9 results on '"Marina Casiraghi"'
Search Results
2. Concerted conformational dynamics and water movements in the ghrelin G protein-coupled receptor
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Laurent J. Catoire, Sonia Cantel, Marjorie Damian, Jean-Alain Fehrentz, Maxime Louet, Antoniel As Gomes, Jean-Louis Banères, Sophie Mary, Paulo R. Batista, David Perahia, Paulo Mascarello Bisch, Mauricio Gs Costa, Khoubaib Ben Haj Salah, Céline M'Kadmi, Marina Casiraghi, Pedro Renault, Severine Denoyelle, Nicolas Floquet, Institut des Biomolécules Max Mousseron [Pôle Chimie Balard] (IBMM), Ecole Nationale Supérieure de Chimie de Montpellier (ENSCM)-Université de Montpellier (UM)-Centre National de la Recherche Scientifique (CNRS), 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), Laboratoire de biologie physico-chimique des protéines membranaires (LBPC-PM (UMR_7099)), Institut de biologie physico-chimique (IBPC (FR_550)), Centre National de la Recherche Scientifique (CNRS)-Centre National de la Recherche Scientifique (CNRS)-Centre National de la Recherche Scientifique (CNRS)-Université de Paris (UP), Department of Molecular and Cellular Physiology [Stanford], Stanford Medicine, Stanford University-Stanford University, Laboratoire de biologie et pharmacologie appliquée (LBPA), Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS)-Ecole Normale Supérieure Paris-Saclay (ENS Paris Saclay), Universidade Federal do Rio de Janeiro (UFRJ), This work was supported by CNRS, Université de Montpellier, Agence Nationale de la Recherche (ANR-17-CE11-0011, ANR-17-CE11-22, ANR-17-CE18-0022), EpiGenMed Labex (post-doctoral fellowship to KBH) and DYNAMO Labex (post-doctoral fellowship to MC). This programreceived funding from the European Union’s Horizon 2020 research and innovation programmeunder the Marie Sklodowska-Curie grant agreement n˚ 799376. Mass spectrometry analyses wereperformed on the instruments located in the IBMM platform of instrumentation, Laboratoire deMesures Physiques (LMP) of Université de Montpellier. We thank GENCI (Grand EquipementNational de Calcul Intensif), CINES (Centre Informatique National de l’Enseignement Supérieur), and IDRIS (Institut du développement et des ressources en informatique scientifique) for computational, ANR-17-CE11-0011,allosig,allostérie, dynamique conformationnelle et signalisation via les RCPG(2017), ANR-17-CE11-0022,GPCteR,Mécanismes moléculaires des régions C-terminales désordonnées et fonctionnelles des RCPG et impact sur les voies de la signalisation cellulaire dépendantes de l'arrestine(2017), and Centre National de la Recherche Scientifique (CNRS)-Université de Montpellier (UM)-Ecole Nationale Supérieure de Chimie de Montpellier (ENSCM)
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QH301-705.5 ,Science ,Growth hormone secretagogue receptor ,Chemical biology ,chemical biology ,010402 general chemistry ,Ligands ,01 natural sciences ,General Biochemistry, Genetics and Molecular Biology ,Receptors, G-Protein-Coupled ,03 medical and health sciences ,Molecular dynamics ,GPCR ,Biochemistry and Chemical Biology ,biochemistry ,Humans ,Biology (General) ,Receptor ,Receptors, Ghrelin ,030304 developmental biology ,G protein-coupled receptor ,chemistry.chemical_classification ,0303 health sciences ,General Immunology and Microbiology ,[SDV.BA]Life Sciences [q-bio]/Animal biology ,General Neuroscience ,digestive, oral, and skin physiology ,E. coli ,General Medicine ,Ghrelin ,0104 chemical sciences ,Amino acid ,[SDV.BBM.BP]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biophysics ,Transmembrane domain ,chemistry ,Biophysics ,Medicine ,signaling ,hydration ,Research Article ,Human ,Signal Transduction - Abstract
International audience; There is increasing support for water molecules playing a role in signal propagation through G protein-coupled receptors (GPCRs). However, exploration of the hydration features of GPCRs is still in its infancy. Here, we combined site-specific labeling with unnatural amino acids to molecular dynamics to delineate how local hydration of the ghrelin receptor growth hormone secretagogue receptor (GHSR) is rearranged upon activation. We found that GHSR is characterized by a specific hydration pattern that is selectively remodeled by pharmacologically distinct ligands and by the lipid environment. This process is directly related to the concerted movements of the transmembrane domains of the receptor. These results demonstrate that the conformational dynamics of GHSR are tightly coupled to the movements of internal water molecules, further enhancing our understanding of the molecular bases of GPCR-mediated signaling.
