Structural constraints for the Crh protein from solid-state NMR experiments

We demonstrate that short, medium and long-range constraints can be extracted from proton mediated, rare-spin detected correlation solid-state NMR experiments for the microcrystalline 10.4 × 2 kDa dimeric model protein Crh. Magnetization build-up curves from cross signals in NHHC and CHHC spectra deliver detailed information on side chain conformers and secondary structure for interactions between spin pairs. A large number of medium and long-range correlations can be observed in the spectra, and an analysis of the resolved signals reveals that the constraints cover the entire sequence, also including inter-monomer contacts between the two molecules forming the domain-swapped Crh dimer. Dynamic behavior is shown to have an impact on cross signals intensities, as indicated for mobile residues or regions by contacts predicted from the crystal structure, but absent in the spectra. Our work validates strategies involving proton distance measurements for large and complex proteins as the Crh dimer, and confirms the magnetization transfer properties previously described for small molecules in solid protein samples

Exploitation de l'anisotropie de déplacement chimique pour l'étude de la géométrie moléculaire et des contacts intermoléculaires par résonance magnétique nucléaire à l'état solide

Ce mémoire traite de l'étude de la géométrie moléculaire et de la nature des contacts intermoléculaires dans les solides organiques et inorganiques grâce à des méthodologies appropriées. La première partie introduit de manière succincte les différentes notions de RMN à l'état solide utilisées dans ce travail. Dans un deuxième chapitre, l'évolution des valeurs principales du tenseur d'écran du carbone-13 et du phosphore-31 au cours de la déprotonation d'acides carboxyliques et de phosphates est corrélée avec leur état d'ionisation, leur conformation et la nature des interactions intermoléculaires dans lesquelles ils sont engagés. Le troisième chapitre est consacré à la caractérisation de la liaison hydrogène forte impliquant les silanols de deux polysilicates de sodium hydratés, grâce à l'emploi de méthodes complémentaires telles que la dynamique de polarisation croisée 1H-29Si et la modulation dipolaire de l'anisotropie de déplacement chimique du 29Si à faible fréquence de rotation.This work presents various methodologies to probe the molecular geometry and the nature of intermolecular contacts in organic and inorganic solids. The first part gives a brief description of basic principles of solid-state NMR that are used through these studies. In the second part, the variation of chemical shielding tensor principal values of carboxy carbon-13 and phosphate phosphorus-31 upon deprotonation is correlated with ionisation state, conformation and nature of the intermolecular interactions. The third part deals with the characterisation of the strong hydrogen bond involving the silanols of sodium hydrous polysilicates, using complementary methods such as 1H-29Si cross-polarisation dynamics and dipolar modulation of the 29Si chemical shift anisotropy at slow spinning frequency.NANCY1-SCD Sciences & Techniques (545782101) / SudocSudocFranceF

Box C/D snoRNPs: solid-state NMR fingerprint of an early-stage 50 kDa assembly intermediate

International audienceMany cellular functions rely on stable protein-only or protein-RNA complexes. Deciphering their assembly mechanism is a key question in cell biology. We here focus on box C/D small nucleolar ribonucleoproteins involved in ribosome biogenesis. The mature particles contain four core proteins and a guide RNA. Despite their relatively simple composition, these particles don't self-assemble in eukaryote and the production of a native and functional particle requires a large number of transient other proteins, called assembly factors. We present here 13C and 15N solid-state NMR assignment of yeast 126-residue core protein Snu13 in the context of its 50 kDa pre-complex with assembly factors Rsa1p:Hit1p. In this sample, only one third of the protein is labelled, leading to a low sensitivity. We could nevertheless obtain assignment data for 91% of the residues. Secondary structure derived from our assignments shows that Snu13p overall structure is maintained in the context of the complex. Chemical shift perturbations are analysed to evaluate Snu13p conformational changes and interaction interface upon binding to its partner proteins. While indirect perturbations are observed in the hydrophobic core, we find other good candidate residues belonging to the interaction interface. We describe the role of some Snu13p N-terminal and C-terminal residues, not identified in previous structural studies. These preliminary results will serve as a basis for future interaction studies, especially by adding RNA, to decipher box C/D snoRNP particles assembly pathway

In situ follow-up of hybrid alginate–silicate microbeads formation by linear rheology

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Monitoring ssDNA Binding to the DnaB Helicase from Helicobacter pylori by Solid-State NMR Spectroscopy

ISSN:1433-7851ISSN:1521-3773ISSN:0570-083

Prion Amyloid Polymorphs -The Tag Might Change It All

International audienceSup35p is a protein from Saccharomyces cerevisiae. It can propagate using a prion-like mechanism, which means that it can recruit non-prion soluble Sup35p into insoluble fibrils. Sup35p is a large protein showing three distinct domains, N, M and an extended globular domain. We have previously studied the conformations of the full-length and truncated NM versions carrying poly-histidine tags on the N-terminus. Comparison with structural data from C-terminally poly-histidine tagged NM from the literature surprisingly revealed discrepancies. Here we investigated fibrils from the untagged, as well as a C-terminally poly-histidine tagged NM construct, using solid-state NMR. We find that the conformation of untagged NM is very close to the N-terminally tagged NM and confirms our previous findings. The C-terminal poly-histidine tag, in contrast, drastically changes the NM fibril structure, and yields data consistent with results obtained previously on this construct. We conclude that the C-terminally located Sup35p globular domain influences the structure of the fibrillar core at the N domain, as previously shown. We further conclude, based on the present data, that small tags on NM C-terminus have a substantial, despite different, impact. Modifications at this remote localization thus shows an unexpected influence on the fibril structure, and importantly also its propensity to induce [PSI+]

Water dynamics in a mesoporous bioactive glass studied by NMR relaxometry

International audienceWater confined in a new class of mesoporous bioactive glasses (MBGs) has been studied by NMR relaxometry technique.Two CaO-SiO2-P2O5 materials have been compared: both have the same chemical composition but they have been made withdifferent methods – standard or microfluidic techniques -, inducing different textural properties (surface areas, porous volume,mesoporous arrangement).NMR is known for being particularly well suited for studying fluids embedded in mesostructured porous materials because, inaddition of being non-invasive and relatively fast, it allows the characterization of molecules dynamics. In this study, we used NMRrelaxometry, which consists in measuring spin relaxation times as a function of the measurement frequency (or equivalently of thestatic magnetic field B0), in order to determine the dynamics of water molecules embedded in the MBGs. The low field domain(10kHz-10MHz proton Larmor frequency) was investigated with measurements done with a fast field-cycling relaxometer, and the socalleddispersion curves (longitudinal relaxation rate as a function of the measurement frequency) were analyzed in order to haveadditional insights on the motion of confined water.The frequency dependence of the proton relaxation rate in MBGs was first studied by comparing the relaxation dispersion detected inHOD (residual proton of heavy water) with that in H2O, the proton low-frequency relaxation is thus shown to be mainly due tointramolecular dipolar interactions. Secondly, dispersion curves were recorded as a function of the filling degree and analyzed in theframe of the two-phase fast exchange model: tiny differences in the dynamical behavior of the water inside the two bioglasses(exchange timescale, hydration layers) could be evidenced