A aterosclerose, doença sistêmica caracterizada pelo acúmulo de lipídios e elementos fibrosos nas artérias, é uma das principais causas de morte em diversos países. Partículas de lipoproteínas de baixa densidade (LDL) oxidadas estão presentes nas lesões aterogênicas, evidenciando a correlação entre estas partículas e a doença aterosclerótica. Este trabalho apresenta estudos de caracterização óptica e estrutural de lipoproteínas humanas nativas e oxidadas in vitro. As caracterizações foram realizadas por meio de diversas técnicas, sendo as principais a Varredura-Z e o espalhamento de raios X a baixos ângulos (SAXS) . Nos estudos de caracterização óptica verificou-se que a resposta não-linear das amostras de LDL está relacionada tanto com o seu conteúdo de antioxidantes, quanto com sua concentração de hidroperóxidos. Com relação à caracterização estrutural, foi proposto um novo método de análise para os dados de SAXS. Neste método, que mostrou-se mais adequando frente àqueles existentes na literatura, a curva de contraste de densidade eletrônica é obtida diretamente da curva de intensidade de espalhamento. Por meio das análises realizadas concluímos que as partículas de LDL apresentam pequenas alterações estruturais apenas quando comparamos a amostra nativa com aquela oxidada por 18 horas. São apresentados ainda alguns resultados exploratórios obtidos, tanto na caracterização óptica quanto na estrutural, para as lipoproteínas de alta densidade (HDL), que apontam para uma maior resistência deste tipo de lipoproteína ao processo oxidação. Por fim, é possível afirmar que a técnica de Varredura-Z é sensível a mudanças que ocorrem no início do processo oxidativo das lipoproteínas, enquanto a técnica de SAXS é sensível a mudanças em estágios mais avançados do mesmo processo. / Atherosclerosis, which is a systemic disease characterized by the accumulation of lipids and fibrous elements in the arteries, is a major cause of death in many countries. Particles of oxidized low density lipoproteins (LDL) are present in atherogenic lesions, showing the correlation between these particles and atherosclerosis. This thesis presents studies of structural and optical characterization of native and in vitro oxidized human lipoproteins. Characterizations were carried out by means of various techniques, the main ones being Z-scan (ZS) and small angle X-ray scattering (SAXS). In the optical characterization studies it was found that the non-linear response of LDL samples is associated with their antioxidant contents and with their concentration of hydroperoxides. With respect to the structural characterization, we propose a new method of analysis for the SAXS data. In this method, which is more suitable than those existing in the literature, the electron density curve is obtained directly from the scattering intensity curve. Through these analyses we conclude that LDL particles exhibit only small structural changes when native LDL sample is compared to the $18$ hours oxidized one. We also present preliminary results, both in structure and in optical characterization, for the high density lipoprotein (HDL), which presents a greater resistance (comparing to LDL) to the oxidation process. Finally, it is possible to say that the ZS technique is sensitive to changes that occur in early stages of the lipoprotein\'s oxidative process, while the SAXS technique is sensitive to changes in the later stages of the same process.

As glicosidases são enzimas que apresentam uma grande variedade de enovelamentos, assim como uma alta especificidade frente a diferentes substratos. Estas enzimas têm em comum a presença de dois resíduos catalíticos, responsáveis pela clivagem das ligações glicosídicas. O uso de glicosidases nas indústrias têxtil e alimentícia, no processamento de polpa de papel e na síntese de oligossacarídeos tem incentivado a engenharia destas proteínas no sentido de melhorar suas propriedades catalíticas e estabilidade. Estudos estruturais das glicosidases têm aumentado nosso entendimento de seus mecanismos de ação catalitica, assim como dos processos de interação proteína-carboidrato. Neste trabalho apresentamos os estudos cristalográficos de duas glicosidases de fungos, sendo elas a beta-galactosidase de Penicillium sp. e a Exo-inulinase de Aspergillis awamori, assim como estudos por espalhamento de raios-X a baixos ângulos (SAXS) da beta-xylosidase de Trichoderma reesei. As estruturas cristalográficas da beta-galactosidase e de seu complexo com galactose foram determinadas pela técnica de substituição isomórfa simples com espalhamento anômalo (SIRAS) até 1.9 A angstron de resolução para a estrutura sem substrato e 2.0 angstron de resolução para o complexo. A estrutura do complexo com galactose foi usada para identificar os resíduos catalíticos, sendo o resíduo Glu 200 identificado como doador de próton e o resíduo Glu 299 como o nucleófílo. As estruturas cristalográficas da Exo-inulinase de Aspergillus awamori e de seu complexo com frutose foram também determinadas pela técnica de substituição isomórfa simples com espalhamento anômalo (SIRAS) até 1.55 angstron e 1.8 angstron de resolução, respectivamente. A partir da estrutura do complexo foi possível identificar os resíduos Asp41 e Glu241 como o nucleófilo e o doador de próton, respectivamente. Além disto, foi possível verificar que o Asp189, o qual faz parte do motivo conservado Arg-Asp-Pro (RDP), é importante no reconhecimento do substrato através de duas pontes de hidrogênio. Com o intuito de obter informações estruturais sobre a P-xylosidase seu envelope foi determinado a partir dos dados do espalhamento de raios-X a baixos ângulos. O envelope da p-xylosidase em solução foi calculado a 20 A de resolução, sendo o raio de giro e a dimensão máxima 36.9 angstron e 90 angstron, respectivamente. Usando algoritmos de reconhecimento de possíveis domínios foi determinado que esta proteína apresenta, além dos dois domínios característicos da família GHF3, um barril TIM e um domínio alfa/beta, um terceiro domínio. A predição da estrutura secundária e os dados de dicroísmo circular indicam que este terceiro domínio apresentaria um enovelamento tipo beta. / Glycosidases belong to a group of enzymes displaying a great variety of protein folds and substrate specificities. Two critically located acidic residues make up the catalytic machinery of these enzymes, responsible for the cleavage of glycosidic bonds. The applications of glycosidases in textile, food, and pulp processing, as well as in catalysts and oligosaccharide synthesis have encouraged the engineering of these proteins in order to obtain improved catalytic properties and stability. Furthermore, structural studies extend our understanding of the catalytic mechanism and the role of glycosidases in the recognition processes of their different substrates. In