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- 2021
3. Author response: Concerted conformational dynamics and water movements in the ghrelin G protein-coupled receptor
- Author
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Céline M'Kadmi, Maxime Louet, Paulo R. Batista, Paulo Mascarello Bisch, Sophie Mary, Nicolas Floquet, Marina Casiraghi, Jean-Alain Fehrentz, Khoubaib Ben Haj Salah, Laurent J. Catoire, Sonia Cantel, Jean-Louis Banères, Antoniel As Gomes, David Perahia, Mauricio Gs Costa, Marjorie Damian, Pedro Renault, and Severine Denoyelle
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Chemistry ,Water Movements ,Dynamics (mechanics) ,Biophysics ,Ghrelin ,G protein-coupled receptor - Published
- 2021
4. Structure of the agonist 12–HHT in its BLT2 receptor-bound state
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Christel Le Bon, Jutta Rieger, Karine Moncoq, Marjorie Damian, Elodie Point, Jean-Louis Banères, Alexandre Pozza, Marina Casiraghi, Fabrice Giusti, Laurent J. Catoire, Physico-chimie moléculaire des membranes biologiques (PCMMB), Centre National de la Recherche Scientifique (CNRS)-Université Paris Diderot - Paris 7 (UPD7), Institut des Biomolécules Max Mousseron [Pôle Chimie Balard] (IBMM), Ecole Nationale Supérieure de Chimie de Montpellier (ENSCM)-Université de Montpellier (UM)-Centre National de la Recherche Scientifique (CNRS), Chimie des polymères (LCP), Institut Parisien de Chimie Moléculaire (IPCM), Centre National de la Recherche Scientifique (CNRS)-Université Pierre et Marie Curie - Paris 6 (UPMC)-Centre National de la Recherche Scientifique (CNRS)-Université Pierre et Marie Curie - Paris 6 (UPMC)-Centre National de la Recherche Scientifique (CNRS), Institut de biochimie et biophysique moléculaire et cellulaire (IBBMC), Université Paris-Sud - Paris 11 (UP11)-Centre National de la Recherche Scientifique (CNRS), Laboratoire de biologie physico-chimique des protéines membranaires (LBPC-PM (UMR_7099)), Institut de biologie physico-chimique (IBPC), Centre National de la Recherche Scientifique (CNRS)-Centre National de la Recherche Scientifique (CNRS)-Université Paris Diderot - Paris 7 (UPD7)-Centre National de la Recherche Scientifique (CNRS), Unité de Chimie et Procédés (UCP), École Nationale Supérieure de Techniques Avancées (ENSTA Paris), Université Paris Diderot - Paris 7 (UPD7)-Centre National de la Recherche Scientifique (CNRS), Université Pierre et Marie Curie - Paris 6 (UPMC)-Institut Parisien de Chimie Moléculaire (IPCM), Université Pierre et Marie Curie - Paris 6 (UPMC)-Centre National de la Recherche Scientifique (CNRS)-Centre National de la Recherche Scientifique (CNRS)-Centre National de la Recherche Scientifique (CNRS), Institut de biologie physico-chimique (IBPC (FR_550)), 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), Institut de Chimie du CNRS (INC)-Université Pierre et Marie Curie - Paris 6 (UPMC)-Centre National de la Recherche Scientifique (CNRS)-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS)-Centre National de la Recherche Scientifique (CNRS), and Université Pierre et Marie Curie - Paris 6 (UPMC)-Centre National de la Recherche Scientifique (CNRS)-Université Pierre et Marie Curie - Paris 6 (UPMC)-Centre National de la Recherche Scientifique (CNRS)-Centre National de la Recherche Scientifique (CNRS)
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Agonist ,Leukotriene B4 ,medicine.drug_class ,Molecular Conformation ,Receptors, Leukotriene B4 ,lcsh:Medicine ,Ligand ,Cell surface receptor ,Ligands ,Article ,03 medical and health sciences ,chemistry.chemical_compound ,Docking (dog) ,Stereochemistry ,medicine ,Humans ,Homology modeling ,lcsh:Science ,Receptor ,Nuclear Magnetic Resonance, Biomolecular ,Unsaturated fatty acid ,ComputingMilieux_MISCELLANEOUS ,030304 developmental biology ,0303 health sciences ,Multidisciplinary ,[SDV.BBM.BS]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Structural Biology [q-bio.BM] ,Chemistry ,Leukotriene receptor ,lcsh:R ,030302 biochemistry & molecular biology ,Molecular Docking Simulation ,[SDV.BBM.BS]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biomolecules [q-bio.BM] ,[CHIM.POLY]Chemical Sciences/Polymers ,Docking (molecular) ,Biophysics ,Fatty Acids, Unsaturated ,lcsh:Q ,Solution-state NMR ,[CHIM.CHEM]Chemical Sciences/Cheminformatics ,Protein Binding - Abstract
