ebel, Debra M Eckert, Leslie E Eisele, Edward Eisenstein, Patrick England, Carlos Escalante, Jeffrey A Fagan, Robert Fairman, Ron M Finn, Wolfgang Fischle, José García de la Torre, Jayesh Gor, Henning Gustafsson, Damien Hall, Stephen E Harding, José G Hernández Cifre, Andrew B Herr, Elizabeth E Howell, Richard S Isaac, Shu-Chuan Jao, Davis Jose, Soon-Jong Kim, Bashkim Kokona, Jack A Kornblatt, Dalibor Kosek, Elena Krayukhina, Daniel Krzizike, Eric A Kusznir, Hyewon Kwon, Adam Larson, Thomas M Laue, Aline Le Roy, Andrew P Leech, Hauke Lilie, Karolin Luger, Juan R Luque-Ortega, Jia Ma, Carrie A May, Ernest L Maynard, Anna Modrak-Wojcik, Yee-Foong Mok, Norbert Mücke, Luitgard Nagel-Steger, Geeta J Narlikar, Masanori Noda, Amanda Nourse, Tomas Obsil, Chad K Park, Jin-Ku Park, Peter D Pawelek, Erby E Perdue, Stephen J Perkins, Matthew A Perugini, Craig L Peterson, Martin G Peverelli, Grzegorz Piszczek, Gali Prag, Peter E Prevelige, Bertrand D E Raynal, Lenka Rezabkova, Klaus Richter, Alison E Ringel, Rose Rosenberg, Arthur J Rowe, Arne C Rufer, David J Scott, Javier G Seravalli, Alexandra S Solovyova, Renjie Song, David Staunton, Caitlin Stoddard, Katherine Stott, Holger M Strauss, Werner W Streicher, John P Sumida, Sarah G Swygert, Roman H Szczepanowski, Ingrid Tessmer, Ronald T Toth, Ashutosh Tripathy, Susumu Uchiyama, Stephan F W Uebel, Satoru Unzai, Anna Vitlin Gruber, Peter H von Hippel, Christine Wandrey, Szu-Huan Wang, Steven E Weitzel, Beata Wielgus-Kutrowska, Cynthia Wolberger, Martin Wolff, Edward Wright, Yu-Sung Wu, Jacinta M Wubben, and Peter Schuck
Subjects: Medicine, Science
Abstract: Analytical ultracentrifugation (AUC) is a first principles based method to determine absolute sedimentation coefficients and buoyant molar masses of macromolecules and their complexes, reporting on their size and shape in free solution. The purpose of this multi-laboratory study was to establish the precision and accuracy of basic data dimensions in AUC and validate previously proposed calibration techniques. Three kits of AUC cell assemblies containing radial and temperature calibration tools and a bovine serum albumin (BSA) reference sample were shared among 67 laboratories, generating 129 comprehensive data sets. These allowed for an assessment of many parameters of instrument performance, including accuracy of the reported scan time after the start of centrifugation, the accuracy of the temperature calibration, and the accuracy of the radial magnification. The range of sedimentation coefficients obtained for BSA monomer in different instruments and using different optical systems was from 3.655 S to 4.949 S, with a mean and standard deviation of (4.304 ± 0.188) S (4.4%). After the combined application of correction factors derived from the external calibration references for elapsed time, scan velocity, temperature, and radial magnification, the range of s-values was reduced 7-fold with a mean of 4.325 S and a 6-fold reduced standard deviation of ± 0.030 S (0.7%). In addition, the large data set provided an opportunity to determine the instrument-to-instrument variation of the absolute radial positions reported in the scan files, the precision of photometric or refractometric signal magnitudes, and the precision of the calculated apparent molar mass of BSA monomer and the fraction of BSA dimers. These results highlight the necessity and effectiveness of independent calibration of basic AUC data dimensions for reliable quantitative studies.
Published: 2015
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3. Structural and functional insights into the activation of the dual incision activity of UvrC, a key player in bacterial NER

Author: Anna Seck, Salvatore De Bonis, Meike Stelter, Mats Ökvist, Müge Senarisoy, Mohammad Rida Hayek, Aline Le Roy, Lydie Martin, Christine Saint-Pierre, Célia M Silveira, Didier Gasparutto, Smilja Todorovic, Jean-Luc Ravanat, Joanna Timmins, Institut de biologie structurale (IBS - UMR 5075), Centre National de la Recherche Scientifique (CNRS)-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Grenoble Alpes (UGA), European Synchrotron Radiation Facility (ESRF), SYstèmes Moléculaires et nanoMatériaux pour l’Energie et la Santé (SYMMES), Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS)-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Instituto de Tecnologia Química e Biológica António Xavier (ITQB), and Universidade Nova de Lisboa = NOVA University Lisbon (NOVA)
Subjects: MESH: DNA Damage, MESH: DNA Repair, MESH: Escherichia coli, [SDV]Life Sciences [q-bio], Genetics, MESH: DNA Helicases, MESH: Endodeoxyribonucleases, MESH: Bacterial Proteins, MESH: DNA, Bacterial, MESH: Deinococcus
Abstract: Bacterial nucleotide excision repair (NER), mediated by the UvrA, UvrB and UvrC proteins is a multistep, ATP-dependent process, that is responsible for the removal of a very wide range of chemically and structurally diverse DNA lesions. DNA damage removal is performed by UvrC, an enzyme possessing a dual endonuclease activity, capable of incising the DNA on either side of the damaged site to release a short single-stranded DNA fragment containing the lesion. Using biochemical and biophysical approaches, we have probed the oligomeric state, UvrB- and DNA-binding abilities and incision activities of wild-type and mutant constructs of UvrC from the radiation resistant bacterium, Deinococcus radiodurans. Moreover, by combining the power of new structure prediction algorithms and experimental crystallographic data, we have assembled the first model of a complete UvrC, revealing several unexpected structural motifs and in particular, a central inactive RNase H domain acting as a platform for the surrounding domains. In this configuration, UvrC is maintained in a ‘closed’ inactive state that needs to undergo a major rearrangement to adopt an ‘open’ active state capable of performing the dual incision reaction. Taken together, this study provides important insight into the mechanism of recruitment and activation of UvrC during NER.
Published: 2023

4. Analytical ultracentrifugation sedimentation velocity for the characterization of recombinant adeno-associated virus vectors sub-populations

Author: Sylvie Saleun, Caroline Mas, Aline Le Roy, Magalie Penaud-Budloo, Oumeya Adjali, Véronique Blouin, Christine Ebel, Laboratoire de Thérapie Génique Translationnelle des Maladies Génétiques / Translational Research in Gene Therapy - UMR_S 1089 (TARGET), Institut National de la Santé et de la Recherche Médicale (INSERM)-Nantes Université - UFR de Médecine et des Techniques Médicales (Nantes Univ - UFR MEDECINE), Nantes Université - pôle Santé, Nantes Université (Nantes Univ)-Nantes Université (Nantes Univ)-Nantes Université - pôle Santé, Nantes Université (Nantes Univ)-Nantes Université (Nantes Univ), Institut de biologie structurale (IBS - UMR 5075), Centre National de la Recherche Scientifique (CNRS)-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), and Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Grenoble Alpes (UGA)
Subjects: AAV vector, AUC, Adeno-associated virus, [SDV]Life Sciences [q-bio], Sedimentation velocity, Analytical ultracentrifugation, Biophysics, Quality control, General Medicine
Abstract: Recombinant adeno-associated virus virus-derived vectors (rAAVs) are among the most used viral delivery system for in vivo gene therapies with a good safety profile. However, rAAV production methods often lead to a heterogeneous vector population, in particular with the presence of undesired empty particles. Analytical ultracentrifugation sedimentation velocity (AUC-SV) is considered as the gold analytical technique allowing the measurement of relative amounts of each vector subpopulation and components like particle aggregates, based on their sedimentation coefficients. This letter presents the principle and practice of AUC experiments for rAAVs characterization. We discuss our results in the framework of previously published works. In addition to classical detection at 260 nm, using interference optics in the ultracentrifuge can provide an independent estimate of weight percentages of the different populations of capsids, and of the genome size incorporated in rAAV particles.
Published: 2023

5. Author Reply to Peer Reviews of Characterization of the REC114-MEI4-IHO1 complex regulating meiotic DNA double-strand break formation

Author: Hamida Laroussi, Ariadna B. Juarez-Martinez, Aline Le Roy, Elisabetta Boeri Erba, Bernard de Massy, and Jan Kadlec
Published: 2023

6. Powerful Avidity with a Limited Valency for Virus-Attachment Blockers on DC-SIGN: Combining Chelation and Statistical Rebinding with Structural Plasticity of the Receptor

Author: Vanessa Porkolab, Martin Lepšík, Stefania Ordanini, Alexander St John, Aline Le Roy, Michel Thépaut, Emanuele Paci, Christine Ebel, Anna Bernardi, Franck Fieschi, Institut de biologie structurale (IBS - UMR 5075), Centre National de la Recherche Scientifique (CNRS)-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Grenoble Alpes (UGA), Centre de Recherches sur les Macromolécules Végétales (CERMAV), Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS)-Université Grenoble Alpes (UGA), Università degli Studi di Milano = University of Milan (UNIMI), School of Chemistry [Leeds], and University of Leeds
Subjects: General Chemical Engineering, [SDV]Life Sciences [q-bio], General Chemistry, Settore CHIM/06 - Chimica Organica
Published: 2023

7. Zwitterionic fluorinated detergents: From design to membrane protein applications

Author: Marine Soulié, Anais Deletraz, Moheddine Wehbie, Florian Mahler, Ilham Bouchemal, Aline Le Roy, Isabelle Petit-Härtlein, Sandro Keller, Annette Meister, Eva Pebay-Peyroula, Cécile Breyton, Christine Ebel, Grégory Durand, Institut des Biomolécules Max Mousseron [Pôle Chimie Balard] (IBMM), Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS)-Université de Montpellier (UM)-Ecole Nationale Supérieure de Chimie de Montpellier (ENSCM), Université de Montpellier (UM), Molecular Biophysics, Technische Universität Kaiserslautern (TU Kaiserslautern), Institut de biologie structurale (IBS - UMR 5075), Centre National de la Recherche Scientifique (CNRS)-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Grenoble Alpes (UGA), University of Kaiserslautern, and Martin-Luther-University Halle-Wittenberg
Subjects: [SDV]Life Sciences [q-bio], MESH: Micelles, MESH: X-Ray Diffraction, General Medicine, MESH: Membrane Proteins, Biochemistry, MESH: Scattering, Small Angle, MESH: Detergents
Abstract: International audience; We report herein the synthesis of zwitterionic sulfobetaine (SB) and dimethylamine oxide (AO) detergents whose alkyl chain is made of either a perfluorohexyl (F6H3) or a perfluoropentyl (F5H5) group linked to a hydrogenated spacer arm. In aqueous solution, the critical micellar concentrations (CMCs) measured by surface tensiometry (SFT) and isothermal titration calorimetry (ITC) were found in the millimolar range (1.3-2.4 mM). The morphologies of the aggregates were evaluated by dynamic light scattering (DLS), analytical ultracentrifugation (AUC), small-angle X-ray scattering (SAXS), and transmission electron microscopy (TEM), demonstrating that the two perfluoropentyl derivatives formed small micelles less than 10 nm in diameter, whereas the perfluorohexyl derivatives formed larger and more heterogeneous micelles. The two SB detergents were able to solubilize synthetic lipid vesicles in a few hours; by contrast, the perfluoropentyl AO induced much faster solubilization, whereas the perfluorohexyl AO did not show any solubilization. All detergents were tested for their abilities to stabilize three membrane proteins, namely, bacteriorhodopsin (bR), the Bacillus subtilis ABC transporter BmrA, and the Streptococcus pneumoniae enzyme SpNOX. The SB detergents outperformed the AO derivatives as well as their hydrogenated analogs in stabilizing these proteins. Among the four new compounds, F5H5SB combines many desirable properties for membrane-protein study, as it is a powerful yet gentle detergent.
Published: 2023

8. Characterization of the REC114-MEI4-IHO1 complex regulating meiotic DNA double-strand break formation

Author: Hamida Laroussi, Ariadna B. Juarez-Martinez, Aline Le Roy, Elisabetta Boeri Erba, Bernard de Massy, and Jan Kadlec
Abstract: Meiotic recombination is initiated by the formation of DNA double-strand breaks (DSBs), essential for fertility and genetic diversity. In the mouse, DSBs are formed by the catalytic TOPOVIL complex consisting of SPO11 and TOPOVIBL. To preserve genome integrity, the activity of the TOPOVIL complex is finely controlled by several meiotic factors including REC114, MEI4 and IHO1, but the underlying mechanism is poorly understood. Here, we report that mouse REC114 forms homodimers, that it associates with MEI4 as a 2:1 heterotrimer that further dimerizes, and that IHO1 forms coiled-coil based tetramers. Using AlphaFold2 modelling combined with biochemical characterization we uncovered the molecular details of these assemblies. Finally, we show that IHO1 directly interacts with the PH domain of REC114 by recognizing the same surface as TOPOVIBL and another meiotic factor ANKRD31. These results provide strong evidence for the existence of a ternary IHO1-REC114-MEI4 complex and show that REC114 is a potential regulatory platform mediating mutually exclusive interactions with several partners.
Published: 2023

