NMR of Large Biomolecular Assemblies
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Development of Innovative isotopic labelling Technologies for NMR
Our team is developing innovative isotopic labelling approaches and cutting-edge NMR experiments to facilitate the characterization by solution NMR spectroscopy of weak interactions as well as high molecular weight assemblies. In the last years, we have succeeded in developing new strategies that allow specific-protonation of any combination of the methyl groups in fully perdeuterated proteins, including the challenging prochiral-specific labelling of the methyl groups of leucine and valine.
Combined with development of optimized NMR experiments, these specific labelling schemes have allowed to detect internuclear interactions up to two orders of magnitude weaker than previously reported NMR approaches. Applications include the identification of pairs of residues stabilized by weak CH/Pi interactions in proteins through the detection of scalar coupling of few hundredths of a hertz and precise long-range distances measurement between protons separated by up to 12 Å. These news protocols are also particularly important to simplify NMR spectra of large assemblies and to increase the number of methyl probes available to investigate the detail of large biomolecular nanomachines. The quality of NMR spectra obtained from such specifically-protonated samples offers the possibility of measuring structural or dynamics information for each individual methyl probe. Part of the team is continuously devoting research efforts in order to extend the cutting edge labelling strategy to tackle more and more challenging systems and to overcome the current size and time limitation of NMR technologies.
Collaborations: O. Hamelin (IRTSV – Grenoble, France); J. Chou (Harvard – Boston, USA)
Funding: HFSP (2004-2007); CNRS (2007); ANR (2009-2012); ERC (2011-2015).
Real Time NMR Studies of Nanomachines in Action.
The study of the structural and functional properties of biomolecular nanomachines remains a considerable practical challenge. Structural studies of such systems combining X-ray crystallography and complementary low-resolution methods (e.g. SAXS, SANS or cryo-EM) usually provide only a static picture of the system and rarely report the kinetic data necessary for a full, atomic resolution understanding of the mode of action.
The recent development in our group of rapid (c.a. 1 s) acquisition 2D NMR experiments for proteins of several hundred kilodaltons has opened up the possibility of studying biological machineries in action. Novel isotope-labelling approaches have permitted the acquisition of more easily interpretable 2D NMR spectra of large protein assemblies. Together with new automated mutation-based assignment strategies, mechanisms of large biological machineries can now be monitored in real time and at atomic resolution. Our team use state of the art of NMR spectroscopy, isotope labelling and automated molecular biology techniques, all developed locally, to characterize self-assembly and functionally important structural rearrangements of large protein assemblies involved in cellular protein quality control systems with size ranging from 200 kDa to 1 MDa.
Funding: ERC (2011-2015); Marie-Curie (2012-2014).
Structure and Interaction in Ribonucleoprotein Particles
The team is studying large protein/RNA complexes involved in microRNAs biogenesis machineries and viral RNA recognition by human protein assemblies with size of several hundred of kilodalton and up to 1 MDa. A continuing difficulty when studying such interactions is the identification of the minimal folded constructs of the protein partner. To resolve this issue, we have developed an interdisciplinary approach for parallel screening of protein constructs that is designed to identify and optimize those amenable for structural characterization. Despite using small-scale (4 mL) expression cultures, high-quality heteronuclear 2D spectra can be recorded within a few minutes to hours (even for proteins of 80 kDa) allowing a quick and convenient assessment of folding state of each target. Structure and interaction of identified individually folded domain are studied by classical and liquid crystal NMR approaches. Together with NMR data, we are using complementary biophysical and biochemical approaches (AUC, ITC, SEC/MALLS) to investigate interactions, and low resolution methods (e.g. SAXS, SANS or cryo-EM) to integrate structural models derived from smaller constructs into full size ribonucleoprotein particles.
Collaborations: J. Palatnik/R. Rasia (IBR- Rosario, Argentina); E. Drouet (UVHCI-Grenoble, France).
Funding: HFSP (2006-2009); ANR (2006-2010); Marie-Curie (2009-2011); ARC (2009-2013); ANRS (2008-2010; 2013-2015).
Publications of the team:
- Benoit M, Imbert L, Palencia A, Perard J, Boisbouvier J. and Plevin M.J. “Structural Characterization of the miRNA Recognition Region of Human TRBP” Nucleic Acids Res doi: 10.1093/nar/gkt086 (2013).
- Ayala I, Hamelin O., Amero C, Pessey O., Plevin M.J., Gans P., Boisbouvier J. “An Optimized Isotopic Labelling Strategy of Isoleucine-g2 Methyl Groups for solution NMR Study of High Molecular Weight Proteins” Chem. Comm. 48,1434-1436 (2012).
- Rasia RM, Brutscher B, Plevin MJ. “Selective isotopic unlabeling of proteins using metabolic precursors : application to NMR assignment of intrinsically disordered proteins“ Chembiochem. 13, 732-9. (2012)
- Burdisso P, Suarez IP, Bologna NG, Palatnik JF, Bersch B, Rasia RM. “Second double-stranded RNA binding domain of Dicer-like ribonuclease 1 : Structural and biochemical characterization“ Biochemistry. 51, 10159-66. (2012).
