Nuclear Magnetic Resonance Spectroscopy

From left to right: Xi Chen (PhD), Eugen Lin (Intern, Imperial College), Cedric Caradeuc, Gildas Bertho, Laura Prieur, Mathieu Baudin, Nicolas Giraud

Team Description

The research group on NMR of Biological Substances is part of the team Bio-Spectroscopies. We develop analytical methods based on Nuclear Magnetic Resonance that we apply to the study of biological systems of major interest. Our research activity covers a wide range of projects ranging from the study of the interaction of small molecules with proteins, to the metabolomic analysis of biological fluids.

We design novel experiments to exceed the sensitivity and resolution limits of traditional NMR analyzes on complex samples. We are also working on the development of modelling and data analysis tools to make the best use of the information obtained on the biological systems under study. 

These projects are carried out in the frame of several collaborations with  laboratories working in the fields of organic chemistry, biochemistry, structural biology or medical research.

 

Research Themes

Methodological developments for ultra-high resolution NMR

We develop original concepts which aim at enhancing resolution in spectra of complex samples, to a point where their analysis becomes a faster, easier, and a more accurate process. 

Since the advent of Fourier transform NMR, a vast number of pulse sequences have been specifically designed, that allow to probe a great number of spin interactions on 1D and 2D spectra. Unfortunately, in most of the systems that are of interest to the scientific community nowadays especially in the biomedical field, the size or the complexity of the molecular architecture which is probed often leads to overcrowded spectra whose resolution is too low to give access to their analytical content.

In this context, we develop new analytical tools based on « pure shift » and « J-edited » NMR methods, and to apply them to challenging chemical and/or biological systems. Some of our most recent developments are described below. 

Pure Shift NMR applied to metabolomics analysis of cancer cells (coll. V. Baud and C. Thieblemont)

We have recently developed a “pure shift” version of the 1H 1D NOESY-presat experiment used for metabolomics. We have successfully applied it to an extra cellular medium taken from a cancer cell line. We have shown that we can detect metabolites in cancer cells, with a significant improvement of their spectral resolution, within an experimental time that is compatible with standard analytical workflows. The analysis of the resulting pure shift data obtained for a series of 40 Diffuse Large B-Cell (DLBCL) exo-metabolomes shows that we can observe individually about 85 % of the 40 metabolites usually reported in literature for extra cellular media in cancer studies. Furthermore, the statistical analyses performed on the “pure shift” dataset show that they are suitable for a clean separation of the groups of cells undergoing different anti-metabolic treatments, despite their lower sensitivity.

A) Pure shift and standard 1H NMR spectra (500 MHz) of an extra cellular medium from a DLBCL cell line treated with a combination of anti-metabolic drugs. B) Regions highlighting the resolution achieved in pure shift data for a collection of 40 DLBCL samples. C) Statistical analyses performed on “standard” and “pure shift” datasets.

Monitoring Conformational Changes in an Enzyme Conversion Inhibitor Using Pure Shift EXSY

We have also achieved recently the acquisition of 2D NMR EXSY spectra with ultrahigh resolution, which allowed us for probing the slow conformational exchange process in a pharmaceutical compound.

The resolution enhancement is achieved by implementing interferogram based PSYCHE homonuclear decoupling to generate a pure shift proton spectrum along the direct domain of the resulting data.

We have shown that although being less sensitive and requiring a longer acquisition time, the quality of pure shift spectra allows for extracting exchange rates values that are coherent with the ones determined by standard approach, on a temperature range that demonstrates the robustness of the chosen homonuclear decoupling method.

The resolution enhancement provided by the simplification of proton lineshape allows for probing a higher number of proton sites whose analysis would have been biased using a standard method. These results open the way to a thorough and accurate study of chemical exchange processes based on a multi-site analysis of  2D pure shift EXSY spectra

Dynamic and structural study of biomolecular interactions

Over the last years, our group has brought several contributions to the field of biomolecular interactions analysis. We develop experimental and theoretical methods for probing both dynamic and structural features of the supramolecular interactions that are underlying several biological processes. 

Describing and understanding supramolecular interactions in large biological or bio-mimetic systems has become a major stake over the recent years. However, despite the considerable progress that have been achieved in this field, probing at an atomic scale protein-protein, protein-nucleic acid, protein-lipid, protein-carbohydrate interactions, or the interactions with small molecules, enzyme substrates and regulators, still constitutes a critical challenge for chemists and biochemists.