9. Powerful avidity with a limited valency for virus-attachment blockers on DC SIGN: Combining chelation and statistical rebinding with structural plasticity of the receptor

Author: Vanessa Porkolab, Martin Lepšík, Stefania Ordanini, Alexander St John, Aline Le Roy, Michel Thépaut, Emanuele Paci, Christine Ebel, Anna Bernardi, and Franck FIESCHI
Abstract: The C-type lectin receptor DC-SIGN has been highlighted as co-receptor for the spike protein of the SARS-CoV-2 virus. A multivalent glycomimetic ligand, Polyman26, has been found to inhibit DCSIGN- dependent trans-infection of SARS-CoV-2. The molecular details underlying avidity generation in such systems remain poorly characterized. In an effort to dissect the contribution of the known multivalent effects - chelation, clustering and statistical rebinding – we studied a series of dendrimer constructs related to Polyman26 with a rod core rationally designed to engage simultaneously two binding sites of the tetrameric DC-SIGN. Binding properties of these compounds have been studied with a range of biophysical techniques, including recently developed Surface Plasmon Resonance oriented-surface methodology. Using molecular modelling we addressed, for the first time, the impact of the carbohydrate recognition domains’ flexibility of the DC-SIGN tetramer on the compounds’ avidity. We were able to gain deeper insight into the role of different binding modes, which in combination produce a construct with a nM affinity despite a limited valency. This multi-faceted experimental-theoretical approach provides detailed understanding of multivalent ligand/multimeric protein interactions which can lead to future predictions. This work opens the way to the development of new virus attachment blockers adapted to different C-type lectin receptors of viruses.
Published: 2022

10. Maltose-Based Fluorinated Surfactants for Membrane-Protein Extraction and Stabilization

Author: Jonathan O. Babalola, Moheddine Wehbie, Florian Mahler, Aline Le Roy, Christine Ebel, Kenechi Kanayo Onyia, Sandro Keller, Anais Deletraz, Carolyn Vargas, Cécile Breyton, Ilham Bouchemal, Grégory Durand, Institut des Biomolécules Max Mousseron [Pôle Chimie Balard] (IBMM), Ecole Nationale Supérieure de Chimie de Montpellier (ENSCM)-Institut de Chimie du CNRS (INC)-Université de Montpellier (UM)-Centre National de la Recherche Scientifique (CNRS), Molecular Biophysics, University of Kaiserslautern [Kaiserslautern], Technische Universität Kaiserslautern (TU Kaiserslautern), Institut de biologie structurale (IBS - UMR 5075), Centre National de la Recherche Scientifique (CNRS)-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Grenoble Alpes (UGA), University of Kaiserslautern, Department of Chemistry, University of Ibadan, and ANR-16-CE92-0001,FLUOR,Tensioactifs Fluorés pour l'Étude de Protéines Membranaires(2016)
Subjects: 02 engineering and technology, 010402 general chemistry, 01 natural sciences, Micelle, Surface tension, Surface-Active Agents, Pulmonary surfactant, Dynamic light scattering, Electrochemistry, Surface Tension, General Materials Science, Thermal stability, Maltose, Micelles, Spectroscopy, [SDV.BBM.BS]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Structural Biology [q-bio.BM], Chemistry, Membrane Proteins, Isothermal titration calorimetry, Surfaces and Interfaces, 021001 nanoscience & nanotechnology, Condensed Matter Physics, 0104 chemical sciences, Membrane, Chemical engineering, Critical micelle concentration, 0210 nano-technology
Abstract: International audience; Two new surfactants, F5OM and F5DM, were designed as partially fluorinated analogues of n-dodecyl-β-D-maltoside (DDM). The micellization properties and the morphologies of the aggregates formed by the two surfactants in water and phosphate buffer were evaluated by NMR spectroscopy, surface tension measurement, isothermal titration calorimetry, dynamic light scattering, small-angle X-ray scattering, and analytical ultracentrifugation. As expected, the critical micellar concentration (cmc) was found to decrease with chain length of the fluorinated tail from 2.1-2.5 mM for F5OM to 0.3-0.5 mM for F5DM, and micellization was mainly entropy-driven at 25 °C. Close to their respective cmc, the micelle sizes were similar for both surfactants, that is, 7 and 13 nm for F5OM and F5DM, respectively, and both increased with concentration forming 4 nm diameter rods with maximum dimensions of 50 and 70 nm, respectively, at a surfactant concentration of ∼30 mM. The surfactants were found to readily solubilize lipid vesicles and extract membrane proteins directly from Escherichia coli membranes. They were found more efficient than the commercial fluorinated detergent F6H2OM over a broad range of concentrations (1-10 mM) and even better than DDM at low concentrations (1-5 mM). When transferred into the two new surfactants, the thermal stability of the proteins bacteriorhodopsin (bR) and FhuA was higher than in the presence of their solubilization detergents and similar to that in DDM; furthermore, bR was stable over several months. The membrane enzymes SpNOX and BmrA were not as active as in DDM micelles but similarly active as in F6OM. Together, these findings indicate both extracting and stabilizing properties of the new maltose-based fluorinated surfactants, making them promising tools in MP applications.
Published: 2021

11. Structural insights into the catalytic cycle of a bacterial multidrug ABC efflux pump

Author: Waqas Javed, Sylvain Vallet, Marie-Pierre Clement, Aline Le Roy, Martine Moulin, Michael Härtlein, Cécile Breyton, Odile Burlet-Schiltz, Julien Marcoux, Cédric Orelle, Christine Ebel, Anne Martel, Jean-Michel Jault, Institut de biologie structurale (IBS - UMR 5075), Centre National de la Recherche Scientifique (CNRS)-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Grenoble Alpes (UGA), 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 Laue-Langevin (ILL), Institut de pharmacologie et de biologie structurale (IPBS), Université Toulouse III - Paul Sabatier (UT3), Université de Toulouse (UT)-Université de Toulouse (UT)-Centre National de la Recherche Scientifique (CNRS), ANR-14-CE09-0024,BmrA-NMX,Révéler les détails conformationnels d'une pompe d'efflux membranaire(2014), ANR-19-CE11-0023,BIOTIFLUX,Dissection du mécanisme moléculaire d'un transporteur d'antibiotiques(2019), ANR-16-CE92-0001,FLUOR,Tensioactifs Fluorés pour l'Étude de Protéines Membranaires(2016), ANR-10-INBS-0005,FRISBI,Infrastructure Française pour la Biologie Structurale Intégrée(2010), ANR-17-EURE-0003,CBH-EUR-GS,CBH-EUR-GS(2017), ANR-10-INBS-0008,ProFI,Infrastructure Française de Protéomique(2010), and European Project: 654000,H2020,H2020-INFRADEV-1-2014-1,SINE2020(2015)
Subjects: History, Polymers and Plastics, [SDV.BBM.BS]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Structural Biology [q-bio.BM], Protein Conformation, multidrug transporter, MESH: Drug Resistance, Multiple, Bacterial, small angle neutron scattering, MESH: Catalysis, Industrial and Manufacturing Engineering, Catalysis, hydrogen deuterium exchange, Adenosine Triphosphate, MESH: Protein Conformation, Structural Biology, Drug Resistance, Multiple, Bacterial, MESH: Adenosine Triphosphate, MESH: ATP-Binding Cassette Transporters, ATP-Binding Cassette Transporters, membrane protein, MESH: Genes, MDR, Business and International Management, Genes, MDR, Molecular Biology, ATP-binding cassette
Abstract: International audience; ABC ("ATP-Binding Cassette") transporters of the type IV subfamily consist of exporters involved in the efflux of many compounds, notably those capable to confer multidrug resistance like the mammalian P-glycoprotein or the bacterial transporter BmrA. They function according to an alternating access mechanism between inward-facing (IF) and outward-facing (OF) conformations, but the extent of physical separation between the two nucleotide-binding domains (NBDs) in different states is still unsettled. Small Angle Neutron Scattering and hydrogen/deuterium exchange coupled to mass spectrometry were used to highlight different conformational states of BmrA during its ATPase cycle. In particular, mutation of the conserved Lysine residue of the Walker-A motif (K380A) captures BmrA in an ATP-bound IF conformation prior to NBD closure. While in the transition-like state induced by vanadate wild-type BmrA is mainly in an OF conformation, the transporter populates only IF conformations in either the apo state or in the presence of ADP/Mg. Importantly, in this post-hydrolytic step, distances between the two NBDs of BmrA seem to be more separated than in the apo state, but they remain shorter than the widest opening found in the related MsbA transporter. Overall, our results highlight the main steps of the catalytic cycle of a homodimeric bacterial multidrug transporter and underline structural and functional commonalities as well as oddities among the type IV subfamily of ABC transporters.
Published: 2022

12. Targeting Tn-Antigen-Positive Human Tumors with a Recombinant Human Macrophage Galactose C-Type Lectin

Author: François Bulteau, Michel Thépaut, Maxime Henry, Amandine Hurbin, Laetitia Vanwonterghem, Corinne Vivès, Aline Le Roy, Christine Ebel, Olivier Renaudet, Franck Fieschi, Jean-Luc Coll, Institut de biologie structurale (IBS - UMR 5075), Centre National de la Recherche Scientifique (CNRS)-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Grenoble Alpes (UGA), Institute for Advanced Biosciences / Institut pour l'Avancée des Biosciences (Grenoble) (IAB), Centre Hospitalier Universitaire [Grenoble] (CHU)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Etablissement français du sang - Auvergne-Rhône-Alpes (EFS)-Centre National de la Recherche Scientifique (CNRS)-Université Grenoble Alpes (UGA), Département de Chimie Moléculaire (DCM), and Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS)-Université Grenoble Alpes (UGA)
Subjects: MESH: HT29 Cells, Pharmaceutical Science, MESH: Spheroids, Cellular, Mice, Nude, MESH: Flow Cytometry, MESH: Recombinant Proteins, 03 medical and health sciences, Mice, 0302 clinical medicine, Spheroids, Cellular, Drug Discovery, Tn antigen, MESH: Mice, Nude, cancer, MESH: Microscopy, Confocal, Animals, Humans, MESH: Animals, Antigens, Tumor-Associated, Carbohydrate, Lectins, C-Type, MESH: Mice, 030304 developmental biology, 0303 health sciences, MESH: Humans, Microscopy, Confocal, MESH: Antigens, Tumor-Associated, Carbohydrate, [SDV.BBM.BS]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Structural Biology [q-bio.BM], Surface Plasmon Resonance, Flow Cytometry, Recombinant Proteins, 3. Good health, MESH: Surface Plasmon Resonance, C-type lectin, A549 Cells, 030220 oncology & carcinogenesis, Molecular Medicine, Female, MESH: A549 Cells, MESH: Female, HT29 Cells, MESH: Neoplasm Transplantation, MESH: Lectins, C-Type, Neoplasm Transplantation
Abstract: International audience; Alterations in glycosylation cause the emergence of tumor-associated carbohydrate antigens (TACAs) during tumorigenesis. Truncation of O-glycans reveals the Thomsen nouveau (Tn) antigen, an N-acetylgalactosamine (GalNAc) frequently attached to serine or threonine amino acids, that is accessible on the surface of cancer cells but not on healthy cells. Interestingly, GalNac can be recognized by macrophage galactose lectin (MGL), a type C lectin receptor expressed in immune cells. In this study, recombinant MGL fragments were tested in vitro for their cancer cell-targeting efficiency by flow cytometry and confocal microscopy and in vivo after administration of fluorescent MGL to tumor-bearing mice. Our results demonstrate the ability of MGL to target Tn-positive human tumors without inducing toxicity. This outcome makes MGL, a fragment of a normal human protein, the first vector candidate for in vivo diagnosis and imaging of human tumors and, possibly, for therapeutic applications.
Published: 2021

13. Structural and functional characterization of DdrC, a novel DNA damage-induced nucleoid associated protein involved in DNA compaction

