Les récepteurs couplés de protéines G (RCPG) sont des capteurs biologiques polyvalents responsables de la majorité des réponses cellulaires aux hormones et neurotransmetteurs ainsi que des sens de la vue, de l'odorat et du goût. La transduction des signaux est associée à un ensemble de changements dans la structure tertiaire des récepteurs entraînant l'activation de partenaires intracellulaires dont les protéines G. La dimérisation est un élément central du mode de fonctionnement des RCPG ; cependant, son influence sur la façon dont le signal est transmis est encore mal définie.Nous avons utilisé ici le récepteur BLT1 du leucotriène B4 comme modèle afin d'analyser les changements de conformation au cours de l'activation. Pour cela, nous avons produit le récepteur suivant une approche qui consiste à l'exprimer dans les corps d'inclusion bactériens puis à le renaturer à l'aide de détergents et/ou surfactants originaux. L'accès au récepteur purifié nous a permis de montrer que la protéine G induit une asymétrie dans les changements de conformation au sein de l'homodimère de BLT1. De plus, nous avons pu établir que l'activation de la protéine G se fait essentiellement par le protomère ayant fixé l'agoniste (cis-activation). Enfin, nous avons montré que la forme monomérique du récepteur est parfaitement capable d'induire l'activation de la protéine G, même si le dimère apporte une modulation de la réponse. Ceci indique qu'un monomère de récepteur possède tous les déterminants moléculaires nécessaires à la transmission du signal. L'ensemble de ces résultats apporte un éclairage nouveau sur la façon dont les dimères de RCPG fonctionnent et peuvent moduler la réponse biologique. / G-protein coupled receptors are versatile biological sensors that are responsible for the majority of cellular responses to hormones and neurotransmitters as well as for the sense of sight, smell and taste. Signal transduction is associated with a set of changes in the tertiary structure of the receptor that are recognized by the associated intracellular partners, in particular the G proteins. There is compelling evidence that GPCR can assemble as dimers but the way these assemblies function at the molecular level is still under investigation.We used here the leukotriene B4 receptor BLT1 as a model to analyze the conformational changes occurring during activation. To this end, we first produced the receptor in E. coli inclusion bodies and subsequently folded it back to its native state in vitro using original membrane mimetics. Using the purified dimeric receptor, we showed that (i) the G protein induces an asymmetric arrangement of the BLT1 homodimer where each of the protomers is in a distinct conformation, and (ii) the G protein is cis-activated, i.e. the protomer that binds the agonist also activates Gα. Finally, we brought evidence that, although the dimer fully activates its G protein partner, the monomer has per se all the molecular determinant for an efficient functioning. All these data are original evidence that sheds light into the way GPCR dimers are activated and in turn modulate G protein-mediated signaling.

Protein phosphorylation is a common post-translational protein modification used in almost all cellular processes. When a phosphate group is added to an amino acid side chain, it may alter the protein conformation and protein-protein interactions due to its size and its negative charge. It may also change the protein function, activity and even localization within the cell. Experimental detection of phosphorylation is still extremely labor demanding and very expensive, even when deploying protein mass spectrometry. For this very reason many bioinformatics scientific groups focus on the prediction of protein phosphorylation sites. Recent analyses of phosphorylation sites studied mainly non-phosphorylated phosphorylation sites and the distribution and representation of amino acids sequentially neighboring them. Since sequentially more distant, but structurally close amino acids can contribute to the recognition of protein substrate by protein kinase, structural environment of phosphorylation sites was studied in this thesis. Furthermore, 3D structures of phosphorylation sites were comprehensively studied for the first time in a phosphorylated state and the results were compared with the results obtained from the analysis of non- phosphorylated sites. Phosphorylation sites were found mostly within...