Deciphering supramolecular interactions between lanthanide complexes and proteins

We have carried out a fruitful collaboration with the group of Dr. Olivier Maury (ENS de Lyon), to develop and understand the properties of Lanthanide derivatives that were shown to interact non-covalently with a broad range of proteins.

In this spirit, we had shown that some Tris(dipicolinate)-lanthanide complexes ([Ln(DPA)3]3- (DPA = dipicolinate = pyridine-2,6-dicarboxylate) interact strongly with specific amino-acids from the proteins, and thus can co-crystallize with them. This approach was demonstrated as a method of choice for incorporating a lanthanide derivative within the protein crystal, hereby exploiting their large anomalous signal for anomalous diffraction in crystallography, alongside with their well-known luminescence property. On the other hand, these lanthanide complexes have also been identified among the most promising non-covalent paramagnetic tags to probe the structure of proteins and peptides by NMR, both in solution and in the solid state. This has been first illustrated on model proteins, then on a protein of unknown structure and finally towards large macromolecular assemblies. The analysis of the binding mode between [Ln(DPA)3]3- and the model proteins has evidenced the existence of a supramolecular effect that mainly involves the tri-anionic complexes and cationic amino-acid residues. Notably, we had identified a preferential supramolecular interaction with arginine residues in protein systems over lysine and histidine, respectively.

Paramagnetic DOSY

Diffusion Ordered NMR SpectroscopY (DOSY) has been shown to be a method of choice for probing a wide range of molecular assemblies by measuring the rate at which they diffuse through the NMR sample, according to their size, or their interaction with their environment. We have implemented diffusion ordered NMR to determine accurately the mobility of paramagnetic tris-dipicolinate lanthanide complexes that are versatile probes of protein structure.
We have shown that the same diffusion coefficient ratios can be measured with an accuracy of 1% using a standard BPPLED pulse sequence, both for diamagnetic and paramagnetic systems, which allows for observing significant –though weak– variations when different species are interacting with the paramagnetic compound.

We have demonstrated that this approach is complementary to classical chemical shift titration experiments, and that it can be applied successfully to probe the supramolecular dynamic interactions between lanthanide complexes and small molecules on the one hand, or to determine rapidly their affinity for a targeted protein.

Metabolomics in the field of Health Sciences

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Dissolution Dynamic Nuclear Polarization (D-DNP) of bio-samples.

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NMR Facility at UMR 8601

Our group is hosting the NMR facility of our Research Unit, including 3 NMR spectrometers (500 MHz to 600 MHz) and a prototype polarizer for Dissolution Dynamic Nuclear Polarization (D-DNP), to improve the sensitivity of solution NMR of bio-samples.

We are members of the Equipex program “CACSICE” (Center for the Analysis of Complex Systems in Complex Environments), and the Equipex program “Paris en resonance”, which provides access to DNP enhanced NMR equipment for users from both academic and industrial laboratories. We have also recently implemented the first NMR facility in France dedicated to metabolomic analyses for Health Sciences “Metabo Paris Santé”.

1. Plainchont, Giraud, N.* et al. Highly Accurate Quantitative Analysis Of Enantiomeric Mixtures From Spatially Frequency Encoded 1H NMR Spectra Anal. Chem. 2018, 90, 1595-1600

2. Collin, S., Giraud, N., Reinaud, O.* et al.A biomimetic strategy for the selective recognition of organophosphates in 100% water: synergies of electrostatic interactions, cavity embedment and metal coordination. Organic Chemistry Frontiers, 2019, 6, 1627-1636

3. M Melikian, B Eluard, G Bertho, V Baud, & N Evrard-Todeschi Model of the Interaction between the NF-κB Inhibitory protein p100 and the E3 ubiquitin ligase β-TrCP based on NMR and Docking Experiments J Chem Inf Model, 2017, 2, 223-233

4. Denis-Quanquin, S., Giraud, N.* et al. Paramagnetic DOSY: an Accurate Tool for the Analysis of the Supramolecular Interactions between Lanthanide Complexes and Proteins. Chem. Eur. J. 2016, 22, 18123-18131

5. M Luck, G Bertho, N Pallet* et al. Rule-Mining for the Early Prediction of Chronic Kidney Disease Based on Metabolomics and Multi-Source Data PLoS One 2016 Nov 18;11(11):e0166905

6.  I Anosova, F Kateb, M Sattler et al. A novel RNA binding surface of the TAM domain of TIP5/BAZ2A mediates epigenetic regulation of rRNA genes Nucleic Acids Res 2015, 43, 5208-5220

Nuclear Magnetic Resonance Spectroscopy

Team Description

 

The research group NMR of Biological Substances is part of the Team Bio-Spectroscopies. We develop analytical methods based on Nuclear Magnetic Resonance that we apply to the study of biological systems of major interest. Our research activity covers a wide range of projects ranging from the study of the interaction of small molecules with proteins, to the metabolomic analysis of biological fluids.