Author: Anne-Sophie Banneville, Claire Bouthier de la Tour, Cécilia Hognon, Joanna Timmins, Pascale Servant, Antonio Monari, Jean-Marie Teulon, François Dehez, Aline Le Roy, Jacques-Philippe Colletier, Jean-Luc Pellequer, Fabrice Confalonieri, Institut de biologie structurale (IBS - UMR 5075), Centre National de la Recherche Scientifique (CNRS)-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Grenoble Alpes (UGA), Institut de Biologie Intégrative de la Cellule (I2BC), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS), Laboratoire de Physique et Chimie Théoriques (LPCT), Institut de Chimie du CNRS (INC)-Université de Lorraine (UL)-Centre National de la Recherche Scientifique (CNRS), Interfaces, Traitements, Organisation et Dynamique des Systèmes (ITODYS (UMR_7086)), Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS)-Université de Paris (UP), The ANR (Agence Nationale de la Recherche) and CGI (Commissariat à l’Investissement d’Avenir) are gratefully acknowledged for their financial support of this work through Labex SEAM (Science and Engineering for Advanced Materials and devices): ANR-11-LABX-086 and ANR-11-IDEX-05-02, ANR-10-INBS-0005,FRISBI,Infrastructure Française pour la Biologie Structurale Intégrée(2010), ANR-10-LABX-0049,GRAL,Grenoble Alliance for Integrated Structural Cell Biology(2010), ANR-17-EURE-0003,CBH-EUR-GS,CBH-EUR-GS(2017), and Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS)-Université Paris Cité (UPCité)
Subjects: 0303 health sciences, DNA Repair, [SDV.BBM.BS]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Structural Biology [q-bio.BM], biology, 030306 microbiology, Chemistry, DNA damage, DNA repair, Deinococcus radiodurans, biology.organism_classification, Cell biology, DNA binding site, 03 medical and health sciences, chemistry.chemical_compound, Bacterial Proteins, Genetics, Nucleoid, A-DNA, Deinococcus, DNA, Circular, Gene, DNA, DNA Damage, 030304 developmental biology
Abstract: Deinococcus radiodurans is a spherical bacterium well-known for its outstanding resistance to DNA-damaging agents. Exposure to such agents leads to drastic changes in the transcriptome of D. radiodurans. In particular, four Deinococcus-specific genes, known as DNA Damage Response genes, are strongly up-regulated and have been shown to contribute to the resistance phenotype of D. radiodurans. One of these, DdrC, is expressed shortly after exposure to γ-radiation and is rapidly recruited to the nucleoid. In vitro, DdrC has been shown to compact circular DNA, circularize linear DNA, anneal complementary DNA strands and protect DNA from nucleases. To shed light on the possible functions of DdrC in D. radiodurans, we determined the crystal structure of the domain-swapped DdrC dimer at a resolution of 2.2 Å and further characterized its DNA binding and compaction properties. Notably, we show that DdrC bears two asymmetric DNA binding sites located on either side of the dimer and can modulate the topology and level of compaction of circular DNA. These findings suggest that DdrC may be a DNA damage-induced nucleoid-associated protein that enhances nucleoid compaction to limit the dispersion of the fragmented genome and facilitate DNA repair after exposure to severe DNA damaging conditions.
Published: 2021

14. The MurG glycosyltransferase provides an oligomeric scaffold for the cytoplasmic steps of peptidoglycan biosynthesis in the human pathogen Bordetella pertussis

Author: Delphine Patin, Dominique Mengin-Lecreulx, Andréa Dessen, Cécile Breyton, Mayara M. Miyachiro, Christine Ebel, Federica Laddomada, Aline Le Roy, Matthew Jessop, Irina Gutsche, Viviana Job, 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), Brazilian Biosciences National Laboratory (LNBio), National Center for Research in Energy and Materials, Institut de Biologie Intégrative de la Cellule (I2BC), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS), AUC, ME, biophysique, ANR-13-BSV8-0015,BACSCREEN,Assemblage et structure de complexes macromoléculaires essentiels pour la biosynthèse de la paroi bactérienne(2013), 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, Cytoplasm, Operon, lcsh:Medicine, Peptidoglycan, N-Acetylglucosaminyltransferases, Bordetella pertussis, Article, Bacterial cell structure, 03 medical and health sciences, chemistry.chemical_compound, 0302 clinical medicine, Bacterial Proteins, X-Ray Diffraction, Biosynthesis, Cell Wall, Catalytic Domain, Scattering, Small Angle, Glycosyltransferase, Humans, Inner membrane, Peptide Synthases, lcsh:Science, chemistry.chemical_classification, Binding Sites, Multidisciplinary, [SDV.BBM.BS]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Structural Biology [q-bio.BM], biology, lcsh:R, Glycosyltransferases, Anti-Bacterial Agents, Cell biology, 030104 developmental biology, Enzyme, chemistry, biology.protein, lcsh:Q, 030217 neurology & neurosurgery, Bacterial Outer Membrane Proteins, Protein Binding
Abstract: Peptidoglycan is a major component of the bacterial cell wall and thus a major determinant of cell shape. Its biosynthesis is initiated by several sequential reactions catalyzed by cytoplasmic Mur enzymes. Mur ligases (MurC, -D, -E, and -F) are essential for bacteria, metabolize molecules not present in eukaryotes, and are structurally and biochemically tractable. However, although many Mur inhibitors have been developed, few have shown promising antibacterial activity, prompting the hypothesis that within the cytoplasm, Mur enzymes could exist as a complex whose architecture limits access of small molecules to their active sites. This suggestion is supported by the observation that in many bacteria, mur genes are present in a single operon, and pairs of these genes often are fused to generate a single polypeptide. Here, we explored this genetic arrangement in the human pathogen Bordetella pertussis and show that MurE and MurF are expressed as a single, bifunctional protein. EM, small angle X-ray scattering (SAXS), and analytical centrifugation (AUC) revealed that the MurE–MurF fusion displays an elongated, flexible structure that can dimerize. Moreover, MurE–MurF interacted with the peripheral glycosyltransferase MurG, which formed discrete oligomers resembling 4- or 5-armed stars in EM images. The oligomeric structure of MurG may allow it to play a bona fide scaffolding role for a potential Mur complex, facilitating the efficient conveyance of peptidoglycan-building blocks toward the inner membrane leaflet. Our findings shed light on the structural determinants of a peptidoglycan formation complex involving Mur enzymes in bacterial cell wall formation.
Published: 2019

15. Biophysical characterization of the complex between the iron-responsive transcription factor Fep1 and DNA

Author: Antimo Cutone, Laura Cervoni, Adriana E. Miele, Christine Ebel, Giovanni Musci, Maria Carmela Bonaccorsi di Patti, Aline Le Roy, 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), Università degli Studi di Roma 'La Sapienza' = Sapienza University [Rome] (UNIROMA), Institut de biologie structurale (IBS - UMR 5075), Centre National de la Recherche Scientifique (CNRS)-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Grenoble Alpes (UGA), Università degli Studi del Molise = University of Molise (UNIMOL), ANR-17-EURE-0003,CBH-EUR-GS,CBH-EUR-GS(2017), 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), Università degli Studi di Roma 'La Sapienza' = Sapienza University [Rome], and Università degli Studi del Molise (Unimol)
Subjects: 0301 basic medicine, 030103 biophysics, Analytical ultracentrifugation sedimentation velocity, differential scanning calorimetry, Fep1, fluorescence spectroscopy, iron sulfur cluster, protein-DNA complex, small angle X-ray scattering, Iron, Protein-DNA complex, Biophysics, Membrane biology, Iron–sulfur cluster, Small angle X-ray scattering, GATA Transcription Factors, Pichia pastoris, Iron sulfur cluster, 03 medical and health sciences, chemistry.chemical_compound, Transcription (biology), Differential scanning calorimetry, Fluorescence spectroscopy, Transcription factor, Zinc finger, biology, [SDV.BBM.BS]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Structural Biology [q-bio.BM], Chemistry, DNA, General Medicine, biology.organism_classification, 030104 developmental biology, Saccharomycetales, Transcription Factors
Abstract: International audience; Fep1 is an iron-responsive GATA-type transcriptional repressor present in numerous fungi. The DNA-binding domain of this protein is characterized by the presence of two zinc fingers of the Cys2-Cys2 type and a Cys-X5-Cys-X8-Cys-X2-Cys motif located between the two zinc fingers, that is involved in binding of a [2Fe-2S] cluster. In this work, biophysical characterization of the DNA-binding domain of Pichia pastoris Fep1 and of the complex of the protein with cognate DNA has been undertaken. The results obtained by analytical ultracentrifugation sedimentation velocity, small-angle X-ray scattering and differential scanning calorimetry indicate that Fep1 is a natively unstructured protein that is able to bind DNA forming 1:1 and 2:1 complexes more compact than the individual partners. Complex formation takes place independently of the presence of a stoichiometric [2Fe-2S] cluster, suggesting that the cluster may play a role in recruiting other protein(s) required for regulation of transcription in response to changes in intracellular iron levels.
Published: 2021

16. Resurrection of Ancestral Malate Dehydrogenases Reveals the Evolutionary History of Halobacterial Proteins : Deciphering Gene Trajectories and Changes in Biochemical Properties

Author: Manolo Gouy, Eric Girard, Bruno Franzetti, Dominique Madern, Mathieu Groussin, Aline Le Roy, Gergely J. Szollosi, Samuel Blanquart, Dynamics, Logics and Inference for biological Systems and Sequences (Dyliss), Inria Rennes – Bretagne Atlantique, Institut National de Recherche en Informatique et en Automatique (Inria)-Institut National de Recherche en Informatique et en Automatique (Inria)-GESTION DES DONNÉES ET DE LA CONNAISSANCE (IRISA-D7), Institut de Recherche en Informatique et Systèmes Aléatoires (IRISA), Université de Rennes (UR)-Institut National des Sciences Appliquées - Rennes (INSA Rennes), Institut National des Sciences Appliquées (INSA)-Institut National des Sciences Appliquées (INSA)-Université de Bretagne Sud (UBS)-École normale supérieure - Rennes (ENS Rennes)-Institut National de Recherche en Informatique et en Automatique (Inria)-CentraleSupélec-Centre National de la Recherche Scientifique (CNRS)-IMT Atlantique (IMT Atlantique), Institut Mines-Télécom [Paris] (IMT)-Institut Mines-Télécom [Paris] (IMT)-Université de Rennes (UR)-Institut National des Sciences Appliquées - Rennes (INSA Rennes), Institut Mines-Télécom [Paris] (IMT)-Institut Mines-Télécom [Paris] (IMT)-Institut de Recherche en Informatique et Systèmes Aléatoires (IRISA), Institut National des Sciences Appliquées (INSA)-Institut National des Sciences Appliquées (INSA)-Université de Bretagne Sud (UBS)-École normale supérieure - Rennes (ENS Rennes)-CentraleSupélec-Centre National de la Recherche Scientifique (CNRS)-IMT Atlantique (IMT Atlantique), Institut Mines-Télécom [Paris] (IMT)-Institut Mines-Télécom [Paris] (IMT), Laboratoire de Biométrie et Biologie Evolutive - UMR 5558 (LBBE), Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-Institut National de Recherche en Informatique et en Automatique (Inria)-VetAgro Sup - Institut national d'enseignement supérieur et de recherche en alimentation, santé animale, sciences agronomiques et de l'environnement (VAS)-Centre National de la Recherche Scientifique (CNRS), Institut de biologie structurale (IBS - UMR 5075), Centre National de la Recherche Scientifique (CNRS)-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Grenoble Alpes (UGA), Biophysics Research Group [Budapest] (ELTE-MTA 'Lendület'), Hungarian Academy of Sciences (MTA), Bioinformatique, phylogénie et génomique évolutive (BPGE), Département PEGASE [LBBE] (PEGASE), Université de Lyon-Université de Lyon-Institut National de Recherche en Informatique et en Automatique (Inria)-VetAgro Sup - Institut national d'enseignement supérieur et de recherche en alimentation, santé animale, sciences agronomiques et de l'environnement (VAS)-Centre National de la Recherche Scientifique (CNRS)-Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-Institut National de Recherche en Informatique et en Automatique (Inria)-VetAgro Sup - Institut national d'enseignement supérieur et de recherche en alimentation, santé animale, sciences agronomiques et de l'environnement (VAS)-Centre National de la Recherche Scientifique (CNRS)-Laboratoire de Biométrie et Biologie Evolutive - UMR 5558 (LBBE), ANR-16-CE11-0011,AlloAnc,Allostérie Ancestrale(2016), Université de Bretagne Sud (UBS)-Institut National des Sciences Appliquées - Rennes (INSA Rennes), Institut National des Sciences Appliquées (INSA)-Université de Rennes (UNIV-RENNES)-Institut National des Sciences Appliquées (INSA)-Université de Rennes (UNIV-RENNES)-Institut National de Recherche en Informatique et en Automatique (Inria)-École normale supérieure - Rennes (ENS Rennes)-Centre National de la Recherche Scientifique (CNRS)-Université de Rennes 1 (UR1), Université de Rennes (UNIV-RENNES)-CentraleSupélec-IMT Atlantique Bretagne-Pays de la Loire (IMT Atlantique), Institut Mines-Télécom [Paris] (IMT)-Institut Mines-Télécom [Paris] (IMT)-Université de Bretagne Sud (UBS)-Institut National des Sciences Appliquées - Rennes (INSA Rennes), Institut National des Sciences Appliquées (INSA)-Université de Rennes (UNIV-RENNES)-Institut National des Sciences Appliquées (INSA)-Université de Rennes (UNIV-RENNES)-École normale supérieure - Rennes (ENS Rennes)-Centre National de la Recherche Scientifique (CNRS)-Université de Rennes 1 (UR1), Centre National de la Recherche Scientifique (CNRS)-Université Grenoble Alpes (UGA)-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: Halobacterium, Proteomics, Malates, halophilic adaptation of proteins, malate dehydrogenase, Euryarchaeota, 010402 general chemistry, AcademicSubjects/SCI01180, 01 natural sciences, Malate dehydrogenase, 03 medical and health sciences, Phylogenetics, Genetics, [SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology, Molecular Biology, Ecology, Evolution, Behavior and Systematics, Discoveries, Phylogeny, 030304 developmental biology, Halobacteriales, 0303 health sciences, biology, Haloarchaea, [SDV.BID.EVO]Life Sciences [q-bio]/Biodiversity/Populations and Evolution [q-bio.PE], AcademicSubjects/SCI01130, ancestral protein resurrection, biology.organism_classification, Archaea, [SDV.MP.BAC]Life Sciences [q-bio]/Microbiology and Parasitology/Bacteriology, Halophile, 0104 chemical sciences, Evolutionary biology, horizontal gene transfer, Adaptation
Abstract: Extreme halophilic Archaea thrive in high salt, where, through proteomic adaptation, they cope with the strong osmolarity and extreme ionic conditions of their environment. In spite of wide fundamental interest, however, studies providing insights into this adaptation are scarce, because of practical difficulties inherent to the purification and characterization of halophilic enzymes. In this work, we describe the evolutionary history of malate dehydrogenases (MalDH) within Halobacteria (a class of the Euryarchaeota phylum). We resurrected nine ancestors along the inferred halobacterial MalDH phylogeny, including the Last Common Ancestral MalDH of Halobacteria (LCAHa) and compared their biochemical properties with those of five modern halobacterial MalDHs. We monitored the stability of these various MalDHs, their oligomeric states and enzymatic properties, as a function of concentration for different salts in the solvent. We found that a variety of evolutionary processes, such as amino acid replacement, gene duplication, loss of MalDH gene and replacement owing to horizontal transfer resulted in significant differences in solubility, stability and catalytic properties between these enzymes in the three Halobacteriales, Haloferacales, and Natrialbales orders since the LCAHa MalDH. We also showed how a stability trade-off might favor the emergence of new properties during adaptation to diverse environmental conditions. Altogether, our results suggest a new view of halophilic protein adaptation in Archaea.
Published: 2021

