Les protéines sont au coeur de la vie. Ce sont d'incroyables nanomachines moléculaires spécialisées et améliorées par des millions d'années d'évolution pour des fonctions bien définies dans la cellule. La structure des protéines, c'est-à-dire l'arrangement tridimensionnel de leurs atomes, est intimement liée à leurs fonctions. L'absence apparente de structure pour certaines protéines est aussi de plus en plus reconnue comme étant tout aussi cruciale. Les protéines amyloïdes en sont un exemple marquant : elles adoptent un ensemble de structures variées difficilement observables expérimentalement qui sont associées à des maladies neurodégénératives. Cette thèse, dans un premier temps, porte sur l'étude structurelle des protéines amyloïdes bêta-amyloïde (Alzheimer) et huntingtine (Huntington) lors de leur processus de repliement et d'auto-assemblage. Les résultats obtenus permettent de décrire avec une résolution atomique les interactions des ensembles structurels de ces deux protéines. Concernant la protéine bêta-amyloïde (AB), nos résultats identifient des différences structurelles significatives entre trois de ses formes physiologiques durant ses premières étapes d'auto-assemblage en environnement aqueux. Nous avons ensuite comparé ces résultats avec ceux obtenus au cours des dernières années par d'autres groupes de recherche avec des protocoles expérimentaux et de simulations variés. Des tendances claires émergent de notre comparaison quant à l'influence de la forme physiologique de AB sur son ensemble structurel durant ses premières étapes d'auto-assemblage. L'identification des propriétés structurelles différentes rationalise l'origine de leurs propriétés d'agrégation distinctes. Par ailleurs, l'identification des propriétés structurelles communes offrent des cibles potentielles pour des agents thérapeutiques empêchant la formation des oligomères responsables de la neurotoxicité. Concernant la protéine huntingtine, nous avons élucidé l'ensemble structurel de sa région fonctionnelle située à son N-terminal en environnement aqueux et membranaire. En accord avec les données expérimentales disponibles, nos résultats sur son repliement en environnement aqueux révèlent les interactions dominantes ainsi que l'influence sur celles-ci des régions adjacentes à la région fonctionnelle. Nous avons aussi caractérisé la stabilité et la croissance de structures nanotubulaires qui sont des candidats potentiels aux chemins d'auto-assemblage de la région amyloïde de huntingtine. Par ailleurs, nous avons également élaboré, avec un groupe d'expérimentateurs, un modèle détaillé illustrant les principales interactions responsables du rôle d'ancre membranaire de la région N-terminal, qui sert à contrôler la localisation de huntingtine dans la cellule. Dans un deuxième temps, cette thèse porte sur le raffinement d'un modèle gros-grain (sOPEP) et sur le développement d'un nouveau modèle tout-atome (aaOPEP) qui sont tous deux basés sur le champ de force gros-grain OPEP, couramment utilisé pour l'étude du repliement des protéines et de l'agrégation des protéines amyloïdes. L'optimisation de ces modèles a été effectuée dans le but d'améliorer les prédictions de novo de la structure de peptides par la méthode PEP-FOLD. Par ailleurs, les modèles OPEP, sOPEP et aaOPEP ont été inclus dans un nouveau code de dynamique moléculaire très flexible afin de grandement simplifier leurs développements futurs. / Proteins are at the center of life. They are formidable molecular nanomachines specialized and optimized during million years of evolution for well-defined functions in the cell. The structure of proteins, meaning the tridimensional setting of their atoms, is closely related to their function. Absence of structure for a subset of proteins is also recognized to be as crucial. Amyloid proteins is a striking example : they fold into an ensemble of various structures hardly observable experimentally that are associated with neurodegenerative diseases. This thesis, firstly, is on the study of the structural ensemble of the amyloid proteins amyloid-beta (Alzheimer) and huntingtin (Huntington) during their folding and aggregation. Our results describe in details, with an atomic resolution, the characteristic interactions present in the structural ensemble of these two proteins. Concerning the amyloid-beta protein (AB), our results show the structural differences between three of its