this work, we describe crystallographic studies of two fungi glycosidases, beta-galactosidase from Penicillium sp and Exo-inulinase from Aspergillis awamori, and the small-angle x-ray scattering (SAXS) studies of another glycosidase, beta-xylosidase (from Trichoderma reesei). The crystallographic structures of j3-galactosidase its complex with galactose were solved by single isomorphous replacement with anomalous scattering (SIRAS) using the quick cryo-soaking technique, at 1.90 angstron and 2.10 angstron resolution, respectively . The X-ray structure of the enzyme-galactose complex was useful in identifying the residue Glu 200 as the proton donor and residue Glu 299 as the nucleophile involved in catalysis. The x-ray structure of exo-inulinase and its complex with fructose were also solved by SIRAS using the quick cryo-soaking technique at 1.55 angstron and 1.8 angstron resolutions, respectively. The solved structure of the enzyme-fructose complex revealed two catalytically important residues, Asp41 and Glu241, as nucleophile and proton donor, respectively. It was also possible to see that residue Asp189, which belongs to the Arg-Asp-Pro motif, provides hydrogen bonds important for substrate recognition. In order to gain structurai insights about the beta-Xylosidase from Trichoderma reesei, we calculated their SAXS envelope. The low resolution shape of this enzyme in solution was obtained fiom synchrotron x-ray scattering data at 20 angstron resolution. The radii of gyration and the maximum dimension of the beta-Xylosidase were calculated to be 36.9 angstron and 90 angstron, respectively. In contrast to the fold of the only structurally characterized member of GHF-3, the beta-D-glucan exohydrolase, which has two distinct domains, the shape of the beta-xylosidase indicates the presence of three domains located in the same plane. Domain recognition algorithms were used to show that the C-terminal part of the mino acid sequence of the protein forms the third domain. Circular dichroism spectroscopy and secondary structure prediction programs show that this additional domain adopts predominantly the B-conformation.

La phosphatidylinositol-4-kinase de type IIIα est une kinase de lipide eukaryote ubiquitaire qui synthétise le phosphatidylinositol-4-phosphate PtdIns(4)P de la membrane plasmique. Ce phosphoinositide est d’autant plus important qu’il tient un rôle majeur dans différentes voies de signalisation cellulaire, le traffic vésiculaire ainsi que dans l’identité des organelles. De plus, la PIK4A humaine est un facteur essentiel pour la réplication du virus de l’hépatite C (VHC). En effet, le recrutement du complexe de réplication du VHC par la protéine virale NS5A à la membrane du reticulum endoplasmique permet la formation d’un réseau membranaire à l’origine de la structuration des complexes de replication viraux.La PI4KA est une kinase imposante (2102 résidus, 240 kDa pour la PI4KA humaine) qui possède un domaine kinase C-terminal d’environ 400 résidus précédé d’un domaine formé de répétitions Armadillo pour lequel aucune fonction n’a été determinée. Le rôle ainsi que le repliement des 1500 résidus N-terminaux de PI4KA ne sont pas connus à ce jour.Afin d’en savoir plus sur la structure tri-dimensionnelle de la PI4KA humaine, nous avons utilisé des outils bio-informatiques afin de délimiter et de modéliser les modules et domaines la composant. Nous avons pu ainsi les exprimer et les produire en bactérie et en cellules d’insecte afin de vérifier nos hypothèses. Nous avons pu conclure que PI4KA est composée de deux modules. Le module N-terminal (1100 résidus), est composé de deux domaines dont un solénoïde α. Les résultats obtenus par diffusion des rayons X aux petits angles (SAXS) nous permettent de définir leur agencement potentiel. Le second module (1000 résidus), le module C-terminal, est l’enzyme-core. Son analyse nous a permis d’identifier une similitude remarquable avec les sérine/thréonine kinases PIKKs, comme mTor, apparentées aux phosphatidylinositol-3-kinases. Nous avons défini au début du module C-terminal de PI4KA trois domaines putatifs que nous avons nommés DI, DII et DIII. Nos collaborateurs ont montré qu'ils sont essentiels à l’activité kinase de la protéine ainsi qu’à la replication du VHC. Le domaine DI a été caractérisé et a permis la validation d’une nouvelle paramétrisation de la molécule de N, N-dimethyl-dodecylamine oxide (LDAO) pour des simulations de dynamique moléculaire. Enfin, la PI4KA humaine dans son entier a été exprimée en cellules d’insecte puis purifiée, et un premier test d’interaction avec les membranes a été initié. / The eukaryotic lipid kinase phosphatidylinositol 4-kinase III alpha is a ubiquitous enzyme that synthesizes the plasma membrane pool of phosphatidylinositol 4-phosphate. This important phosphoinositide has key roles in different signalization pathways, vesicular traffic and cellular compartment identity. Moreover, PI4KA is an essential factor for hepatitis C virus (HCV) replication. Indeed, PI4KA's interaction with the non-structural HCV protein NS5A at the endoplasmic reticulum membrane leads to formation of a “membranous web” giving to the membrane the signature necessary to the formation of viral replication machineryPI4KA is a large protein (2102 residues, 240 kDa for human PI4KA) with the kinase domain making up the ca 400 C-terminal residues preceded by an Armadillo domain for which no function is known. There is essentially no structural information about the 1500 N-terminal residues and no clue as to the function of most of this region of PI4KA.We use computational methods in order to delineate fragments of human PI4KA amenable to soluble production in Escherichia coli and insect cells. We clone and express these fragments and evaluate the soluble fraction of each construction. Our results further suggest that PI4KA can be described as a two-module protein. The N-terminal module (1100 residues), is composed of two domains which one is an alpha solenoid. Their potential arrangement was defined by small angle X-ray scattering (SAXS).The second module (1000 residues), the C-terminal module, is the core enzyme. Its analysis leads us to identify similarities with the serine/threonine kinases PIKKs, as mTor, homologous to phosphatidylinositol-3-kinases. Three putative domains were delineate at the beginning of this C-terminal module. We name the DI, DII and DIII. Our collaborators have shown their necessity to the kinase activity of PI4KA and the HCV replication. DI domain was characterized and allowed the validation of a new parametrization of the N, N-dimethyl-dodecylamine oxide molecule (LDAO) for simulation of molecular dynamics. Finally, the full-length human PI4KA was expressed in insect cells, purified and a first interaction experiment with membranes have been initiated.

