Environ 40 pour cent du protéome humain est composé d'importantes régions dépliées. Ces protéines intrinsèquement désordonnées (PID) n'adoptent pas de structures secondaires et tertiaires stables mais échantillonnent un vaste paysage conformationnel. Malgré cela, elles sont aujourd'hui connues pour intervenir dans de nombreux processus biologiques ou pathologiques. À l'instar des eucaryotes, les virus -- surtout les virus à ARN -- ont eux aussi recours aux propriétés particulières des PID pour effectuer les interactions nécessaires à leur réplication. Les paramyxovirus, comme le virus de la rougeole, sont des virus à ARN simple brin de polarité négative et environ 10 pour cent de leur génome de 15 à 18 kilobases code pour des régions dépliées. Cette thèse détaille l'étude de deux protéines virales directement impliquées dans la réplication, la nucléoprotéine et la phosphoprotéine. Elles interagissent l'une avec l'autre et sont composées à la fois de régions dépliées et repliées. Des données à résolution atomique ont été obtenues en spectroscopie par Résonance Magnétique Nucléaire (RMN) en ce qui concerne les parties désordonnées, et en cristallographie pour ce qui est des parties repliées. Les résultats apportent un nouvel aperçu du rôle du désordre conformationnel dans la transcription et la réplication des paramyxovirus. / About 40 percent of the human proteome contains large disordered regions. These intrinsically disordered proteins (IDPs) do not adopt stable secondary and tertiary structures, but sample a large conformational space. In spite of that, they are now known to be involved in many physiological as well as pathological processes. Following the example of eukaryotes, viruses -- especially RNA viruses -- benefit from the particular features of IDPs in their replication machinery. Paramyxoviruses, that includes Measles virus, are single stranded, negative sense RNA viruses and about 10 percent of their 15 to 18 kilobase RNA genome is known to encode for disordered regions. This thesis focuses on the study of two different proteins of paramyxoviruses, namely the nucleoprotein and the phosphoprotein that are directly involved in the replication of the viral genome. They interact with each other and are composed of folded and disordered domains. Atomic resolution information is obtained about the structure and dynamics of these proteins using a combination of Nuclear Magnetic Resonance (NMR) spectroscopy measurements for the disordered parts and X-ray crystallography for the folded domains. The results provide novel insight into the role of conformational disorder in transcription and replication of paramyxoviruses.

Les protéines intrinsèquement désordonnées sont caractérisées par un manque de structure 3D stable et sont biologiquements actives dans cet état. La spectroscopie RMN est la méthode de choix pour leurs études à une résolution atomiques, car la cristallographie aux rayons X ne permet pas leur étude en raison de leur caractère hautement dynamique.Cependant, l'étude par spectroscopie RMN de ces protéines est difficiles à cause du grand nombre de recouvrement entre les signaux dans le spectre résultant de l'absence d'un réseau de liaison hydrogène qui pourrait stabiliser la structure et permettre d'obtenir une dispersion des signaux plus élevé. Un autre problème est la sensibilité expérimentale car souvent le temps de mesure est limité en raison de leur prédisposition à la dégradation protéolytique. Dans la première partie de cette thèse les protéines intrinsèquement désordonnées sont introduites. La deuxième partie porte sur la spectroscopie RMN des protéines intrinsèquement désordonnées, des expériences RMN de type BEST-TROSY sont présentées et sont montrées comme étant bien adapté pour l'étude de protéines intrinsèquement désordonnées, en particulier pour celle avec une grande étendue de structure résiduelle. Des expériences 3D BEST-TROSY sont présentées pour leur attribution, une version proline-éditée permet d'aider à l'identification de ce type d'acide aminé et enfin l'expérience HETex-BEST-TROSY qui permet une mesure rapide des taux de change de