La présence d’argile dans les granulats (sables et graviers), influencent considérablement les propriétés rhéologiques des bétons. Considérant les évolutions techniques apportées au béton ces dernières années, la compréhension des mécanismes de nocivité des argiles est donc d’un intérêt majeur sur le plan industriel. Au cours de cette étude, nous avons scindé la problématique en deux parties, la première s’attardant plus spécifiquement sur les mécanismes d’adsorption aux surfaces des argiles. Une analyse fines des interactions polymere-argile par IR, SAXS-WAXS et mesure d’adsorption a alors permis de valider l’adsorption des superplastifiants par les argiles et le rôle bénéfique de l’utilisation de molécule cationique (F25) pour empêcher leur fixation. La détermination d’un processus d’adsorption par échange cationique de ces molécules explique d’une part leur effet inertant et d’autre part la restructuration des particules d’argile à l’échélle du micron comme du nanométre. Dans un deuxième temps, une étude rhéologique des systèmes granulaires a permis de caractériser l’effet intrinséque de argiles sur les propriétés d’écoulement de ces systèmes. L’étude des suspensions d’argile a démontré le rôle de la rétention d’eau par les argiles. L’anisotropie des particules, caractérisée au moyen de différentes techniques (MET, SAXS-WAXS), permet d’expliquer cette rétention d’eau / Clay minerals which can be found in granular materials strongly influence the rheological properties of fresh concrete. Due to the recent technical evolution of concrete, the understanding of the effect of clays in such systems takes on a significant industrial interest. This study was divided into two part, the first one dealing with the adsorption mechanisms of two polymers onto clay surfaces: a superplasticizer (PCP) and a polycation (F25). IR and SAXS-WAXS analysis together with adsorption measurment revealed that superplasticizer are adsorbed by clays and that the used of polycation inhibites clays-PCP interactions. The adsorption process of F25 onto clays by cationic exchange explains the preferential adsorption of cationic compounds rather than PCP and the restructuration of clay particles at the nano and micronic scale during the adsorption. In a second part, the rheological study of granular suspensions revealed a strong effect of clay particles on the flow behaviors of such systems. The rheological study of pure clay suspensions allowed the modelisation of the effect of clays on granular suspensions using excluded volume. In this case, the anistropic features of clays particles characterized by various technics (TEM, SAXS-WAXS) explains water retention properties of such nanometric minerals

Les carbones désordonnés nanoporeux sont des supports efficaces pour le piégeage de polluants y compris à l’état de traces dans les eaux usées. Le phénomène d’adsorption à l’origine de la rétention des molécules est cependant complexe car dépendant d’une multitude de facteurs : structure, morphologie et charge de la surface carbonée d’une part,taille/forme et polarité de la molécule d’autre part, l’ensemble étant dépendant du pH et de la concentration. Pourune meilleure compréhension du phénomène, il est important de pouvoir étudier séparément certains paramètres.Dans la perspective d’étudier le phénomène d’adsorption en milieu aqueux sur des carbones nanoporeux à structure et morphologie modèle, des structures lamellaires nanoporeuses de type carbone turbostratique ont été générées numériquement en langage C++ avec le calcul de la fonction de distribution radiale ou de paires. L’adsorption gazeuse d’une molécule non polaire ou polaire puis de deux molécules polaires (H2O/CO2) et (H2O/C6H6O)a été simulée par la méthode Grand Canonique Monte Carlo sur ce support modèle (Isotherme d’adsorption,chaleur d’adsorption, densité des molécules adsorbées) en fonction de la température. Les temps de calcul ont été drastiquement diminués en développant des codes parallèles optimisés sous MPI C++. L’influence de la forme etde la distribution en taille des pores a été mise en évidence en simulant l’adsorption sur la structure d’un carbone activé déjà obtenue par reconstruction 3D de type RMC. Enfin, d’un point de vue expérimental, l’intercalation d’ions tetraalkylammonium par voie électrochimique dans des carbones lamellaires (HOPG et graphite) a été explorée en vue d’obtenir des carbones lamellaires nanoporeux (≈1 nm). La structure a été caractérisée par diffraction des rayons X. / Disordered nanoporous carbons are the good materials for capturing pollutants, including traces in wastewater. The phenomenon of adsorption at the origin of the retention of molecules is complex. However, depending on a multitude of factors : structure, morphology and loading of the carbonaceous surface, on the one hand, size/shapeand polarity of the molecule, on the other hand, the whole being dependent on pH and concentration. For a better understanding of the phenomenon, it is important to be able to study some parameters separately. In order to study the phenomenon of adsorption in aqueous medium on nanoporous carbons with structure and model morphology, nanoporous slit structures of turbostratic carbon type were generated numerically in C ++ language with thecalculation of the radial distribution function or pairs. The gas adsorption of a nonpolar or polar molecule and then oftwo polar molecules (H2O/CO2) and (H2O/C6H6O) was simulated by Grand Canonical Monte Carlo method on this model support (adsorption isotherm, adsorption heat, density of adsorbed molecules) as a function of temperature.The runtime has been drastically reduced by developing parallel codes optimized under MPI C ++. The influence of the shape and the pore size distribution was demonstrated by simulating the adsorption on the structure of an activated carbon already obtained by 3D reconstruction of the RMC type. Finally, from an experimental point of view, the intercalation of tetraalkylammonium ions electrochemically in slit carbons (HOPG and graphite) was explored in order to obtain nanoporous lamellar carbons ( ≈1 nm). The structure was characterized by X-ray diffraction.

