Denis Courtier-Murias

Comprehensive multiphase NMR spectroscopy: Basic experimental approaches to differentiate phases in heterogeneous samples

Heterogeneous samples, such as soils, sediments, plants, tissues, foods and organisms, often contain liquid-, gel- and solid-like phases and it is the synergism between these phases that determine their environmental and biological properties. Studying each phase separately can perturb the sample, removing important structural information such as chemical interactions at the gel-solid interface, kinetics across boundaries and conformation in the natural state. In order to overcome these limitations a Comprehensive Multiphase-Nuclear Magnetic Resonance (CMP-NMR) probe has been developed, and is introduced here, that permits all bonds in all phases to be studied and differentiated in whole unaltered natural samples. The CMP-NMR probe is built with high power circuitry, Magic Angle Spinning (MAS), is fitted with a lock channel, pulse field gradients, and is fully susceptibility matched. Consequently, this novel NMR probe has to cover all HR-MAS aspects without compromising power handling to permit the full range of solution-, gel- and solid-state experiments available today. Using this technology, both structures and interactions can be studied independently in each phase as well as transfer/interactions between phases within a heterogeneous sample. This paper outlines some basic experimental approaches using a model heterogeneous multiphase sample containing liquid-, gel- and solid-like components in water, yielding separate (1)H and (13)C spectra for the different phases. In addition, (19)F performance is also addressed. To illustrate the capability of (19)F NMR soil samples, containing two different contaminants, are used, demonstrating a preliminary, but real-world application of this technology. This novel NMR approach possesses a great potential for the in situ study of natural samples in their native state.

Comparison of nuclear magnetic resonance methods for the analysis of organic matter composition from soil density and particle fractions

Environmental context The association of specific organic matter (OM) compounds with clay mineral surfaces is believed to protect these compounds from degradation and thus result in long-term protection in soil. The molecular-level composition of soil OM associated with soil fractions was measured and compared using solid-state 13C nuclear magnetic resonance (NMR) and solution-state 1H NMR methods. Combining these methods allowed more detailed characterisation of OM associated with different soil fractions and will improve the understanding of OM dynamics in soil. Abstract Organic matter (OM) associated with fine soil fractions is hypothesised to be protected from complete biodegradation by soil microbes. It is therefore important to understand the structure and stage of decomposition of OM associated with various soil fractions. Solid-state 13C nuclear magnetic resonance (NMR) spectroscopy has been used extensively to investigate the OM composition of soils and soil fractions. Solution-state 1H NMR spectroscopy has not been used as much but is an emerging tool for analysing soil OM because 1H NMR spectra are often better resolved and provide information that complements the structural information obtained from solid-state 13C NMR experiments. This study compares one-dimensional solution-state 1H NMR and solid-state 13C NMR methods for assessing the degradation and composition of OM in three different soils, and their light and clay-size fractions. The alkyl/O-alkyl degradation parameter was consistent across all NMR methods and showed that OM associated with clay-size fractions were at more advanced stages of degradation as compared to that in light density soil fractions. Solution-state 1H and diffusion edited (DE) 1H NMR results showed the presence of high concentrations of microbial-derived peptidoglycan and peptide side-chains in clay-sized fractions. Lignin was also identified in clay-sized fractions using solid-state 13C and solution-state 1H NMR techniques. The combination of solid-state 13C and solution-state 1H NMR methods provides a more detailed analysis of OM composition and thereby facilitates a better understanding of the fate and preservation of OM in soil.

Experimental verification of force fields for molecular dynamics simulations using Gly-Pro-Gly-Gly.

Experimental NMR verification of MD simulations using 12 different force fields (AMBER, CHARMM, GROMOS, and OPLS-AA) and 5 different water models has been undertaken to identify reliable MD protocols for structure and dynamics elucidations of small open chain peptides containing Gly and Pro. A conformationally flexible tetrapeptide Gly-Pro-Gly-Gly was selected for NMR (3)J-coupling, chemical shift, and internuclear distance measurements, followed by their calculations using 2 μs long MD simulations in water. In addition, Ramachandran population maps for Pro-2 and Gly-3 residues of GPGG obtained from MD simulations were used for detailed comparisons with similar maps from the protein data bank (PDB) for large number of Gly and Pro residues in proteins. The MD simulations revealed strong dependence of the populations and geometries of preferred backbone and side chain conformations, as well as the time scales of the peptide torsional transitions on the force field used. On the basis of the analysis of the measured and calculated data, AMBER99SB is identified as the most reliable force field for reproducing NMR measured parameters, which are dependent on the peptide backbone and the Pro side chain geometries and dynamics. Ramachandran maps showing the dependence of conformational populations as a function of backbone ϕ/ψ angles for Pro-2 and Gly-3 residues of GPGG from MD simulations using AMBER99SB, AMBER03, and CHARMM were found to resemble similar maps for Gly and Pro residues from the PDB survey. Three force fields (AMBER99, AMBER99ϕ, and AMBER94) showed the least satisfactory agreement with both the solution NMR and the PDB survey data. The poor performance of these force fields is attributed to their propensity to overstabilize helical peptide backbone conformations at the Pro-2 and Gly-3 residues. On the basis of the similarity of the MD and PDB Ramachandran plots, the following sequence of transitions is suggested for the Gly backbone conformation: α(L) ⇆ β(PR) ⇆ β(S) ⇆ β(P) ⇆ α, where backbone secondary structures α(L) and α are associated with helices and turns, β(P) and β(PR) correspond to the left- and right-handed polyproline II structures and β(S) denotes the fully stretched backbone conformation. Compared to the force field dependence, less significant, but noteworthy, variations in the populations of the peptide backbone conformations were observed. For different solvent models considered, a correlation was noted between the number of torsional transitions in GPGG and the water self-diffusion coefficient on using TIP3P, TIP4P, and TIP5P models. In addition to MD results, we also report DFT derived Karplus relationships for Gly and Pro residues using B972 and B3LYP functionals.

