Modélisation moléculaire par homologie des protéines : ses applications en Biologie et en Bioinformatique

Mes activités de recherche se focalisent sur l’étude structurale de la fonction des protéines tant au point de vue expérimental que théorique et plus particulièrement l’aspect de la reconnaissance intermoléculaire. Notre système de référence est celui de la reconnaissance antigène-anticorps. Ce système est le prototype parfait, de part sa fonction, mais également grâce à la dynamique de ce processus lié au concept de maturation. Aucours de ma thèse de doctorat, nous avons mesuré les constantes d’affinité, à l’équilibre et en cinétique, entre des anticorps monoclonaux et un antigène multivalent (virus de la mosaïque du tabac, VMT). De manière inattendue, nos résultats ont démontré une coopérativité négative dans la liaison d’anticorps au VMT. A partir de mon premier stage postdoctoral et jusqu’à aujourd’hui, ma recherche s’est orientée vers l’aspect structuralde la reconnaissance ; incluant le développement de fonctions énergétiques et de techniques d’analyse des structures tridimensionnelles, la construction de modèles moléculaires et l’assemblage de ligand dans leur recepteur.Ce mémoire résume mes cinq dernières années d’activités scientifiques dans le domaine de la modélisation moléculaire, bien que la parution de publications ne reflète guère ce laps de temps. La modélisation moléculaire est une discipline récente qui nécessite, à mon sens, une introduction formelle. Dans ce mémoire on définit la modélisation moléculaire comme l’ensemble des techniques qui permettent d’étudier la fonction d’unemolécule grâce à la connaissance de sa structure tridimensionnelle. Ces techniques incluent la modélisation par homologie, les méthodes de simulations, les méthodes d’assemblage (docking), les méthodes d’étude du repliement ab-initio des protéines. Les approches spectroscopiques comme la résonance magnétique nucléaire (RMN) ou le dichroisme circulaire et la microscopie électronique (ME) sont également inclues. Au sens littéral notre définition intègre également la diffraction des rayons X (RX).L’objectif de ce mémoire est de décrire en détail une de ces techniques : la modélisation par homologie. Ce mémoire focalise principalement sur les protéines bien que la modélisation moléculaire s’applique aussi bien aux acides nucléiques, aux sucres ou aux lipides. En dépit de la jeunesse de cette discipline, il est pratiquement impossible de la couvrir en son intégralité. Malgré une recherche bibliographique approfondie, elle est certainement incomplète due à l’interdisciplinarité de cette technique qui couvre tous les champs de recherches, de la théorie fondamentale à la médecine. Ce mémoire espère présenter l’impact de la modélisation par homologie dans la biologie moderne de la manière la plus juste.Ce mémoire comprend quatre parties. Après une introduction générale, on présentera en détail les diverses approches employées pour la modélisation par homologie. Les contributions personnelles à cette discipline seront enfin exposées. La perspective d’évolution de la modélisation moléculaire sera finalement brièvement discutée

AFMBioMed Conference: Paris, France, August 2011

International audienceThe fourth edition of the AFMBioMed Conference, Paris 2011 was organized by the Institut Curie, INSERM, and the Life Science Division of the CEA (DSV). The conference was held at the Institut Curie, Paris, France on 23–26 August and chaired by Dr Simon Scheuring

Combined small angle X-ray solution scattering with atomic force microscopy for characterizing radiation damage on biological macromolecules

Background Synchrotron radiation facilities are pillars of modern structural biology. Small-Angle X-ray scattering performed at synchrotron sources is often used to characterize the shape of biological macromolecules. A major challenge with high-energy X-ray beam on such macromolecules is the perturbation of sample due to radiation damage. Results By employing atomic force microscopy, another common technique to determine the shape of biological macromolecules when deposited on flat substrates, we present a protocol to evaluate and characterize consequences of radiation damage. It requires the acquisition of images of irradiated samples at the single molecule level in a timely manner while using minimal amounts of protein. The protocol has been tested on two different molecular systems: a large globular tetremeric enzyme (β-Amylase) and a rod-shape plant virus (tobacco mosaic virus). Radiation damage on the globular enzyme leads to an apparent increase in molecular sizes whereas the effect on the long virus is a breakage into smaller pieces resulting in a decrease of the average long-axis radius. Conclusions These results show that radiation damage can appear in different forms and strongly support the need to check the effect of radiation damage at synchrotron sources using the presented protocol

Conserved pseudoknots in lncRNA MEG3 are essential for stimulation of the p53 pathway