La "cryopolymérisation" permet d'obtenir des gels de polymère macroporeux ou "cryogels". Cette méthode a été utilisée pour la synthèse d'hydrogels thermosensibles à base de pNIPA. La température critique TC correspondant à la transition de volume a été déterminée par des mesures de taux de gonflement et par DSC. La macroporosité (distribution de la taille des pores et épaisseur des parois) et son évolution en fonction de T ont été étudiées par la microscopie biphotonique donnant des informations à l'échelle du µm à plusieurs dizaines de µm. La diffusion de rayons X (SAXS et WAXS) a été utilisée pour caractériser la structure multi-échelle (de quelques dixièmes à quelques dizaines de nm) du gel constituant les parois des macropores. Les courbes de diffusion ont été décrites analytiquement. L'évolution des dix paramètres contenus dans l'équation a été étudiée en fonction de T et discutée. Enfin, des expériences utilisant les phonons hyperfréquences générés par la technique des réseaux transitoires avec détection hétérodyne (HD-TG) ont été réalisées. Ces mesures ont permis de déterminer la vitesse de propagation de l'onde ultra-sonore (à 340 MHz), son atténuation, et la constante de diffusion thermique à différentes températures. / "Cryopolymerisation" yields macroporous gels named "cryogels". The method was used to synthesise thermosensitive pNIPA based hydrogels. The critical temperature TC corresponding to the volume phase transition was determined by swelling ratio measurements and DSC. The macroporosity (pore size distribution and wall thickness) and its change with temperature, was investigated by two-photon microscopy yielding information at the micrometer scale (a few tenths to tens of micrometers). X-ray scattering (SAXS and WAXS) was used to characterise the multi-scale structure of the gel forming the pore walls. The scattering curves were described analytically. The variation with temperature of the 10 parameters contained in the equation was investigated and discussed. Finally, heterodyne detected transient grating experiments were performed on a bulk pNIPA gel. These measurements allowed the determination of the speed of the ultrasonic wave (at 340 MHz), its attenuation and the thermal diffusion constant in the gel at different temperatures.

Les petites GTPases de la famille Arf, régulateurs majeurs du trafic membranaire, sont activé par plusieurs familles de facteurs d’échange nucléotidiques (ArfGEFs). Les ArfGEFs jouent un rôle essentiel dans l’intégration des signaux de régulation qui conduisent à l’activation d’Arf au niveau de compartiments cellulaires spécifiques, cependant les mécanismes par lesquels ils ciblent les Arfs activés aux membranes spécifiques et leur coordination avec l’échange de nucléotide reste peu comprise. Nous utilisons ici la cristallographie et la reconstitution des activités ArfGEF sur des membranes artificielles pour analyser ces mécanismes pour un ArfGEF humain atypique, impliqués dans l’endocytose de récepteurs et associé à l’invasion tumorale dans de nombreuses cellules cancéreuses. Les membres de cette famille ont été décrits comme des GEFs spécifique d’Arf6, et comporte un domaine de type PH après leur domaine Sec7. Dans la deuxième partie de ma thèse, nous voulions savoir comment les isoformes Arf1 et Arf6 achevaient leurs fonctions dans la cellule. Arf1 et Arf6 sont très similaires: elles possèdent plus de 60% d’identité de séquence, et des études structurales ont montré que la surface qu’ils utilisent pour interagir avec leurs régulateurs et effecteurs est essentiellement identique en séquence et en structure. Cependant, elles ont des fonctions différentes dans la cellule et des propriétés différentes in vitro, pour lesquelles aucune donnée structurale n’a donné d’explications. Nous utilisons ici la cristallographie, le SAXS et la RMN pour comprendre la différence entre ces deux isoformes. / Small GTPases of the Arf family, which are pivotal regulators of membrane traffic in eukaryotes, are activated by several families of guanine nucleotide exchange factors (ArfGEFs). ArfGEfs play a key role in processing upstream regulatory signals that lead to Arf activation onto specific subcellular compartments, yet the mechanisms by which they target activated Arfs to specific membranes and their coordination with nucleotide exchange remain poorly understood. Here we used X-ray crystallography and reconstitution of ArfGEF activities on artificial membranes to analyze these mechanisms for an atypical human ArfGEF, involved in receptor endocytosis and associated with tumour invasion in various cancer cells. Members of this family have been described as Arf6-specific GEFs, and carry a PH-like domain downstream their Sec7 domain. In a second part of the work we wanted to know how the isoforms Arf1 and Arf6 achieve exquisitely specific functions in cells. Arf1 and Arf6 are highly similar: they have over 60% sequence identity, and structural studies have shown that the surfaces they use to interact with regulators and effectors are essentially identical in sequence and structure. Yet, they have non-overlapping functions in cells. Arf1 is a major regulator of most aspects of vesicular traffic, while Arf6 is restricted to the plasma membrane where it acts at the crossroads of trafficking and cytoskeleton functions (D'Souza-Schorey and Chavrier 2006). Consistent with their cellular specificities, Arf1 and Arf6 also have distinctive biochemical properties in vitro, for which no straightforward structural explanation has been put forward. Here we used X-ray crystallography, synchrotron SAXS experiments and NMR to assess the difference between these two isoforms.