17. TETRALEC, Artificial Tetrameric Lectins: A Tool to Screen Ligand and Pathogen Interactions

Author: Sabine Mayer-Lambertz, Corinne Vivès, Bernd Lepenies, Franck Fieschi, João T. Monteiro, Christine Ebel, Michel Thépaut, Niels-Christian Reichardt, Aline Le Roy, Sonia Serna, Silvia Achilli, Institut de biologie structurale (IBS - UMR 5075), Centre National de la Recherche Scientifique (CNRS)-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Grenoble Alpes (UGA), Immunology Unit and Research Center for Emerging Infections and Zoonoses, University of Veterinary Medicine Hannover, CIC biomaGUNE, Glycotechnology Laboratory, Biomedical Research Networking Center in Bioengineering, Biomaterials and Nanomedicine (CIBER-BBN), Instituto de Salud Carlos III [Madrid] (ISC)-ministerio de ciencia e innovacion, Department of Biomolecular Systems [Potsdam], Max Planck Institute of Colloids and Interfaces, Max-Planck-Gesellschaft-Max-Planck-Gesellschaft, and ANR-17-EURE-0003,CBH-EUR-GS,CBH-EUR-GS(2017)
Subjects: 0301 basic medicine, Glycan, Recombinant Fusion Proteins, glycan array, Ligands, 01 natural sciences, Catalysis, Article, Flow cytometry, law.invention, lcsh:Chemistry, Inorganic Chemistry, 03 medical and health sciences, C-type lectin, Confocal microscopy, law, Candida albicans, medicine, pathogen recognition, Lectins, C-Type, Physical and Theoretical Chemistry, Receptor, lcsh:QH301-705.5, Molecular Biology, Spectroscopy, biology, medicine.diagnostic_test, [SDV.BBM.BS]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Structural Biology [q-bio.BM], 010405 organic chemistry, Chemistry, Organic Chemistry, Lectin, General Medicine, multivalency, Ligand (biochemistry), Flow Cytometry, In vitro, eye diseases, 0104 chemical sciences, Computer Science Applications, Immunoglobulin Fc Fragments, DC-SIGNR, 030104 developmental biology, lcsh:Biology (General), lcsh:QD1-999, Biochemistry, biology.protein
Abstract: International audience; C-type lectin receptor (CLR)/carbohydrate recognition occurs through low affinity interactions. Nature compensates that weakness by multivalent display of the lectin carbohydrate recognition domain (CRD) at the cell surface. Mimicking these low affinity interactions in vitro is essential to better understand CLR/glycan interactions. Here, we present a strategy to create a generic construct with a tetrameric presentation of the CRD for any CLR, termed TETRALEC. We applied our strategy to a naturally occurring tetrameric CRD, DC-SIGNR, and compared the TETRALEC ligand binding capacity by synthetic N- and O-glycans microarray using three different DC-SIGNR constructs i) its natural tetrameric counterpart, ii) the monomeric CRD and iii) a dimeric Fc-CRD fusion. DC-SIGNR TETRALEC construct showed a similar binding profile to that of its natural tetrameric counterpart. However, differences observed in recognition of low affinity ligands underlined the importance of the CRD spatial arrangement. Moreover, we further extended the applications of DC-SIGNR TETRALEC to evaluate CLR/pathogens interactions. This construct was able to recognize heat-killed Candida albicans by flow cytometry and confocal microscopy, a so far unreported specificity of DC-SIGNR. In summary, the newly developed DC-SIGNR TETRALEC tool proved to be useful to unravel novel CLR/glycan interactions, an approach which could be applied to other CLRs.
Published: 2020

18. Interdomain Flexibility within NADPH Oxidase Suggested by SANS Using LMNG Stealth Carrier

Author: Annelise Vermot, Anne L. Martel, Corinne Vivès, Sergei Grudinin, Michael Härtlein, Aline Le Roy, Michel Thépaut, Isabelle Petit-Hartlein, Christine Ebel, Martine Moulin, Susan M.E. Smith, Cécile Breyton, Franck Fieschi, Institut de biologie structurale (IBS - UMR 5075), Centre National de la Recherche Scientifique (CNRS)-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Grenoble Alpes (UGA), Sciences et Société, Historicité, Éducation et Pratiques (EA S2HEP), École normale supérieure de Lyon (ENS de Lyon)-Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon, Institut Laue-Langevin (ILL), Algorithms for Modeling and Simulating Nanosystems [2018-...] (NANO-D-POST [2018-2020]), Inria Grenoble - Rhône-Alpes, Institut National de Recherche en Informatique et en Automatique (Inria)-Institut National de Recherche en Informatique et en Automatique (Inria), Department of Molecular and Cellular Biology [Kennesaw], Kennesaw State University (KSU), ANR-17-CE11-0013,BANDIT,d'une Nox bactérienne à la conception d'outil de criblage(2017), ANR-16-CE92-0001,FLUOR,Tensioactifs Fluorés pour l'Étude de Protéines Membranaires(2016), École normale supérieure - Lyon (ENS Lyon)-Université Claude Bernard Lyon 1 (UCBL), ILL, Algorithms for Modeling and Simulation of Nanosystems (NANO-D), Institut National de Recherche en Informatique et en Automatique (Inria)-Institut National de Recherche en Informatique et en Automatique (Inria)-Laboratoire Jean Kuntzmann (LJK), Institut National de Recherche en Informatique et en Automatique (Inria)-Centre National de la Recherche Scientifique (CNRS)-Université Grenoble Alpes (UGA)-Institut polytechnique de Grenoble - Grenoble Institute of Technology (Grenoble INP ), Université Grenoble Alpes (UGA)-Centre National de la Recherche Scientifique (CNRS)-Université Grenoble Alpes (UGA)-Institut polytechnique de Grenoble - Grenoble Institute of Technology (Grenoble INP ), and Université Grenoble Alpes (UGA)
Subjects: 0303 health sciences, Molecular model, [SDV.BBM.BS]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Structural Biology [q-bio.BM], Biophysics, Membrane Proteins, NADPH Oxidases, Dehydrogenase, Articles, Neopentyl glycol, Small-angle neutron scattering, Transmembrane protein, 03 medical and health sciences, chemistry.chemical_compound, Crystallography, Neutron Diffraction, 0302 clinical medicine, chemistry, Docking (molecular), Scattering, Small Angle, Linker, Integral membrane protein, Oxidation-Reduction, 030217 neurology & neurosurgery, 030304 developmental biology
Abstract: International audience; Small angle neutron scattering (SANS) provides a method to obtain important low-resolution information for integral membrane proteins (IMPs), challenging targets for structural determination. Specific deuteration furnishes a "stealth" carrier for the solubilized IMP. We used SANS to determine a structural envelope of SpNOX, the Streptococcus pneumoniae NADPH oxidase (NOX), a prokaryotic model system for exploring structure and function of eukaryotic NOXes. SpNOX was solubilized in the detergent lauryl maltose neopentyl glycol, which provides optimal SpNOX stability and activity. Using deuterated solvent and protein, the lauryl maltose neopentyl glycol was experimentally undetected in SANS. This affords a cost-effective SANS approach for obtaining novel structural information on IMPs. Combining SANS data with molecular modeling provided a first, to our knowledge, structural characterization of an entire NOX enzyme. It revealed a distinctly less compact structure than that predicted from the docking of homologous crystal structures of the separate transmembrane and dehydrogenase domains, consistent with a flexible linker connecting the two domains.
Published: 2020

19. Fluorinated diglucose detergents for membrane-protein extraction

Author: Gildas Nyame Mendendy Boussambe, Florian Mahler, Anass Jawhari, Carolyn Vargas, Grégory Durand, Pierre Guillet, Aline Le Roy, Damien Cornut, Marine Soulié, Christine Ebel, Sandro Keller, Anaïs Marconnet, Françoise Bonneté, Institut des Biomolécules Max Mousseron [Pôle Chimie Balard] (IBMM), Ecole Nationale Supérieure de Chimie de Montpellier (ENSCM)-Institut de Chimie du CNRS (INC)-Université de Montpellier (UM)-Centre National de la Recherche Scientifique (CNRS), Molecular Biophysics, Technische Universität Kaiserslautern (TU Kaiserslautern), 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), CHEM2STAB, Avignon Université (AU), 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), CALIXAR, and ANR-16-CE92-0001,FLUOR,Tensioactifs Fluorés pour l'Étude de Protéines Membranaires(2016)
Subjects: 0301 basic medicine, chemistry.chemical_classification, Aggregation number, [SDV.BBM.BS]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Structural Biology [q-bio.BM], Halogenation, Detergents, Extraction (chemistry), Membrane Proteins, Isothermal titration calorimetry, Calorimetry, Micelle, General Biochemistry, Genetics and Molecular Biology, 03 medical and health sciences, 030104 developmental biology, Membrane, Solubility, chemistry, Pulmonary surfactant, Dynamic light scattering, Organic chemistry, Molecular Biology, Micelles, Alkyl
Abstract: International audience; Fluorinated surfactants have scarcely been explored for the direct extraction of proteins from membranes because fluorination is believed to abrogate detergency. However, we have recently shown that a commercially available fluorinated surfactant readily solubilizes lipid membranes, thereby suggesting that fluorination per se does not interfere with detergent activity. In this work, we developed new fluorinated surfactants that exhibit detergency in terms of both lipid-vesicle solubilization and membrane-protein extraction. The compounds made and tested contain two glucose moieties as polar headgroup, a hydrogenated thioether linker, and a perfluorinated alkyl tail with either 4, 6, or 8 carbon atoms. The physicochemical properties of the micelles formed by the three fluorinated surfactants were evaluated by NMR spectroscopy, surface tensiometry, isothermal titration calorimetry, dynamic light scattering, small-angle X-ray scattering, and analytical ultracentrifugation. At 25 °C, micellization was mainly entropy-driven, and the CMC values were found to decrease with chain length of the fluorinated tail, whereas the aggregation number increased with chain length. Remarkably, all three surfactants were found to solubilize lipid vesicles and extract a broad range of proteins from Escherichia coli membranes. These findings demonstrate, for the first time, that nonionic fluorinated surfactants could be further exploited for the direct extraction and solubilization of membrane proteins.
Published: 2018