G Protein-Coupled receptors represent the main communicating pathway for signals from the outside to the inside of most of eukaryotic cells. They define the largest family of integral membrane receptors at the surface of the cells and constitute the main target of the current drugs on the market. The low affinity leukotriene receptor BLT2 is a receptor involved in pro- and anti-inflammatory pathways and can be activated by various unsaturated fatty acid compounds. We present here the NMR structure of the agonist 12–HHT in its BLT2-bound state and a model of interaction of the ligand with the receptor based on a conformational homology modeling associated with docking simulations. Put into perspective with the data obtained with leukotriene B4, our results illuminate the ligand selectivity of BLT2 and may help define new molecules to modulate the activity of this receptor.
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- 2020
5. Specific cardiolipin–SecY interactions are required for proton-motive force stimulation of protein secretion
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Bruno Miroux, Ian Collinson, Robin A. Corey, Marina Casiraghi, Euan Pyle, William J. Allen, Daniel W. Watkins, Ignacio Arechaga, Argyris Politis, Biotechnology and Biological Sciences Research Council (UK), Wellcome Trust, Centre National de la Recherche Scientifique (France), Imperial College London, Laboratoire de biologie physico-chimique des protéines membranaires (LBPC-PM (UMR_7099)), Institut de biologie physico-chimique (IBPC (FR_550)), Centre National de la Recherche Scientifique (CNRS)-Centre National de la Recherche Scientifique (CNRS)-Centre National de la Recherche Scientifique (CNRS)-Université de Paris (UP), Department of Biochemistry, School of Medical Sciences, ANR-11-LABX-0011,DYNAMO,Dynamique des membranes transductrices d'énergie : biogénèse et organisation supramoléculaire.(2011), ANR-17-CE09-0007,GenCaps,Bioproduction de capsules fonctionnelles génétiquement encodées(2017), and Universidad de Cantabria
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0301 basic medicine ,SecYEG ,Membrane lipids ,Molecular Dynamics Simulation ,Biology ,Molecular dynamics ,Motor protein ,03 medical and health sciences ,chemistry.chemical_compound ,Native mass spectrometry ,ATP hydrolysis ,Escherichia coli ,Cardiolipin ,Thermotoga maritima ,[SDV.BBM.BC]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biochemistry [q-bio.BM] ,Bacterial Secretion Systems ,ComputingMilieux_MISCELLANEOUS ,Protein translocation ,Multidisciplinary ,Chemiosmosis ,Escherichia coli Proteins ,Proton-Motive Force ,Biological Sciences ,biology.organism_classification ,Transmembrane protein ,Transport protein ,030104 developmental biology ,chemistry ,Biochemistry ,Biophysics ,SEC Translocation Channels - Abstract
The transport of proteins across or into membranes is a vital biological process, achieved in every cell by the conserved Sec machinery. In bacteria, SecYEG combines with the SecA motor protein for secretion of preproteins across the plasma membrane, powered by ATP hydrolysis and the transmembrane proton-motive force (PMF). The activities of SecYEG and SecA are modulated by membrane lipids, particularly cardiolipin (CL), a specialized phospholipid known to associate with a range of energy-transducing machines. Here, we identify two specific CL binding sites on the Thermotoga maritima SecA–SecYEG complex, through application of coarse-grained molecular dynamics simulations. We validate the computational data and demonstrate the conserved nature of the binding sites using in vitro mutagenesis, native mass spectrometry, biochemical analysis, and fluorescence spectroscopy of Escherichia coli SecYEG. The results show that the two sites account for the preponderance of functional CL binding to SecYEG, and mediate its roles in ATPase and protein transport activity. In addition, we demonstrate an important role for CL in the conferral of PMF stimulation of protein transport. The apparent transient nature of the CL interaction might facilitate proton exchange with the Sec machinery, and thereby stimulate protein transport, by a hitherto unexplored mechanism. This study demonstrates the power of coupling the high predictive ability of coarse-grained simulation with experimental analyses, toward investigation of both the nature and functional implications of protein–lipid interactions., This work was funded by the Biotechnology and Biological Sciences Research Council (Grants BB/M003604/1, BB/I008675/1, and BB/N015126/1) and the Wellcome Trust (Grants 104632 and 109854/Z/15/Z). The mass spectrometry work was supported by the Centre National de la Recherche Scientifique, INSERM (Grant “DYNAMO,” ANR-11-LABEX-0011-01 to M.C.) and the Région Ile de France for cofunding the Le Service d’Analyse des Médicaments et Métabolites Mass Spectrometry Facility at IPSIT. E.P. is the recipient of an Imperial College London Institute of Chemical Biology Engineering and Physical Sciences Research Council (EPSRC) Centre for Doctoral Training studentship.