Le objectif de ma thèse était la caractérisation moléculaire de la P des virus Nipah et Hendra (BL4) du genre Henipavirus. Le génome est encapsidé par la N qui sert de substrat pour la transcription et la réplication. La polymérase est composée par la L e son cofacteur la P. La P est composée d’un domaine N-terminal (PNT) désordonné et un domaine C-terminal (PCT) constitué d’une alternance de régions désordonnés et ordonnés (PMD domaine de multimerization). J'ai étudié PMD, PCT et PNT utilisant le «cross-linking», le CD, le SAXS, la RMN et la modélisation moléculaire. J'ai montré que le PMD du Hendra et Nipah sont un coiled-coil triméreric. La région PCT, est également un trimère en solution. Les protéines P des henipavirus constituent à ce jour le seul exemple de protéines P paramyxovirales ayant une organisation trimérique. En utilisant le SAXS, j'ai obtenu une description de Hendra PNT en tant qu’ensemble conformationnel. J'ai entrepris la caractérisation de la PNT par RMN. J’ai divisée la PNT avec l’approche divide et impera (PNT1,2,3,4). J’ai pu réaliser des expériences permettant l’attribution de PNT1, et j’ai également effectué des mesures de relaxation (R1, R2 et NOE) sur les fragments PNT1, PNT2 et PNT3. Les résultats issus des travaux effectués ont ouvert la voie vers l’obtention d’une description atomistique de la PNT en tant qu'ensemble conformationnel. Ces informations avec les informations structurales que j’ai sur PCT, PMD et XD, devraient conduire à une description atomistique de la P entière en tant qu’ensemble conformationnel. Ces informations structurales détaillées constitueront aussi un socle pour des approches antivirales rationnelles. / The objective of my PhD project was the molecular characterization of the P protein from the Nipah and Hendra viruses (BL4) belonging to the Henipavirus genus. The genome is encapsidated by the N that is the substrate for transcription and replication. The polymerase is made up the L and its cofactor the P. The P protein consists of an intrinsically disordered N-terminal domain (PNT), and a C-terminal domain (PCT) made of alternating disordered and ordered domain (PMD or P multimerization domain). I investigated the PMD, PCT and PNT regions, using cross-linking, AUC, CD, SAXS, NMR and molecular modeling. I showed that Hendra and Nipah PMD are a trimeric coiled-coil in solution. The Henipavirus proteins constitute so far the unique examples of a trimeric organization in paramyxoviral P proteins. The PCT is a trimer as well. Using SAXS, I obtained an ensemble description of PNT. To obtain site-specific information that improve SAXS-based models, I undertook the characterization of Hendra PNT by NMR. The latter was divided using the “divide et impera” approach to get four fragments (PNT1,2,3,4). Experiments for the assignment have been performed for PNT1. R1, R2 and NOE were carried out on PNT1,2,3. Altogether the results laid the basis for achieving an atomic-resolution conformational ensemble description of Hendra PNT. This information, combined with structural information that I collected on PCT, PMD and XD, is expected to lead an atomistic ensemble description of the full-length P, which would represent the first, such a description of a paramyxoviral P protein. This detailed structural information will also constitute an asset for rational antiviral approaches.