Les bactéries utilisent un mode de communication appelé quorum sensing pour régulerl’expression des gènes en fonction de la densité de population et contrôler ainsi de façonmulticellulaire des processus tels que la sporulation, la compétence ou la virulence. Chez les bactériesà Gram-positif, le quorum sensing repose principalement sur la production, la sécrétion et la détectionde petits peptides de signalisation.Le projet porte sur l’étude du système quorum sensing: NprR/NprX chez Bacillus cereus, oùNprR est l’effecteur qui reconnait spécifiquement le peptide de signalisation NprX. NprR est uneprotéine bi-fonctionnelle. Seule, elle agit en tant qu’inhibiteur de la sporulation, en complexe avecNprX, elle perd sa fonction initiale au profit d’une activité facteur de transcription impliquée dans lavirulence. NprR appartient à une famille d’effecteurs de quorum sensing appelée RNPP (Rap, NprR,PlcR et PrgX) encore mal caractérisée au niveau structural. Mon projet de thèse a consisté en l’analysestructure-fonction du système NprR/NprX.Pour comprendre la régulation fonctionnelle de NprR par NprX, des études en solution (SECMALSet DLS) ont permis de mettre en évidence un switch moléculaire qui repose sur un changementd’oligomérisation. Ainsi NprX fait basculer NprR d’une conformation Apo dimérique à uneconformation compléxée tétramérique.L’étude structurale par cristallographie a aboutit à la résolution de la structure du complexeNprR/NprX. L’analyse de ce tétramère suggère la reconnaissance de 2 sites distincts sur l’ADN.L’étude structurale par SAXS, a quant à elle, permis de proposer une conformation dimérique de laforme Apo NprR, modèle conforté grâce à une étude par mutagénèse dirigée des résidus d’interface. Ils’agit d’un mode de dimérisation semblable à celui des protéines Rap (membres de la famille RNPP).La caractérisation par ITC de l’interaction NprR/NprX avec différentes formes du peptide,ainsi que l’analyse de la poche de fixation du complexe, ont permis de mieux comprendre la spécificitéd’interaction et de mettre en évidence deux résidus clés de l’effecteur : l’Asn275 essentielle à lafixation du peptide et l’Arg 126 essentielle à l’activation de la fonction facteur de transcription.Ces travaux ont contribué à une meilleure compréhension du système quorum sensingNprR/NprX grâce à l’élucidation du switch moléculaire contrôlé par NprX mais aussi à une meilleureconnaissance de la famille d’effecteurs RNPP. / Bacteria use a communication mode named quorum sensing to regulate gene expression depending on the population density and thus to control processes such as sporulation, competence or virulence in a multicellular manner. In Gram-positive bacteria, the quorum sensing relies mostly on the production, the secretion and the detection of small signaling peptides. The project focuses on the study of the quorum sensing system NprR/NprX in Bacillus cereus, where NprR is the effector, which recognizes specifically the signaling peptide NprX. NprR is a bi-functional protein. In the absence of peptide, it acts as a sporulation inhibitor while in complex with NprX, it acts as transcription factor implicated in virulence. NprR belongs to a family of quorum sensing effectors named RNPP (for the first identified members: Rap, NprR, PlcR and PrgX) still not well characterized at a structural level. My PhD project consisted to perform the structure/function analysis of the NprR/NprX system. To understand the functional regulation of NprR by NprX, I carried out different studies in solution (SEC-MALS and DLS). These results allowed me to highlight a molecular switch based on a changing of the oligomerisation state of the protein. NprX binding switches NprR from an Apo dimeric conformation to a tetrameric complex. The structural study by crystallography led to the resolution of the tetrameric NprR/NprX complex structure. The analysis of this tetramer suggests the recognition of 2 DNA binding sites. The structural study of the dimeric conformation of Apo NprR by SAXS, allowed me to propose a model similar to that of the Rap dimers (members of RNPP family). This model is supported by a directed mutagenesis study of interface residues. The characterization by ITC of the NprR/NprX interaction with different forms of the peptide, as well as the analysis of the binding pocket in the complex, led to a better understanding of the specificity of the interaction. Two key residues of the effector were highlighted: Asn275, essential to peptide binding and Arg126, essential to the activation of the transcription factor function. These results have contributed to a better understanding of the NprR/NprX quorum sensing system thanks to the elucidation of the molecular switch controlled by NprX but also in a better knowledge of the RNPP effectors family.