- Published
- 2018
6. Specific cardiolipin-SecY interactions are required for proton-motive-force stimulation of protein secretion
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Argyris Politis, Bruno Miroux, Ian Collinson, Robin A. Corey, Ignacio Arechaga, Euan Pyle, Marina Casiraghi, and William J. Allen
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0303 health sciences ,010304 chemical physics ,Chemiosmosis ,Membrane lipids ,01 natural sciences ,Transport protein ,Motor protein ,03 medical and health sciences ,chemistry.chemical_compound ,chemistry ,Membrane protein ,0103 physical sciences ,Cardiolipin ,Biophysics ,Cardiolipin binding ,SEC Translocation Channels ,030304 developmental biology - Abstract
The transport of proteins across or into membranes is a vital biological process, achieved in every cell by the conserved Sec machinery. In bacteria, SecYEG combines with the SecA motor protein for secretion of pre-proteins across the plasma membrane, powered by ATP hydrolysis and the trans-membrane proton-motive-force (PMF). The activities of SecYEG and SecA are modulated by membrane lipids, particularly by cardiolipin, a specialised phospholipid known to associate with a range of energy-transducing machines. Here, we identify two specific cardiolipin binding sites on theThermotoga maritimaSecA-SecYEG complex, through application of coarse-grained molecular dynamics simulations. We validate the computational data and demonstrate the conserved nature of the binding sites usingin vitromutagenesis, native mass spectrometry and biochemical analysis ofEscherichia coliSecYEG. The results show that the two sites account for the preponderance of functional cardiolipin binding to SecYEG, and mediate its roles in ATPase and protein transport activity. In addition, we demonstrate an important role for cardiolipin in the conferral of PMF-stimulation of protein transport. The apparent transient nature of the CL interaction might facilitate proton exchange with the Sec machinery and thereby stimulate protein transport, by an as yet unknown mechanism. This study demonstrates the power of coupling the high predictive ability of coarse-grained simulation with experimental analyses, towards investigation of both the nature and functional implications of protein-lipid interactions.Significance StatementMany proteins are located in lipid membranes surrounding cells and cellular organelles. The membrane can impart important structural and functional effects on the protein, making understanding of this interaction critical. Here, we apply computational simulation to the identification of conserved lipid binding sites on an important highly conserved bacterial membrane protein, the Sec translocase (SecA-SecYEG), which uses ATP and the proton motive force (PMF) to secrete proteins across the bacterial plasma membrane. We experimentally validate and reveal the conserved nature of these binding sites, and use functional analyses to investigate the biological significance of this interaction. We demonstrate that these interactions are specific, transient, and critical for both ATP- and PMF- driven protein secretion.
