Etudes des assemblages biomoléculaires par RMN

L’étude des assemblages biomoléculaires de grande taille a été initiée au cours de la thèse de R. Sounier (bourse ministère de la recherche) dont j’ai assuré la formation et la co-direction depuis octobre 2004 avec Dr. Jean-Pierre Simorre (IBS, Grenoble). Deux premiers articles issusde ce travail ont déjà été publiés (Sounier et al. 2007; Schanda et al. 2007) et deux autres, avec R. Sounier en premier auteur, sont actuellement en préparation. Ces travaux ont débouché sur l’étude RMN hors d’équilibre de NanoMachines hyperthermophiles de plusieurs centaines de kDa. Ce dernier projet a été confié au Dr. C Amero qui a rejoint l’équipe en juin 2008, pour être formé sous ma direction à la RMN des assemblages supramoléculaires.J’ai également encadré, plusieurs étudiants en Master de Biologie de l’UJF. Ils ont été impliqués dans la mise au point de protocoles de protonation spécifique des méthyles des isoleucines pour K. Treche (2005) et des alanines pour N. Usé (2007). Deux étudiants en Masters de Physique ont été impliqués dans les développements pour l’attribution des ponts disulfures pour A. Sollier (1999) et M Falges (2006) pour la mesure rapide de RDC 1H-1H. Plus récemment, j’ai également formé J. Retel, un étudiant hollandais effectuant son stage de Master Life Science & Technology (programme commun des universités de Leiden et de Delft, NL 2007). Son travail de stage consiste à généraliser les outils développés lors de la thèse de Remy Sounier. Cette implication dans la formation des étudiants de Master a été validée par quatre articles dans lesquels les étudiants de Master sont co-auteurs (Boisbouvier et al. 2000; Scanda et al. 2007; Ayala et al., in press; Sounier et al., en préparation) et vient compléter l’expérienced’enseignement en Licence et Agrégation que j’ai acquise pendant deux ans de monitorat

Advanced isotopic labeling for the NMR investigation of challenging proteins and nucleic acids

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CH3-specific NMR assignment of alanine, isoleucine, leucine and valine methyl groups in high molecular weight proteins using a single sample

International audienceA new strategy for the NMR assignment of aliphatic side-chains in large perdeuterated proteins is proposed. It involves an alternative isotopic labeling protocol, the use of an out-and-back C-13-C-13 TOCSY experiment ((H)C-TOCSY-C-TOCSY-(C)H) and an optimized non-uniform sampling protocol. It has long been known that the non-linearity of an aliphatic spin-system (for example Ile, Val, or Leu) substantially compromises the efficiency of the TOCSY transfers. To permit the use of this efficient pulse scheme, a series of optimized precursors were designed to yield linear C-13 perdeuterated side-chains with a single protonated CH3 group in these three residues. These precursors were added to the culture medium for incorporation into expressed proteins. For Val and Leu residues, the topologically different spin-systems introduced for the pro-R and pro-S methyl groups enable stereospecific assignment. All CH3 can be simultaneously assigned on a single sample using a TOCSY experiment. It only requires the tuning of a mixing delay and is thus more versatile than the relayed COSY experiment. Enhanced resolution and sensi-tivity can be achieved by non-uniform sampling combined with the removal of the large J(CC) coupling by deconvolution prior to the processing by iterative soft thresholding. This strategy has been used on malate synthase G where a large percentage of the CH3 groups could be correlated directly up to the backbone Ca. It is anticipated that this robust combined strategy can be routinely applied to large proteins

Pi7, an orphan peptide from the scorpion Pandinus imperator: a 1H-NMR analysis using a nano-NMR Probe.

International audienceThe three-dimensional solution structure of a novel peptide, Pi7, purified from the venom of the scorpion Pandinus imperator, and for which no specific receptor has been found yet, was determined by two-dimensional homonuclear proton NMR methods from a nanomole amount of compound using a nano-nmr probe. Pandinus imperator peptide 7 does not block voltage-dependent K(+)-channels and does not displace labeled noxiustoxin from rat brain synaptosomal membranes. The toxin has 38 amino acid residues and, similarly to Pi1, is stabilized by four disulfide bridges (Cys6-Cys27, Cys12-Cys32, Cys16-Cys34, and Cys22-Cys37). In addition, the lysine at position 26 crucial for potassium-channel blocking is replaced in Pi7 by an arginine. Tyrosine 34, equivalent to Tyr36 of ChTX is present, but the N-terminal positions 1 and 2 are occupied by two acidic residues Asp and Glu, respectively. The dihedral angles and distance restraints obtained from measured NMR parameters were used in structural calculations in order to determine the conformation of the peptide. The disulfide-bridge topology was established using distance restraints allowing ambiguous partners between S atoms combined with NMR-derived structural information. The structure is organized around a short alpha-helix spanning residues Thr9 to Thr20/Gly21 and a beta-sheet. These two elements of secondary structure are stabilized by two disulfide bridges, Cys12-Cys32 and Cys16-Cys34. The antiparallel beta-sheet is composed of two strands extending from Asn22 to Cys34 with a tight turn at Ile28-Asn29 in contact with the N-terminal fragment Ile4 to Cys6

