Institut de Biologie StructuraleGrenoble / France

Contact person(s) related to this article / BOISBOUVIER Jerome / FAVIER Adrien

NMR of Large Biomolecular Assemblies

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Team Members

Jérôme Boisbouvier (CNRS Researcher)
Pierre Gans (CEA Researcher)
Isabel Ayala (CNRS Engineer)
Lionel Imbert (CNRS Engineer)
Pavel Macek (Researcher co-funded by NMR-Bio)
Rida Awad (CNRS engineer)
Faustine Henot (PhD)
Ricarda Törner (PhD)

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).