La squalénisation est la base d’une nouvelle et très prometteuse nanotechnologie. Le concept repose sur l’observation que la conjugaison d’un analogue nucléosidique ayant une activité thérapeutique à une molécule de squalène conduit à la formation spontanée dans l’eau de nanoparticules, de diamètre compris entre 100 et 300 nm, montrant une activité très supérieure à celle de l’analogue nucléosidique seul. Au cours de cette thèse nous avons cherché à comprendre les relations entre la nature de la paire drogue-terpénoide, la structure des nanoparticules et leur activité biologique. Pour cela, d’une part différents nucléosides et analogues nucléosidiques ont été couplés de façon covalente au squalène et d’autre part la gemcitabine a été couplée à des dérivés terpénoides de longueurs croissantes. L’organisation supramoléculaire de ces composés a été déterminée par diffusion des rayons X aux petits angles et cryo-microscopie électronique. L’influence des conditions de nanoprécipitation sur la structure des nanoparticules a été étudiée. L’impact de l’organisation supramoléculaire des nanoparticules sur leur internalisation cellulaire et leur cytotoxicité a été mis en évidence pour certaines lignées. / Squalenoylation is a new and very promising nanotechnology. The concept consists in coupling nucleoside derivatives that have therapeutic activity to a molecule of squalene: whatever the nucleoside, nanoparticles with a diameter around 100 - 300 nm are spontaneously obtained upon nanoprecipitation of the nucleolipid in an aqueous medium. These nanoparticles display an increased biological activity compared with the nucleoside. The aim of this PhD thesis was to understand the link between the nature of the drug-terpenoid pair, the supramolecular structure of nanoparticles and the biological activity.Therefore, in a first part, several nucleosides and nucleoside derivatives were covalently coupled to squalene, and in a second part gemcitabine was coupled to terpenoid chains with increasing length. The supramolecular organization of the nanoparticles was determined by Small Angle X-rays Scattering and cryogenic transmission electronic microscopy. The influence of the condition of nanoprecipitation on the supramolecular structure of nanoparticles was studied. The impact of the supramolecular organization on the cell internalization and cytotoxicity was highlighted for some cell lines.

O uso desenfreado dos recursos naturais durante as últimas décadas têm impactado drasticamente o meio ambiente, direcionando a humanidade a investir no desenvolvimento de tecnologias para produção sustentável e ecológica de novas fontes de energia renovável e de produtos verdes. Nesse âmbito, o uso de resíduos derivados da biomassa vegetal tem sido apresentado como uma promissora alternativa à substituição de combustíveis, componentes químicos e polímeros de origem fóssil. Esse material é barato, abundante e não compete direta ou indiretamente com a segurança alimentar. Hoje, mais de 200 compostos químicos e biopolímeros de valor agregado podem ser obtidos a partir do processamento de material lignocelulósico. Todavia, essa tecnologia ainda não é plenamente desenvolvida, afetando sua competitividade econômica, sendo que o maior custo atribui-se à despolimerização enzimática dos polissacarídeos que formam a parede celular vegetal (PCV). Essa etapa requer preparados enzimáticos compostos por diversas enzimas, que agem sinergicamente sobre a complexa estrutura da PCV. Dentre essas enzimas, as pectinases e xilanases desempenham um importante papel na desconstrução dos polímeros pécticos e da hemicelulose. O presente trabalho objetivou o estudo funcional e estrutural de diferentes classes de pectinases e xilanases com potencial biotecnológico, no intuito de contribuir para o desenvolvimento pleno da despolimerização enzimática da PCV. Dentro dessa perspectiva, foram estudadas: uma pectina metilesterase (Sl-PME) e uma endo-poligalacturonase (Sl-EndoPG) do inseto Sphenophorus levis; uma exo-poligalacturonase (Bl-ExoPG) de Bacillus licheniformis; uma xilanase GH10 (MT-Xyn10) e duas GH11 (MT-Xyn11a e MT-Xyn11b) identificadas no metatranscriptoma de um consórcio microbiótico derivado de compostagem de bagaço de cana-de-açúcar. A estrutura cristalográfica da Sl-PME evidenciou alta semelhança com outra PME de inseto. Também foi concluído que as PMEs de inseto são mais similares às bacterianas, quando comparadas às fúngicas e vegetais, principalmente em relação ao sulco catalítico. Além disso, PMEs de inseto, exclusivamente, apresentam uma permutação circular, possívelmente realcionada a um evento de transferência horizontal. A Bl-ExoPG apresentou-se monomérica em solução, com atividade ótima em pH neutro a 60°C, sendo estável em uma ampla faixa de pH (5-10) e com considerável termoestabilidade em elevadas temperaturas. Essa enzima, também, apresentou especificidade por pectina não-metilada, liberando unicamente monômeros de ácido galacturônico. As três xilanases estudadas apresentaram-se monoméricas em solução, com maior atividade entre 30 e 45°C e pHs de 6 a 9, retendo atividade acima de 50% nos pHs 5 e 10. Além disso, todas elas apresentam especificidade por xilano, sendo que a MT-Xyn10 apresentou, também, alta atividade sobre arabinoxilano. A MT-Xyn10 apresentou um conjunto de propriedades enzimáticas bastante atrativas às aplicações industriais, uma vez que é altamente estável em uma ampla faixa de pH (4-10), termoestável em temperaturas de até 