A família proteína quinases C (PKC) é composta por dez isoenzimas, as quais são capazes de fosforilar resíduos de serina e treonina. A ativação dessas quinases envolve mudanças conformacionais, como a remoção do pseudo-substrato do sítio ativo e associação dessas enzimas com lipídeos em membranas biológicas. Além disso, três fosforilações são importantes para a maturação/ enovelamento da enzima e não estão associadas com o estado de ativação das cPKCs. Apesar dessas quinases estarem envolvidas em vários processos patológicos, como carcinogênese e doenças cardiovasculares, ainda não se estabeleceu a relação entre estado de ativação das PKCs com essas doenças. Isso se deve, em parte, à ausência de ferramentas que possibilitam a distinção das formas ativas e inativas das PKCs. Na presente tese, baseando-se em mudanças conformacionais sofridas pelas PKCs durante o processo de ativação, dois anticorpos contra cPKCs ativas foram racionalmente desenvolvidos, sendo um anticorpo policlonal (anti-C2Cat) e outro monoclonal (4.8E). O anticorpo anti-C2Cat foi desenvolvido a partir de imunização de coelhos com um peptídeo localizado na região de interação entre os domínios C2 e catalítico na PKC inativa. Já o anticorpo monoclonal 4.8E foi produzido após a imunização de camundongos Balb/ C com extrato de proteínas proveniente de células HEK293T superexpressando formas constitutivamente ativas da PKCβI. A seletividade de anti-C2Cat e 4.8E por cPKCs ativas foi demonstrada por ensaios de ELISA e de imunoprecipitação, sendo que os anticorpos sempre apresentaram maior afinidade por cPKCs ativas purificadas, superexpressas ou mesmo as endógenas. O anticorpo anti-C2Cat foi capaz de monitorar a dinâmica espaço-temporal da ativação das cPKCs em linhagens de neuroblastoma (Neuro-2A e SK-N-SH) estimuladas com PMA, morfina, ATP ou glutamato por diferentes tempos. Ainda, um maior conteúdo de cPKCs ativas foi detectado por anti-C2Cat na linhagem de câncer de mama MDA-MB-231 (triplo- negativa) do que em células MCF-7 (ER+). Em acordo com esses dados, anti-C2Cat identificou uma maior ativação de cPKCs em tumores mais agressivos de câncer de mama (subtipo triplo-negativo) do que em tumores menos agressivos (ER+, subtipo luminal). Os anticorpos conformacionais anti-C2Cat e 4.8E foram aplicados para elucidar vias de sinalização que levam à carcinogênese em células MDA-MB-231, por meio da realização de ensaios de co-imunoprecipitação, seguida pela identificação das proteínas por espectrometria de massas. Usando essa abordagem, os resultados sugerem que as cPKCs ativas possam estar envolvidas com a tradução de proteínas envolvidas na migração celular, como actina. Em conjunto, os resultado obtidos na presente tese demonstram duas formas racionais de desenvolver anticorpos contra cPKCs ativas, sendo que algumas aplicações para estas ferramentas foram demonstradas. Estratégias baseadas em mudanças conformacionais, similares às apresentadas aqui, poderão ser utilizadas para a produção racional de anticorpos contra outras quinases ou proteínas / The protein kinase C family (PKC) is composed of ten isoenzymes, which are capable of phosphorylating serine and threonine amino acid residues. PKC activation involves conformational changes, such as removing the pseudo-substrate from the active site and binding of the enzyme to lipids in biological membranes. In addition, PKC undergoes three phosphorylations that are important for the maturation/ folding of the enzyme and are not linked with activation status. Despite the fact that these kinases are involved in various pathological processes, such as carcinogenesis and cardiovascular disease, a relationship between PKC activation status with these diseases has not yet been established. This is partly due to the lack of tools to detect active PKC in tissue samples. In this thesis, based on conformational changes suffered by PKC during its activation, two antibodies against active cPKCs were rationally developed; a polyclonal antibody (anti-C2Cat) and a monoclonal (4.8E). Anti-C2Cat was produced after immunization of rabbits with a peptide located at the interface between the C2 and catalytic domains of cPKCs in an inactive PKC. The monoclonal antibody 4.8E was produced after immunization of Balb/C mice with total lysates from HEK293T cells overexpressing constitutively active forms of PKCβI. The anti-C2Cat and 4.8E specificity by active cPKCs was demonstrated by ELISA and immunoprecipitation assays, where the antibodies always showed higher affinity to active cPKCs. Anti-C2Cat was able to detect the temporal and spatial dynamics of cPKC activation upon receptor (morphine, ATP or glutamate) or phorbol ester stimulation in neuroblastoma lines (Neuro-2A and SK-N-SH). Futhermore, anti-C2Cat is able to detect active PKC in human tissues. Higher levels of active cPKC were observed in the more aggressive triple negative breast cancer tumors as compared to the less aggressive estrogen receptor positive tumors. Also, both antibodies were applied to study signaling pathways that lead to carcinogenesis in MDA-MB-231 cells by performing co-immunoprecipitation and mass spectrometry. Using this approach, the results suggest that active cPKCs may be involved in translation of proteins involved in cell migration, such as actin. Taken together, the results obtained in this thesis showed two rational ways to develop antibodies against active cPKCs and some applications for these tools were demonstrated. Strategies based on conformational changes, similar to those presented herein may be used for rational production of antibodies against other kinases and proteins.