20. Role of Phosphorylation in Moesin Interactions with PIP2-Containing Biomimetic Membranes

Author: Christine Ebel, Aline Le Roy, Quentin Lubart, Mathieu Kowalski, Hélène Vitet, Catherine Picart, Fabien Dalonneau, Marianne Weidenhaupt, Morgane Lourdin, Laboratoire des matériaux et du génie physique (LMGP ), Institut National Polytechnique de Grenoble (INPG)-Institut polytechnique de Grenoble - Grenoble Institute of Technology (Grenoble INP )-Institut de Chimie du CNRS (INC)-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), Département de Chimie Moléculaire (DCM), Institut de Chimie du CNRS (INC)-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 )-Institut National Polytechnique de Grenoble (INPG)-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: 0301 basic medicine, [SDV.BBM.BS]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Structural Biology [q-bio.BM], 030102 biochemistry & molecular biology, Chemistry, Viral budding, Moesin, Biophysics, macromolecular substances, Plasma protein binding, 03 medical and health sciences, 030104 developmental biology, Ezrin, Radixin, Microtubule, Phosphorylation, lipids (amino acids, peptides, and proteins), Cytoskeleton
Abstract: International audience; Moesin, a protein of the ezrin, radixin, and moesin family, which links the plasma membrane to the cytoskeleton, is involved in multiple physiological and pathological processes, including viral budding and infection. Its interaction with the plasma membrane occurs via a key phosphoinositide, the phosphatidyl(4,5)inositol-bisphosphate (PIP2), and phosphorylation of residue T558, which has been shown to contribute, in cellulo, to a conformationally open protein. We study the impact of a double phosphomimetic mutation of moesin (T235D, T558D), which mimics the phosphorylation state of the protein, on protein/PIP2/microtubule interactions. Analytical ultracentrifugation in the micromolar range showed moesin in the monomer and dimer forms, with wild-type (WT) moesin containing a slightly larger fraction (∼30%) of dimers than DD moesin (10-20%). Only DD moesin was responsive to PIP2 in its micellar form. Quantitative cosedimentation assays using large unilamellar vesicles and quartz crystal microbalance on supported lipid bilayers containing PIP2 reveal a specific cooperative interaction for DD moesin with an ability to bind two PIP2 molecules simultaneously, whereas WT moesin was able to bind only one. In addition, DD moesin could subsequently interact with microtubules, whereas WT moesin was unable to do so. Altogether, our results point to an important role of these two phosphorylation sites in the opening of moesin: since DD moesin is intrinsically in a more open conformation than WT moesin, this intermolecular interaction is reinforced by its binding to PIP2. We also highlight important differences between moesin and ezrin, which appear to be finely regulated and to exhibit distinct molecular behaviors.
Published: 2018

21. Assemblies of lauryl maltose neopentyl glycol (LMNG) and LMNG-solubilized membrane proteins

Author: Jérôme Dupuy, Anne L. Martel, Isabelle Petit-Hartlein, Charles-Adrien Arnaud, Michel Thépaut, Franck Fieschi, Christine Hajjar, Cécile Breyton, Lionel Porcar, Waqas Javed, Jean-Michel Jault, Christine Ebel, Aline Le Roy, Cédric Orelle, Annelise Vermot, Institut de biologie structurale (IBS - UMR 5075), Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Grenoble Alpes (UGA)-Centre National de la Recherche Scientifique (CNRS), Institut Laue-Langevin (ILL), ILL, 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 structurale (IBS - UMR 5075 ), 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), 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), ANR-17-CE11-0013,BANDIT,d'une Nox bactérienne à la conception d'outil de criblage(2017), and ANR-16-CE92-0001,FLUOR,Tensioactifs Fluorés pour l'Étude de Protéines Membranaires(2016)
Subjects: Detergent, Size-exclusion chromatography, Biophysics, 010402 general chemistry, 01 natural sciences, Biochemistry, Neopentyl glycol, Micelle, Rods, Glycols, 03 medical and health sciences, chemistry.chemical_compound, Membrane proteins, Centrifugation, Particle Size, Maltose, Micelles, 030304 developmental biology, 0303 health sciences, Molecular Structure, biology, [SDV.BBM.BS]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Structural Biology [q-bio.BM], Bacteriorhodopsin, Cell Biology, 0104 chemical sciences, Kinetics, [SDV.BBM.BS]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biomolecules [q-bio.BM], Solubility, Membrane protein, chemistry, LMNG, biology.protein, Homogeneity, Protein quaternary structure, Bacterial outer membrane, Crystallization, Hydrophobic and Hydrophilic Interactions
Abstract: International audience; Laurylmaltose neopentylglycol (LMNG) bears two linked hydrophobic chains of equal length and two hydrophilic maltoside groups. It arouses a strong interest in the field of membrane protein biochemistry, since it was shown to efficiently solubilize and stabilize membrane proteins often better than the commonly used dodecylmaltopyranoside (DDM), and to allow structure determination of some challenging membrane proteins. However, LMNG was described to form large micelles, which could be unfavorable for structural purposes. We thus investigated its auto-assemblies and the association state of different membrane proteins solubilized in LMNG by analytical ultracentrifugation, size exclusion chromatography coupled to light scattering, centrifugation on sucrose gradient and/or small angle scattering. At high concentrations (in the mM range), LMNG forms long rods, and it stabilized the membrane proteins investigated herein, i.e. a bacterial multidrug transporter, BmrA; a prokaryotic analogous of the eukaryotic NADPH oxidases, SpNOX; an E. coli outer membrane transporter, FhuA; and the halobacterial bacteriorhodopsin, bR. BmrA, in the Apo and the vanadate-inhibited forms showed reduced kinetics of limited proteolysis in LMNG compared to DDM. Both SpNOX and BmrA display an increased specific activity in LMNG compared to DDM. The four proteins form LMNG complexes with their usual quaternary structure and with usual amount of bound detergent. No heterogeneous complexes related to the large micelle size of LMNG alone were observed. In conditions where LMNG forms assemblies of large size, FhuA crystals diffracting to 4.0 Å were obtained by vapor diffusion. LMNG large micelle size thus does not preclude membrane protein homogeneity and crystallization.
Published: 2019

22. Structure of an H1-Bound 6-Nucleosome Array Reveals an Untwisted Two-Start Chromatin Fiber Conformation

Author: Ali Hamiche, Manu S. Shukla, Jan Bednar, Hervé Menoni, Isabel Garcia-Saez, Dimitrios A. Skoufias, Ramachandran Boopathi, Marjolaine Noirclerc-Savoye, Carlo Petosa, Stefan Dimitrov, Aline Le Roy, Dimitar Angelov, Lama Soueidan, 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 biologie et modélisation de la cellule (LBMC UMR 5239), École normale supérieure de Lyon (ENS de Lyon)-Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Institute for Advanced Biosciences / Institut pour l'Avancée des Biosciences (Grenoble) (IAB), Centre Hospitalier Universitaire [Grenoble] (CHU)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Etablissement français du sang - Auvergne-Rhône-Alpes (EFS)-Centre National de la Recherche Scientifique (CNRS)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019]), First Faculty of Medicine Charles University [Prague], Institut de Génétique et de Biologie Moléculaire et Cellulaire (IGBMC), Université de Strasbourg (UNISTRA)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Institut d'oncologie/développement Albert Bonniot de Grenoble (INSERM U823), Université Joseph Fourier - Grenoble 1 (UJF)-CHU Grenoble-EFS-Institut National de la Santé et de la Recherche Médicale (INSERM), ANR-16-CE12-0013,EpiVarZ,Analyses intégratives du variant d'histone H2A.Z au niveau moléculaire, fonctionnel et structural(2016), ANR-18-CE12-0010,ZFun,Fonctions biologiques de l'histone variant H2A.Z(2018), ANR-17-CE11-0019,Chrom3D,Le rôle de l'histone H1 et de CENP-C dans la structure et les propriétés épigénétiques de la chromatine(2017), Département de Génomique Fonctionnelle et Cancer, Laboratoire de Biologie Moléculaire de la Cellule (LBMC), Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-École normale supérieure - Lyon (ENS Lyon)-Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon, 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), École normale supérieure - Lyon (ENS Lyon)-Université Claude Bernard Lyon 1 (UCBL), and Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Université de Strasbourg (UNISTRA)
Subjects: 0301 basic medicine, Models, Molecular, Protein Conformation, alpha-Helical, Genetic Vectors, Stacking, Gene Expression, Biology, Crystallography, X-Ray, histone H1, nucleosome array, Histones, 03 medical and health sciences, Xenopus laevis, Prophase, Histone H1, 30-nm fiber, Escherichia coli, Nucleosome, Animals, Humans, Protein Interaction Domains and Motifs, [SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology, Fiber, Cloning, Molecular, crystallography, Molecular Biology, Chromatin Fiber, Binding Sites, Nucleosome Assembly Protein 1, 030102 biochemistry & molecular biology, Hydroxyl Radical, Cryoelectron Microscopy, Osmolar Concentration, Cell Biology, DNA, Recombinant Proteins, Chromatin, Nucleosomes, [SDV.BBM.BS]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biomolecules [q-bio.BM], linker histone, 030104 developmental biology, Helix, Biophysics, chromatin, cryo-EM, Protein Conformation, beta-Strand, Protein Multimerization, Protein Binding
Abstract: International audience; Chromatin adopts a diversity of regular and irregular fiber structures in vitro and in vivo. However, how an array of nucleosomes folds into and switches between different fiber conformations is poorly understood. We report the 9.7 Å resolution crystal structure of a 6-nucleosome array bound to linker histone H1 determined under ionic conditions that favor incomplete chromatin condensation. The structure reveals a flat two-start helix with uniform nucleosomal stacking interfaces and a nucleosome packing density that is only half that of a twisted 30-nm fiber. Hydroxyl radical footprinting indicates that H1 binds the array in an on-dyad configuration resembling that observed for mononucleosomes. Biophysical, cryo-EM, and crosslinking data validate the crystal structure and reveal that a minor change in ionic environment shifts the conformational landscape to a more compact, twisted form. These findings provide insights into the structural plasticity of chromatin and suggest a possible assembly pathway for a 30-nm fiber.
Published: 2018

23. Therapeutic effects of proteoliposomes on X-linked chronic granulomatous disease: proof of concept using macrophages differentiated from patient-specific induced pluripotent stem cells