50°C e sua ação catalítica produz diversos xilo-oligossacarídeos de alto valor agregado. A análise da estrutura cristalográfica da MT-Xyn11a revela três particularidades estruturais, compartilhadas com a MT-Xyn11b, mas não descritas para outras GH11. Dentre essas particularidades, um loop parece limitar o acesso do substrato ao sítio catalítico, contribuindo diretamente para a baixa afinidade ao substrato apresentada por essas duas enzimas. / Decades of unbridled use of natural resources have drastically affected the global environment, driving humanity to invest in the development of novel technologies for production of sustainable and ecofriendly renewable energy sources and green products. In this context, plant biomass residues have been presented as a promising alternative to fuels, chemicals and polymers derived from fossil reserves. This feedstock is abundant, cheap and does not compete directly or indirectly with food security. Today, more than 200 value-added chemicals and biopolymers can be generated by processing lignocellulosic material. However, this technology is not fully developed yet; its major costs stem from the enzymatic depolymerization of the polysaccharides that constitute the plant cell wall (PCW). This step requires enzymatic cocktails composed of several enzymes that synergistically deconstruct the complex PCW. Among these enzymes, pectinases and xylanases play an important role in the depolymerization of pectic polymers and hemicellulose. The present work is a functional and structural study of different classes of pectinase and xylanases with biotechnological potential. It intends to contribute to the full development of PCW enzymatic depolymerization. With this perspective, we studied a pectin methylesterase (Sl-PME) and an endo-polygalacturonase (Sl-EndoPG) from the insect Sphenophorus levis; an exo-polygalacturonase (Bl-ExoPG) from Bacillus licheniformis; a GH10 xylanase (MT-Xyn10); and two GH11 xylanases (MT-Xyn11a and MT-Xyn11b) from the metatranscriptome of sugarcane bagasse compost-derived microbial consortia. The Sl-PME crystallographic structure showed high similarity with other insect PME. It was also concluded that insect PMEs are more similar to bacterial PMEs than fungi or plant PMEs, especially in relation to the catalytic groove. Moreover, insect PMEs exclusively presented a circular permutation that is possibly related to an event of horizontal gene transfer. Bl-ExoPG is monomeric in solution, with optimal activity on neutral pH and 60°C, being stable in a wide pH range (5-10) and with considerable thermostability at high temperatures. This enzyme, also presented specificity for non-methylated pectin substrates, releasing only monomers of galacturonic acid as catalytic product. All three xylanases studied here are monomeric in solution, with optimal activity between 30°C and 45°C and between pHs 6 and 9, retaining more than 50% of original activity in the pHs 5 and 10. Besides, they all showed specificity for xylan, and MT-Xyn10 also showed high activity on arabinoxylan. MT-Xyn10 revealed a set of enzymatic properties attractive for industrial applications, such as high stability in a wide pH range (4-10), thermostability up to 50°C and released products that are high value-added xilo-oligosaccharides. The MT-Xyn11a crystallographic structure revealed three structural particularities shared with MT-Xyn11b, but not previously described in other GH11. Among these particularities, a loop seems to limit the substrate access to the catalytic site, contributing to the low enzyme affinity presented by both MT-Xyn11a and MT-Xyn11b.

Este trabalho visou investigar as interações entre enzimas e polímeros em solução e a adsorção das mesmas sobre superfícies sólidas e para isto foi dividido em duas partes distintas. Na primeira parte a influência do poli(etileno glicol) (PEG), polímero considerado inerte e utilizado em muitos processosbiotecnológicos, na atividade enzimática e na conformação estrutural de enzimas foi estudada através de medidas de espectrofotometria- UV, calorimetria e espalhamento de raio-X de baixo ângulo (SAXS). Foram escolhidas neste estudo as enzimas glicose-6-fosfato desidrogenase (G-6-PDH) e hexoquinase (HK), que são enzimas largamente aplicadas em análises clínicas na determinação de glicose no sangue, e também a enzima álcool desidrogenase (AD), utilizada para determinação de concentração de álcool. Foram obtidos resultados quantitativos, numa faixa de baixa concentração de enzima, que indicam uma forte influência de PEG na atividade das enzimas estudadas. Medidas de calorimetria revelaram que PEG interage não só com a enzima em estudo mas também com a coenzima NADP+. Numa faixa de concentração maior, os resultados de SAXS mostraram que PEG exerce também um efeito significativo no processo de agregação das enzimas. Acima de tudo, foi evidenciado neste estudo que PEG não pode ser tratado como um polímero inerte, pois ele interfere na atividade e conformação de enzimas. As enzimas são macromoléculas complexas e PEG interage de forma diferenciada com cada enzima, merecendo atenção especial caso a caso. Na segunda parte do trabalho, o estudo da adsorção de hexoquinase (HK) e creatina fosfoquinase (CPK) sobre lâminas de silício foi realizado através de medidas de ângulo de contato, elipsometria in situ e microscopia de força atômica (AFM) em água. A CPK é uma enzima bastante utilizada em kits de determinação de creatina no sangue e no diagnóstico de