Institut de Chimie Moléculaire et des Matériaux d'Orsay

Laboratoire de RMN en milieu orienté - LRMN



 This project is a long term collaboration with the group of Olivier Maury, at Ecole Normale Superieure de Lyon, and is currently funded by the Agence Nationale de la Recherche (Project Ln23).

Introduction: lanthanides and protein structure determination

 Describing and understanding supramolecular interactions in large biological 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. To address this issue, macromolecular crystallography (MX) has shown to be one of the most powerful techniques in so far as it allows to visualize biological structures at the atomic level. Nowadays, determining the 3D structure of the molecule of interest by MX includes two main methods. The majority of the new macromolecular structures deposited to date in the Protein Data Bank (PDB) are solved by molecular replacement, which requires that the structure of an homolog protein is already available. If no homolog model exists, experimental phasing remains a method of choice for determining the structure of the original macromolecule. Accordingly, about 20% of the PDB deposited X-rays structures have been solved using methods based on anomalous scattering such as single–wavelength (SAD) or multi-wavelength (MAD) anomalous diffraction. In this context, lanthanide ions are probably among the most promising elements for anomalous diffraction, due to their large anomalous signal.

Deciphering supramolecular interactions between lanthanide complexes and proteins

  Recently, we have proposed to directly incorporate a lanthanide derivative within the crystal through supramolecular non covalent interactions. In this spirit, we have recently shown that some lanthanide complexes interact strongly with specific amino-acids from the proteins, and thus co-crystallize with them. Tris(dipicolinate)-lanthanide complexes ([Ln(DPA)3]3- (DPA = dipicolinate = pyridine-2,6-dicarboxylate) have exhibited a high potential for the determination of protein structures alongside with well-known luminescence property, allowing a direct detection of their incorporation into the protein crystals. This has been first illustrated on model proteins, then on a protein of unknown structure and finally towards large macromolecular assemblies.

Identification of a preferential interaction between the lanthanide complex and cationaic amino-acid residues such as arginine.

 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. In particular, in the case of HEWL a preferential hydrogen-bonding network is created with arginine side chains, that also plays a central role in the crystallization process. We have thus aimed at getting a deeper insight in the comprehension of this supramolecular effect. To that end, we have first reported a statistical study of the three cationic amino-acids (R, K and H) behavior, in three different derived protein structures (Hen egg-white lysozyme HEWL, Urate Oxidase from Aspergillus flavus, Thaumatine from Thaumatococcus daniellii).

Modeling the interaction between Ln(dpa)3 and Arginine side chain ...

 This analysis has provided evidence for their bio-availability toward [Ln(DPA)3]3- complexes. Then, in order to bring to light the detail of such interaction mechanism that is extremely difficult to quantify at the protein level, we have introduced simple molecular models that mimic arginine, histidine and lysine side chains, namely ethylguanidinium (EtGua+), imidazolium (Imz+) and ethylammonium (EtNH3+) respectively. We have reported an X-ray diffraction analysis of [EtGua]3[Tb(DPA)3].2H2O, [Imz]3[Tb(DPA)3].3H2O and [EtNH3]3[Tb(DPA)3].3H2O crystals that has confirmed the existence of a supramolecular effect, and provided the description of the resulting hydrogen-bond networks in each crystal packing.

Multi-dimensional error analysis in chemical shift titration experiments ...

 Furthermore, the strength of these networks has been quantified by ab initio calculations while throwing off crystal packing contribution. Finally, these supramolecular interactions have been thermodynamically quantified in aqueous solution, using 1H NMR titration experiments. To that end, an adapted fitting procedure has been implemented toward an accurate determination and comparison of free energy values for the three different ammonium models, while taking into account the experimental uncertainty. This procedure has yielded affinity constants together with 3D uncertainty. An affinity sequence for [Ln(DPA)3]3- has been unambiguously evidenced : EtGua+ > EtNH3+ > Imz+, explaining the preferential supramolecular interaction with arginine residues in protein systems over lysine and histidine, respectively.

Establishment of an affinity sequence EtGua+ > EtNH3+ > Imz+...

Probing supramolecular interactions by Diffusion Ordered NMR SpectroscopY (DOSY)

  Diffusion ordered NMR 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.

Principle of a DOSY experiment. The sample is spatially encoded by pulsed field gradients, so that the NMR signal of any molecule diffusing from one region to another during the analysis can be monitored.

 We have implemented diffusion oredered 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.

DOSY Spectra of the free species [Na]3[Lu(DPA)3] (green) and [EtGua][Cl] (blue), and of the adduct [EtGua]3[Lu(DPA)3] (red)

 We demonstrate 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.

Application of paramagnetic DOSY NMR to the rapid determination of the affinity of a non labeled protein for a given lanthanide complex


(1) Pompidor, G., D'Aléo, A., Vicat, J., Toupet, L., Giraud N.*, Kahn, R.* & Maury, O.*, Ang. Chem. Int. Ed. 47 (18):3388-3391 (2008)
D’Aléo, A., Dumont, E., Maury, O. & Giraud, N.* , Magn. Res. Chem., 51 (10): 641–648 (2013)
(3) Dumont, E., Pompidor, G., D’Aléo, A., Vicat, J., Toupet, L., Maury, O. & Giraud, N.* , Phys. Chem. Chem. Phys., 15 (41), 18235-18242 (2013)
(4) Denis-Quanquin, S., Riobé, F., Delsuc, M.A., Maury, O. & Giraud, N.*, Chemistry - A European Journal, in Press (2016)

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