132 research outputs found
Modélisation moléculaire par homologie des protéines : ses applications en Biologie et en Bioinformatique
Mes activitĂ©s de recherche se focalisent sur lâĂ©tude structurale de la fonction des protĂ©ines tant au point de vue expĂ©rimental que thĂ©orique et plus particuliĂšrement lâaspect de la reconnaissance intermolĂ©culaire. Notre systĂšme de rĂ©fĂ©rence est celui de la reconnaissance antigĂšne-anticorps. Ce systĂšme est le prototype parfait, de part sa fonction, mais Ă©galement grĂące Ă la dynamique de ce processus liĂ© au concept de maturation. Aucours de ma thĂšse de doctorat, nous avons mesurĂ© les constantes dâaffinitĂ©, Ă lâĂ©quilibre et en cinĂ©tique, entre des anticorps monoclonaux et un antigĂšne multivalent (virus de la mosaĂŻque du tabac, VMT). De maniĂšre inattendue, nos rĂ©sultats ont dĂ©montrĂ© une coopĂ©rativitĂ© nĂ©gative dans la liaison dâanticorps au VMT. A partir de mon premier stage postdoctoral et jusquâĂ aujourdâhui, ma recherche sâest orientĂ©e vers lâaspect structuralde la reconnaissance ; incluant le dĂ©veloppement de fonctions Ă©nergĂ©tiques et de techniques dâanalyse des structures tridimensionnelles, la construction de modĂšles molĂ©culaires et lâassemblage de ligand dans leur recepteur.Ce mĂ©moire rĂ©sume mes cinq derniĂšres annĂ©es dâactivitĂ©s scientifiques dans le domaine de la modĂ©lisation molĂ©culaire, bien que la parution de publications ne reflĂšte guĂšre ce laps de temps. La modĂ©lisation molĂ©culaire est une discipline rĂ©cente qui nĂ©cessite, Ă mon sens, une introduction formelle. Dans ce mĂ©moire on dĂ©finit la modĂ©lisation molĂ©culaire comme lâensemble des techniques qui permettent dâĂ©tudier la fonction dâunemolĂ©cule grĂące Ă la connaissance de sa structure tridimensionnelle. Ces techniques incluent la modĂ©lisation par homologie, les mĂ©thodes de simulations, les mĂ©thodes dâassemblage (docking), les mĂ©thodes dâĂ©tude du repliement ab-initio des protĂ©ines. Les approches spectroscopiques comme la rĂ©sonance magnĂ©tique nuclĂ©aire (RMN) ou le dichroisme circulaire et la microscopie Ă©lectronique (ME) sont Ă©galement inclues. Au sens littĂ©ral notre dĂ©finition intĂšgre Ă©galement la diffraction des rayons X (RX).Lâobjectif de ce mĂ©moire est de dĂ©crire en dĂ©tail une de ces techniques : la modĂ©lisation par homologie. Ce mĂ©moire focalise principalement sur les protĂ©ines bien que la modĂ©lisation molĂ©culaire sâapplique aussi bien aux acides nuclĂ©iques, aux sucres ou aux lipides. En dĂ©pit de la jeunesse de cette discipline, il est pratiquement impossible de la couvrir en son intĂ©gralitĂ©. MalgrĂ© une recherche bibliographique approfondie, elle est certainement incomplĂšte due Ă lâinterdisciplinaritĂ© de cette technique qui couvre tous les champs de recherches, de la thĂ©orie fondamentale Ă la mĂ©decine. Ce mĂ©moire espĂšre prĂ©senter lâimpact de la modĂ©lisation par homologie dans la biologie moderne de la maniĂšre la plus juste.Ce mĂ©moire comprend quatre parties. AprĂšs une introduction gĂ©nĂ©rale, on prĂ©sentera en dĂ©tail les diverses approches employĂ©es pour la modĂ©lisation par homologie. Les contributions personnelles Ă cette discipline seront enfin exposĂ©es. La perspective dâĂ©volution de la modĂ©lisation molĂ©culaire sera finalement briĂšvement discutĂ©e
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 23â26 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 (ÎČ-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 (âkissing loopsâ). 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
Fifteen years of Servitude et Grandeur to the application of a biophysical technique in medicine: The tale of AFMBioMed
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