Nas duas últimas décadas, houve um enorme aumento no número de estruturas proteicas resolvidas, e entre elas há uma variedade imensa de proteínas com mais de uma conformação observada. Essa quantidade incontestável de dados experimentais corroboram a hipótese de que cada proteína exista num espaço conformacional próprio, onde ela possa adotar inúmeras conformações, umas mais distintas ou estáveis que outras. Essas conformações estão distribuídas nesse espaço de acordo com sua energia potencial, que pode ser definida como uma superfície cheia de rugosidades, poços e barreiras energéticas. Duas conformações distantes nesse espaço são muito diferentes entre si, enquanto que duas conformações próximas são mais semelhantes. Da mesma forma, se distinguem os movimentos necessários para passar de uma conformação à outra. Para uma proteína passar de um estado a outro, geralmente identificados com grandes mudanças conformacionais, é necessário um movimento coletivo. Por ser de grande amplitude, esse tipo de movimento ocorre com baixa frequência, e dificilmente é observado em simulações clássicas de dinâmica molecular. Assim, existem métodos dedicados à obtenção destes movimentos, como a análise de modos normais, os modelos de redes elásticas e a análise de componentes principais. Neste trabalho, adaptamos o método de transformada de Fourier para recuperar modos harmônicos que compõem uma trajetória simulada suficientemente longa para analisar três proteínas distintas quanto a seus movimentos biológicos de importância funcional. Uma é a DEA, cuja simetria hexagonal observamos influenciar nos modos coletivos e na transição entre estados. Outra é a MosR, que simulamos em seus dois estados diferentes, oxidado ou reduzido, para encontrar como a oxidação é capaz de impedir os movimentos coletivos que levam à conformação ligada ao DNA. Nestas duas proteínas, observamos que nenhum modo por si só é responsável pela transição entre as conformações experimentais, mas que eles dependem de outros modos ou outras mudanças conformacionais ocorrendo de forma combinada. A terceira proteína analisada é um regulador transcricional, assim como a MosR, a ElrR, cuja estrutura é conhecida somente na forma apo. Neste trabalho, construímos modelos da ElrR ligada ao DNA pela combinação linear de modos harmônicos para modelar um possível ligante na nova conformação do sítio alostérico. As amplitudes usadas nessa combinação foram obtidas pelo método de mínimos quadrados, visando minimizar o desvio em relação somente às coordenadas que as hélices de reconhecimento devem apresentar para se ligar ao sítio de DNA. Este prognóstico foi feito pela análise metódica das estruturas de 27 reguladores transcricionais, homodiméricos com o motivo HTH, em complexo com DNA. Essa análise também nos permitiu descrever a estereoquímica do encaixe das hélices de reconhecimento nos sulcos maiores do DNA com novos parâmetros geométricos, intimamente relacionados com a simetria do complexo, com a sequência de resíduos das hélices de reconhecimento e com a sequência de bases do sítio de DNA, de forma a auxiliar na modelagem de novos complexos. / There was an enormous increase in the deposited protein structures in the past two decades, among them there is a great variety of proteins with more than one observed conformation. This undenieble amount of experimental data ratify the hypothesis that each protein posseses its own conformational space, where it can adopt countless conformations, some more distinct or stable than others. These conformations are distributed in the space according to its potential energy, which maybe defined as a rough landscape fulled with energetic wells and barriers. Two conformations lying apart from each other in this landscape do not carry much resemblances, while neighbouring conformations are very similar. The motions required to get one conformation to another are just as distinguishable. There must be a collective motion inbetween two states of a protein, commonly characterized by large conformation changes. This type of motion is related to large amplitudes and low frequencies, thus it is hardly seen in classical molecular dynamics simulations. Therefore, there are dedicated methods to obtain these motions, as normal modes analysis, elastic network models and essential dynamics. In this work we adapted the method of Fourier transform filtering to retrieve harmonic modes that compose a simulated trajectory and thus analise the biological motions with functional importance of three distinct proteins. One is DEA, which hexagonal symmetry was observed to affect its collective motions and the transition between biological states. Another protein is MosR, which we simulated in two different states, oxidized or reduced, to learn how the formation of a disulphide bridge is able to preclude the collective motions that lead to a DNA-binding conformation. With these two proteins we observed that no mode by itself is responsible for the transition between experimental conformations, and they actually depend on other conformational changes occurring in a combined manner. The third protein that we analised, ElrR, is a transcriptional regulator, like MosR, which structure is known only on its apo form. Hence in this work we built models of ElrR bound to DNA by the linear combination of harmonic modes aiming to model a ligand that would fit in the allosteric site upon the conformational changes driven by the collective motions. The amplitudes we used in this method were calculated by the least square method to minimize the deviation to the positions of the recognition helices when bound to the DNA. This prognostic of the target position of the recognition helices was made upon the methodical analysis of 27 structures of homodimeric transcriptional regulators, that present the Helix-Turn-Helix motif, complexed with DNA. This approach allowed us to describe the stereochemical fitting of the recognition helices into the DNA major grooves with new geometrical parameters intimatelly related to the symmetry of the complex, the residue sequence of the recognition helices and the base sequence of the DNA site, providing thus support to model new complexes.