desordens musculares. Este trabalho revelou que o mecanismo de adsorção de CPK sobre silício depende fortemente do pH. Em pH 4, 7 ou 9 CPK adsorveu mantendo a mesma conformação que tinha em solução. Medidas de espectrofotometria UV-Vis revelaram uma mudança no pH ótimo para atividade enzimática de CPK de 6,8 para 9 após adsorção. A HK imobilizada em esferas de vidro mostrou atividade maior do que HK imobilizada nas placas. A reutilização das esferas e placas recobertas com HK foi testada e observou-se que as atividades das enzimas adsorvidas no substrato esférico foram mantidas. Entretanto, nas placas revestidas a atividade foi perdida. As enzimas imobilizadas sobre esferas puderam ser reutilizadas pelo menos 3 vezes, mantendo a atividade por um período de até 3 semanas. / This work aimed to investigate the interactions between enzymes and polymers in solution and also the adsorption behavior of these enzymes on solid surfaces. For that reason it was divided into two parts. In the first part, the influence of poly(ethylene glycol) (PEG), a polymer considered inert and utilized in several biotechnological processes, on the enzymatic activity and structure of the enzyme was studied by means of UV spectrophotometry, calorimetric titration, circular dichroism (CD) and small angle X-ray scattering (SAXS). Glucose-6-phosphate dehydrogenase (G-6-PDH) and hexokinase (HK) were chosen because of their large application in clinical analysis for determination of glucose in the blood strain. Alcohol dehydrogenase (AD), which is widely used to determine alcohol concentration in various samples, was also used. Quantitative results, in a low enzyme concentration range, indicated a strong influence of PEG on the enzymes activity. The calorimetric measurements revealed no favorable interactions between enzyme and polymer, but indicated favorable interactions between PEG and co-enzyme NADP+. In a higher concentration range, SAXS results showed that PEG also exerts a significant effect on the enzyme aggregation process. This work showed that PEG shall no longer be treated as an inert polymer since it interferes in the enzyme activity and structure. The enzymes are complex macromolecules and PEG interacts differently with each one, deserving special attention in each case. In the second part of the work, the adsorption behavior of creatine phosphokinase (CPK) and hexokinase (HK) onto silicon wafers was studied by means of contact angle measurements, in situ ellipsometry and atomic force microscopy (AFM) in water. CPK was chosen due to its large application on the diagnosis of several muscle disorders. This work revealed that the adsorption mechanism of CPK on silicon surfaces is strongly dependent on pH. At pH 4, 6.8 or 9, CPK adsorbed keeping the same conformation as in solution. pectrophotometric measurements revealed a shift on the optimum pH from 6,8 to 9 upon CPK adsorption. HK adsorbed onto glass beads showed higher activity than HK immobilized on silicon wafers. HK covered glass beads could also be reused three times and for a period of at least three weeks. In the contrary, HK covered silicon wafers could not be reused. For practical purposes, HK covered glass beads showed to be a better “biosensor" than HK covered silicon wafers.

Les polykétides sont des molécules complexes possédant des rôles thérapeutiques divers (antibiotiques, anticancéreux, immunosuppresseurs, etc..). Ces composés sont synthétisés par les polykétides synthases (PKS) qui présentent donc un intérêt certain. Une stratégie prometteuse et en développement, appelée biologie synthétique, consiste en l’ingénierie protéique des PKS pour produire de nouveaux polykétides. Cependant, un prérequis au succès de cette méthode est la compréhension du fonctionnement des PKS. En effet, les PKS sont des systèmes complexes composés de plusieurs sous-unités. Celles-ci comportent chacune un ou plusieurs modules responsable d’un cycle d’extension du polykétide. Les modules sont composés également de plusieurs domaines assurant chacun un rôle biosynthétique. Il est ainsi nécessaire de comprendre comment s’opère la communication entre domaines au sein d’un module. Pour cela, nous avons étudié le module 5 de la PKS synthétisant la virginiamycine M par une approche combinant la diffusion des rayons X aux petits angles (SAXS), la résonance magnétique nucléaire (RMN) et la modélisation par homologie. Ainsi nous sommes parvenus à caractériser la structure en solution du module 5, mais également à positionner les structures à haute résolution des domaines à l’intérieur de l’enveloppe SAXS. De plus, notre étude de la dynamique du module 5 a montré que les deux domaines acyl carrier protein (ACP) composant le module semblent non-équivalents, et que l’ACP5b doit être doté d’une certaine mobilité afin d’être fonctionnel, ceci dû à la flexibilité du linker reliant les deux ACP. L’interaction entre les sous-unités consécutives est également primordiale pour assurer la fidélité de la synthèse des polykétides. Ces interactions sont assurées, au moins en partie, par des domaines de petite taille appelés « domaine de docking (DD) ». Jusqu’alors, les DD avaient été caractérisés uniquement chez les PKS de type cis-AT. Nous avons caractérisé une nouvelle classe de DD, la première chez les PKS trans-AT. Grâce à une approche pluridisciplinaire, nous avons montré que l’un des DD constitue probablement une protéine intrinsèquement désordonnée (IDP) : son interaction avec le DD partenaire