We design novel experiments to exceed the sensitivity and resolution limits of traditional NMR analyzes on complex samples. We are also working on the development of modelling and data analysis tools to make the best use of the information obtained on the biological systems under study. 

These projects are carried out in the frame of several collaborations with  laboratories working in the fields of organic chemistry, biochemistry, structural biology or medical research.

 

 

 

Research Themes

 

Methodological developments for ultra-high resolution NMR

We develop original concepts which aim at enhancing resolution in spectra of complex samples, to a point where their analysis becomes a faster, easier, and a more accurate process. 

Since the advent of Fourier transform NMR, a vast number of pulse sequences have been specifically designed, that allow to probe a great number of spin interactions on 1D and 2D spectra. Unfortunately, in most of the systems that are of interest to the scientific community nowadays especially in the biomedical field, the size or the complexity of the molecular architecture which is probed often leads to overcrowded spectra whose resolution is too low to give access to their analytical content.

In this context, we develop new analytical tools based on « pure shift » and « J-edited » NMR methods, and to apply them to challenging chemical and/or biological systems. Some of our most recent developments are described below. 

Pure Shift NMR applied to metabolomics analysis of cancer cells (coll. V. Baud and C. Thieblemont)

We have recently developed a “pure shift” version of the 1H 1D NOESY-presat experiment used for metabolomics. We have successfully applied it to an extra cellular medium taken from a cancer cell line. We have shown that we can detect metabolites in cancer cells, with a significant improvement of their spectral resolution, within an experimental time that is compatible with standard analytical workflows. The analysis of the resulting pure shift data obtained for a series of 40 Diffuse Large B-Cell (DLBCL) exo-metabolomes shows that we can observe individually about 85 % of the 40 metabolites usually reported in literature for extra cellular media in cancer studies. Furthermore, the statistical analyses performed on the “pure shift” dataset show that they are suitable for a clean separation of the groups of cells undergoing different anti-metabolic treatments, despite their lower sensitivity.

A) Pure shift and standard 1H NMR spectra (500 MHz) of an extra cellular medium from a DLBCL cell line treated with a combination of anti-metabolic drugs. B) Regions highlighting the resolution achieved in pure shift data for a collection of 40 DLBCL samples. C) Statistical analyses performed on “standard” and “pure shift” datasets.

Monitoring Conformational Changes in an Enzyme Conversion Inhibitor Using Pure Shift EXSY

We have also achieved recently the acquisition of 2D NMR EXSY spectra with ultrahigh resolution, which allowed us for probing the slow conformational exchange process in a pharmaceutical compound.

The resolution enhancement is achieved by implementing interferogram based PSYCHE homonuclear decoupling to generate a pure shift proton spectrum along the direct domain of the resulting data.

We have shown that although being less sensitive and requiring a longer acquisition time, the quality of pure shift spectra allows for extracting exchange rates values that are coherent with the ones determined by standard approach, on a temperature range that demonstrates the robustness of the chosen homonuclear decoupling method.

The resolution enhancement provided by the simplification of proton lineshape allows for probing a higher number of proton sites whose analysis would have been biased using a standard method. These results open the way to a thorough and accurate study of chemical exchange processes based on a multi-site analysis of  2D pure shift EXSY spectra

Dynamic and structural study of biomolecular interactions

Over the last years, our group has brought several contributions to the field of biomolecular interactions analysis. We develop experimental and theoretical methods for probing both dynamic and structural features of the supramolecular interactions that are underlying several biological processes. 

Describing and understanding supramolecular interactions in large biological or bio-mimetic systems has become a major stake over the recent years. However, despite the considerable progress that have been achieved in this field, probing at an atomic scale protein-protein, protein-nucleic acid, protein-lipid, protein-carbohydrate interactions, or the interactions with small molecules, enzyme substrates and regulators, still constitutes a critical challenge for chemists and biochemists.

Deciphering supramolecular interactions between lanthanide complexes and proteins

We have carried out a fruitful collaboration with the group of Dr. Olivier Maury (ENS de Lyon), to develop and understand the properties of Lanthanide derivatives that were shown to interact non-covalently with a broad range of proteins.

