Bénédicte Elena

Experimental aspects of proton NMR spectroscopy in solids using phase-modulated homonuclear dipolar decoupling

In this paper we demonstrate experimentally that the continuously phase-modulated homonuclear decoupling sequence DUMBO-1 is suitable for high-resolution proton NMR spectroscopy of rigid solids. Over a wide range of experimental conditions, we show on the model sample L-alanine as well as on small peptides that proton linewidths of less than 0.5 ppm can be obtained under DUMBO-1 decoupling. In particular the DUMBO-1 sequence yields well resolved proton spectra both at slow and fast MAS. The DUMBO-1 decoupling scheme can in principle be inserted in any multi-nuclear or multi-dimensional solid-state NMR experiment which requires a high-resolution 1H dimension. An example is provided with the 13C-1H MAS-J-HMQC experiment.

Powder crystallography by combined crystal structure prediction and high-resolution 1H solid-state NMR spectroscopy.

A fast method for crystal structure determination using crystal structure prediction and solid-state (1)H NMR is presented. This technique does not need any prior knowledge except the chemical formula; resonance assignment is not necessary. Starting from an ensemble of predicted crystal structures for powdered thymol, comparison between experimental and calculated (1)H solid-state isotropic NMR chemical shifts is sufficient to determine which predicted structure corresponds to the powder under study. The same approach using proton-proton spin-diffusion data is successful and can be used for cross-validation.

Metabotyping of Caenorhabditis elegans reveals latent phenotypes

Assigning functions to every gene in a living organism is the next challenge for functional genomics. In fact, 85–90% of the 19,000 genes of the nematode Caenorhabditis elegans genome do not produce any visible phenotype when inactivated, which hampers determining their function, especially when they do not belong to previously characterized gene families. We used 1H high-resolution magic angle spinning NMR spectroscopy (1H HRMAS-NMR) to reveal the latent phenotype associated to superoxide dismutase (sod-1) and catalase (ctl-1) C. elegans mutations, both involved in the elimination of radical oxidative species. These two silent mutations are significantly discriminated from the wild-type strain and from each other. We identify a metabotype significantly associated with these mutations involving a general reduction of fatty acyl resonances from triglycerides, unsaturated lipids being known targets of free radicals. This work opens up perspectives for the use of 1H HRMAS-NMR as a molecular phenotyping device for model organisms. Because it is amenable to high throughput and is shown to be highly informative, this approach may rapidly lead to a functional and integrated metabonomic mapping of the C. elegans genome at the systems biology level.

Statistical Recoupling Prior to Significance Testing in Nuclear Magnetic Resonance Based Metabonomics

Significance testing is a crucial step in metabolic biomarker recovery from the metabolome-wide latent variables computed by multivariate statistical analysis. In this study we propose an algorithm based on the landscape of the covariance/correlation ratio of consecutive variables along the chemical shift axis to restore, prior to significance testing, the spectral dependency and recouple variables in clusters which correspond to physical, chemical, and biological entities: statistical recoupling of variables (SRV). Variables are associated into a series of clusters, which are then considered as individual objects for the control of the false discovery rate. Compared to classical procedures, it is found that SRV allows efficient recovery of statistically significant metabolic variables. The proposed SRV method when associated with the Benjamini-Yekutieli correction retains a low level of significant variables in the noise areas of the nuclear magnetic resonance (NMR) spectrum, close to that observed using the conservative Bonferroni correction (false positive rate), while also allowing successful identification of statistically significant metabolic NMR signals in cases where the classical procedures of Benjamini-Yekutieli and Benjamini-Hochberg (false discovery rate) fail. This procedure improves the interpretability of latent variables for metabolic biomarker recovery.

Metabolic profiling strategy of Caenorhabditis elegans by whole-organism nuclear magnetic resonance.

In this study, we present a methodology for metabotyping of C. elegans using 1H high resolution magic angle spinning (HRMAS) whole-organism nuclear magnetic resonance (NMR). We demonstrate and characterize the robustness of our metabolic phenotyping method, discriminating wild-type N2 from mutant sod-1(tm776) animals, with the latter being an otherwise silent mutation, and we identify and quantify several confounding effects to establish guidelines to ensure optimal quality of the raw data across time and space. We monitor the sample stability under experimental conditions and examine variations arising from effects that can potentially confuse the biological interpretation or prevent the automation of the protocol, including sample culture (breeding of the worms by two biologists), sample preparation (freezing), NMR acquisition (acquisition by different spectroscopists, acquisition in different facilities), and the effect of the age of the animals. When working with intact model organisms, some of these exogenous effects are shown to be significant and therefore require control through experimental design and sample randomization.

Correlation of fast and slow chemical shift spinning sideband patterns under fast magic-angle spinning

A new two-dimensional solid-state NMR experiment, which correlates slow and fast chemical shift anisotropy sideband patterns is proposed. The experiment, dubbed ROSES, is performed under fast magic-angle spinning and leads to an isotropic spectrum in the directly detected omega(2) dimension. In the evolution dimension omega(1), the isotropic chemical shift is reduced by a factor S, and spinning sidebands are observed spaced by a scaled effective spinning speed omega(R)/S. These spinning sidebands patterns are not identical to those observed with standard slow magic-angle spinning experiments. Chemical shift anisotropy parameters can be accurately extracted with standard methods from these spinning sideband patterns. The experiment is demonstrated with carbon-13 experiments on powdered samples of a dipeptide and a cyclic undecapeptide, cyclosporin-A.

Methyl Proton Contacts Obtained Using Heteronuclear Through-Bond Transfers in Solid-State NMR Spectroscopy

A two-dimensional proton-mediated carbon-carbon correlation experiment that relies on through-bond heteronuclear magnetization transfers is demonstrated in the context of solid-state NMR of proteins. This new experiment, dubbed J-CHHC by analogy to the previously developed dipolar CHHC techniques, is shown to provide selective and sensitive correlations in the methyl region of 2D spectra of crystalline organic compounds. The method is then demonstrated on a microcrystalline sample of the dimeric protein Crh (2 x 10.4 kDa). A total of 34 new proton-proton contacts involving side-chain methyl groups were observed in the J-CHHC spectrum, which had not been observed with the conventional experiment. The contacts were then used as additional distance restraints for the 3D structure determination of this microcrystalline protein. Upon addition of these new distance restraints, which are in large part located in the hydrophobic core of the protein, the root-mean-square deviation with respect to the X-ray structure of the backbone atom coordinates of the 10 best conformers of the new ensemble of structures is reduced from 1.8 to 1.1 A.