Author: Sandra Cortès, Marie José Stasia, Jean-Luc Lenormand, Julie Brault, Guillaume Vaganay, Aline Le Roy, Université Grenoble Alpes - UFR Pharmacie (UGA UFRP), Université Grenoble Alpes [2016-2019] (UGA [2016-2019]), Synthelis, 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), Techniques de l'Ingénierie Médicale et de la Complexité - Informatique, Mathématiques et Applications, Grenoble - UMR 5525 (TIMC-IMAG), Institut polytechnique de Grenoble - Grenoble Institute of Technology (Grenoble INP )-VetAgro Sup - Institut national d'enseignement supérieur et de recherche en alimentation, santé animale, sciences agronomiques et de l'environnement (VAS)-Centre National de la Recherche Scientifique (CNRS)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019]), 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, Pharmaceutical Science, chronic granulomatous disease, Granulomatous Disease, Chronic, law.invention, Cell membrane, chemistry.chemical_compound, Chronic granulomatous disease, MESH: Granulomatous Disease, Chronic, International Journal of Nanomedicine, NADPH oxidase complex, law, protein therapy, Drug Discovery, MESH: Phagocytosis, Candida, Original Research, Oxidase test, NADPH oxidase, [SDV.BBM.BS]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Structural Biology [q-bio.BM], biology, MESH: Reactive Oxygen Species, Cell Differentiation, Genetic Diseases, X-Linked, General Medicine, MESH: Protein Subunits, 3. Good health, medicine.anatomical_structure, Child, Preschool, Recombinant DNA, Nicotinamide adenine dinucleotide phosphate, MESH: Proteolipids, MESH: Cell Differentiation, induced pluripotent stem cells, Proteolipids, Biophysics, Bioengineering, MESH: Induced Pluripotent Stem Cells, MESH: NADPH Oxidases, Biomaterials, 03 medical and health sciences, Phagocytosis, proteoliposomes, MESH: Candida, MESH: Genetic Diseases, X-Linked, medicine, Humans, MESH: Humans, Macrophages, MESH: Child, Preschool, Organic Chemistry, Cell Membrane, MESH: Macrophages, NADPH Oxidases, medicine.disease, Molecular biology, Protein Subunits, 030104 developmental biology, chemistry, Membrane protein, Immunology, biology.protein, Reactive Oxygen Species, MESH: Cell Membrane
Abstract: Julie Brault,1,2 Guillaume Vaganay,3 Aline Le Roy,4–6 Jean-Luc Lenormand,1 Sandra Cortes,3 Marie José Stasia1,2 1UMR CNRS 5525, University of Grenoble Alpes, Grenoble, France; 2CGD Diagnosis and Research Centre, University Hospital Centre of Grenoble Alpes, Grenoble, France; 3Synthelis SAS, La Tronche, France; 4IBS, University of Grenoble Alpes, Grenoble, France; 5CNRS, IBS, University Grenoble Alpes, Grenoble, France; 6CEA, IBS, University of Grenoble Alpes, Grenoble, France Abstract: Chronic granulomatous disease (CGD) is a rare inherited immunodeficiency due to dysfunction of the phagocytic nicotinamide adenine dinucleotide phosphate (NADPH) oxidase complex leading to severe and recurrent infections in early childhood. The main genetic form is the X-linked CGD leading to the absence of cytochrome b558 composed of NOX2 and p22phox, the membrane partners of the NADPH oxidase complex. The first cause of death of CGD patients is pulmonary infections. Recombinant proteoliposome-based therapy is an emerging and innovative approach for membrane protein delivery, which could be an alternative local, targeted treatment to fight lung infections in CGD patients. We developed an enzyme therapy using recombinant NOX2/p22phox liposomes to supply the NADPH oxidase activity in X0-linked CGD (X0-CGD) macrophages. Using an optimized prokaryotic cell-free protein synthesis system, a recombinant cytochrome b558 containing functional hemes was produced and directly inserted into the lipid bilayer of specific liposomes. The size of the NOX2/p22phox liposomes was estimated to be around 700nm. These proteoliposomes were able to generate reactive oxygen species (ROS) in an activated reconstituted cell-free NADPH oxidase activation assay in the presence of recombinant p47phox, p67phox and Rac, the cytosolic components of the NADPH oxidase complex. Furthermore, using flow cytometry and fluorescence microscopy, we demonstrated that cytochrome b558 was successfully delivered to the plasma membrane of X0-CGD-induced pluripotent stem cell (iPSC)-derived macrophages. In addition, NADPH oxidase activity was restored in X0-CGD iPSC-derived macrophages treated with NOX2/p22phox liposomes for 8h without any toxicity. In conclusion, we confirmed that proteoliposomes provide a new promising technology for the delivery of functional proteins to the membrane of targeted cells. This efficient liposomal enzyme replacement therapy will be useful for future treatment of pulmonary infections in CGD patients refractory to conventional anti-infectious treatments. Keywords: protein therapy, proteoliposomes, chronic granulomatous disease, NADPH oxidase, induced pluripotent stem cells, macrophages&nbsp
Published: 2017

24. MIB–MIP is a mycoplasma system that captures and cleaves immunoglobulin G

Author: Alain Blanchard, Laurent Coquet, Pascal Sirand-Pugnet, Joerg Jores, Yonathan Arfi, Sanjay Vashee, Stéphane Claverol, Carmelo Di Primo, Laetitia Minder, Aline Le Roy, Christine Ebel, Biologie du fruit et pathologie (BFP), Université Bordeaux Segalen - Bordeaux 2-Institut National de la Recherche Agronomique (INRA)-Université Sciences et Technologies - Bordeaux 1, Institut Européen de Chimie et de Biologie, Institut de biologie structurale (IBS - UMR 5075), Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Grenoble Alpes (UGA)-Centre National de la Recherche Scientifique (CNRS), Polymères Biopolymères Surfaces (PBS), Université de Rouen Normandie (UNIROUEN), Normandie Université (NU)-Normandie Université (NU)-Centre National de la Recherche Scientifique (CNRS)-Institut national des sciences appliquées Rouen Normandie (INSA Rouen Normandie), Normandie Université (NU)-Institut National des Sciences Appliquées (INSA)-Institut National des Sciences Appliquées (INSA), Centre Génomique Fonctionnelle Bordeaux [Bordeaux] (CGFB), Institut Polytechnique de Bordeaux-Université de Bordeaux Ségalen [Bordeaux 2], J Craig Venter Institute, International Livestock Research Institute, Université Bordeaux Segalen - Bordeaux 2-Institut National de la Recherche Agronomique (INRA)-Université Sciences et Technologies - Bordeaux 1 (UB), Institut de biologie structurale (IBS - UMR 5075 ), Centre National de la Recherche Scientifique (CNRS)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA), Normandie Université (NU)-Normandie Université (NU)-Institut national des sciences appliquées Rouen Normandie (INSA Rouen Normandie), Institut National des Sciences Appliquées (INSA)-Normandie Université (NU)-Institut National des Sciences Appliquées (INSA)-Institut Normand de Chimie Moléculaire Médicinale et Macromoléculaire (INC3M), Université de Caen Normandie (UNICAEN), Normandie Université (NU)-Normandie Université (NU)-École Nationale Supérieure d'Ingénieurs de Caen (ENSICAEN), Normandie Université (NU)-Université Le Havre Normandie (ULH), Normandie Université (NU)-Université de Rouen Normandie (UNIROUEN), Normandie Université (NU)-Institut national des sciences appliquées Rouen Normandie (INSA Rouen Normandie), Institut National des Sciences Appliquées (INSA)-Normandie Université (NU)-Institut National des Sciences Appliquées (INSA)-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS)-Université de Caen Normandie (UNICAEN), Normandie Université (NU)-École Nationale Supérieure d'Ingénieurs de Caen (ENSICAEN), Normandie Université (NU)-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS)-Centre National de la Recherche Scientifique (CNRS), J. Craig Venter Institute, Université Sciences et Technologies - Bordeaux 1-Université Bordeaux Segalen - Bordeaux 2-Institut National de la Recherche Agronomique (INRA), Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Centre National de la Recherche Scientifique (CNRS), Institut national des sciences appliquées Rouen Normandie (INSA Rouen Normandie), Institut National des Sciences Appliquées (INSA)-Normandie Université (NU)-Institut National des Sciences Appliquées (INSA)-Normandie Université (NU)-Institut de Chimie du CNRS (INC)-Institut Normand de Chimie Moléculaire Médicinale et Macromoléculaire (INC3M), Institut de Chimie du CNRS (INC)-École Nationale Supérieure d'Ingénieurs de Caen (ENSICAEN), Institut National des Sciences Appliquées (INSA)-Normandie Université (NU)-Institut National des Sciences Appliquées (INSA)-Université Le Havre Normandie (ULH), Normandie Université (NU)-Centre National de la Recherche Scientifique (CNRS)-Université de Caen Normandie (UNICAEN), Normandie Université (NU)-Centre National de la Recherche Scientifique (CNRS), and Université Sciences et Technologies - Bordeaux 1-Institut National de la Recherche Agronomique (INRA)-Université Bordeaux Segalen - Bordeaux 2
Subjects: 0301 basic medicine, medicine.medical_treatment, 030106 microbiology, mycoplasmas, medicine.disease_cause, Immunoglobulin G, Microbiology, 03 medical and health sciences, Immune system, Bacterial Proteins, medicine, Gene, neoplasms, Serine protease, Multidisciplinary, Protease, biology, [SDV.BBM.BS]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Structural Biology [q-bio.BM], Mycoplasma mycoides, protease, Mycoplasma, Biological Sciences, biology.organism_classification, Virology, 3. Good health, [SDV.BBM.BS]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biomolecules [q-bio.BM], 030104 developmental biology, Multiprotein Complexes, biology.protein, Antibody, immunoglobulin, Protein Binding
Abstract: International audience; Mycoplasmas are "minimal" bacteria able to infect humans, wildlife, and a large number of economically important livestock species. Mycoplasma infections include a spectrum of clinical manifestations ranging from simple fever to fulminant inflammatory diseases with high mortality rates. These infections are mostly chronic, suggesting that mycoplasmas have developed means to evade the host immune response. Here we present and functionally characterize a two-protein system from Mycoplasma mycoides subspecies capri that is involved in the capture and cleavage of IgG. The first component, Mycoplasma Ig binding protein (MIB), is an 83-kDa protein that is able to tightly bind to the Fv region of a wide range of IgG. The second component, Mycoplasma Ig protease (MIP), is a 97-kDa serine protease that is able to cleave off the VH domain of IgG. We demonstrate that MIB is necessary for the proteolytic activity of MIP. Cleavage of IgG requires a sequential interaction of the different partners of the system: first MIB captures the IgG, and then MIP is recruited to the MIB-IgG complex, enabling protease activity. MIB and MIP are encoded by two genes organized in tandem, with homologs found in the majority of pathogenic mycoplasmas and often in multiple copies. Phylogenetic studies suggest that genes encoding the MIB-MIP system are specific to mycoplasmas and have been disseminated by horizontal gene transfer. These results highlight an original and complex system targeting the host immunoglobulins, playing a potentially key role in the immunity evasion by mycoplasmas.
Published: 2016

25. A multilaboratory comparison of calibration accuracy and the performance of external references in analytical ultracentrifugation