Made available in DSpace on 2014-12-17T15:41:55Z (GMT). No. of bitstreams: 1 IdyllaST_DISSERT.pdf: 1649846 bytes, checksum: b16782ef605bdc7a1d3225b281a6f2b3 (MD5) Previous issue date: 2011-11-08 / Chitosan nanoparticles have been used in several systems for the controlled release of drugs. The aim of this study was to obtain and characterize chitosan nanoparticles prepared by the method of coacervation / precipitation using sodium sulfate at different concentrations as the crosslinking agent. The characterization was done using zeta potential and small angle Xray scattering, SAXS. The dispersions of chitosan were obtained at pH 1 and pH = 3. The results of zeta potential at pH = 1 ranged from +64.8 to +29.27 mV and for pH = 3 they varied from +72.4 to +23.48 mV, indicating that the chain of chitosan is positively charged in acidic pH and the behavior of nanoparticles in terms of surface charge was independent of pH. However, the results indicated a dependence of particle size in relation to pH. This difference in behavior was explained by the influence of enthalpic and entropic components / Nanopart?culas de quitosana t?m sido utilizadas em v?rios sistemas destinados a libera??o controlada de f?rmacos. O objetivo desse trabalho foi a obten??o e caracteriza??o de nanopart?culas de quitosana atrav?s do m?todo de coacerva??o/precipita??o utilizando o sulfato de s?dio em diferentes concentra??es como agente reticulante. A caracteriza??o foi feita utilizando potencial zeta e espalhamento de pequenos ?ngulos, SAXS. As dispers?es de quitosana foram obtidas a pH=1 e pH=3. Os resultados do potencial zeta variaram no pH = 1 de +64,8 a +29,27 mV e no pH = 3 de +72,4 a +23,48 mV, indicando que a cadeia de quitosana fica carregada positivamente em pH ?cido e que comportamento das nanopart?culas em termos de carga superficial foi independente do pH. Entretanto, os resultados indicaram depend?ncia do tamanho de part?cula em rela??o ao pH. Essa diferen?a em termos de comportamento foi explicada pela influ?ncia dos componentes ent?lpicos e entr?picos

Virtuellement tous les habitats terrestres sont dominés par les angiospermes, ou plantes à fleurs. Leur capacité à coloniser de nouveaux habitats et supplanter une autre espèce est due à l'avènement d'une nouvelle structure reproductrice – la fleur. La fleur uni les organes mâles et femelles dans une structure compacte et contient la graine. Les plantes à fleurs ne sont pas seulement le type dominant des plantes terrestres, mais sont également la principale source de nourriture et l'habitat de tous les animaux, y compris les humains. En termes d'évolution, les fleurs sont considérées comme un développement récent. Elles ont fait l'objet de spéculations depuis l'époque de Charles Darwin qui à nommé l’évolution dominante et la diversification des plantes à fleurs comme «un abominable mystère» en raison de l'absence d'une transition en douceur de la non-floraison vers la floraison des plantes dans le registre fossile. Avec le séquençage de plusieurs génomes de gymnospermes (semences de plantes non-florales), d’angiospermes basals et de plantes à fleurs supérieures, certaines familles de gènes jouant un rôle central dans le développement et l'évolution de la fleur ont été identifiées. Notre recherche se concentre sur une de ces familles de régulateurs de niveau supérieur appelée « famille de facteur de transcription MADS » (TF). Cette famille de TF permet d'orchestrer le développement des fleurs. Nous nous sommes intéressés à la compréhension des mécanismes moléculaires de la famille des MADS et à la façon dont ces protéines sont capables de contrôler les fonctions de reproduction complexes.Ce projet intègre différentes techniques biophysiques comme la cristallographie aux rayons X, la diffusion des rayons X aux petits angles (SAXS) et la microscopie à force atomique (AFM) afin d’étudier les interactions protéine-protéine et protéine-ADN des FT MADS. Aucune étude n’a, à ce jour, porté sur les mécanismes moléculaires des FT MADS en utilisant cette approche structurale intégrée.Un obstacle important dans l'étude des FT MADS a été l’expression des protéines recombinantes et leur purification. Dans ce projet, les protocoles de purification de plusieurs recombinants FT MADS entières ont été établis, permettant la caractérisation structurale et biochimique des protéines dans leurs intégralités. La structure aux rayons X du domaine d'oligomérisation de la protéine de la famille MADS, SEPALLATA3 (SEP3) est présenté et utilisé comme modèle pour comprendre les motifs d'oligomérisation de la famille élargie et les bases moléculaires des interactions protéine-protéine. Des solutions de structures9provenant d'études SAXS de AGAMOUS (AG) et de la phase végétative courte (SVP) sont présentées et complétés par la caractérisation biochimique de leur état d'oligomérisation.Afin d'étudier les interactions protéine-ADN, des procédés complémentaires ont été utilisés. Une propriété importante des FT MADS est leur capacité à modifier la structure de l'ADN grâce à la formation de boucles d'ADN. De manière hypothétique, les FT MADS oligomérisent et fixent l'ADN sur deux sites différents, bouclant potentiellement l'ADN. En utilisant l'AFM, la première preuve directe de la formation de boucle d'ADN par SEP3 est obtenue. Les caractéristiques de liaison d'ADN de SVP ont été étudiées par analyse de décalage de mobilité électrophorétique (EMSA), par thermophorèseà échelle microscopique (MST) et par AFM. Contrairement au cas de SEP3, l’EMSA et l’AFM ont montrés que SVP est un dimère et présente différents modes de liaison à l'ADN.Ces données fournissent une base atomique et structurale de la fonction des FT MADS. Sur la base de ce travail, nous commençons à comprendre l’oligomérisation et certaines spécificités déterminantes de liaison à l'ADN. Ces études montrent comment les FT MADS s’oligomérisent. / Virtually all terrestrial habitats are dominated by angiosperms, or flowering plants. Their success in colonizing new habitats and supplanting other species is due to the advent of a complex reproductive structure – the flower. The flower unites the male and female organs into one compact structure and encloses the seed. Flowering plants are not only the dominant type of land plants, but also are the primary source of food and habitat for all animals, including humans. In evolutionary terms, flowers are considered a recent development and have been a subject of speculation from the time of Charles Darwin who termed the dominant rise and diversification of flowering plants as “an abominable mystery”* due to the lack of a smooth transition from non-flowering to flowering plants in the fossil record. With the sequencing of multiple genomes from gymnosperms (non-flowering seed plants), basal angiosperms and higher flowering plants, certain gene families have been identified which play a central role in the development and evolution of the flower. My research focuses on one such family of high-level regulators, the MADS transcription factor (TF) family. This TF family helps to orchestrate flower development among other functions. As such, there is great interest in understanding the molecular mechanisms of the MADS family and how these proteins are able to control complex reproductive pathways.This project integrates different biophysical techniques including x-ray crystallography, small angle x-ray scattering (SAXS) and atomic force microscopy (AFM) to investigate protein-protein and protein-DNA interactions of MADS TFs. No studies to date have investigated the molecular mechanisms of MADS TFs using this integrated structural approach.One important hurdle in the study of the MADS TFs has been recombinant protein expression and purification. In this project, recombinant purification protocols for several full length MADS TFs were established, allowing the structural and biochemical characterisation of the proteins. The crystal structure of the oligomerisation domain of the MADS family protein SEPALLATA3 (SEP3) is presented and used as a template for understanding the oligomerisation patterns of the larger family and the molecular basis for protein-protein interactions. Investigation of solution structures, derived from SAXS studies, of AGAMOUS (AG) and SHORT VEGETATIVE PHASE (SVP) along with biochemical characterisation of their oligomerisation states are also presented.In order to study protein-DNA interactions, complementary methods were used. An important putative property of the MADS TFs is their ability to change the structure of DNA through the formation of DNA loops. MADS TFs are hypothesized to oligomerise and bind DNA at two different sites, potentiating looping of DNA. Using AFM, the first direct evidence of DNA looping by SEP3 is described. The DNA binding characteristics of SVP were studied using electrophoretic mobility shift assay (EMSA), microscale thermophoresis (MST) and AFM. Unlike SEP3, SVP is dimeric and thus exhibits different DNA-binding patterns.The data presented here provide an atomic and structural basis for MADS TF function. Based on this work, we now are beginning to understand some of the oligomerisation and DNA-binding specificity determinants. These studies demonstrate how the MADS TFs oligomerise and the results show that we can disrupt oligomerisation and potentially DNA-binding very specifically through the introduction of point mutations. Future work will investigate the in vivo consequences of altered oligomerisation and how this affects different developmental programs in plant reproduction and floral organ morphogenesis.*Letter from Charles Darwin to Joseph Dalton Hooker, written 22 July 1879 (Source: Cambridge University Library DAR 95: 485 – 488) (Friedman, 2009b).