solvants. Dans la troisième partie de cette thèse ces expériences RMN sont appliquées pour l'étude de la région intrinsèquement désordonnés (domaines 2 et 3) de la protéine NS5A du virus de l'hépatite C (VHC). La structure secondaire résiduel présente dans le fragment de la protéine est analysée. La comparaison des données RMN sur trois constructions de la protéine de différentes longueurs ainsi que les données de SAXS permettent l'identification des interactions transitoires à longue portée entre les différentes régions de cette protéine. En outre, les modes de liaison de ce fragment de protéine à Bin1 domaine SH3 sont analysés. Enfin, les résultats préliminaires obtenus sur l'étude de la phosphorylation de NS5A du VHC par certaines kinases, qui ont été montrées comme biologiquement pertinents, sont présentés. / Intrinsically disordered proteins are characterized by a lack of a stable, 3D structure and fulfill their biological role as such. NMR spectroscopy is the method of choice for their atomic resolution studies, as X-ray crystallography is not amenable to them due to their highly dynamic character.However, NMR spectroscopic studies of these proteins are challenging, because of the high extent of signal overlap in the spectra, resulting from the absence of a hydrogen-bonding network that would lead to structuring and higher signal dispersion. A further problem is experimental sensitivity as often measurement time is limited due to their predisposition for proteolytic degradation. In the fist part of this thesis intrinsically disordered proteins are introduced. The second part focuses on NMR spectroscopy of IDPs, BEST-TROSY-type NMR methods are presented and are shown to be well suited for large IDPs, especially for those with high extent of residual structure. 3D BEST-TROSY experiments are presented for assignment, a proline-edited version for aiding amino acid-type identification, and the HETex-BEST-TROSY experiment that allows rapid measurement of solvent exchange rates. In the third part of this thesis NMR methods are applied for study of the entire intrinsically disordered region (domains 2 and 3) of NS5A protein of hepatitis C virus. The residual secondary structure in this protein fragment is analyzed. Comparison of NMR data on three protein constructs of different lengths together with SAXS data allows identification of transient long range interactions between different regions of this protein. Furthermore, the binding modes of this protein fragment to Bin1 SH3 domain are analyzed. Finally, the preliminary results obtained on investigation of phosphorylation of NS5A of HCV by certain kinases, reported to be biologically relevant, are presented

Orientador: Jorg Kobarg / Tese (doutorado) - Universidade Estadual de Campinas, Instituto e Biologia / Made available in DSpace on 2018-08-14T00:02:08Z (GMT). No. of bitstreams: 1 Bressan_GustavoCosta_D.pdf: 16510013 bytes, checksum: 30f3887b16b18caf89b81d091caca8d7 (MD5) Previous issue date: 2009 / Resumo: A proteína Ki-1/57 foi descoberta através da reação cruzada do anticorpo monoclonal Ki-1 em células do linfoma de Hodgkin. Foi demonstrado previamente que Ki-1/57 sofre fosforilação por PKCs e metilação por PRMT1, uma arginino metiltransferase que modula diversas proteínas ligadoras a RNA. Nesse trabalho, é mostrada a interação de Ki-1/57 com sondas de RNA e com proteínas envolvidas no controle de splicing de pré-mRNA. O seu envolvimento no controle de splicing foi confirmado em ensaios de cotransfecção em células de mamíferos. Análises de microscopia de confocal mostraram a localização da construção EGFP-Ki-1/57 em diferentes corpúsculos nucleares de forma dependente da metilação celular. Essas regiões compreendem nucléolos, speckles, corpos de Cajal e GEMS, conhecidamente envolvidas na biogênese, maturação ou armazenamento de complexos de processamento de RNA/pré-RNA no núcleo. Análises a partir de construções truncadas sugeriram o N-terminal de Ki-1/57 como importante para a interação com proteínas reguladoras de splicing e localização nos corpúsculos nucleares, enquanto o C-terminal como necessário e suficiente