Author: Tomas Obsil, Helmut Cölfen, Anna Modrak-Wójcik, José García de la Torre, Caitlin I. Stoddard, Ute Curth, Thomas M. Laue, Geeta J. Narlikar, Szu Huan Wang, Carlos Alfonso, Carlos R. Escalante, Jia Ma, Gali Prag, Masanori Noda, Katherine Stott, Klaus Richter, Carrie A. May, Peter Schuck, Fumio Arisaka, Edward Eisenstein, Stephen J. Perkins, Ilan Attali, John P. Sumida, Catherine T. Chaton, Erby E. Perdue, Davis Jose, David Staunton, Matthew A. Perugini, Jin Ku Park, Amanda Nourse, Peter E. Prevelige, Craig L. Peterson, Norbert Mücke, Satoru Unzai, Peter D. Pawelek, Jayesh Gor, Arne C. Rufer, Rose Rosenberg, Hauke Lilie, Martin G. Peverelli, Yee-Foong Mok, Martin Wolff, Jacinta M. Wubben, Luitgard Nagel-Steger, Peter H. von Hippel, Karolin Luger, Edward Wright, Holger M. Strauss, Lenka Rezabkova, Agnieszka Bzowska, Aline Le Roy, Steven E. Weitzel, Jonathan B. Chaires, Elena Krayukhina, Donald F. Becker, Alexandra S. Solovyova, Alison E. Ringel, Chad A. Brautigam, Dalibor Kosek, David J. Scott, Kimberly A. Crowley, Cynthia Wolberger, Soon-Jong Kim, Adam G. Larson, Leslie E. Eisele, Susumu Uchiyama, Gregory J. Bedwell, Javier Seravalli, Bertrand Raynal, Ron M. Finn, Hyewon Kwon, Tabot M. D. Besong, Marina Bakhtina, Ahmet Bekdemir, Shu-Chuan Jao, Bashkim Kokona, Catherine Birck, Roman H. Szczepanowski, Andrew B. Herr, Christine Wandrey, Stephen E. Harding, Daniel D Krzizike, David L. Bain, José G. Hernández Cifre, Grzegorz Piszczek, Renjie Song, Stephan Uebel, Tina Daviter, Damien Hall, Debra M. Eckert, Patrick England, Ingrid Tessmer, Eric Kusznir, Ernest L. Maynard, William L. Dean, Werner Streicher, Christine Ebel, Anna Vitlin Gruber, Beata Wielgus-Kutrowska, Rodolfo Ghirlando, Yu Sung Wu, Ana I. Díez, Olwyn Byron, Robert Fairman, William Brennerman, Henning Gustafsson, Richard Stefan Isaac, Wolfgang Fischle, Elizabeth E. Howell, Ronald T. Toth, Sarah G. Swygert, Ashutosh Tripathy, Arthur J. Rowe, Chad K. Park, Jeffrey A. Fagan, Keith D. Connaghan, Huaying Zhao, Jack A. Kornblatt, Andrew P. Leech, Juan Román Luque-Ortega, BGI Shenzhen, Laboratoire d'Informatique pour l'Entreprise et les Systèmes de Production (LIESP), Université Lumière - Lyon 2 (UL2)-École Centrale de Lyon (ECL), Université de Lyon-Université de Lyon-Université Claude Bernard Lyon 1 (UCBL), 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), Innovative Computing Laboratory [Knoxville] (ICL), The University of Tennessee [Knoxville], Institut de Génétique et de Biologie Moléculaire et Cellulaire (IGBMC), Université de Strasbourg (UNISTRA)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), EDF R&D (EDF R&D), EDF (EDF), Physikalische Chemie, Universität Konstanz, Photonic Crystal Fibres group, University of Bath [Bath], Institut de biologie structurale (IBS - UMR 5075), Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Grenoble Alpes (UGA)-Centre National de la Recherche Scientifique (CNRS), Observatoire Astronomique de l'Université de Genève (ObsGE), Université de Genève (UNIGE), Biophysique des macromolécules et leurs interactions, Institut Pasteur [Paris]-Centre National de la Recherche Scientifique (CNRS), Laboratory of Chromatin Biochemistry [Göttingen], Max Planck Institute for Biophysical Chemistry (MPI-BPC), Max-Planck-Gesellschaft-Max-Planck-Gesellschaft, Stellenbosch University, Department of Biochemistry, The Centre for Ornithology, School of Biosciences, College of Life and Environmental Sciences, University of Alabama at Birmingham [ Birmingham] (UAB), Département de Chimie Moléculaire - Chimie Inorganique Redox Biomimétique (DCM - CIRE), Université Joseph Fourier - Grenoble 1 (UJF)-Institut de Chimie Moléculaire de Grenoble (ICMG)-Centre National de la Recherche Scientifique (CNRS)-Université Grenoble Alpes (UGA), Hormone Research Center, Chonnam National University, Anglophonie : Communautés, Ecritures (ACE ), Université de Rennes 2 (UR2), Université de Rennes (UNIV-RENNES)-Université de Rennes (UNIV-RENNES), Institut de Chimie des Milieux et Matériaux de Poitiers (IC2MP), Université de Poitiers-Centre National de la Recherche Scientifique (CNRS), Department of Animal Science, Iowa State University (ISU), Université Laval, School of Mathematics [Bristol], University of Bristol [Bristol], Chimie et Biocatalyse, Laboratoire de Physique des Solides (LPS), Université Paris-Sud - Paris 11 (UP11)-Centre National de la Recherche Scientifique (CNRS), Department of marine Ecology, University of Gothenburg (GU), Laboratoire de physique de la matière condensée (LPMC), École polytechnique (X)-Centre National de la Recherche Scientifique (CNRS), Pharma Research Discovery Chemistry, F. Hoffmann-La Roche AG, University of KwaZulu-Natal (UKZN), Institute for Cell and Molecular Biosciences, Newcastle University [Newcastle], Changzhou University, Institut für Geologie und Paläontologie, Westfälische Wilhelms-Universität Münster (WWU), Eindhoven University of Technology [Eindhoven] (TU/e), Department of Biochemistry and Biophysics, UNC Macromolecular Interactions Facility, University of North Carolina [Chapel Hill] (UNC), University of North Carolina System (UNC)-University of North Carolina System (UNC), Institute of Botany, University of Innsbruck, NSF Center for EUV Science and Technology, NSF, Sustainable Energy Economics, Department of mathematics and MOE-LSC, Shanghai Jiao Tong University [Shanghai], Science et Technologie du Lait et de l'Oeuf (STLO), Institut National de la Recherche Agronomique (INRA)-AGROCAMPUS OUEST, Platforms of the Grenoble Instruct centre (ISBG, CNRS-CEA-UJF-EMBL), ANR-10-INBS-0005-02,FRISBI,Infrastructure Française pour la Biologie Structurale Intégrée(2010), ANR-10- LABX-49-01,Labex GRAL,Labex GRAL, Beijing Genomics Institute [Shenzhen] (BGI), Institut National des Sciences Appliquées de Lyon (INSA Lyon), Institut National des Sciences Appliquées (INSA)-Université de Lyon-Institut National des Sciences Appliquées (INSA)-Université de Lyon-Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université Lumière - Lyon 2 (UL2)-École Centrale de Lyon (ECL), Université de Lyon, Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Université de Strasbourg (UNISTRA), Institut de biologie structurale (IBS - UMR 5075 ), 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), Département de Chimie Moléculaire (DCM), Centre National de la Recherche Scientifique (CNRS)-Institut de Chimie du CNRS (INC)-Université Joseph Fourier - Grenoble 1 (UJF)-Centre National de la Recherche Scientifique (CNRS)-Institut de Chimie du CNRS (INC)-Université Joseph Fourier - Grenoble 1 (UJF), Institut national des sciences de l'Univers (INSU - CNRS)-Centre National de la Recherche Scientifique (CNRS)-Université de Poitiers-Institut de Chimie du CNRS (INC), Université Laval [Québec] (ULaval), Centre National de la Recherche Scientifique (CNRS)-Université Paris-Sud - Paris 11 (UP11), Institut national d'enseignement supérieur pour l'agriculture, l'alimentation et l'environnement (Institut Agro)-Institut national d'enseignement supérieur pour l'agriculture, l'alimentation et l'environnement (Institut Agro), ANR-10-INBS-0005,FRISBI,Infrastructure Française pour la Biologie Structurale Intégrée(2010), ANR-10-LABX-0049,GRAL,Grenoble Alliance for Integrated Structural Cell Biology(2010), Stott, Katherine [0000-0002-4014-1188], Apollo - University of Cambridge Repository, Centre National de la Recherche Scientifique (CNRS)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA), Université de Genève = University of Geneva (UNIGE), Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS), Département de Chimie Moléculaire - Chimie Inorganique Redox (DCM - CIRE), Université Joseph Fourier - Grenoble 1 (UJF)-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS)-Université Joseph Fourier - Grenoble 1 (UJF)-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS), Université de Poitiers-Institut national des sciences de l'Univers (INSU - CNRS)-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS), F. Hoffmann-La Roche [Basel], University of KwaZulu-Natal [Durban, Afrique du Sud] (UKZN), Westfälische Wilhelms-Universität Münster = University of Münster (WWU), and Leopold Franzens Universität Innsbruck - University of Innsbruck
Subjects: Accuracy and precision, Analytical chemistry, lcsh:Medicine, Magnification, 02 engineering and technology, bcs, Standard deviation, Analytical Ultracentrifugation, 03 medical and health sciences, Range (statistics), Calibration, ddc:610, lcsh:Science, 030304 developmental biology, Mathematics, 0303 health sciences, Field flow fractionation, Multidisciplinary, [SDV.BBM.BS]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Structural Biology [q-bio.BM], lcsh:R, Reproducibility of Results, 021001 nanoscience & nanotechnology, 3. Good health, ddc, Data set, [SDV.BBM.BS]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biomolecules [q-bio.BM], ddc:540, lcsh:Q, 0210 nano-technology, Ultracentrifugation, Research Article
Abstract: 30 p.-14 fig. Zhao, Huaying, et alt., Analytical ultracentrifugation (AUC) is a first principles based method to determine absolute sedimentation coefficients and buoyant molar masses of macromolecules and their complexes, reporting on their size and shape in free solution. The purpose of this multi-laboratory study was to establish the precision and accuracy of basic data dimensions in AUC and validate previously proposed calibration techniques. Three kits of AUC cell assemblies containing radial and temperature calibration tools and a bovine serum albumin (BSA) reference sample were shared among 67 laboratories, generating 129 comprehensive data sets. These allowed for an assessment of many parameters of instrument performance, including accuracy of the reported scan time after the start of centrifugation, the accuracy of the temperature calibration, and the accuracy of the radial magnification. The range of sedimentation coefficients obtained for BSA monomer in different instruments and using different optical systems was from 3.655 S to 4.949 S, with a mean and standard deviation of (4.304 ± 0.188) S (4.4%). After the combined application of correction factors derived from the external calibration references for elapsed time, scan velocity, temperature, and radial magnification, the range of s-values was reduced 7-fold with a mean of 4.325 S and a 6-fold reduced standard deviation of ± 0.030 S (0.7%). In addition, the large data set provided an opportunity to determine the instrument-to-instrument variation of the absolute radial positions reported in the scan files, the precision of photometric or refractometric signal magnitudes, and the precision of the calculated apparent molar mass of BSA monomer and the fraction of BSA dimers. These results highlight the necessity and effectiveness of independent calibration of basic AUC data dimensions for reliable quantitative studies.
Published: 2015

26. AUC and Small-Angle Scattering for Membrane Proteins

Author: Cécile Breyton, Peter Schuck, Béatrice Schaack, Christine Ebel, Aline Le Roy, and Kai Wang
Subjects: Analytical Ultracentrifugation, Chromatography, Membrane protein, Small-angle X-ray scattering, Chemistry, Amphiphile, Biophysics, Ultracentrifuge, Lipid bilayer, Small-angle neutron scattering, Macromolecule
Abstract: Analytical ultracentrifugation is a key tool to assess homogeneity of membrane protein samples, to determine protein association state and detergent concentration, and to characterize protein-protein equilibrium. Combining absorbance and interference detections gives information on the amount of the detergent and lipid bound to proteins. Changing the solvent density affects specifically the buoyancy of each of the different components, and can also be used to gain information on particle composition and interaction. We will present the related tools, recently implemented in the softwares Sedphat (sedfitsedphat.nibib.nih.gov/software) and Gussi (http://biophysics.swmed.edu/MBR/software.html), which help to measure the amount of detergent bound to the protein, and ascertain the protein association state within the protein-detergent complex. In addition, fluorescence detection allows focusing specifically on a labeled component within a complex mixture. We present two examples of sedimentation velocity experiments, allowing on one hand to evidence complex formation between an unpurified GFP-labeled protein and a membrane protein, and on the other hand to characterize fluorescent lipid vesicles. Small-angle X-ray and neutron scattering are techniques that give insights into the structure and conformation of macromolecules in solution. However, the detergents used to purify membrane protein are often imperfectly masked due to their amphipathic character. Particular strategies addressing membrane proteins were recently proposed, which are shortly presented.
Published: 2015

27. Unique DC-SIGN Clustering Activity of a Small Glycomimetic: A Lesson for Ligand Design

Author: Ieva Sutkeviciute, José J. Reina, Angela Berzi, Michel Thépaut, Sara Sattin, Aline Le Roy, Javier Rojo, John D. McGeagh, Mario Clerici, Sergei Grudinin, Anna Bernardi, Christine Ebel, Franck Fieschi, Jörg Weiser, 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), Department of Chemistry, Università degli Studi di Milano = University of Milan (UNIMI), Department of Biomedical and Clinical Sciences, Anterio Consult & Research GmbH, Algorithms for Modeling and Simulation of Nanosystems (NANO-D), Inria Grenoble - Rhône-Alpes, Institut National de Recherche en Informatique et en Automatique (Inria)-Institut National de Recherche en Informatique et en Automatique (Inria)-Laboratoire Jean Kuntzmann (LJK), Université Pierre Mendès France - Grenoble 2 (UPMF)-Université Joseph Fourier - Grenoble 1 (UJF)-Institut polytechnique de Grenoble - Grenoble Institute of Technology (Grenoble INP )-Centre National de la Recherche Scientifique (CNRS)-Université Pierre Mendès France - Grenoble 2 (UPMF)-Université Joseph Fourier - Grenoble 1 (UJF)-Institut polytechnique de Grenoble - Grenoble Institute of Technology (Grenoble INP )-Centre National de la Recherche Scientifique (CNRS), Glycosystems Laboratory, Instituto de Investigaciones Químicas (IIQ), Universidad de Sevilla / University of Sevilla-Consejo Superior de Investigaciones Científicas [Madrid] (CSIC)-Universidad de Sevilla / University of Sevilla-Consejo Superior de Investigaciones Científicas [Madrid] (CSIC), Department of Physiopatology and Transplantation, University of Milan (DEPT), Fondazione Don Carlo Gnocchi, Marie Curie ITN FP7 project CARMUSYS (PITN-GA-2008-213592), ANR-11-MONU-0006,PEPSI,Expansions Polynomiales de Structures de Protéines et Interactions(2011), Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Centre National de la Recherche Scientifique (CNRS), University of Milan, Centre National de la Recherche Scientifique (CNRS)-Institut polytechnique de Grenoble - Grenoble Institute of Technology (Grenoble INP )-Université Joseph Fourier - Grenoble 1 (UJF)-Université Pierre Mendès France - Grenoble 2 (UPMF)-Centre National de la Recherche Scientifique (CNRS)-Institut polytechnique de Grenoble - Grenoble Institute of Technology (Grenoble INP )-Université Joseph Fourier - Grenoble 1 (UJF)-Université Pierre Mendès France - Grenoble 2 (UPMF), Consejo Superior de Investigaciones Científicas [Madrid] (CSIC)-Universidad de Sevilla-Consejo Superior de Investigaciones Científicas [Madrid] (CSIC)-Universidad de Sevilla, European Commission (EC), and Agence Nationale de la Recherche. France
Subjects: ligand design, C-Type Lectin, Stereochemistry, protein-protein interactions, HIV Infections, Receptors, Cell Surface, 010402 general chemistry, Crystallography, X-Ray, 01 natural sciences, Biochemistry, Clustering, Protein–protein interaction, 03 medical and health sciences, Protein structure, Glycomimetic, C-type lectin, Biomimetics, Potency, Humans, Lectins, C-Type, Binding site, Isothermal microcalorimetry, 030304 developmental biology, 0303 health sciences, Binding Sites, biology, Molecular Structure, [SDV.BBM.BS]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Structural Biology [q-bio.BM], Ligand, Chemistry, HIV, General Medicine, 3. Good health, 0104 chemical sciences, Protein Structure, Tertiary, molecular modelling, DC-SIGN, Drug Design, Mannosides, Glycomimetics, biology.protein, Biophysics, HIV-1, Analytical ultracentrifugation, Molecular Medicine, Thermodynamics, pharmacology, Cell Adhesion Molecules, Ultracentrifugation
Abstract: DC-SIGN is a dendritic cell-specific C-type lectin receptor that recognizes highly glycosylated ligands expressed on the surface of various pathogens. This receptor plays an important role in the early stages of many viral infections, including HIV, which makes it an interesting therapeutic target. Glycomimetic compounds are good drug candidates for DC-SIGN inhibition due to their high solubility, resistance to glycosidases, and nontoxicity. We studied the structural properties of the interaction of the tetrameric DC-SIGN extracellular domain (ECD), with two glycomimetic antagonists, a pseudomannobioside (1) and a linear pseudomannotrioside (2). Though the inhibitory potency of 2, as measured by SPR competition experiments, was 1 order of magnitude higher than that of 1, crystal structures of the complexes within the DC-SIGN carbohydrate recognition domain showed the same binding mode for both compounds. Moreover, when conjugated to multivalent scaffolds, the inhibitory potencies of these compounds became uniform. Combining isothermal titration microcalorimetry, analytical ultracentrifugation, and dynamic light scattering techniques to study DC-SIGN ECD interaction with these glycomimetics revealed that 2 is able, without any multivalent presentation, to cluster DC-SIGN tetramers leading to an artificially overestimated inhibitory potency. The use of multivalent scaffolds presenting 1 or 2 in HIV trans-infection inhibition assay confirms the loss of potency of 2 upon conjugation and the equal efficacy of chemically simpler compound 1. This study documents a unique case where, among two active compounds chemically derived, the compound with the lower apparent activity is the optimal lead for further drug development
Published: 2014