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- 2017
7. NMR Spectroscopy for the Characterization of GPCR Energy Landscapes
- Author
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Jean-Louis Banères, Marina Casiraghi, and Laurent Catoire
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Ligand efficiency ,Chemistry ,Allosteric regulation ,Biophysics ,Energy landscape ,Context (language use) ,Nuclear magnetic resonance spectroscopy ,Signal transduction ,Conformational ensembles ,G protein-coupled receptor - Abstract
G protein-coupled receptor (GPCR)-mediated signal transduction has a central role in human physiology and implication in many diseases. Despite the tremendous number of X-ray crystallography structures published in the past decade, the molecular mechanisms of ligand-dependent signaling remain to be completed. In particular, very little information is available concerning the implication of receptor dynamics and conformational changes on GPCR ligand efficiency and coupling. In this context, mapping the conformational landscape of GPCRs, and how it is modulated by the membrane environment and allosteric and signaling partners, is fundamental in order to gain a clear picture of how the signaling mechanism proceeds. Solution-state nuclear magnetic resonance (NMR) is a powerful technique to study GPCR energy landscapes, i.e., conformational ensembles along activation and inactivation pathway, and associated kinetic barriers.
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- 2017
8. Functional Modulation of a G Protein-Coupled Receptor Conformational Landscape in a Lipid Bilayer
- Author
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Elodie Point, Karine Moncoq, Marina Casiraghi, Ewen Lescop, Jean-Louis Banères, Marjorie Damian, Daniel Lévy, Eric Guittet, Nelly Morellet, Laurent Catoire, Jacky Marie, Physico-chimie moléculaire des membranes biologiques (PCMMB), Université Paris Diderot - Paris 7 (UPD7)-Centre National de la Recherche Scientifique (CNRS), Institut des Biomolécules Max Mousseron [Pôle Chimie Balard] (IBMM), Centre National de la Recherche Scientifique (CNRS)-Institut de Chimie du CNRS (INC)-Université de Montpellier (UM)-Ecole Nationale Supérieure de Chimie de Montpellier (ENSCM), Institut de Chimie des Substances Naturelles (ICSN), Centre National de la Recherche Scientifique (CNRS)-Institut de Chimie du CNRS (INC), Laboratoire de biologie physico-chimique des protéines membranaires (LBPC-PM (UMR_7099)), Institut de biologie physico-chimique (IBPC (FR_550)), Centre National de la Recherche Scientifique (CNRS)-Centre National de la Recherche Scientifique (CNRS)-Université Paris Diderot - Paris 7 (UPD7)-Centre National de la Recherche Scientifique (CNRS), BioImaging Cell and Tissue Core Facility (PICT-IBiSA), Institut Curie [Paris], Ecole Nationale Supérieure de Chimie de Montpellier (ENSCM)-Université de Montpellier (UM)-Centre National de la Recherche Scientifique (CNRS), Centre National de la Recherche Scientifique (CNRS), Centre National de la Recherche Scientifique (CNRS)-Université Paris Diderot - Paris 7 (UPD7), Institut de biologie physico-chimique (IBPC), and Institut Curie
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0301 basic medicine ,Agonist ,Models, Molecular ,medicine.drug_class ,G protein ,Stereochemistry ,Protein Conformation ,[SDV]Life Sciences [q-bio] ,Lipid Bilayers ,Receptors, Leukotriene B4 ,010402 general chemistry ,Ligands ,01 natural sciences ,Biochemistry ,Catalysis ,03 medical and health sciences ,Colloid and Surface Chemistry ,Allosteric Regulation ,medicine ,[SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology ,Receptor ,Lipid bilayer ,G protein-coupled receptor ,Chemistry ,Leukotriene B4 receptor ,General Chemistry ,0104 chemical sciences ,Coupling (electronics) ,030104 developmental biology ,Membrane ,Biophysics ,Signal Transduction - Abstract
International audience; Mapping the conformational landscape of G protein-coupled receptors (GPCRs), and in particular how this landscape is modulated by the membrane environment, is required to gain a clear picture of how signaling proceeds. To this end, we have developed an original strategy based on solution-state nuclear magnetic resonance combined with an efficient isotope labeling scheme. This strategy was applied to a typical GPCR, the leukotriene B4 receptor BLT2, reconstituted in a lipid bilayer. Because of this, we are able to provide direct evidence that BLT2 explores a complex landscape that includes four different conformational states for the unliganded receptor. The relative distribution of the different states is modulated by ligands and the sterol content of the membrane, in parallel with the changes in the ability of the receptor to activate its cognate G protein. This demonstrates a conformational coupling between the agonist and the membrane environment that is likely to be fundamental for GPCR signaling.