para a ligação a RNA poliuridina e localização citoplasmática. Por outro lado, essas duas regiões pareceram atuar em conjunto no processamento do gene E1A. Similarmente a hnRNPQ, Ki-1/57 e outras proteínas funcionalmente relacionadas, SFRS9 é mostrada como alvo de metilação por PRMT1. A inibição da metilação resultou em um aumento do número de células apresentando localização da construção EGFP-SFRS9 no interior de nucléolos, mostrando a importância dessa modificação para a localização subnuclear de SFRS9. As características estruturais de Ki-1/57 também foram investigadas através de diferentes abordagens. Análises por SAXS, gel filtração analítica e ultracentrifugação analítica indicaram uma estrutura bastante alongada e flexível para a construção C-terminal 6xhis-(122-413)Ki-1/57. Ensaios de proteólise limitada também sugeriram uma baixa composição de núcleos hidrofóbicos estáveis e compactos. A capacidade de Ki-1/57 em sofrer enovelamento induzido após a interação com ligantes também foi monitorada em experimentos de dicroísmo circular. Embora não tenha sido observada nenhuma alteração estrutural após a incubação de 6xhis-(122-413)Ki-1/57 com o RNA poliuridina, a adição de TFE foi capaz de promover pequenos ganhos de elementos de estrutura secundária regular. Esses dados, juntamente com predições computacionais, sugerem que Ki-1/57 é uma nova proteína intrinsecamente desordenada, o que pode explicar o elevado número de diferentes proteínas parceiras que ela é capaz de interagir. / Abstract: The Ki-1/57 protein has been discovered through the cross reactivity of the monoclonal antibody Ki-1 in Hodgkin lymphoma cells. Previously, it was demonstrated that Ki-1/57 undergoes phosphorylation by PKCs and methylation by PRMT1, an arginine methyltransferase that modulates many RNA binding proteins. Here, the interaction of Ki-1/57 with RNA polyuridine and proteins involved in pre-mRNA splicing control are shown. Its involvement in splicing regulation was confirmed by cotransfection assays in mammalian cells. Confocal microscopy analyses revealed the localization of EGFP-Ki-1/57 at different nuclear bodies, depending on the cellular methylation status. These regions include nucleoli, speckles, Cajal bodies and GEMS, which are all known to be involved in biogenesis, maturation or storing of RNA/pre-mRNA processing complexes in the nucleus. Analysis from experiments with truncated forms of Ki-1/57 suggested its N-terminus as important for its interaction with splicing proteins and localization at nuclear bodies. In turn, its C-terminus was seen as necessary and sufficient for the cytoplasmic localization and polyuridine RNA binding. However, these two regions seemed to be required working together for an efficient splicing activity on E1A gene. Similarly to hnRNPQ, Ki-1/57 and others functionally related proteins, SFRS9 is shown here as a target for methylation by PRMT1. The inhibition of this activity resulted in increase in the number of cells showing EGFP-SFRS9 in the nucleoli, suggesting the importance of methylation for the subnuclear localization of SFRS9. The structural characteristics of Ki-1/57 also have been investigated through different approaches. Analyses by SAXS, analytical gel filtration and analytical ultracentrifugation techniques suggested a very elongated and flexible structure for the C-terminal construct (122-413)Ki-1/57. Also, limited proteolysis analysis suggested a low composition of stable and compact hydrophobic cores. The ability of Ki-1/57 in suffering binding-induced folding was also investigated. Although no structural modification has been observed after incubating (122-413)Ki-1/57 with a polyuridine RNA, the addition of the TFE probe was able to promote a small gain of regular secondary structural elements. These findings, together with different computational predictions, pointed out that Ki-1/57 is a novel intrinsically unstructured protein. This could explain the wide array of protein partners with which it is able to interact. / Doutorado / Bioquimica / Doutor em Biologia Funcional e Molecular