- Plevin MJ, Boisbouvier J. “Isotope-Labelling of Methyl Groups for NMR Studies of Large Proteins.” Book chapter in Recent Developments in Biomolecular NMR, Royal Society of Chemistry, Biomolecular Science Series, DOI:10.1039/9781849735391 (2012).
- Benoit MP, Plevin MJ. “Backbone resonance assignments of the micro-RNA precursor binding region of human TRBP “ Biomol NMR Assign. doi : 10.1007/s12104-012-9416-8 (2012)
- Plevin MJ, Hamelin O, Boisbouvier J., Gans P “A simple biosynthetic method for stereospecific resonance assignment of prochiral methyl groups in proteins” J. Biomol NMR 49, 61-67 (2011).
- Desjardins G, Bonneau E, Girard N, Boisbouvier J, Legault P “NMR structure of the A730 loop of the Neurospora VS ribozyme: insights into the formation of the active site” Nucleic Acids Res. 9, 4427-4437 (2011).
- Amero CD, Durá MA, Noirclerc-Savoye M, Perollier A, Gallet B, Plevin MJ, Vernet T, Franzetti B, Boisbouvier J. “A Systematic Mutagenesis-driven Strategy for Site-Resolved NMR Studies of Supramolecular Assemblies” J. Biomol NMR. 50, 229-236 (2011).
- Rasia RM, Lescop E, Palatnik JF, Boisbouvier J., Brutscher B “Rapid measurement of Residual Dipolar Couplings J. Biomol NMR 51, 369-378 (2011).
- Schanda P., Huber M., Boisbouvier J., Meier B.H., Ernst M. “Solid-state NMR measurements of asymmetric dipolar couplings provide insight into protein side-chain motion” Angew. Chem. Int. Ed. 50, 11005-11009 (2011).
- Gans P, Hamelin O, Sounier R, Ayala I, Dura MA, Amero C, Noirclerc-Savoye M, Franzetti B, Plevin MJ, Boisbouvier J. “Stereospecific Isotopic Labeling of Methyl Groups for NMR Studies of High Molecular Weight Proteins” Angew. Chem. Int. Ed. 49, 1958-1862 (2010).
- Plevin MJ, Bryce DL, Boisbouvier J. “Direct Detection of CH/p Interactions in Proteins” Nature Chem. 2, 466-471 (2010).
- Rasia RM, Mateos J, Bologna NG, Burdisso P, Imbert L, Palatnik JF, Boisbouvier J. “Structure and RNA interactions of the plant MicroRNA processing-associated protein HYL1” Biochemistry 49, 8237-8239 (2010).
- Gans P, Boisbouvier J. “Le marquage isotopique spécifique : Un outil pour repousser les frontières de la RMN biomoléculaire” Actualité Chimique 347, 5-11 (2010).
- Ayala I, Sounier R, Use N, Gans P, Boisbouvier J. “An efficient protocol for the complete incorporation of methyl specifically protonated alanine in perdeuterated protein” J. Biomol. NMR 43, 111-119 (2009).
- Rasia R, Noirclerc-Savoye M, Gallet B, Bologna N, Plevin M, Blanchard L, Palatnik J, Brutscher B, Vernet T, Boisbouvier J. “Parallel screening and optimization of protein constructs for structural studies” Protein Science 18, 434-439 (2009).
- Perard J, Rasia R, Medenbach J, Ayala I, Boisbouvier J, Drouet E, Baudin F. “Initiation factor eIF3 subunit b interacts with HCV IRES RNA through its N-terminal RNA recognition motif“ FEBS Letters 583, 570-574 (2009).
- Amero C, Schanda P, Dura A, Ayala I, Marion D, Franzetti B, Brutscher B, Boisbouvier J. “Fast Two Dimensional NMR Spectroscopy of High Molecular Weight Protein Assemblies”. J. Am. Chem. Soc. 131, 3448-9 (2009).
- Farjon J, Boisbouvier J, Schanda P, Simorre JP, Pardi, A, Brutscher B. “New sensitive longitudinal relaxation optimized NMR experiments for the study of RNA in solution“.J. Am. Chem. Soc 131, 8571-8577 (2009).
- Van Melckebeke H, Devany M, Di Primo C, Beaurain F, Toulmé JJ, Bryce DL, Boisbouvier J. “Liquid Crystal NMR Structure of HIV TAR RNA Bound to its SELEX RNA Aptamer Reveals the Origins of the High Stability of the Complex“ Proc. Natl. Acad. Sci. USA 105, 9210-5 (2008).
- Sounier R, Blanchard L, Wu Z, Boisbouvier J. “High-Accuracy Distance Measurement Between Remote Methyls in Specifically Protonated Proteins” J. Am. Chem. Soc. 129, 472-3 (2007).
- Schanda P, Lescop E, Falge M, Sounier R, Boisbouvier J, Brutscher B. “Sensitivity-optimized experiment for the measurement of residual dipolar couplings between amide protons” J. Biomol. NMR 38, 47-55 (2007).
- Palatnik J, Wollmann H, Schommer C, Schwab R, Boisbouvier J, Rodriguez R, Warthmann N, Allen E, Dezulian T, Huson D, Carrington J, Weigel D. “Sequence and expression differences underlie functional specialization of arabidopsis microRNAs miR159 and miR319” Dev. Cell 13, 115-25 (2007).