La Beta-2-microglobuline est une protéine de 12kDa, impliquée dans une maladie dûe à un mauvais repliement: l'amylose liée à la dialyse. Elle constitue donc un modèle pour la formation de fibrilles amyloides et pour le repliement des protéines. La B2M est un objet à la fois difficile et fructueux à étudier. La production de B2M est complexe et demande une optimisation important pour obtenir une protéine correctement repliée et atteindre des rendements approprié pour des études de RMN et SAXS. Le repliement de la B2M est sensible au solvent, à la température, à la concentration et souvent aux conditions de préparation. Pourtant notre étude, à l'aide de plusieurs méthodes biophysiques, a pu révéler plusieurs faits essentiels de son mécanisme de repliement et de la structure et propriétés des intermédiaires. Un premier résultat est que le repliement et l'oligomérisation sont deux processus concourants. Une découverte majeure est l'existence d'un équilibre monomère oligomère entre deux états I1 et I2 intermédiaires du repliement. Détecté indirectement à l'aide de RMN temps réel comme SOFAST, I2 a été directement charactérisé en SAXS: Il s'agit probablement d'un dimère. Les états intermédiaires de repliement de B2M avaient été pointés comme favorisant la formation de fibrilles: cela s'explique facilement avec l'existence d'un intermédiaire dimérique. Une combinaison de méthodes biophysiques permet la caractérisation de cet équilibre monomère-oligomère. En SAXS, puis confirmé en RMN, la stoichiométrie de l'équilibre est celle d'un monomère-dimère. Des travaux complémentaires utilisant les techniques développées pour cette étude pourront servir à caractériser plus finement cet équilibre. L'étude approfondie du repliement de B2M pousse les techniques biophysiques dans leurs retranchements: la sensibilité et le temps d'acquisition pour la RMN, la polydispersité pour le SAXS. Pourtant dans les deux cas un grand oligomère I3, qui disparait en quelques minutes, a pu être détecté, ce qui fut confirmé par UV-Fluo. La caractérisation d'I3 demandera des dévelopements méthodologiques supplémentaires, ainsi qu'un nouveau plan d'expérience. D'autres méthodes comme la spectrométrie de masse nano-ESI pourraient représenter des sources d'information utiles. S'attaquer aux limites des méthodes biophysiques pousse au développement méthodologique. Ainsi pour étudier la structure et dynamique d'I1, la méthode d'acquisition continue des données a permis l'attribution des résonnances de cette espèce qui a une demi vie de quelques dizaines de minutes. Un échange conformationnel a été découvert pour l'état I1 du mutant W60G, en développant une méthode de relaxation RMN: R2-BEST-TROSY. Les méthodes développées pour cette étude pourront servir des études sur le repliement d'autres protéines, mais aussi dans d'autres contextes où la demi-vie des objets étudiés est courte, comme dans les expérience RMN intracellulaires. Cette étude est évidemment éloignée d'une application directe dans le combat contre les maladies du mauvais repliement des protéines. Pour autant, la découverte d'états intermédiaires oligomériques souligne que l'oligomérisation et le repliement ne devraient pas être étudiés séparément, mais sont des processus liés. Les développements méthodologiques de cette étude pourront aussi être appliqués à d'autres protéines comme à d'autres contexte. Il est donc permis d'espérer que ces questionnements et développements permettront d'avancer vers une meilleure compréhension de ces maladies. / Beta-2-microglobulin is a 12kDa protein, involved in a misfolding disease: dialysis related amyloidosis. It is therefore a model for amyloid fibril formation and protein folding studies. B2M is both a fruitful and difficult object of study. B2M production is complex, requiring optimization to obtain a well folded protein and reach yields suitable to NMR and SAXS studies. B2M folding is highly sensitive on buffer, temperature, concentration and often preparation conditions. Yet our studies, using several biophysical methods, revealed several essential facts of the folding mechanism and of the structure and dynamics of folding intermediates. A first outcome of our studies is that folding and oligomerization are co-existing processes. A major finding is the existence of a monomer-oligomer equilibrium between I1 and I2 folding intermediate states. Indirectly detected using real time NMR methods like SOFAST, I2 was directly detected and characterized using SAXS: I2 is likely to be a dimer. Folding intermediate states of B2M had been shown to favor fibril formation: this is easily explained by the existence of a dimeric folding intermediate state with an important population. A combination of biophysical methods allows the characterization of this monomer-oligomer equilibrium. Using SAXS, and later confirmed by NMR relaxation experiments, stoichiometry is shown to be a monomer-dimer equilibrium. Further work based on the methodology applied to the folding of the W60G-B2M mutant, including a further optimization of the sensitivity of the experiment, will give a sharper picture of the I1-I2 equilibrium for the WT protein, and may provide information on the timescale of the equilibrium. The thorough study of the folding of B2M pushes biophysical methods to their limits: sensitivity