The Golgi complex is an organelle responsible for receiving synthesized cargo from the endoplasmic reticulum for subsequent post-translations modifications, sorting and secretion. A family of proteins named Golgi Reassembly and Stacking Proteins (GRASP) is essential for the correct assembly and laterally tethering of the Golgi cisternae, a necessary structuration to keep this organelle working correctly. The GRASP structure is mainly composed of two regions: an N-terminal formed by two PDZ domains connected by a short loop (GRASP domain) and a non-conserved C-terminal region, rich in serine and proline residues. Although there are now a few crystal structures solved for the N-terminal domain, it is surprising to notice that no information is currently available regarding a full-length protein or even about dynamic and structural differences between the two PDZs in solution, which is the main functional region of this protein. Using a full-length GRASP model, we were capable of detecting the coexistence of regular secondary structures and large amounts of disordered regions. The overall structure is less compact than a regular globular protein and the high structural flexibility makes its hydrophobic core more accessible to solvent. GRASP coexist in a dynamic conformational ensemble of a µs-ms timescale. Our results indicate an unusual behavior of GRASP in solution, closely resembling a class of collapsed intrinsically disordered proteins called molten globule. We report here also the disorder-to-order transition propensities for a native molten globule-like protein in the presence of different mimetics of cell conditions. Changes in the dielectric constant (such as those experienced close to the membrane surface) seem to be the major factor capable of inducing several disorder-to-order transitions in GRASP, which seems to show very distinct behavior when in conditions that mimic the vicinity of the membrane surface as compared to those found when free in solution. Other folding factors such as molecular crowding, counter ions, pH and phosphorylation exhibit lower or no effect on GRASP secondary structure and/or stability. This is the first study focusing on understanding the disorder-to-order transitions of a molten globule structure without the need for any mild denaturing condition. Regarding the PDZs that form the GRASP domain, we observed that GRASPs are formed by a more unstable and flexible PDZ1 and much more stable and structurally well-behaved PDZ2. More than that, many of the unstable regions found in PDZ1 are in the predicted binding pocket, suggesting a structural promiscuity inside this domain that correlates with the functional promiscuity of interacting with multiple protein partners. This thesis presents the first structural characterization of a full-length GRASP, the first model of how GRASPs (or any molten globule-like protein) can be modulated by the cell during different cell functionalities and the first work in the community proving that the established idea that both PDZs are structurally equivalent is not completely right / O complexo de Golgi é um organela responsável pela recepção de carga sintetizada no retículo endoplasmático e por subsequente modificações pós-traducionais, classificação e secreção. Uma família de proteínas chamada Golgi Reassembly and Stacking Proteins (GRASP) é essencial para o correto empilhamento das cisternas e conexões laterais das pilhas do complexo de Golgi, uma estruturação necessária para manter essa organela funcionando corretamente. A estrutura das GRASPs é composta de duas regiões principais: uma extensão N-terminal formado por dois domínios PDZ conectados por um loop (domínio GRASP) e uma região C-terminal não conservada, rica em resíduos de serina e prolina. Embora existam algumas estruturas cristalográficas resolvidas para o domínio N-terminal, é surpreendente notar que não havia nenhuma informação na literatura sobre a construção inteira de um GRASP, ou mesmo um estudo detalhado sobre os PDZs no N-terminal em solução, que é a principal região funcional dessa proteína. Usando um modelo de GRASP em sua construção completa, fomos capazes de detectar a coexistência