induirait son repliement. Nous avons résolu la structure RMN d’une protéine de fusion entre les deux DD, affichant une nouvelle topologie pour une interaction protéine-protéine. Cette interface de docking a ensuite été replacée dans son contexte modulaire par SAXS. Contrairement aux autres DD qui ne forment qu’une seule interface de docking, ces DD forment deux complexes de docking à l’intersection des deux sous-unités. Nos données SAXS nous avons également permis de proposer un modèle de l’interface créée entre deux sous-unités PKS dans laquelle une chambre réactionnelle est formée, qui pourrait servir à protéger des intermédiaires réactifs de polykétide. Des enzymes post-PKS interviennent suite à la synthèse du polykétide pour maturer ce dernier. Cette étape est d’une importance considérable puisqu’elle confère la structure et l’activité finale du polykétide. Dans ce contexte, nous avons mené une étude structure/fonction de l’enzyme post-PKS catalysant la macrocyclisation de l’antibiotique lankacidine C. Après un phasage par SAD sur la protéine séléniée, nous avons résolu la structure de l’enzyme en complexe avec des analogues de substrats, puis procéder à la conception de mutants pour résoudre la structure de l’enzyme avec son substrat naturel / Polyketides are structurally complex molecules which exhibit diverse therapeutic activities (antibiotic, antitumor, immunosuppressant…). These compounds and the enzymes responsible for their synthesis, the polyketide synthases (PKSs), are thus of significant biomedical interest. An emerging though promising strategy for the generation of novel polyketides is the engineering of the PKS proteins, an approach called synthetic biology. Nevertheless, a fundamental understanding of the mode of operation of the PKS enzymes is directly correlated with the success of this methodology. Indeed, PKSs are molecular-scale assembly lines which are composed of several subunit, each of which includes one or several modules catalyzing a polyketide elongation cycle. The module themselves are composed of several domains each with a specific role in the biosynthesis. It is therefore necessary to understand how the domains within a module communicate with each other. To address this question, we studied module 5 of the PKS responsible for virginiamycin M using an approach combining small angle X-ray scattering (SAXS), nuclear magnetic resonance (NMR) and homology modeling. This strategy allowed us to characterize the solution structure of module 5, but additionally to position high-resolution domain structures inside the SAXS envelopes. We also studied the dynamics of module 5, demonstrating that the two component acyl carrier proteins (ACPs) appear to be non-equivalent, and that the function of ACP5b is likely dependent on the mobility conferred on it by the flexible linker between the two domains. The interaction between the consecutive subunits is also critical to ensuring the fidelity of polyketide synthesis. This communication is assured, at least in part, by small domains called docking domains (DD), which have to date only been characterized from cis-AT PKS. Here, we have identified a new class of DD, the first shown to be present in trans-AT PKSs. Using a multidisciplinary approach, we have demonstrated that the N-terminal DD (VirFG NDD) is likely to be an intrinsically disordered protein (IDP), whose interaction with its partner DD (VirA CDD) induces its folding. We have solved the NMR structure of a fusion protein between the two DD, revealing a new topology for a protein-protein interaction. This docking interface was then placed into its modular context by SAXS. In contrast to the other classes of DD which form a single docking complex, this new type of DD gives rise to two docking interfaces at the intersubunit junction. Finally, our SAXS data have allowed us to propose a model for the complete interface between two PKS subunits in which a reaction chamber is formed, which may allow reactive polyketide intermediates to be protected. Post-PKS enzymes catalyze maturation of the polyketide after its release from the last module of the PKS. This processing is critical as it yields the final structure and activity of the polyketide. In this context, we conducted a structure/function study of the post-PKS enzyme catalyzing the macrocyclisation of the antibiotic lankacidine C. After phasing by SAD using a seleniated protein, we solved the structure of the enzyme in complex with substrate analogues. We then proceeded to site-directed mutagenesis of specific residues, in order to solve the structure of the enzyme in complex with its natural substrate

Líquidos iônicos (LIs) são sais que se encontram no estado líquido em temperaturas menores que 100ºC e que vêm ganhando protagonismo na área chamada química verde, prometendo: substituir solventes nocivos ao meio ambiente, aprimorar componentes eletrônicos, favorecer biocatálises dentre outros. Sua alta estabilidade e baixa toxicidade são frequentemente afirmadas, porém, devem ainda ser melhor investigadas. Com o objetivo de implementar o entendimento da interação dos líquidos iônicos com sistemas de relevância biológica, realizamos um estudo sistemático acerca da interação de 3 diferentes líquidos iônicos anfifílicos de mesma cabeça polar e diferentes caudas carbônicas ([C10mim][Cl], [C12mim][Cl] e [C14mim][Cl]) com 3 diferentes proteínas modelo, através das técnicas de absorção óptica, fluorescência, dicroísmo circular (CD) e espalhamento