28. Analytical Ultracentrifugation and Size-Exclusion Chromatography Coupled with Light Scattering for the Characterization of Membrane Proteins in Solution

Author: Christine Ebel, Aline Le Roy, Cécile Breyton, 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), Isabelle Mus-Veteau, Thomas, Frank, Centre National de la Recherche Scientifique (CNRS)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), and Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)
Subjects: 0303 health sciences, Chromatography, Molar mass, [SDV.BBM.BS]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Structural Biology [q-bio.BM], 010304 chemical physics, [SDV.BBM.BS] Life Sciences [q-bio]/Biochemistry, Molecular Biology/Structural Biology [q-bio.BM], Chemistry, Size-exclusion chromatography, Analytical chemistry, 01 natural sciences, Light scattering, Absorbance, Analytical Ultracentrifugation, 03 medical and health sciences, Membrane protein, Dynamic light scattering, 0103 physical sciences, Refractive index, 030304 developmental biology
Abstract: International audience; Analytical ultracentrifugation (AUC) sedimentation velocity (SV) with absorbance and interference detection and size-exclusion chromatography coupled with static and dynamic light scattering, absorbance, and refractive index detections (SEC/MALS) are two techniques that combine separation and analysis, in an absolute manner, of the mass and size of macromolecules in solution. We present here how they can be applied to the study of membrane proteins. We describe briefly the principles of the species separation, what the detection systems used measures, some theoretical background, the steps for data analysis with the example of the outer membrane protein FhuA, and emphasize the complementarity between these two techniques.
Published: 2014

29. Sedimentation Velocity Analytical Ultracentrifugation in Hydrogenated and Deuterated Solvents for the Characterization of Membrane Proteins

Author: Christine Ebel, Jonathan Sarwan, Jean-Michel Jault, Hugues Nury, Aline Le Roy, and Benjamin Wiseman
Subjects: Analytical Ultracentrifugation, 0303 health sciences, 03 medical and health sciences, Chromatography, Deuterium, Membrane protein, Solubilization, Chemistry, 030303 biophysics, 030304 developmental biology
Abstract: This chapter is a step-by-step protocol for setting up, realizing, and analyzing sedimentation velocity experiments in hydrogenated and deuterated solvents, in the context of the characterization of membrane protein, in terms of homogeneity, association state, and amount of bound detergent, based on a real case study of the membrane protein BmrA solubilized in n-Dodecyl-β-D-Maltopyranoside) detergent.
Published: 2013

30. Characterization of the elongasome core PBP2 : MreC complex of Helicobacter pylori

Author: El Ghachi, Meriem, Matteï, Pierre-Jean, Ecobichon, Chantal, Martins, Alexandre, Hoos, Sylviane, Schmitt, Christine, Colland, Frédéric, Ebel, Christine, Prévost, Marie-Christine, Gabel, Frank, England, Patrick, Dessen, Andréa, Boneca, Ivo G., Biologie et Génétique de la Paroi bactérienne - Biology and Genetics of Bacterial Cell Wall (BGPB), Institut Pasteur [Paris] (IP), Institut National de la Santé et de la Recherche Médicale (INSERM), Institut de biologie structurale et microbiologie (IBSM), Université de la Méditerranée - Aix-Marseille 2-Université Paul Cézanne - Aix-Marseille 3-Université de Provence - Aix-Marseille 1-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Biophysique des Macromolécules et de leurs Interactions, Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS), Microscopie ultrastructurale (plate-forme), Hybrigenics [Paris], Hybrigenics, Meriem El Ghachi was supported by a Roux fellowship (Institut Pasteur), ERC starting Grant (PGNfromSHAPEtoVIR n°202283) and INSERM. Ivo G. Boneca was supported by a Roux fellowship (Institut Pasteur) and a post-doctoral Fellowship from the Fundação de Ciências e Tecnologia, Portugal. Work in the group of IGB was supported by ERC starting grant (PGNfromSHAPEtoVIR n°202283). Work in the Dessen laboratory was supported by the Fondation pour la Recherche Médicale (FRM DEQ20090515390) and the European Commission (LSHM-CT-2004–512138). Pierre-Jean Matteï received a PhD fellowship from the Rhône Alpes region., We would like to acknowledge the technical help of Christian Vanderbergh with the production of the rabbit polyclonal antibodies, Hybrigenics for the Yeast two-hybrid screen, as well as Adam Round (BioSAXS beamline ID14-3, ESRF Grenoble, Partnership for Structural Biology) for help with SAXS data collection and analysis. We thank Aline Le Roy (analytical ultracentrifugation platform, IBS, PSB) for access to and assistance with AUC measurements., and European Project: 202283,EC:FP7:ERC,ERC-2007-StG,PGNFROMSHAPETOVIR(2008)
Subjects: MESH: Penicillin-Binding Proteins, MESH: Molecular Sequence Data, Microbial Viability, Helicobacter pylori, [SDV]Life Sciences [q-bio], Molecular Sequence Data, MESH: Sequence Alignment, MESH: Amino Acid Sequence, biochemical phenomena, metabolism, and nutrition, MESH: Two-Hybrid System Techniques, Protein Structure, Tertiary, MESH: Protein Structure, Tertiary, Bacterial Proteins, Two-Hybrid System Techniques, polycyclic compounds, MESH: Helicobacter pylori, MESH: Protein Binding, Penicillin-Binding Proteins, Amino Acid Sequence, MESH: Bacterial Proteins, Sequence Alignment, MESH: Microbial Viability, Protein Binding
Abstract: International audience; The definition of bacterial cell shape is a complex process requiring the participation of multiple components of an intricate macromolecular machinery. We aimed at characterizing the determinants involved in cell shape of the helical bacterium Helicobacter pylori. Using a yeast two-hybrid screen with the key cell elongation protein PBP2 as bait, we identified an interaction between PBP2 and MreC. The minimal region of MreC required for this interaction ranges from amino acids 116 to 226. Using recombinant proteins, we showed by affinity and size exclusion chromatographies and surface plasmon resonance that PBP2 and MreC form a stable complex. In vivo, the two proteins display a similar spatial localization and their complex has an apparent 1:1 stoichiometry; these results were confirmed in vitro by analytical ultracentrifugation and chemical cross-linking. Small angle X-ray scattering analyses of the PBP2 : MreC complex suggest that MreC interacts directly with the C-terminal region of PBP2. Depletion of either PBP2 or MreC leads to transition into spherical cells that lose viability. Finally, the specific expression in trans of the minimal interacting domain of MreC with PBP2 in the periplasmic space leads to cell rounding, suggesting that the PBP2/MreC complex formation in vivo is essential for cell morphology.
Published: 2011

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30 results on '"Aline Le Roy"'

1. Molecular recognition of Escherichia coli R1-type core lipooligosaccharide by DC-SIGN

2. A multilaboratory comparison of calibration accuracy and the performance of external references in analytical ultracentrifugation.

3. Structural and functional insights into the activation of the dual incision activity of UvrC, a key player in bacterial NER

4. Analytical ultracentrifugation sedimentation velocity for the characterization of recombinant adeno-associated virus vectors sub-populations

5. Author Reply to Peer Reviews of Characterization of the REC114-MEI4-IHO1 complex regulating meiotic DNA double-strand break formation

6. Powerful Avidity with a Limited Valency for Virus-Attachment Blockers on DC-SIGN: Combining Chelation and Statistical Rebinding with Structural Plasticity of the Receptor

7. Zwitterionic fluorinated detergents: From design to membrane protein applications

8. Characterization of the REC114-MEI4-IHO1 complex regulating meiotic DNA double-strand break formation

9. Powerful avidity with a limited valency for virus-attachment blockers on DC SIGN: Combining chelation and statistical rebinding with structural plasticity of the receptor

10. Maltose-Based Fluorinated Surfactants for Membrane-Protein Extraction and Stabilization

11. Structural insights into the catalytic cycle of a bacterial multidrug ABC efflux pump

12. Targeting Tn-Antigen-Positive Human Tumors with a Recombinant Human Macrophage Galactose C-Type Lectin

13. Structural and functional characterization of DdrC, a novel DNA damage-induced nucleoid associated protein involved in DNA compaction

14. The MurG glycosyltransferase provides an oligomeric scaffold for the cytoplasmic steps of peptidoglycan biosynthesis in the human pathogen Bordetella pertussis

15. Biophysical characterization of the complex between the iron-responsive transcription factor Fep1 and DNA

16. Resurrection of Ancestral Malate Dehydrogenases Reveals the Evolutionary History of Halobacterial Proteins : Deciphering Gene Trajectories and Changes in Biochemical Properties

17. TETRALEC, Artificial Tetrameric Lectins: A Tool to Screen Ligand and Pathogen Interactions

18. Interdomain Flexibility within NADPH Oxidase Suggested by SANS Using LMNG Stealth Carrier

19. Fluorinated diglucose detergents for membrane-protein extraction

20. Role of Phosphorylation in Moesin Interactions with PIP2-Containing Biomimetic Membranes

21. Assemblies of lauryl maltose neopentyl glycol (LMNG) and LMNG-solubilized membrane proteins

22. Structure of an H1-Bound 6-Nucleosome Array Reveals an Untwisted Two-Start Chromatin Fiber Conformation

23. Therapeutic effects of proteoliposomes on X-linked chronic granulomatous disease: proof of concept using macrophages differentiated from patient-specific induced pluripotent stem cells

24. MIB–MIP is a mycoplasma system that captures and cleaves immunoglobulin G

25. A multilaboratory comparison of calibration accuracy and the performance of external references in analytical ultracentrifugation

26. AUC and Small-Angle Scattering for Membrane Proteins

27. Unique DC-SIGN Clustering Activity of a Small Glycomimetic: A Lesson for Ligand Design

28. Analytical Ultracentrifugation and Size-Exclusion Chromatography Coupled with Light Scattering for the Characterization of Membrane Proteins in Solution

29. Sedimentation Velocity Analytical Ultracentrifugation in Hydrogenated and Deuterated Solvents for the Characterization of Membrane Proteins

30. Characterization of the elongasome core PBP2 : MreC complex of Helicobacter pylori

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