Quantum Mechanical and NMR Studies of Ring Puckering and cis/trans-Rotameric Interconversion in Prolines and Hydroxyprolines

Nuclear magnetic resonance (NMR) and quantum mechanical (QM) studies have been carried out for proline (Pro) containing peptides: N-acetyl-l-proline (AcProOH) and N-acetyl-4-hydroxy-l-proline (AcHypOH). Preliminary results of variable temperature NMR measurements for Gly-Pro-Gly-Gly (GPGG), Val-Ala-Pro-Gly (VAPG), and Ala-Pro-Gly-Trp amide acetate salt (APGW) are also reported. The effect of solvent (D(2)O, DMSO-d(6) and CD(3)CN) on the pyrrolidine ring conformation and cis/trans-rotamerisation along the amide bond preceding Pro was investigated by temperature dependent NMR followed by detailed transition state (TS) searches for both conformational equilibria using QM methods. The results revealed the energetic characteristics of the TS, which were in satisfactory agreement with NMR, and the corresponding TS geometries, which are not available from experiment. The most remarkable feature of the cis/trans-rotamerisation is that the amide nitrogen in AcProOH and AcHypOH adopts a tetrahedral geometry in the TS. Various HF, DFT, and MP2 calculations together with implicit solvation modeling were employed in order to identify the most suitable QM protocols for reliable predictions of the geometry and the relative energies of the conformations of Pro and Hyp containing peptides in aqueous solution. Solution NMR results were used for the verification of the reliability of the QM predictions. The results indicate that the MP2 calculations combined with implicit solvation models are reasonably accurate in reproducing NMR measured populations of four different conformations of either AcProOH or AcHypOH in different solvents, whereas HF and DFT B3LYP calculations were significantly less accurate.

In-Situ Molecular-Level Elucidation of Organofluorine Binding Sites in a Whole Peat Soil

The chemical nature of xenobiotic binding sites in soils is of vital importance to environmental biogeochemistry. Interactions between xenobiotics and the naturally occurring organic constituents of soils are strongly correlated to environmental persistence, bioaccessibility, and ecotoxicity. Nevertheless, because of the complex structural and chemical heterogeneity of soils, studies of these interactions are most commonly performed indirectly, using correlative methods, fractionation, or chemical modification. Here we identify the organic components of an unmodified peat soil where some organofluorine xenobiotic compounds interact using direct molecular-level methods. Using (19)F→(1)H cross-polarization magic angle spinning (CP-MAS) nuclear magnetic resonance (NMR) spectroscopy, the (19)F nuclei of organofluorine compounds are used to induce observable transverse magnetization in the (1)H nuclei of organic components of the soil with which they interact after sorption. The observed (19)F→(1)H CP-MAS spectra and dynamics are compared to those produced using model soil organic compounds, lignin and albumin. It is found that lignin-like components can account for the interactions observed in this soil for heptafluoronaphthol (HFNap) while protein structures can account for the interactions observed for perfluorooctanoic acid (PFOA). This study employs novel comprehensive multi-phase (CMP) NMR technology that permits the application of solution-, gel-, and solid-state NMR experiments on intact soil samples in their swollen state.

HR-MAS NMR Spectroscopy: A Practical Guide for Natural Samples

High Resolution Magic Angle Spinning (HR-MAS) NMR spectroscopy is a versatile technique that provides high resolution NMR data on samples containing solutions, gels and swellable solids. In addition to providing structural information HR-MAS can also be used to investigate interfaces (for example organic structures at the solid-aqueous soil interface), processes such as swelling/flocculation, kinetic transfer between gel/liquid phases, as well as conformation and molecular interactions in situ. As such, HRMAS has potential in a diversity of fields including organic, biological, environmental, and medical research. This manuscript focuses on the application of HR-MAS to intact natural samples. A range of 1D and 2D NMR experiments are reviewed and compared in terms of general performance on a range of samples. Therefore, this practical guide and review should be a useful reference and starting point for those wishing to apply HR-MAS NMR. While this article focuses on natural samples, common key 1D and 2D experiments are considered which may be of interest to readers with diverse research interests.