and acquisition time for NMR, polydispersity for SAXS. Yet in both cases a large oligomer (I3) that disappears within minutes was detected, and confirmed using UV-fluo. Characterization of I3 will demand further methodological developments, a new experimentation plan including a full dilution scale, or double jump experiments, for example. A question that arises is the comparison of this large oligomer and oligomeric intermediate states that are populated during the formation of fibrils. Other biophysical methods, such as ESI mass spectroscopy, may be an interesting input. Tackling the limits of biophysical methods leads to methodological developments. For example, to study the structure and dynamics of I1, the continuous data acquisition method allowed the assignment of this species that has a half-lifetime of tens of minutes. A conformational exchange was discovered for the I1 state of the W60G-B2M mutant, through the development of a spin relaxation measurement experiment: R2-BEST-TROSY. The methods developed for this study may be later used to study the folding and folding intermediate states of other proteins, such as alpha-lactalbumin , or in other contexts in which the short lifetime of the protein is an issue, as for in-cell NMR experiments. Our studies are of course far from an application or a concrete result in the fight against misfolding diseases such as dialysis related amyloidosis or Parkinson's. But the discovery of oligomeric folding intermediate states underlines that oligomerization (including fibril formation) and folding should not be studied separately, and are processes that are closely related. Methodological developments included in our work can be applied to other proteins as well as other contexts. Hopefully these questionings and developments will constitute a step forward a better understanding of this diseases.

A aterosclerose, doença sistêmica caracterizada pelo acúmulo de lipídios e elementos fibrosos nas artérias, é uma das principais causas de morte em diversos países. Partículas de lipoproteínas de baixa densidade (LDL) oxidadas estão presentes nas lesões aterogênicas, evidenciando a correlação entre estas partículas e a doença aterosclerótica. Este trabalho apresenta estudos de caracterização óptica e estrutural de lipoproteínas humanas nativas e oxidadas in vitro. As caracterizações foram realizadas por meio de diversas técnicas, sendo as principais a Varredura-Z e o espalhamento de raios X a baixos ângulos (SAXS) . Nos estudos de caracterização óptica verificou-se que a resposta não-linear das amostras de LDL está relacionada tanto com o seu conteúdo de antioxidantes, quanto com sua concentração de hidroperóxidos. Com relação à caracterização estrutural, foi proposto um novo método de análise para os dados de SAXS. Neste método, que mostrou-se mais adequando frente àqueles existentes na literatura, a curva de contraste de densidade eletrônica é obtida diretamente da curva de intensidade de espalhamento. Por meio das análises realizadas concluímos que as partículas de LDL apresentam pequenas alterações estruturais apenas quando comparamos a amostra nativa com aquela oxidada por 18 horas. São apresentados ainda alguns resultados exploratórios obtidos, tanto na caracterização óptica quanto na estrutural, para as lipoproteínas de alta densidade (HDL), que apontam para uma maior resistência deste tipo de lipoproteína ao processo oxidação. Por fim, é possível afirmar que a técnica de Varredura-Z é sensível a mudanças que ocorrem no início do processo oxidativo das lipoproteínas, enquanto a técnica de SAXS é sensível a mudanças em estágios mais avançados do mesmo processo. / Atherosclerosis, which is a systemic disease characterized by the accumulation of lipids and fibrous elements in the arteries, is a major cause of death in many countries. Particles of oxidized low density lipoproteins (LDL) are present in atherogenic lesions, showing the correlation between these particles and atherosclerosis. This thesis presents studies of structural and optical characterization of native and in vitro oxidized human lipoproteins. Characterizations were carried out by means of various techniques, the main ones being Z-scan (ZS) and small angle X-ray scattering (SAXS). In the optical characterization studies it was found that the non-linear response of LDL samples is associated with their antioxidant contents and with their concentration of hydroperoxides. With respect to the structural characterization, we propose a new method of analysis for the SAXS data. In this method, which is more suitable than those existing in the literature, the electron density curve is obtained directly from the scattering intensity curve. Through these analyses we conclude that LDL particles exhibit only small structural changes when native LDL sample is compared to the $18$ hours oxidized one. We also present preliminary results, both in structure and in optical characterization, for the high density lipoprotein (HDL), which presents a greater resistance (comparing to LDL) to the oxidation process. Finally, it is possible to say that the ZS technique is sensitive to changes that occur in early stages of the lipoprotein\'s oxidative process, while the SAXS technique is sensitive to changes in the later stages of the same process.