- Published
- 2016
9. Synthesis, Characterization and Applications of a Perdeuterated Amphipol
- Author
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Fabrice Giusti, Laurent Catoire, Shuo Qian, Thomas G. Watkinson, J.-L. Popot, Marina Casiraghi, Jutta Rieger, Antonio N. Calabrese, Sheena E. Radford, Alison E. Ashcroft, Physico-chimie moléculaire des membranes biologiques (PCMMB), Université Paris Diderot - Paris 7 (UPD7)-Centre National de la Recherche Scientifique (CNRS), Institut Parisien de Chimie Moléculaire (IPCM), Université Pierre et Marie Curie - Paris 6 (UPMC)-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS), Centre National de la Recherche Scientifique (CNRS), Sorbonne Université (SU), Institut de biologie physico-chimique (IBPC (FR_550)), Oak Ridge National Laboratory [Oak Ridge] (ORNL), UT-Battelle, LLC, Astbury Centre for Structural Molecular Biology, University of Leeds, Centre National de la Recherche Scientifique (CNRS)-Université Paris Diderot - Paris 7 (UPD7), Chimie des polymères (LCP), Centre National de la Recherche Scientifique (CNRS)-Université Pierre et Marie Curie - Paris 6 (UPMC)-Centre National de la Recherche Scientifique (CNRS)-Université Pierre et Marie Curie - Paris 6 (UPMC)-Centre National de la Recherche Scientifique (CNRS), Université Pierre et Marie Curie - Paris 6 (UPMC)-Centre National de la Recherche Scientifique (CNRS), Centre National de la Recherche Scientifique (CNRS)-Université Pierre et Marie Curie - Paris 6 (UPMC), Institut de biologie physico-chimique (IBPC), and Sorbonne Université (SU)-Centre National de la Recherche Scientifique (CNRS)
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Magnetic Resonance Spectroscopy ,Physiology ,Polymers ,030310 physiology ,Size-exclusion chromatography ,Lipid Bilayers ,Biophysics ,Analytical chemistry ,Nuclear Overhauser effect ,Neutron scattering ,03 medical and health sciences ,chemistry.chemical_compound ,Surface-Active Agents ,[CHIM]Chemical Sciences ,Isopropylamine ,[SDV.BBM.BC]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biochemistry [q-bio.BM] ,ComputingMilieux_MISCELLANEOUS ,030304 developmental biology ,0303 health sciences ,Molar mass ,Propylamines ,Staining and Labeling ,[SDV.BBM.BS]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Structural Biology [q-bio.BM] ,Polyacrylic acid ,Deuterium Exchange Measurement ,Membrane Proteins ,Water ,Cell Biology ,Nuclear magnetic resonance spectroscopy ,Deuterium ,[SDV.BBM.BC]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biomolecules [q-bio.BM] ,NMR spectra database ,Solutions ,[SDV.BBM.BP]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biophysics ,[SDV.BBM.BS]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biomolecules [q-bio.BM] ,[CHIM.POLY]Chemical Sciences/Polymers ,chemistry ,Solubility ,Physical chemistry ,Hydrophobic and Hydrophilic Interactions - Abstract
Amphipols are short amphipathic polymers that can substitute for detergents at the hydrophobic surface of membrane proteins (MPs), keeping them soluble in the absence of detergents while stabilizing them. The most widely used amphipol, known as A8-35, is comprised of a polyacrylic acid (PAA) main chain grafted with octylamine and isopropylamine. Among its many applications, A8-35 has proven particularly useful for solution-state NMR studies of MPs, for which it can be desirable to eliminate signals originating from the protons of the surfactant. In the present work, we describe the synthesis and properties of perdeuterated A8-35 (perDAPol). Perdeuterated PAA was obtained by radical polymerization of deuterated acrylic acid. It was subsequently grafted with deuterated amines, yielding perDAPol. The number-average molar mass of hydrogenated and perDAPol, ~4 and ~5 kDa, respectively, was deduced from that of their PAA precursors, determined by size exclusion chromatography in tetrahydrofuran following permethylation. Electrospray ionization-ion mobility spectrometry-mass spectrometry measurements show the molar mass and distribution of the two APols to be very similar. Upon neutron scattering, the contrast match point of perDAPol is found to be ~120% D2O. In (1)H-(1)H nuclear overhauser effect NMR spectra, its contribution is reduced to ~6% of that of hydrogenated A8-35, making it suitable for extended uses in NMR spectroscopy. PerDAPol ought to also be of use for inelastic neutron scattering studies of the dynamics of APol-trapped MPs, as well as small-angle neutron scattering and analytical ultracentrifugation.
- Published
- 2014
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