physiological forms during its first aggregation steps in an aqueous environment. We have then compared these results with those obtained during the past few years by several other research groups using various experimental and simulation protocols. Clear trends come out of this comparison regarding the influence of AB physiological form on its structural ensemble during its first aggregation steps. Their distinct aggregation pathways are rationalized by the identified differences. For their part, the identified similarities offer targets for therapeutical compounds disrupting the aggregation of the neurotoxic oligomers. Concerning the huntingtin protein, we identify the structural ensemble of its functional region at its N-terminal in an aqueous environment and in a phospholipid membrane. In agreement with the available experimental results on the global structure of this region in aqueous solution, our results reveal the dominant interactions, at an atomic precision, in its structural ensemble as well as the influence of its neighboring regions. We have also characterized the stability and the growth of nanotube-like structures that could occur during the aggregation of the amyloid region of huntingtin. Moreover, we have developed, in collaboration with a group of experimentalists, a precise model describing the main membrane interactions of huntingtin N-terminal, which serves as a membrane anchor that controls the localization of huntingtin in the cell. Secondly, this thesis is on the refinement of a coarse-grained model (sOPEP) and on the development of a new all-atom model (aaOPEP) that are both based on the coarse-grained OPEP force field, commonly used to study protein folding and amyloid protein aggregation. The goal behind the optimization of these models is to improve the de novo structure prediction of the PEP-FOLD method. These three models -- OPEP, sOPEP and aaOPEP -- are now also implemented in a new molecular dynamics software that we have developed specifically to greatly ease their future developments.

Septinas constituem uma família de proteínas de ligação a GTP que foram inicialmente identificadas em levedura Saccharomyces cerevisiae, mas também estão presentes em outros eucariotos com exceção de plantas. Septinas são purificadas de leveduras, Drosophila e cérebros de mamíferos na forma de filamentos, porém o mecanismo através do qual acorre a formação destes filamentos ainda não é muito bem compreendido. Septinas são constituídas de três regiões principais: um N-terminal variável, um domínio central GTPase altamente conservado e um domínio coiled-coil C-terminal. O gene SEPT4 foi identificado por M. Tanaka e colaboradores a partir do cDNA de cérebro humano e apresentou duas distintas transcrições: Bradeiona ? e ?. Interessantemente, além de cérebro e coração, as proteínas Bradeiona Α e Β. são detectadas somente em câncer do cólon, reto, próstata e melanoma maligno. Neste trabalho, o gene da proteína Bradeiona Β foi subclonado em um vetor de expressão bacteriano, produzido em E. coli e purificado com sucesso. O espectro de dicroísmo circular (CD) mostrou o perfil característico de proteínas com hélices a na estrutura secundária. Resultados de cromatografia de exclusão molecular (SEC) e espalhamento dinâmico de luz (DLS) indicam que a septina Bradeina foi produzida na forma de um estável oligômero com características monodispersivas, que foi subseqüentemente cristalizado em PEG6000. A atividade GTPase da Bradeiona Β foi comprovada através da técnica de eletroforese capilar (CE), mostrando-se absolutamente dependente de íons Mg2+. Inibição da atividade GTPase foi verificada em altas concentrações de Mg2+ (maiores que 5 mM). Com a finalidade de caracterizar os domínios preditos da Bradeiona Β (Fragmento Conservado e domínio GTPase), essas regiões foram previamente definidas, expressas em E. cozi e purificadas com sucesso. Resultados de CD, SEC, espectroscopia de fluorescência e NMR-600MHz indicam que o FC foi produzido na forma de um estável monômero com pouca estrutura secundária regular. Resultados de DLS e CD indicam que a fusão 6xHis-DGTPase foi produzida na forma de um oligômero com a presença de hélices a na estrutura secundária. A fusão 6xHis-DGTPase mostrou-se instável a altas concentrações na ausência de imidazol. A atividade GTPase da fusão GST+DGTPase foi comprovada, similarmente a Bradeiona , através da técnica de