As glicosidases são enzimas que apresentam uma grande variedade de enovelamentos, assim como uma alta especificidade frente a diferentes substratos. Estas enzimas têm em comum a presença de dois resíduos catalíticos, responsáveis pela clivagem das ligações glicosídicas. O uso de glicosidases nas indústrias têxtil e alimentícia, no processamento de polpa de papel e na síntese de oligossacarídeos tem incentivado a engenharia destas proteínas no sentido de melhorar suas propriedades catalíticas e estabilidade. Estudos estruturais das glicosidases têm aumentado nosso entendimento de seus mecanismos de ação catalitica, assim como dos processos de interação proteína-carboidrato. Neste trabalho apresentamos os estudos cristalográficos de duas glicosidases de fungos, sendo elas a beta-galactosidase de Penicillium sp. e a Exo-inulinase de Aspergillis awamori, assim como estudos por espalhamento de raios-X a baixos ângulos (SAXS) da beta-xylosidase de Trichoderma reesei. As estruturas cristalográficas da beta-galactosidase e de seu complexo com galactose foram determinadas pela técnica de substituição isomórfa simples com espalhamento anômalo (SIRAS) até 1.9 A angstron de resolução para a estrutura sem substrato e 2.0 angstron de resolução para o complexo. A estrutura do complexo com galactose foi usada para identificar os resíduos catalíticos, sendo o resíduo Glu 200 identificado como doador de próton e o resíduo Glu 299 como o nucleófílo. As estruturas cristalográficas da Exo-inulinase de Aspergillus awamori e de seu complexo com frutose foram também determinadas pela técnica de substituição isomórfa simples com espalhamento anômalo (SIRAS) até 1.55 angstron e 1.8 angstron de resolução, respectivamente. A partir da estrutura do complexo foi possível identificar os resíduos Asp41 e Glu241 como o nucleófilo e o doador de próton, respectivamente. Além disto, foi possível verificar que o Asp189, o qual faz parte do motivo conservado Arg-Asp-Pro (RDP), é importante no reconhecimento do substrato através de duas pontes de hidrogênio. Com o intuito de obter informações estruturais sobre a P-xylosidase seu envelope foi determinado a partir dos dados do espalhamento de raios-X a baixos ângulos. O envelope da p-xylosidase em solução foi calculado a 20 A de resolução, sendo o raio de giro e a dimensão máxima 36.9 angstron e 90 angstron, respectivamente. Usando algoritmos de reconhecimento de possíveis domínios foi determinado que esta proteína apresenta, além dos dois domínios característicos da família GHF3, um barril TIM e um domínio alfa/beta, um terceiro domínio. A predição da estrutura secundária e os dados de dicroísmo circular indicam que este terceiro domínio apresentaria um enovelamento tipo beta. / Glycosidases belong to a group of enzymes displaying a great variety of protein folds and substrate specificities. Two critically located acidic residues make up the catalytic machinery of these enzymes, responsible for the cleavage of glycosidic bonds. The applications of glycosidases in textile, food, and pulp processing, as well as in catalysts and oligosaccharide synthesis have encouraged the engineering of these proteins in order to obtain improved catalytic properties and stability. Furthermore, structural studies extend our understanding of the catalytic mechanism and the role of glycosidases in the recognition processes of their different substrates. In this work, we describe crystallographic studies of two fungi glycosidases, beta-galactosidase from Penicillium sp and Exo-inulinase from Aspergillis awamori, and the small-angle x-ray scattering (SAXS) studies of another glycosidase, beta-xylosidase (from Trichoderma reesei). The crystallographic structures of j3-galactosidase its complex with galactose were solved by single isomorphous replacement with anomalous scattering (SIRAS) using the quick cryo-soaking technique, at 1.90 angstron and 2.10 angstron resolution, respectively . The X-ray structure of the enzyme-galactose complex was useful in identifying the residue Glu 200 as the proton donor and residue Glu 299 as the nucleophile involved in catalysis. The x-ray structure of exo-inulinase and its complex with fructose were also solved by SIRAS using the quick cryo-soaking technique at 1.55 angstron and 1.8 angstron resolutions, respectively. The solved structure of the enzyme-fructose complex revealed two catalytically important residues, Asp41 and Glu241, as nucleophile and proton donor, respectively. It was also possible to see that residue Asp189, which belongs to the Arg-Asp-Pro motif, provides hydrogen bonds important for substrate recognition. In order to gain structurai insights about the beta-Xylosidase from Trichoderma reesei, we calculated their SAXS envelope. The low resolution shape of this enzyme in solution was obtained fiom synchrotron x-ray scattering data at 20 angstron resolution. The radii of gyration and the maximum dimension of the beta-Xylosidase were calculated to be 36.9 angstron and 90 angstron, respectively. In contrast to the fold of the only structurally characterized member of GHF-3, the beta-D-glucan exohydrolase, which has two distinct domains, the shape of the beta-xylosidase indicates the presence of three domains located in the same plane. Domain recognition algorithms were used to show that the C-terminal part of the mino acid sequence of the protein forms the third domain. Circular dichroism spectroscopy and secondary structure prediction programs show that this additional domain adopts predominantly the B-conformation.