de estruturas secundárias regulares e grandes quantidades de regiões desordenadas. A estrutura é menos compacta do que uma proteína globular e a alta flexibilidade estrutural torna o seu núcleo hidrofóbico mais acessível ao solvente. GRASPs coexistem em um conjunto conformacional dinâmico numa escala de tempo característico de s-ms. Nossos resultados indicam um comportamento incomum da GRASP em solução, similar à de uma classe de proteínas intrinsicamente desordenadas colapsadas conhecidas como glóbulos fundidos. Nós relatamos também as propensões de transição estrutural do tipo desordem-ordem para uma proteína glóbulo fundido nativa, induzidas pela presença de diferentes miméticos de condições celulares especificas. A mudança na constante dielétrica do meio (como as experimentadas próximas à superfície da membrana biológica) é o principal modulador estrutural, capaz de induzir múltiplas transições desordem-ordem na GRASP, sugerindo um comportamento muito distinto quando em condições que imitam a vizinhança da superfície da membrana em comparação com os encontrados quando livre em solução. Outros fatores de enovelamento, tais como o molecular crowding, contra-ions, pH e a fosforilação exibem efeitos menores (ou nenhum) na estrutura secundária e/ou estabilidade da GRASP. Este é o primeiro estudo focado na compreensão das transições desordem-ordem em uma estrutura do tipo glóbulo fundido sem que houvesse a necessidade de qualquer condição desnaturante. Em relação aos PDZs que formam o domínio GRASP, observamos que as GRASPs são formadas por um PDZ1 mais instável e flexível e um PDZ2 muito mais estável e estruturalmente bem comportado. Mais do que isso, muitas das regiões instáveis encontradas no PDZ1 estão no predito bolsão de ligação, sugerindo uma promiscuidade estrutural dentro desse domínio que se correlaciona com a promiscuidade funcional de interação com múltiplos parceiros proteicos. É apresentado nesta tese a primeira caracterização estrutural de uma GRASP em sua forma completa, o primeiro modelo de como as GRASPs (ou qualquer proteína em forma de glóbulo fundido) pode ser modulada estruturalmente pela célula durante diferentes funcionalidades e o primeiro trabalho na comunidade provando que a estabelecido ideia de que ambos os PDZs são estruturalmente equivalentes não é completamente correta

The discovery of intrinsically disordered proteins has led to a paradigm shift in protein science. Many disordered proteins have regions that can transform from a disordered state to an ordered. Those regions are called protean segments. Many intrinsically disordered proteins are involved in diseases, including Alzheimer's disease, Parkinson's disease and Down's syndrome, which makes them prime targets for medical research. As protean segments often are the functional part of the proteins, it is of great importance to identify those regions. This report presents Proteus, a new predictor for protean segments. The predictor uses Random Forest (a decision tree ensemble classifier) and is trained on features derived from amino acid sequence and conservation data. Proteus compares favourably to state of the art predictors and performs better than the competition on all four metrics: precision, recall, F1 and MCC. The report also looks at the differences between protean and non-protean regions and how they differ between the two datasets that were used to train the predictor.

Les protéines intrinsèquement désordonnées (IDP), dépourvues d’une structure rigide et stable, constituent une classe de protéines diverses et fonctionnellement importantes. La résonance magnétique nucléaire (RMN) est une technique spectroscopique bien établie pour caractériser les propriétés conformationnelles et dynamiques des IDP avec une résolution atomique. L’espace conformationnel, en général large et varié, des IPD en fait une cible difficile pour la biologie structurale dont le but est de déterminer avec précision et exactitude les propriétés structurales, dynamique et physico-chimiques qui sous-tendent la fonction des macromolécules biologiques. Ce manuscrit présente une étude biophysique détaillée