de raios-X a baixos ângulos (SAXS). Para Tanto, utilizamos as proteínas BSA e HSA (Albuminas de Soro Bovino e Humano, respectivamente) além da lisozima. Observamos a supressão da fluorescência das proteínas em todos os casos analisados, onde a diminuição da intensidade correspondeu a, para as proteínas BSA, HSA e lisozima, respectivamente, (55±3)%, (16.1±0.8)% e (4.1±0.2)%, em presença de 0.6mM de [C14mim][Cl], (38±2)%, (13.2±0.7)% e (0.6±0.1)% em presença de 0.6mM de [C12mim][Cl] e (11.0±0.5)%, (9.2±0.5)% e (0.0±0.1)% em presença de 0.6mM de [C10mim][Cl]. Os espectros de absorbância e fluorescência de todos os sistemas nos indicam uma interação de contato entre as proteínas e os líquidos iônicos. Constatamos também o deslocamento do pico de fluorescência, das proteínas BSA e HSA, para menores comprimentos de onda (blue-shift), na medida em que a concentração de LI era aumentada. O máximo deslocamento () alcançado correspondeu a (21±1)nm para ambas albuminas, enquanto que a lisozima não apresentou deslocamento significativo. O blue-shift pode ser explicado pela aproximação das cadeias carbônicas e formações de pontes de hidrogênio nas proximidades dos triptofanos. De acordo com a técnica de SAXS, evidenciamos o aumento do raio de giro das proteínas, na medida em que adicionamos LIs. O raio de giro da BSA, da HSA e lisozima em ausência de LI são (29±1)Å, (30±1)Å e (15±1)Å, respectivamente, e passam para (46±1)Å, (44±1)Å e (20±1)Å respectivamente, em presença de 0.6mM de [C14mim][Cl]. As curvas de SAXS também apresentaram o indício da formação de estruturas micelares a partir de uma dada concentração. Além da alteração em sua estrutura terciária, os dados de CD indicam uma leve perda de estrutura secundária de ambas as albuminas (BSA e HSA), passando de 80 para 65% de -hélice em ausência e presença de 0.6mM de [C14mim][Cl], respectivamente. Sugerimos que as interações das proteínas com os líquidos iônicos, embora inicialmente movidas por forças eletroestática, possuem como principal fator o efeito hidrofóbico, portanto quanto maior a cadeia carbônica do LI maior é sua interação com a proteína. Tal interação causa o desenovelamento das proteínas e formação de um complexo e estruturas micelares a altas concentrações de LI. Acreditamos que este trabalho traz novas informações acerca da interação dos LIs com proteínas modelo, indicando sua capacidade de alterar a conformação das mesmas. / Ionic liquids (ILs) are salts that are liquid at temperatures smaller than 100 ° C and are gaining prominence in the so-called green chemistry, promising: replace harmful solvents to the environment, improve electronic components, and favor biocatalysis, among others. Its high stability and low toxicity are often asserted; nevertheless, they are ascribed to ILs due to its small volatility. With the aim of improving the understanding of the interaction of ILs with biological relevant systems, we conducted a systematic study of the interaction of three different ionic liquids of the same polar head and different paraffinic tails ([C10mim][Cl], [C12mim][Cl] and [C14mim][Cl]) with three different model proteins, through the techniques of optical absorption, fluorescence, circular dicrhoism (CD) and small angle X-ray scattering (SAXS). To do so, we use BSA and HSA proteins (Bovine Serum Albumin and the Human Serum Albumin, respectively) and lysozyme. We observed fluorescence quenching, of all studied proteins, where the decrease in the fluorescence was (for BSA, HAS and lysozyme, respectively): (55 ± 3)%, (16.1 ± 0.8)% to (4.1 ± 0.2 )% in the presence of 0.6mm [C14mim][Cl], (38 ± 2)%, (13.2 ± 0.7)% to (0.6 ± 0.1)% in the presence of 0.6mm [C12mim][Cl] and ( 11.0 ± 0.5)% (9.2 ± 0.5)% and (0.0 ± 0.1)% in the presence of 0.6mm [C10mim][Cl]. UV-vis absorbance spectra and fluorescence indicate all systems in a contact interaction between proteins and ionic liquids. We also note the shift of the fluorescent peak of BSA and HSA proteins for shorter wavelengths (blue-shift), as the IL content was increased. The maximum shift () achieved corresponded to (21 ± 1) nm for both albumins, whereas no significant displacement was observed for lysozyme. The blue-shift can be explained by the approach of carbon chains and formation of hydrogen bonds in the vicinity of tryptophan. SAXS data indicate an increasing in the proteins radius of gyration value as ILs was added in the solution. The turning radius of BSA, HSA and lysozyme in the absence of IL are (29 ± 1) Å, (30 ± 1) Å and (15 ± 1) Å, respectively, and go to (46 ± 1) Å, ( 44 ± 1) Å and (20 ± 1) Å, respectively, in the presence of 0.6mm [C14mim][Cl]. The SAXS curves also show evidence of the formation of micellar structures from a given concentration. Besides the change in its tertiary structure, the CD data indicates a slight loss of secondary structure of both albumins (BSA and HSA), from 80 to 65% of -helix in the absence and presence of 0.6mm [C14mim][Cl], respectively. We suggest that the interactions of the protein with the ionic liquid, although initially driven by electrostatic forces, have a major factor hydrophobic effect and thus the higher the carbon chain of greater IL is its interaction with the protein. This interaction causes unfolding of the protein and formation of a micellar structures at high concentrations of IL. We believe this work provides new information about the interaction of ILs with model proteins, indicating its ability to alter the conformation of the same.