A hemoglobina extracelular de Glossoscolex paulistus (HbGp) possui estrutura oligomérica composta por 144 cadeias globínicas e 36 cadeias linkers, que não possuem heme, formando uma bicamada hexagonal. Estudos mostraram que a HbGp possui uma alta estabilidade á variação de pH e presença de agentes desnaturantes, tais como, surfactantes e ureia, a 25°C. Com esses conhecimentos prévios, o presente estudo tem por objetivo avaliar a estabilidade térmica da HbGp 0,5-3,0 mg/mL, nas formas oxi-, meta- e cianometa-, em diferentes valores de pH. O efeito do SDS na estabilidade térmica da oxi-HbGp 0,5 e 3,0 mg/mL, em função do pH, será investigado também. Esses estudos foram realizados usando as técnicas de absorção óptica, dicroísmo circular (CD), espalhamento de luz dinâmico (DLS) e espalhamento de raios X a baixo ângulo (SAXS). Os resultados de absorção óptica e CD revelam que o processo de desnaturação da oxi- e cianometa-HbGp, no pH 7,0, envolve a formação das espécies oxidadas aquo-meta-HbGp e hemicromo. O processo de desnaturação é próximo ao modelo de dois estados, com uma temperatura crítica (Tc) de 58-59 °C. No pH ácido, a proteína agrega a partir de 58 °C. A cinética de agregação da oxi-HbGp, no pH 7,0, é dependente da concentração de proteína e da temperatura. Os dados de DLS mostram que a meta- e cianometa-HbGp 0,5 mg/mL, pH 7,0, desnaturam a 48 ± 1 e 56 ± 1 °C, respectivamente. Em pH alcalino, a proteína dissocia parcialmente antes de desnaturar, e o aumento da concentração de proteína faz aumentar o valor de Tc para a cianometa-HbGp. Dados de SAXS mostram que a oxi- e meta-HbGp, pH 7,0, desnaturam a 60 °C, apresentando valores de Rg=143±1 Å e Dmax=450±1 Å, enquanto que a cianometa-HbGp se mantém estável, com valores de Rg=107±1 Å e Dmax=300±1 Å. As análises das curvas p(r) mostram uma porcentagem crescente de dodecâmero e tetrâmero em solução, em relação à fração de protreína íntegra e de subunidades maiores, com o aumento do pH e da temperatura. As análises baseadas no programa OLIGOMER são similares às baseadas na função p(r). A presença do SDS induz a dissociação da oxi-HbGp 0,5 mg/mL pH 7,0. Entretanto, com 3,0 mg/mL de proteína a dissociação é parcial, com a sobreposição dos processos de dissociação, desnaturação e agregação, com o aumento da temperatura. No pH 5,0, o SDS promove a agregação da oxi-HbGp em temperaturas menores. As constantes cinéticas de dissociação da oxi-HbGp 0,5 mg/mL, pH 7,0 aumentam de (0,53±0,07)x10-4 s-1 para (2,1±0,2)x10-4 s-1 na presença de 0,4 e 0,6 mmol/L de SDS a 40 °C, respectivamente. Na temperatura de 42 °C a constante aumenta 2,6 vezes, com 0,6 mmol/L de SDS, comparada a 40 °C. A oxi-HbGp 3,0 mg/ml na presença de 0,6 mmol/L de SDS, dissocia parcialmente em tempos menores com o aumento da temperatura antes de agregar. Portanto, esses estudos mostram que a estabilidade térmica da HbGp é sensível ao aumento de pH e presença de SDS. A ordem de estabilidade térmica em pH alcalino é dado por: cianometa->oxi->meta-HbGp. Alem disso, o processo de desnaturação é governado pelo valor do pH e dependente da concentração de proteína em solução. / The extracellular hemoglobin Glossoscolex paulistus of (HbGp) has an oligomeric structure composed by 144 globin chains and 36 non globin chains (named linkers), forming a hexagonal bilayer. HbGp presents a high stability reagarding pH variation and the presence of denaturing agents, such as, for example, urea and surfactant, at 25°C. In this way, the present studies aim to evaluate the thermal stability for oxy-, meta- and cyanomet-HbGp 0.5-3.0 mg/ml, at different pH values. The SDS effect on the thermal stability of oxy-HbGp 0.5 and 3.0 mg / mL is also investigated. Optical absorption, circular dichroism (CD), dynamic light scattering (DLS) and small angle X-ray scattering (SAXS) techniques were emplayed for these studies. The results based on the optical absorption and CD spectroscopies show that the denaturation process for oxy- and cyanomet-HbGp, at pH 7.0, involves the formation of oxidized species, such as aquo-met-HbGp and hemichrome. This denaturation process is very close to a two-state model, with a critical temperature (Tc) of 58-59 °C. However, in the acidic pH, the aggregation of protein occurs at 58 °C. The aggregation process kinetics for oxy-HbGp, pH 7.0, is dependent on the protein concentration and temperature. DLS data show that meta- and cyanomet-HbGp, 0.5 mg/mL, pH 7.0, undergo denaturation at 48 ± 1 and 56 ± 1 ° C, respectively. At alkaline pH, two HbGp forms undergo partial dissociation before denaturation, and at higher protein concentration, an increase of Tc values for cyanomet-HbGp is observed. SAXS results show that the denaturation of oxy-and met-HbGp occur at 60 °C, presenting Rg=143±1 Å and Dmax=450±15 Å, while cyanomet-HbGp remains stable with Rg =107±1 Å and Dmax= 300±10Å, at this temperature. The p(r) curves analysis show the increase of dodecamer and tetramer percentages in solution, with increase of pH and temperature. The results using the OLIGOMER program are similar to the p(r) data analysis. For oxy-HbGp 0.5 mg/mL pH 7.0, in the presence of SDS, oligomeric dissociation before denaturation is observed. However, with 3.0 mg/ml of protein the dissociation process is slower, showing an overlap of the dissociation, denaturation and aggregation processes in the system, with increase of temperature. At pH 5.0, SDS promotes the aggregation of oxy-HbGp at lower temperatures, as compared to the absence of surfactant. The kinetic dissociation constant values for oxy-HbGp 0.5 mg/mL increase from (0.53 ± 0.07) x10-4 s-1 to (2.1 ± 0.2) x10-4 s-1, in the presence of 0.4 and 0.6 mmol/L SDS at 40 ° C, respectively. At 42 °C the dissociation constant value increases 2.6-fold, with 0.6 mmol/L SDS, as compared to 40 °C. For oxy-HbGp 3.0 mg/ml, in the presence of 0.6 mmol/L SDS, the oligomeric dissociation is smallest occurring in shorter times with increasing temperature before aggregation. Therefore, these studies show that the thermal stability of HbGp is sensitive to the pH variation and the presence of SDS. At alkaline pH, the order of thermal stability is the following: cyanomet->oxy->met-HbGp. Furthermore, the denaturation process is governed by the pH value, being dependent on the protein concentration in solution.