de la région intrinsèquement désordonnée (IDR) du facteur de transcription Engrailed-2, avant tout par RMN. Après une présentation de cette homéoprotéine, nous décrivons les protocoles d’expression et de purification de cette protéine isotopiquement marquée. Nous introduisons ensuite une nouvelle approche pour la caractérisation des mouvements pico- et nanoseconde des protéines intrinsèquement désordonnées à partir de données de relaxation des spins nucléaires enregistrées à plusieurs champs magnétiques. Les effets de relaxation paramagnétique (PRE) ont été utilisés pour identifier des interactions transitoires entre la région désordonnée et l’homéodomaine d’Engrailed-2. L’interaction d’Engrailed-2 avec l’ADN a été étudiée en détail en utilisant l’anisotropie de fluorescence sur une série de constructions de la protéine, afin de mettre en lumière le rôle de la partie désordonnée dans l’interaction avec l’ADN. Nous avons également employé la résonance paramagnétique électronique pour tenter de détecter une interaction potentielle entre le noyau hydrophobe de l’hexapeptide dans la région désordonnée et l’homéodomaine. Les couplages dipolaires résiduels (RDC) dans les paires 1H-15N, Cα-Hα et Cα-C′ ont également été mesurés sur des échantillons d’Engrailed en milieu anisotrope. Ces données seront essentielles pour reconstituer l’espace conformationnel d’Engrailed 2. L’ensemble des approches présentées a permis de constituer un socle solide de connaissances qui permettent de mieux comprendre les propriétés conformationnelles, dynamiques et fonctionnelles de l’IDR d’Engrailed-2. / Intrinsically Disordered Proteins (IDPs), which lack a stable rigid structure constitute a large and functionally important class of proteins. Nuclear Magnetic Resonance (NMR) is a well-established technique to characterize the structural and dynamical features of IDPs at atomic resolution. The broad conformational space of IDPs makes them challenging targets for structural biology to define their precise structural features and motions, the physical and chemical properties that underlie their biological functions. The present thesis establishes biophysical investigation of the disordered region of the transcription factor Engrailed-2 (13.5 kDa) primarily by NMR. After describing the protocol of expression and purification of the isotopically labeled protein, we present a novel approach to characterize the pico – nano second motions in IDPs using nuclear spin relaxation data at multiple fields. Paramagnetic Relaxation Enhancements (PREs) are used to identify transient long-range interactions between the disordered region and the folded homeodomain of Engrailed-2. Binding to DNA was studied by fluorescence anisotropy and highlights the role of the disordered region in the DNA binding. We used Electron Paramagnetic Resonance (EPR) to probe the potential interaction between the hydrophobic cluster (hexapeptide) in the disordered region and the homeodomain. The one-bond 1H-15N, Cα-Hα and Cα-C′ residual dipolar couplings (RDCs) measured for Engrailed-2 provide important constraints for the refinement of the conformational space of Engrailed_2. All these approaches provide valuable insights in understanding the structural, dynamical and functional properties of this IDP.

De nombreuses protéines associées aux maladies neurodégénératives humaines sont intrinsèquement désordonnées. Ce sont des protéines qui sont dépourvues de structure tertiaire ou secondaire stable dans des conditions physiologiques. Plus précisément, les protéines intrinsèquement désordonnées (IDPs) subissent diverses changements conformationnels entre la pelote aléatoire, des conformations hélicoïdales et des structures en feuillet-β, ces deux dernières étant généralement impliquées dans la reconnaissance protéine-protéine. Parmi une vingtaine de peptides amyloïdogènes connus liés aux maladies dégénératives humaines, notre étude porte sur deux protéines désordonnées: le peptide Amyloïde-β (Aβ) associé à la maladie d'Alzheimer et l'Islet Amyloid Polypeptide (IAPP) impliqué dans le diabète de type II. Aβ possède deux alloformes courants