The electromagnetic absorption properties of plasmonic nanostructures were utilized to develop mesoscopic sites for highly efficient photothermal generation steam, SERS biosensing, and light-triggered cellular delivery uptake. Plasmonic nanostructures embedded in common thermal solutions produces vapor without the requirement of heating the fluid volume. When particles are dispersed in water at ambient temperature, energy is directed primarily to vaporization of water into steam, with a much smaller fraction resulting in heating of the fluid. Solar illuminated aqueous nanoparticle solution can drive water-ethanol distillation, yielding fractions significantly richer in ethanol content than simple thermal distillation and also produced saturated steam destroying Geobacillus stearothermophilus bacteria in a compact solar powered autoclave. Subwavelength biosensing sites were developed using the plasmonic properties of gold nanoshells to investigate the properties of aptamer (DNA) target complexes. Nanoshells are tunable core-shell nanoparticles whose resonant absorption and scattering properties are dependent on core/shell thickness ratio. Nanoshells were used to develop a label free detection method using SERS to monitor conformational change induced by aptamer target binding. The conformational changes to the aptamers induced by target binding were probed by monitoring the aptamer SERS spectra reproducibility. Furthermore, nanoshells can serve as a nonviral light-controlled delivery vector for the precise temporal and spatial control of molecular delivery in vitro. The drug delivery concept using plasmonic vectors was shown using a monolayer of ds-DNA attached to the nanoshell surface and the small molecular “parcel” intercalated inside ds-DNA loops. DAPI, a fluorescent dye, was used as the molecular parcel to visualize the release process in living cells. Upon laser illumination at the absorption resonance the nanoshell converts photon energy into heat producing a local temperature gradient that induces DNA dehybridization, releasing the intercalated molecules.

Les structures cristallographiques de macromolécules comme les protéines, obtenues par la biologie structurale sont des modèles statiques. Or, c'est la flexibilité et la dynamique de ces macromolécules qui sont généralement responsables de leurs fonctions. La simulation permet d'explorer cette flexibilité lors de différents phénomènes qui ont lieu dans ces systèmes : une réaction chimique, des interactions avec une petite molécule… Simuler de tels phénomènes est un défi car la dynamique moléculaire classique ne permet pas de les observer. Des algorithmes permettent d'accélérer l'échantillonnage des dynamiques pour lever cette limitation et de calculer les barrières d'activation pour de tels phénomènes. Simultanément, le choix du niveau de calcul est crucial car il faut concilier la taille importante des systèmes, la nature des interactions et les phénomènes électroniques impliqués. Dans ce travail, différentes méthodes, dont principalement la métadynamique soit au niveau classique ou quantique, ou encore en combinant les deux niveaux quantique/classique, seront utilisées pour modéliser quatre processus complexes : des changements de conformations d'une protéine, des interactions entre métalloprotéine et inhibiteur, des réactions en solution et dans une enzyme. / Crystallographic structures of macromolecules, such as proteins, obtained by structural biology are static models. However, flexibility and dynamics of macromolecules are generally responsible for their functions. Modeling allows us to explore this flexibility in different phenomena that take place in these systems: a chemical reaction, interaction with a small molecule... Modeling such phenomena is a challenge because they cannot be observed by classical molecular dynamics. Algorithms can accelerate sampling of dynamics to simulate these events and calculate their activation barriers. Simultaneously, the choice of the level of calculation is crucial because it must merge with the size of the systems, the nature of interactions and the electronic phenomena involved.In this thesis, some methods, mainly metadynamics at classical level, quantum or the hybrid quantum/classical level, will be used to model four complex processes: conformational changes of proteins, metalloprotein/inhibitor interactions, reactivity in solution and enzymatic reactivity.

