Blanc SIMI 7 - Blanc - SIMI 7 - Chimie moléculaire, organique, de coordination, catalyse et chimie biologique

Lanthanide complexes as Trojan Horse for proteins structural studies – Ln23

Ln23 or Lanthanide complexes as Trojan Horse for proteins structural studies

The Ln23 project proposes to exploit lanthanide complexes noted [Ln], as Trojan Horses for structural determination of proteins by developing original and straightforward methodologies for three complementary structural techniques, namely X-ray crystallography including crystallogenesis, Small-Angle X-ray Scattering (SAXS) and solid-state NMR. Ultimately, the dissemination of these new integrative tool in the structural biology community.

Design of lanthanide based additives for protein crystallography

The objective of the project is to design lanthanide-based coordination complexes for the determination of protein structures using three complementary techniques: X-ray diffraction, SAXS (Small Angle X-rays scattering) and NMR. This requires finding a family of lanthanide complexes that is (i) stable, (ii) soluble, (iii) able of interacting with proteins and (iii) luminescent, in order to take advantage of their anomalous diffusion properties for diffraction and paramagnetism for NMR. <br />The project was initially divided into three tasks: <br />Task A is the core of the project: it consists in discovering a family of complexes meeting the specifications mentioned above, obtaining a sufficient quantity of model protein structures to identify complex-protein interactions and quantifying these interactions using small model molecules of increasing complexity. <br />Task B is methodological. It consists in using the complex-protein system for several analytical methods: X-ray diffraction of course, but also diffusion techniques (SANS and SAXS), NMR and trying to understand crystallization mechanisms. <br />Task C concerns the applications of this original methodology for the determination of the structure of unknown proteins of increasing complexity: (i) unknown proteins of biological interest, (ii) membrane proteins and (iii) large protein assemblies.

The methodology of this project consists in synthesizing stable and water-soluble lanthanide complexes and then testing their nucleation and phasing properties in the presence of model proteins. This loop was made many times before discovering the crystallophore. This compound was then further investigated for its supramolecular protein interaction properties using X-ray diffraction (synchrotron) and theoretical approaches (QM-MM method). In parallel new NMR analysis methodology have been implemented, in particular titrations of complex/Protein interactions by paramagnetic DOSY. This tool is important to understand the nucleation mechanism.
Given the exceptional potential of crystallophore, national and international collaborations have been established to validate our discoveries by other laboratories. Once this step was validated, a company was founded to produce and market this compound.

The Ln23 project has led to the discovery of an exceptional additive for protein crystallography whose significance is not yet fully apprehended. This lanthanide complex is the best nucleating and phasing additive currently known and saves considerable time if used routinely for protein crystallography. All proofs of concept were established during the project, the product has been on the market since 2017. This product has clearly the potential to become a new standard for protein crystallography, if the habits of the crystallographer community change.


1. S. Denis-Quanquin, F. Riobé, M.A. Delsuc, O. Maury, N. Giraud Chem. Eur. J. 2016, 22, 18123-18131
2. S. Engilberge, F. Riobé, S. Di Pietro, L. Lassalle, N. Coquelle, C. Arnaud, D. Madern, C. Breyton, O. Maury,* E. Girard*. Chem. Science. 2017, 8, 5909-5917.
3. B. Vögeli, S. Engilberge, E. Girard, F. Riobé, O. Maury, T. J. Erb, S. Shima, T. Wagner. PNAS 2018, 115, 3380-3385
4. S. Engilberge, F. Riobé, T. Wagner, S. Di Pietro, C. Breyton, B. Franzetti, S. Shima, E. Girard,* E. Dumont, O. Maury* Chem. Eur. J. 2018, 24, 9739-9746. r
5. Crystallophore, an efficient additive for protein crystal structure determination. IUCRj. submitted 2018.
6. I. Bernhardsgrütter, B. Vögeli, T. Wagner, P. Dominik, N. S. Cortina, G. Bange,S. Engilberger, F. Riobé, O. Maury, E. Girard, S. Shima, J. Zarzycki, T. J. Erb. Nature Chem. Biol. Submitted 2018
7. Complexe de lanthanide pour la cristallisation de protéine et la détermination de leur structure cristallographique. S. Engilberge, E. Girard, O. Maury, F. Riobé, Fr. Demande (2017), FR 3045606 A1 20170623 PCT Int. Appl. (2017), WO 2017103545 A1 20170622. Licence d’exploitation avec la société Polyvalan
8. Large scale synthesis of FR39 complex D. Pitrat, J.-C. Mulatier, S. Di Pietro, F. Riobé, E. Girard and O. Maury. Enveloppe SOLEAU ref. 557323 déposée le 28/01/2016.
9. Nouveaux complexes d’ions métalliques pour la cristallisation macromolécules biologiques et la détermination de leur structure cristallographique C. Chapelle, S. Engilberge, E. Girard, O. Maury, F. Riobé, Fr. Demande (2017) FR1770467 déposé le 06/05/2017