de 40 et 42 résidus, tandis que IAPP est une hormone peptidique de 37 résidus. Les agrégats de Aβ sont toxiques pour les cellules du cerveau, tandis que la fibrillisation de IAPP affecte les cellules-β du pancréas. Le mécanisme d'agrégation de ces deux peptides reste encore mal connu, mais il a été proposé qu’en solution, ces peptides visitent différentes conformations, l'une d'entre elles étant riche en feuillets-β. Cela conduirait à l’oligomérisation de ces peptides, par le biais d’interactions feuillet-β / feuillet-β et, éventuellement, à la formation de fibrilles. Le but de notre étude est de mieux caractériser la dynamique conformationnelle de ces deux peptides, dans leur forme monomérique et oligomérique. Comprendre les premières étapes de leur agrégation est crucial pour le développement de nouvelles molécules thérapeutiques efficaces contre ces protéines amyloïdes. / Many proteins associated with human neurodegenerative diseases are intrinsically disordered. They are proteins which lack stable tertiary or secondary structure under physiological conditions. More specifically, intrinsically disordered proteins (IDPs) undergo various structural conversions between random coil, helical conformations and β-strand structures, these two latter being generally involved in protein-protein recognition. Among about twenty known amyloidogenic peptides related to human degenerative diseases, we focus our study on two disordered proteins: the Amyloid-β peptide (Aβ) associated to the Alzheimer’s disease and the Islet Amyloid Polypeptide (IAPP) involved in type II diabetes. Aβ has two common alloforms of 40 and 42 residues in length, meanwhile IAPP is a 37-residues peptide hormone. Aggregates of Aβ are toxic to the brain cells, meanwhile IAPP fibrillization affects the pancreatic β-cells. The aggregation mechanism of these two peptides is not known in detail, but it was proposed that in solution, these peptides visit various conformations, one of them being rich in β-strands. This would lead to peptide oligomerization, through β-strand / β-strand interactions and eventually to the fibril formation. The aim of our study is to provide insights into the conformational dynamics of these two peptides in monomeric and oligomeric forms. Understanding the early steps of their aggregation is crucial for the development of new effective therapeutic molecules against these amyloid proteins.De nombreuses protéines associées aux maladies neurodégénératives humaines sont intrinsèquement désordonnées. Ce sont des protéines qui sont dépourvues de structure tertiaire ou secondaire stable dans des conditions physiologiques. Plus précisément, les protéines intrinsèquement désordonnées (IDPs) subissent diverses changements conformationnels entre la pelote aléatoire, des conformations hélicoïdales et des structures en feuillet-β, ces deux dernières étant généralement impliquées dans la reconnaissance protéine-protéine. Parmi une vingtaine de peptides amyloïdogènes connus liés aux maladies dégénératives humaines, notre étude porte sur deux protéines désordonnées: le peptide Amyloïde-β (Aβ) associé à la maladie d'Alzheimer et l'Islet Amyloid Polypeptide (IAPP) impliqué dans le diabète de type II. Aβ possède deux alloformes courants de 40 et 42 résidus, tandis que IAPP est une hormone peptidique de 37 résidus. Les agrégats de Aβ sont toxiques pour les cellules du cerveau, tandis que la fibrillisation de IAPP affecte les cellules-β du pancréas. Le mécanisme d'agrégation de ces deux peptides reste encore mal connu, mais il a été proposé qu’en solution, ces peptides visitent différentes conformations, l'une d'entre elles étant riche en feuillets-β. Cela conduirait à l’oligomérisation de ces peptides, par le biais d’interactions feuillet-β / feuillet-β et, éventuellement, à la formation de fibrilles. Le but de notre étude est de mieux caractériser la dynamique conformationnelle de ces deux peptides, dans leur forme monomérique et oligomérique. Comprendre les premières étapes de leur agrégation est crucial pour le développement de nouvelles molécules thérapeutiques efficaces contre ces protéines amyloïdes.