The pH-dependence of organofluorine binding domain preference in dissolved humic acid.

In this study we explore the relationship between solution pH and the distribution of the binding interactions at different domains of a dissolved humic acid (HA) for three xenobiotics: pentafluoroaniline (PFA), pentafluorophenol (PFP), and hexafluorobenzene (HFB). The components of HA where xenobiotic interactions occur are identified using the (1)H{(19)F} Reverse Heteronuclear Saturation Transfer Difference (RHSTD) Nuclear Magnetic Resonance (NMR) spectroscopy experiment. At low pH, PFA and PFP interact preferentially with aromatic components of HA. Increasing pH reduces this preference. Conversely, HFB interacts with all components of HA equally, across the entire pH range. The possible roles of both aromatic-specific interactions and conformational changes of HA behind these observations are explored. It is shown that T-oriented π-π interactions at π-electron accepting HA structures are slightly stronger for PFA and PFP than for HFB. Using DOSY NMR it is shown that the pH-dependence of the interactions is correlated with changes in the conformation of the carbohydrate components of HA rather than with the aromatic components. It is argued that the observed preference for aromatic HA is caused by restricted access to the non-aromatic components of HA at low pH. These HA components form tightly bound hydrophobic domains due to strong inter- and intra-molecular hydrogen bonds. At high pH, these structures open up, making them more available for interactions with polar compounds.

Soil Organic Matter in Its Native State: Unravelling the Most Complex Biomaterial on Earth

Since the isolation of soil organic matter in 1786, tens of thousands of publications have searched for its structure. Nuclear magnetic resonance (NMR) spectroscopy has played a critical role in defining soil organic matter but traditional approaches remove key information such as the distribution of components at the soil-water interface and conformational information. Here a novel form of NMR with capabilities to study all physical phases termed Comprehensive Multiphase NMR, is applied to analyze soil in its natural swollen-state. The key structural components in soil organic matter are identified to be largely composed of macromolecular inputs from degrading biomass. Polar lipid heads and carbohydrates dominate the soil-water interface while lignin and microbes are arranged in a more hydrophobic interior. Lignin domains cannot be penetrated by aqueous solvents even at extreme pH indicating they are the most hydrophobic environment in soil and are ideal for sequestering hydrophobic contaminants. Here, for the first time, a complete range of physical states of a whole soil can be studied. This provides a more detailed understanding of soil organic matter at the molecular level itself key to develop the most efficient soil remediation and agricultural techniques, and better predict carbon sequestration and climate change.

Comprehensive multiphase NMR: a promising technology to study plants in their native state

Nuclear magnetic resonance (NMR) spectroscopy is arguably one the most powerful tools to study the interactions and molecular structure within plants. Traditionally, however, NMR has developed as two separate fields, one dealing with liquids and the other dealing with solids. Plants in their native state contain components that are soluble, swollen, and true solids. Here, a new form of NMR spectroscopy, developed in 2012, termed comprehensive multiphase (CMP)‐NMR is applied for plant analysis. The technology composes all aspects of solution, gel, and solid‐state NMR into a single NMR probe such that all components in all phases in native unaltered samples can be studied and differentiated in situ. The technology is evaluated using wild‐type Arabidopsis thaliana and the cellulose‐deficient mutant ectopic lignification1 (eli1) as examples. Using CMP‐NMR to study intact samples eliminated the bias introduced by extraction methods and enabled the acquisition of a more complete structural and metabolic profile; thus, CMP‐NMR revealed molecular differences between wild type (WT) and eli1 that could be overlooked by conventional methods. Methanol, fatty acids and/or lipids, glutamine, phenylalanine, starch, and nucleic acids were more abundant in eli1 than in WT. Pentaglycine was present in A. thaliana seedlings and more abundant in eli1 than in WT. Copyright

Tracking the Fate of Microbially Sequestered Carbon Dioxide in Soil Organic Matter

The microbial contribution to soil organic matter (SOM) has recently been shown to be much larger than previously thought and thus its role in carbon sequestration may also be underestimated. In this study we employ (13)C ((13)CO₂) to assess the potential CO₂ sequestration capacity of soil chemoautotrophic bacteria and combine nuclear magnetic resonance (NMR) with stable isotope probing (SIP), techniques that independently make use of the isotopic enrichment of soil microbial biomass. In this way molecular information generated from NMR is linked with identification of microbes responsible for carbon capture. A mathematical model is developed to determine real-time CO₂ flux so that net sequestration can be calculated. Twenty-eight groups of bacteria showing close homologies with existing species were identified. Surprisingly, Ralstonia eutropha was the dominant group. Through NMR we observed the formation of lipids, carbohydrates, and proteins produced directly from CO₂ utilized by microbial biomass. The component of SOM directly associated with CO₂ capture was calculated at 2.86 mg C (89.21 mg kg(-1)) after 48 h. This approach can differentiate between SOM derived through microbial uptake of CO₂ and other SOM constituents and represents a first step in tracking the fate and dynamics of microbial biomass in soil.