CE. Novamente, verificou-se dependência de íons Mg2+ (para a atividade catalítica) e inibição em altas concentrações de Mg2+. A fusão GST+DGTPase também foi capaz de hidrolisar ATP. Espera-se que as informações relatadas neste estudo proporcionem um alicerce para estudos estruturais/funcionais futuros das proteínas Bradeiona Α e Βoutras septinas. / Septins form a class of eukaryoyic guanine nucleotide-binding proteins that were first identified in budding yeast. Septins purified from yeast, Drosophila and mammalian brain form filaments, however the mechanism by which the filaments assemble is unclear. Septins have a highly conserved structure, which includes a central GTP-binding domain, a variable N-terminal region, and most septins also contain a coiled coil domain at the Cterminus. Bradeion p is one of the splice variants of the human septin gene, SEPT4, recently isolated by expression screening of an adult human brain cDNA library. The Bradeion gene resides at 17q23, and has been shown to present specific expression in both human colorectal cancer, urologic cancers and malignant melanoma. In order to characterize the GTPase activity of Bradeion Β , the protein was successfully expressed in E. coli and purified. The recombinant protein was characterized by circular dichroism (CD), dynamic light scattering (DLS) and a novel non-radioactive enzyme assay, which utilizes capillary electrophoresis (EC) to monitor GTP hydrolysis. The CD spectrum exhibited the typical shape characteristic of the presence of helical elements of secondary structure and the DLS pattern was indicative of a monodisperse solution, which was readily crystallized in the presence of PEG6000. GTP hydrolysis was shown to be Mg2+ dependent within the low millimolar range but at 5 mM was inhibitory. In order to characterize the predicted domains of Bradeion Β, these defined regions were successfully expressed in E. cozi and purified. The CD spectrum of CF exhibited the shape typically found for non-regular structure. The results of fluorescence spectroscopy, gel filtration (SEC) and NMR-600MHz also corroborate with the CD results indicating an irregular structure. The fusion protein 6xHis-DGTPase exhibited a CD spectrum with the typical shape characteristic of the presence of helical elements but was show to be instable at high concentrations in the absence of imidazole. To characterize the GTPase activity of the fusion protein GST+DGTPase, the CE technique was used to monitor GTP hydrolysis. Analysis by CE showed that GST+DGTPase was functional, since both GTP and ATP hydrolysis was observed in a Mg2+ dependent manner. This work provides novel approaches, which should aid in the fbture study of the structure and fùnction of Bradeion Α e Β, others septins and related GTPases.

Orientador: José Roberto Ruggiero / Banca: Pedro Geraldo Pascutti / Banca: José Maria Pires / Banca: Alexandre Suman de Araújo / Banca: Sabrina Thais Broggio Costa / Resumo: Peptídeos antimicrobianos são moléculas biologicamente ativas que, geralmente, tem as membranas fosfolipídicas como alvo primário. Resultados de diferentes técnicas experimentais têm sugerido que esses peptídeos permeabilizam as membranas pela formação de poros. Parte dos peptídeos caracterizados apresentam especificidade de disrupção para membranas de bactérias, em detrimento das membranas dos hospedeiros. Essa característica tem atraído a atenção da comunidade científica internacional, porque indica que estas moléculas podem ser modelos para o desenvolvimento de novos antibióticos, portanto o entendimento do mecanismo de ação, ou seja, do mecanismo de formação de poro, tem extrema importância. Simulações por Dinâmica Molecular foram produzidas para investigarmos o impacto que peptídeos antimicrobianos da família Mastoparano tem sobre membranas lipídicas modelo. Dois cenários foram explorados: (i) de baixa concentração peptídeo/lipídeo, P/L=1/128, que consistia de simulações fine-grained das interações de um peptídeo com uma bicamada pura de 128 lipídeos aniônicos (POPG) ou zwiteriônicos (POPC); (ii) de alta concentração, P/L=1/21, que abordava as interações de seis peptídeos com uma bicamada mista de 128 lipídeos POPC/POPG (1/1) usando uma modelagem coarse-grained. Tomando o peptídeo MP1 como caso paradigmático, verificamos que em baixo P/L é possível sugerir que sua característica seletiva surge da capacidade de coordenar e perturbar maior