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Institut de Biologie StructuraleGrenoble / France

Contact person(s) related to this article / BRUTSCHER Bernhard / FAVIER Adrien

Team: Liquid-state NMR methods

Team members

Isabel Ayala (protein engineer)
Bernhard Brutscher (CEA research director)
Nina-Eleni Christou (PhD student)
Adrien Favier (research engineer)
Alicia Vallet (NMR platform engineer)

If you want to contact team members, please use the generic e-mail address :
firstname(dot)lastname(at)ibs(dot)fr

Presentation

A major research theme of our team is the development of optimized NMR pulse sequences and spectral analysis tools for biomolecular applications. Recent efforts have focused on the development of fast multidimensional data acquisition techniques, especially polarization-enhanced fast-pulsing methods (SOFAST, BEST, …). Such methods not only prove useful for speeding up NMR data collection, but they also provide new opportunities for site-resolved kinetic real-time NMR investigation. We are particularly interested in applying our fast NMR techniques, in combination with other biophysical methods, to kinetic events such as the folding of proteins and nucleic acids, the structural and dynamic characterization of transiently populated excited states, the influence of chaperone proteins on the folding kinetics, as well as light-induced protein conformational dynamics in light-sensing proteins. We have also assembled dedicated hardware for in-situ (in the NMR magnet) fast mixing of solutions and sample illumination by laser light.

Recent publications

  • Favier, A., and B. Brutscher. 2019. NMRlib: user-friendly pulse sequence tools for Bruker NMR spectrometers. J. Biomol. NMR. 73: 199–211.
  • Vallet, A., A. Favier, and B. Brutscher. 2019. Aromatic SOFAST-HMBC for proteins at natural 13C abundance. J. Magn. Reson. 300: 95–102.
  • Christou, N.E., and B. Brutscher. 2018. BEST and SOFAST experiments for resonance assignment of histidine and tyrosine side chains in 13C/15N labeled proteins. J. Biomol. NMR. 72: 115–124.
  • Rennella, E., T.S. Sára, M. Juen, C. Wunderlich, L. Imbert, Z. Solyom, A. Favier, I. Ayala, K. Weinhäupl, P. Schanda, R. Konrat, C. Kreutz, and B. Brutscher. 2017. RNA binding and chaperone activity of the E. coli cold-shock protein CspA. Nucleic Acids Res. : 1–14.
  • Franco, R., S. Gil-Caballero, I. Ayala, A. Favier, and B. Brutscher. 2017. Probing conformational exchange dynamics in a short-lived protein folding intermediate by real-time relaxation-dispersion NMR. J. Am. Chem. Soc. 139.
  • Franco, R., A. Favier, P. Schanda, and B. Brutscher. 2017. Optimized fast mixing device for real-time NMR applications. J. Magn. Reson. 281: 125–129.

Recent applications of our NMR methods

Light-induced conformational dynamics in PTFPs
Photo-transformable Fluorescent Proteins (PTFPs) are essential tools for super-resolution (SR) microscopy. Rational fluorescent protein engineering exploits the mechanistic information available from structural studies, mainly X-ray crystallography, in order to design new PTFP variants with improved properties for particular applications. Here we apply NMR spectroscopy in solution to investigate the light-induced changes in conformational dynamics of rsFolder, a reversibly switchable fluorescent protein. The dynamic view offered by NMR highlights protein regions that comprise potentially interesting mutation points for future mutagenesis campaigns.
Collaborations: D. Bourgeois (IBS Grenoble)
Real-Time NMR Investigation of Transiently-Populated Protein States
Identification and characterization of a monomer-dimer exchange process in the excited state of b2 microglobulin by a combination of real-time NMR and CPMG relaxation-dispersion NMR. This conformational state has been shown previously in our team to be 7-times more aggregation prone than the native state.
Collaborations: A.Corazza, G. Esposito (Univ. of Udine, Italy); V. Forge (iRTSV, CEA Grenoble, France)
Transient structure in an intrinsically disordered viral protein
The non-structural protein 5A (NS5A) from the hepatitis C virus plays an important role in viral replication and particle formation. The C-terminal 250 residues are highly disordered, and display numerous interaction sites for other viral and host proteins. Our NMR study revealed the presence of transient α-helical structures in 4 regions of the peptide sequence that are partly stabilized by long-range tertiary interactions. Two of these transient α-helical regions form a non-canonical binding motive for low affinity binding to SH3 domains, and partial compaction upon phosphorylation of the protein.
Collaborations: D. Willbold (FZ Jülich, Germany)
RNA conformational transitions and RNA chaperone activity
Chaperone proteins assist RNA in adopting their functionally relevant states. Little is known, however, about the mechanistic details of the RNA chaperone activity. We have used a combination of real-time and steady-state NMR methods to decipher the RNA duplex formation pathways of two complementary RNA hairpins, as well as the chaperone activity of the small bacterial cold shock protein CspA on this refolding reaction.
Collaborations: C. Kreutz (Innsbruck, Austria), R. Konrat (Vienna, Austria)