Specific labeling and assignment strategies of valine methyl groups for NMR studies of high molecular weight proteins

International audienceThe specific protonation of valine and leucine methyl groups in proteins is typically achieved by overexpressing proteins in M9/D2O medium supplemented with either labeled α-ketoisovalerate for the labeling of the four prochiral methyl groups or with 2-acetolactate for the stereospecific labeling of the valine and leucine side chains. However, when these labeling schemes are applied to large protein assemblies, significant overlap between the correlations of the valine and leucine methyl groups occurs, hampering the analysis of 2D methyl-TROSY spectra. Analysis of the leucine and valine biosynthesis pathways revealed that the incorporation of labeled precursors in the leucine pathway can be inhibited by the addition of exogenous l-leucine-d10. We exploited this property to label stereospecifically the pro-R and pro-S methyl groups of valine with minimal scrambling to the leucine residues. This new labeling protocol was applied to the 468 kDa homododecameric peptidase TET2 to decrease the complexity of its NMR spectra. All of the pro-S valine methyl resonances of TET2 were assigned by combining mutagenesis with this innovative labeling approach. The assignments were transferred to the pro-R groups using an optimally labeled sample and a set of triple resonance experiments. This improved labeling scheme enables us to overcome the main limitation of overcrowding in the NMR spectra of prochiral methyl groups, which is a prerequisite for the site-specific measurement of the structural and dynamic parameters or for the study of interactions in very large protein assemblies

Spectral editing of intra- and inter-chain methyl–methyl NOEs in protein complexes

International audienceSpecific isotopic labeling of methyl groups in a perdeuterated protein background enables the detection of long range NOEs in proteins or high molecular weight complexes. We introduce here an approach, combining an optimized isotopic labeling scheme with a specifically tailored NMR pulse sequence, to distinguish between intramolecular and intermolecular NOE connectivities. In hetero-oligomeric complexes, this strategy enables sign encoding of intra-subunit and inter-subunit NOEs. For homo-oligomeric assemblies, our strategy allows the specific detection of intra-chain NOEs in high resolution 3D NOESY spectra. The general principles, possibilities and limitations of this approach are presented. Applications of this approach for the detection of intermolecular NOEs in a hetero-hexamer, and the assignment of methyl 1H and 13C resonances in a homo-tetrameric protein complex are shown

Sensitivity-optimized experiment for the measurement of residual dipolar couplings between amide protons

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Backbone and methyl resonances assignment of the 87 kDa prefoldin from Pyrococcus horikoshii

International audiencePrefoldin is a heterohexameric protein assembly which acts as a co-chaperonin for the well conserved Hsp60 chaperonin, present in archaebacteria and the eukaryotic cell cytosol. Prefoldin is a holdase, capturing client proteins and subsequently transferring them to the Hsp60 chamber for refolding. The chaperonin family is implicated in the early stages of protein folding and plays an important role in proteostasis in the cytosol. Here, we report the assignment of 1HN, 15N, 13C', 13Cα, 13Cβ, 1Hmethyl, and 13Cmethyl chemical shifts of the 87 kDa prefoldin from the hyperthermophilic archaeon Pyrococcus horikoshii, consisting of two α and four β subunits. 100% of the [13C, 1H]-resonances of Aβ, Iδ1, Iδ2, Tγ2, Vγ2 methyl groups were successfully assigned for both subunits. For the β subunit, showing partial peak doubling, 80% of the backbone resonances were assigned. In the α subunit, large stretches of backbone resonances were not detectable due to slow (μs-ms) time scale dynamics. This conformational exchange limited the backbone sequential assignment of the α subunit to 57% of residues, which corresponds to 84% of visible NMR signals