número de lipídeos em membrana aniônica comparada à neutra. Essa capacidade fica acentuada nas simulações com membrana mista, onde a atração dos lipídeos aniônicos pelos peptídeos catiônicos guiou a separação local e a formação de domínios de lipídeos aniônicos, o que facilitou o afinamento local da membrana e a formação de poro transmembrânico. Esses achados ajudam a explicar como peptídeos / Abstract: Antimicrobial peptides are biologically active molecules that, usually, have the phospholipid membranes as a primary target. Results from different experimental techniques have suggested these peptides permeabilize membranes by the pore formation. Part of the characterized peptides have specificity of disruption for bacterial membranes, instead of host membrane. This feature has attracted the attention of the international scientific community, because it indicates that these molecules can be models for the development of novel antibiotics, so understanding the mechanism of action, ie, the mechanism of pore formation, is extremely important. Molecular dynamics simulations were performed to investigate the impact of antimicrobial peptides from the Mastoparano family have on model lipid membranes. Two scenarios were explored: (i) of low peptide/lipid concentration, P/L=1/128, which consisted of fine-grained simulations of the interactions of a peptide with a pure bilayer of 128 anionic (POPG) or zwitterionic (POPC) lipids; (ii) of high concentration, P/L=1/21, which addressed the interactions of six peptides with a mixed bilayer of 128 POPC/POPG (1/1) lipids, using a coarse-grained modeling. Taking the MP1 peptide as a paradigmatic case, we found that in low P/L is possible to suggest that its selective feature arises of its ability to coordinate and disturb large number of lipids in the anionic membrane compared to neutral one. This ability is accentuated in simulations with mixed membrane, where the attraction of the anionic lipids by the cationic peptides led to the local segregation and formation of POPG lipid domains, which facilitated the local thinning of the membrane and the formation of transmembrane pore. These findings help to explain how short peptides, such as MP1, are able of forming pores in a membrane whose thickness is larger than the length of the peptide / Doutor

Septinas constituem uma família de proteínas de ligação a GTP que foram inicialmente identificadas em levedura Saccharomyces cerevisiae, mas também estão presentes em outros eucariotos com exceção de plantas. Septinas são purificadas de leveduras, Drosophila e cérebros de mamíferos na forma de filamentos, porém o mecanismo através do qual acorre a formação destes filamentos ainda não é muito bem compreendido. Septinas são constituídas de três regiões principais: um N-terminal variável, um domínio central GTPase altamente conservado e um domínio coiled-coil C-terminal. O gene SEPT4 foi identificado por M. Tanaka e colaboradores a partir do cDNA de cérebro humano e apresentou duas distintas transcrições: Bradeiona ? e ?. Interessantemente, além de cérebro e coração, as proteínas Bradeiona Α e Β. são detectadas somente em câncer do cólon, reto, próstata e melanoma maligno. Neste trabalho, o gene da proteína Bradeiona Β foi subclonado em um vetor de expressão bacteriano, produzido em E. coli e purificado com sucesso. O espectro de dicroísmo circular (CD) mostrou o perfil característico de proteínas com hélices a na estrutura secundária. Resultados de cromatografia de exclusão molecular (SEC) e espalhamento dinâmico de luz (DLS) indicam que a septina Bradeina foi produzida na forma de um estável oligômero com características monodispersivas, que foi subseqüentemente cristalizado em PEG6000. A atividade GTPase da Bradeiona Β foi comprovada através da técnica de eletroforese capilar (CE), mostrando-se absolutamente dependente de íons Mg2+. Inibição da atividade GTPase foi verificada em altas concentrações de Mg2+ (maiores que 5 mM). Com a finalidade de caracterizar os domínios preditos da Bradeiona Β (Fragmento Conservado e domínio GTPase), essas regiões foram previamente definidas, expressas em E. cozi e purificadas com sucesso. Resultados de CD, SEC, espectroscopia de fluorescência e NMR-600MHz indicam que o FC foi produzido na forma de um estável monômero com pouca estrutura secundária regular. Resultados de DLS e CD indicam que a fusão 6xHis-DGTPase foi produzida na forma