Indiana University-Purdue University Indianapolis (IUPUI) / Intrinsically disordered proteins (IDPs) are abundant in nature, being more prevalent in the proteomes of eukaryotes than those of bacteria or archaea. As introduced in Chapter I, these proteins, or portions of these proteins, lack stable equilibrium structures and instead have dynamic conformations that vary over time and population. Despite the lack of preformed structure, IDPs carry out many and varied molecular functions and participate in vital biological pathways. In particular, IDPs play important roles in cellular signaling that is, in part, enabled by the ability of IDPs to mediate molecular recognition. In Chapter II, the role of intrinsic disorder in molecular recognition is examined through two example IDPs: p53 and 14-3-3. The p53 protein uses intrinsically disordered regions at its N- and C-termini to interact with a large number of partners, often using the same residues. The 14-3-3 protein is a structured domain that uses the same binding site to recognize multiple intrinsically disordered partners. Examination of the structural details of these interactions highlights the importance of intrinsic disorder and induced fit in molecular recognition. More generally, many intrinsically disordered regions that mediate interactions share similar features that are identifiable from protein sequence. Chapter IV reviews several models of IDP mediated protein-protein interactions that use completely different parameterizations. Each model has its relative strengths in identifying novel interaction regions, and all suggest that IDP mediated interactions are common in nature. In addition to the biologic importance of IDPs, they are also practically important in the structural study of proteins. The presence of intrinsic disordered regions can inhibit crystallization and solution NMR studies of otherwise well-structured proteins. This problem is compounded in the context of high throughput structure determination. In Chapter III, the effect of IDPs on structure determination by X-ray crystallography is examined. It is found that protein crystals are intolerant of intrinsic disorder by examining existing crystal structures from the PDB. A retrospective analysis of Protein Structure Initiative data indicates that prediction of intrinsic disorder may be useful in the prioritization and improvement of targets for structure determination.

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L’étude de la structure de complexes non-covalents présente un intérêt fondamental dans la recherche en chimie des protéines. Le premier objectif est de caractériser les interactions physico-chimiques sur lesquelles repose l’adoption d’une structure tridimensionnelle fonctionnelle par un édifice multimoléculaire. Le second objectif est de mettre en évidence les changements structuraux induits par le phénomène de complexation, et leur influence sur la fonction du système. Le couplage entre la spectrométrie de masse et la mobilité ionique (IM/MS) est une technique d’étude structurale en phase gazeuse, dont le principe repose sur la séparation d’ions selon leur forme et leur rapport masse sur charge, et qui permet en outre de mesurer leurs sections efficaces de diffusion. Grâce à cette technique, nous avons réalisé l’étude structurale de trois complexes non-covalents : l’agrégation de molécules de tanin sur la protéine salivaire humaine IB5, la fixation du ligand Ac2KAA sur la vancomycine, et la complexation de cations métalliques sur des polymères poly-lactide. L’évolution des sections efficaces en fonction de la taille du système ou de l’état de complexation met en évidence la présence de transitions structurales. De plus, utilisé avec de la modélisation moléculaire ou de la spectroscopie laser, le couplage IM/MS s’avère pertinent pour caractériser les interactions responsables de la stabilisation de tels complexes. Ces travaux de thèse montrent que cette technique , au-delà du simple aspect analytique (séparation d’isomères), peut également être utilisée au sein d’études plus globales, mettant en jeu plusieurs techniques afin de résoudre la structure de systèmes complexes / Knowing the structure of non-covalent complexes is essential to understand many biological processes. The first step is the characterization of the interactions leading to the adoption of a functional tridimensional structure by a multimeric assembly. The second step consists of underlining the structural modifications induced by the complexation, and their influence on the system’s function. The Ion Mobility/Mass Spectrometry (IM/MS) is a gas-phase method that is used to separate ions according to their geometry and their masse-to-charge ratio. IM/MS also provides insights on their intrinsic properties, by measuring their collision cross sections. Using this method, we have studied the structure of three different non-covalent complexes: the aggregation of tannins on the human salivary protein IB-5, the fixation of a small ligand (Ac2KAA) on vancomycin, and the complexation between metallic cations and poly-lactid polymers. The evolution of the collision cross-sections as a function of the size of the system or the complexation state clearly shows structural transitions. Moreover, combined with molecular modeling or laser spectroscopy, the IM/MS technique reveals to be a powerful tool to characterize the relevant interactions in such systems. This work proves that IM/MS, besides a powerful analytical aspect, can also be used in global studies that involve several structural methods to resolve the structure of large multimeric assemblies