The tremendous success of determining the complete determination of human genome in 2000 opened the way for an even wider research area: structural genomics, that consists in determining protein structures, a crucial milestone in the process of understanding the relationship between their structure and functions. Due to the high number, and also the variety of existing proteins, this is a huge enterprise, yet just at its beginning, and for which we do not figure out the impact in terms of scientific and medical benefits. Nowadays, the two tools of choice for this structural determination are crystallography and nuclear magnetic resonance spectroscopy (NMR). These two complementary techniques have their own advantages and limitations: (i) NMR allows the analysis of proteins in solution but requires an isotopic enrichment, (ii) crystallography allows a more rapid determination of structures, but remains dependent on the preparation of crystals of good quality, a step still poorly controlled. Nowadays any technological achievement that helps improving the performances of NMR as well as protein crystallography will of course constitute a major step forward in the quest to understand complex biochemical processes involving proteins. Unfortunately, most of the research effort that is currently made to develop these two powerful analytical tools focuses on the development of very large instruments to the detriment of the research for new methodologies.
The Ln23 project proposes to exploit lanthanide complexes (noted [Ln] ), and their intrinsic physical properties (paramagnetism, anomalous scattering, luminescence ...) as Trojan Horses for the structural determination of proteins, by developing original and straightforward methodologies for three complementary structural techniques, namely X-ray crystallography including crystallogenesis, Small-Angle X-ray Scattering and solid-state NMR. Ultimately, the dissemination of these new integrative tool in the structural biology community will result is a significant gain of time and/or accuracy for protein structure determination and will allow an optimal use of the existing large equipments.
The core of the Ln23 project is based on the comprehension and quantification of the supramolecular interactions occurring between [Ln] and protein, an actually empty field of research. To that end, a family of [Ln] will be rationally designed featuring various charge, shape, hydrophilic or lipophilic balance and hydrogen bond donating or accepting ability. The interaction of these complexes with a homogeneous panel of proteins belonging to the malate deshydrogenase family and featuring similar shape but different surface electrostatic charge will be studied using high throughput methods. Finally the binding sites will be identified and the thermodynamic strength of these interactions will be estimated using models of increased complexity.
Based on the deep understanding of these interactions, it will be possible to co-crystallize [Ln] with proteins, allowing new developments in X-ray crystallography or solid state NMR crystallography based on anomalous scattering or paramagnetic intrinsic properties of f-elements respectively. Furthermore, it will be possible to avoid these interactions and to locate selectively the [Ln] into the solvent channels giving rise to low resolution structural method (MASC) based on solvent contrast.
It is important to emphasis the integrative character of this new tool since the control of supramolecular [Ln]-protein interactions will allow on the one hand important technical developments in the field of structural methods, and on the other hand to address rapidly biologically relevant applications for soluble proteins to amphiphilic membrane proteins and finally towards large molecular assemblies.

Project coordinator

Monsieur Olivier Maury (Laboratoire de chimie de l'ENS Lyon)

The author of this summary is the project coordinator, who is responsible for the content of this summary. The ANR declines any responsibility as for its contents.


ENSL Laboratoire de chimie de l'ENS Lyon
ICMMO Institut de Chimie Moléculaire et des Matériaux d'Orsay

Help of the ANR 445,827 euros
Beginning and duration of the scientific project: December 2013 - 48 Months

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