de um oligômero com a presença de hélices a na estrutura secundária. A fusão 6xHis-DGTPase mostrou-se instável a altas concentrações na ausência de imidazol. A atividade GTPase da fusão GST+DGTPase foi comprovada, similarmente a Bradeiona , através da técnica de CE. Novamente, verificou-se dependência de íons Mg2+ (para a atividade catalítica) e inibição em altas concentrações de Mg2+. A fusão GST+DGTPase também foi capaz de hidrolisar ATP. Espera-se que as informações relatadas neste estudo proporcionem um alicerce para estudos estruturais/funcionais futuros das proteínas Bradeiona Α e Βoutras septinas. / Septins form a class of eukaryoyic guanine nucleotide-binding proteins that were first identified in budding yeast. Septins purified from yeast, Drosophila and mammalian brain form filaments, however the mechanism by which the filaments assemble is unclear. Septins have a highly conserved structure, which includes a central GTP-binding domain, a variable N-terminal region, and most septins also contain a coiled coil domain at the Cterminus. Bradeion p is one of the splice variants of the human septin gene, SEPT4, recently isolated by expression screening of an adult human brain cDNA library. The Bradeion gene resides at 17q23, and has been shown to present specific expression in both human colorectal cancer, urologic cancers and malignant melanoma. In order to characterize the GTPase activity of Bradeion Β , the protein was successfully expressed in E. coli and purified. The recombinant protein was characterized by circular dichroism (CD), dynamic light scattering (DLS) and a novel non-radioactive enzyme assay, which utilizes capillary electrophoresis (EC) to monitor GTP hydrolysis. The CD spectrum exhibited the typical shape characteristic of the presence of helical elements of secondary structure and the DLS pattern was indicative of a monodisperse solution, which was readily crystallized in the presence of PEG6000. GTP hydrolysis was shown to be Mg2+ dependent within the low millimolar range but at 5 mM was inhibitory. In order to characterize the predicted domains of Bradeion Β, these defined regions were successfully expressed in E. cozi and purified. The CD spectrum of CF exhibited the shape typically found for non-regular structure. The results of fluorescence spectroscopy, gel filtration (SEC) and NMR-600MHz also corroborate with the CD results indicating an irregular structure. The fusion protein 6xHis-DGTPase exhibited a CD spectrum with the typical shape characteristic of the presence of helical elements but was show to be instable at high concentrations in the absence of imidazole. To characterize the GTPase activity of the fusion protein GST+DGTPase, the CE technique was used to monitor GTP hydrolysis. Analysis by CE showed that GST+DGTPase was functional, since both GTP and ATP hydrolysis was observed in a Mg2+ dependent manner. This work provides novel approaches, which should aid in the fbture study of the structure and fùnction of Bradeion Α e Β, others septins and related GTPases.

Advanced fluorescence techniques were used to explore tree distinct topics concerning biological membrane and their interactions. Following thesis is according to the topic divided into three parts: 1) Ionic effects were studied employing time dependent fluorescence shift experiments and molecular dynamic simulations. Combination of these two approaches are suitable to reveal characteristic like mobility and hydration of particular bilayer segment, lipid packing or ion binding sites. Halide anions were reported to adsorb to the cationic lipid bilayer specifically, altering membrane mobility and organization. Changes in observed parameters follows Hofmeister order. Their effect is mediated either by direct ionic interaction (soft, polarizable ions) as well as via alteration of water structure (hard, non-polarizable ions) in proximity of ion molecule. Further, divalent calcium was shown to bind strongly to neutral and negatively charged lipid bilayers. Several types of binding sites depending on calcium concentration were identified. 2) Two complementary lipopeptides, CPK and CPE, incorporated into distinct lipid bilayers serve as a minimal model inducing membrane fusion. Effectiveness of fusion event might be influenced by lipopeptide-membrane and lipopeptide-lipopeptide interaction. To reveal...

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