In this spirit, we had shown that some Tris(dipicolinate)-lanthanide complexes ([Ln(DPA)3]3- (DPA = dipicolinate = pyridine-2,6-dicarboxylate) interact strongly with specific amino-acids from the proteins, and thus can co-crystallize with them. This approach was demonstrated as a method of choice for incorporating a lanthanide derivative within the protein crystal, hereby exploiting their large anomalous signal for anomalous diffraction in crystallography, alongside with their well-known luminescence property. On the other hand, these lanthanide complexes have also been identified among the most promising non-covalent paramagnetic tags to probe the structure of proteins and peptides by NMR, both in solution and in the solid state. This has been first illustrated on model proteins, then on a protein of unknown structure and finally towards large macromolecular assemblies. The analysis of the binding mode between [Ln(DPA)3]3- and the model proteins has evidenced the existence of a supramolecular effect that mainly involves the tri-anionic complexes and cationic amino-acid residues. Notably, we had identified a preferential supramolecular interaction with arginine residues in protein systems over lysine and histidine, respectively.

Paramagnetic DOSY

Diffusion Ordered NMR SpectroscopY (DOSY) has been shown to be a method of choice for probing a wide range of molecular assemblies by measuring the rate at which they diffuse through the NMR sample, according to their size, or their interaction with their environment. We have implemented diffusion ordered NMR to determine accurately the mobility of paramagnetic tris-dipicolinate lanthanide complexes that are versatile probes of protein structure.
We have shown that the same diffusion coefficient ratios can be measured with an accuracy of 1% using a standard BPPLED pulse sequence, both for diamagnetic and paramagnetic systems, which allows for observing significant –though weak– variations when different species are interacting with the paramagnetic compound.

We have demonstrated that this approach is complementary to classical chemical shift titration experiments, and that it can be applied successfully to probe the supramolecular dynamic interactions between lanthanide complexes and small molecules on the one hand, or to determine rapidly their affinity for a targeted protein.

Metabolomics in the field of Health Sciences

Your content goes here. Edit or remove this text inline or in the module Content settings. You can also style every aspect of this content in the module Design settings and even apply custom CSS to this text in the module Advanced settings.

Dissolution Dynamic Nuclear Polarization (D-DNP) of bio-samples.

Your content goes here. Edit or remove this text inline or in the module Content settings. You can also style every aspect of this content in the module Design settings and even apply custom CSS to this text in the module Advanced settings.

NMR Facility at UMR 8601

Our group is hosting the NMR facility of our Research Unit, including 3 NMR spectrometers (500 MHz to 600 MHz) and a prototype polarizer for Dissolution Dynamic Nuclear Polarization (D-DNP), to improve the sensitivity of solution NMR of bio-samples.

We are members of the Equipex program “CACSICE” (Center for the Analysis of Complex Systems in Complex Environments), and the Equipex program “Paris en resonance”, which provides access to DNP enhanced NMR equipment for users from both academic and industrial laboratories. We have also recently implemented the first NMR facility in France dedicated to metabolomic analyses for Health Sciences “Metabo Paris Santé”.

1. Plainchont, Giraud, N.* et al. Highly Accurate Quantitative Analysis Of Enantiomeric Mixtures From Spatially Frequency Encoded 1H NMR Spectra Anal. Chem. 2018, 90, 1595-1600

2. Collin, S., Giraud, N., Reinaud, O.* et al.A biomimetic strategy for the selective recognition of organophosphates in 100% water: synergies of electrostatic interactions, cavity embedment and metal coordination. Organic Chemistry Frontiers, 2019, 6, 1627-1636

3. M Melikian, B Eluard, G Bertho, V Baud, & N Evrard-Todeschi Model of the Interaction between the NF-κB Inhibitory protein p100 and the E3 ubiquitin ligase β-TrCP based on NMR and Docking Experiments J Chem Inf Model, 2017, 2, 223-233

4. Denis-Quanquin, S., Giraud, N.* et al. Paramagnetic DOSY: an Accurate Tool for the Analysis of the Supramolecular Interactions between Lanthanide Complexes and Proteins. Chem. Eur. J. 2016, 22, 18123-18131

5. M Luck, G Bertho, N Pallet* et al. Rule-Mining for the Early Prediction of Chronic Kidney Disease Based on Metabolomics and Multi-Source Data PLoS One 2016 Nov 18;11(11):e0166905

6.  I Anosova, F Kateb, M Sattler et al. A novel RNA binding surface of the TAM domain of TIP5/BAZ2A mediates epigenetic regulation of rRNA genes Nucleic Acids Res 2015, 43, 5208-5220