Funding Information: Work in the Marcia lab is partly funded by the Agence Nationale de la Recherche (ANR-15-CE11-0003-01), the Agence Nationale de Recherche sur le Sida et les H?patites Virales (ANRS, ECTZ18552), and ITMO Cancer (18CN047-00). The Marcia lab uses the platforms of the Grenoble Instruct Center (ISBG UMS 3518 CNRS-CEA-UJF-EMBL) with support from FRISBI (ANR-10-INSB-05-02) and GRAL (ANR-10-LABX-49-01) within the Grenoble Partnership for Structural Biology (PSB). IBS acknowledges integration into the Interdisciplinary Research Institute of Grenoble (IRIG, CEA). This work acknowledges the AFM platform at the IBS.Long non-coding RNAs (lncRNAs) are key regulatory molecules, but unlike with other RNAs, the direct link between their tertiary structure motifs and their function has proven elusive. Here we report structural and functional studies of human maternally expressed gene 3 (MEG3), a tumor suppressor lncRNA that modulates the p53 response. We found that, in an evolutionary conserved region of MEG3, two distal motifs interact by base complementarity to form alternative, mutually exclusive pseudoknot structures (“kissing loops”). Mutations that disrupt these interactions impair MEG3-dependent p53 stimulation in vivo and disrupt MEG3 folding in vitro. These findings provide mechanistic insights into regulation of the p53 pathway by MEG3 and reveal how conserved motifs of tertiary structure can regulate lncRNA biological function.Publisher PDFPeer reviewe

Pilot in vivo toxicological investigation of boron nitride nanotubes

Boron nitride nanotubes (BNNTs) have attracted huge attention in many different research fields thanks to their outstanding chemical and physical properties. During recent years, our group has pioneered the use of BNNTs for biomedical applications, first of all assessing their in vitro cytocompatibility on many different cell lines. At this point, in vivo investigations are necessary before proceeding toward realistic developments of the proposed applications. In this communication, we report a pilot toxicological study of BNNTs in rabbits. Animals were injected with a 1 mg/kg BNNT solution and blood tests were performed up to 72 hours after injection. The analyses aimed at evaluating any acute alteration of hematic parameters that could represent evidence of functional impairment in blood, liver, and kidneys. Even if preliminary, the data are highly promising, as they showed no adverse effects on all the evaluated parameters, and therefore suggest the possibility of the realistic application of BNNTs in the biomedical field

Computational Reconstruction of Multidomain Proteins Using Atomic Force Microscopy Data

SummaryClassical structural biology techniques face a great challenge to determine the structure at the atomic level of large and flexible macromolecules. We present a novel methodology that combines high-resolution AFM topographic images with atomic coordinates of proteins to assemble very large macromolecules or particles. Our method uses a two-step protocol: atomic coordinates of individual domains are docked beneath the molecular surface of the large macromolecule, and then each domain is assembled using a combinatorial search. The protocol was validated on three test cases: a simulated system of antibody structures; and two experimentally based test cases: Tobacco mosaic virus, a rod-shaped virus; and Aquaporin Z, a bacterial membrane protein. We have shown that AFM-intermediate resolution topography and partial surface data are useful constraints for building macromolecular assemblies. The protocol is applicable to multicomponent structures connected in the polypeptide chain or as disjoint molecules. The approach effectively increases the resolution of AFM beyond topographical information down to atomic-detail structures

Standardized nanomechanical atomic force microscopy procedure (SNAP) for measuring soft and biological samples

We present a procedure that allows a reliable determination of the elastic (Young's) modulus of soft samples, including living cells, by atomic force microscopy (AFM). The standardized nanomechanical AFM procedure (SNAP) ensures the precise adjustment of the AFM optical lever system, a prerequisite for all kinds of force spectroscopy methods, to obtain reliable values independent of the instrument, laboratory and operator. Measurements of soft hydrogel samples with a well-defined elastic modulus using different AFMs revealed that the uncertainties in the determination of the deflection sensitivity and subsequently cantilever's spring constant were the main sources of error. SNAP eliminates those errors by calculating the correct deflection sensitivity based on spring constants determined with a vibrometer. The procedure was validated within a large network of European laboratories by measuring the elastic properties of gels and living cells, showing that its application reduces the variability in elastic moduli of hydrogels down to 1%, and increased the consistency of living cells elasticity measurements by a factor of two. The high reproducibility of elasticity measurements provided by SNAP could improve significantly the applicability of cell mechanics as a quantitative marker to discriminate between cell types and conditions

Special collection for the ninth AFM BioMed conference

International audienc