Nas duas últimas décadas, houve um enorme aumento no número de estruturas proteicas resolvidas, e entre elas há uma variedade imensa de proteínas com mais de uma conformação observada. Essa quantidade incontestável de dados experimentais corroboram a hipótese de que cada proteína exista num espaço conformacional próprio, onde ela possa adotar inúmeras conformações, umas mais distintas ou estáveis que outras. Essas conformações estão distribuídas nesse espaço de acordo com sua energia potencial, que pode ser definida como uma superfície cheia de rugosidades, poços e barreiras energéticas. Duas conformações distantes nesse espaço são muito diferentes entre si, enquanto que duas conformações próximas são mais semelhantes. Da mesma forma, se distinguem os movimentos necessários para passar de uma conformação à outra. Para uma proteína passar de um estado a outro, geralmente identificados com grandes mudanças conformacionais, é necessário um movimento coletivo. Por ser de grande amplitude, esse tipo de movimento ocorre com baixa frequência, e dificilmente é observado em simulações clássicas de dinâmica molecular. Assim, existem métodos dedicados à obtenção destes movimentos, como a análise de modos normais, os modelos de redes elásticas e a análise de componentes principais. Neste trabalho, adaptamos o método de transformada de Fourier para recuperar modos harmônicos que compõem uma trajetória simulada suficientemente longa para analisar três proteínas distintas quanto a seus movimentos biológicos de importância funcional. Uma é a DEA, cuja simetria hexagonal observamos influenciar nos modos coletivos e na transição entre estados. Outra é a MosR, que simulamos em seus dois estados diferentes, oxidado ou reduzido, para encontrar como a oxidação é capaz de impedir os movimentos coletivos que levam à conformação ligada ao DNA. Nestas duas proteínas, observamos que nenhum modo por si só é responsável pela transição entre as conformações experimentais, mas que eles dependem de outros modos ou outras mudanças conformacionais ocorrendo de forma combinada. A terceira proteína analisada é um regulador transcricional, assim como a MosR, a ElrR, cuja estrutura é conhecida somente na forma apo. Neste trabalho, construímos modelos da ElrR ligada ao DNA pela combinação linear de modos harmônicos para modelar um possível ligante na nova conformação do sítio alostérico. As amplitudes usadas nessa combinação foram obtidas pelo método de mínimos quadrados, visando minimizar o desvio em relação somente às coordenadas que as hélices de reconhecimento devem apresentar para se ligar ao sítio de DNA. Este prognóstico foi feito pela análise metódica das estruturas de 27 reguladores transcricionais, homodiméricos com o motivo HTH, em complexo com DNA. Essa análise também nos permitiu descrever a estereoquímica do encaixe das hélices de reconhecimento nos sulcos maiores do DNA com novos parâmetros geométricos, intimamente relacionados com a simetria do complexo, com a sequência de resíduos das hélices de reconhecimento e com a sequência de bases do sítio de DNA, de forma a auxiliar na modelagem de novos complexos. / There was an enormous increase in the deposited protein structures in the past two decades, among them there is a great variety of proteins with more than one observed conformation. This undenieble amount of experimental data ratify the hypothesis that each protein posseses its own conformational space, where it can adopt countless conformations, some more distinct or stable than others. These conformations are distributed in the space according to its potential energy, which maybe defined as a rough landscape fulled with energetic wells and barriers. Two conformations lying apart from each other in this landscape do not carry much resemblances, while neighbouring conformations are very similar. The motions required to get one conformation to another are just as distinguishable. There must be a collective motion inbetween two states of a protein, commonly characterized by large conformation changes. This type of motion is related to large amplitudes and low frequencies, thus it is hardly seen in classical molecular dynamics simulations. Therefore, there are dedicated methods to obtain these motions, as normal modes analysis, elastic network models and essential dynamics. In this work we adapted the method of Fourier transform filtering to retrieve harmonic modes that compose a simulated trajectory and thus analise the biological motions with functional importance of three distinct proteins. One is DEA, which hexagonal symmetry was observed to affect its collective motions and the transition between biological states. Another protein is MosR, which we simulated in two different states, oxidized or reduced, to learn how the formation of a disulphide bridge is able to preclude the collective motions that lead to a DNA-binding conformation. With these two proteins we observed that no mode by itself is responsible for the transition between experimental conformations, and they actually depend on other conformational changes occurring in a combined manner. The third protein that we analised, ElrR, is a transcriptional regulator, like MosR, which structure is known only on its apo form. Hence in this work we built models of ElrR bound to DNA by the linear combination of harmonic modes aiming to model a ligand that would fit in the allosteric site upon the conformational changes driven by the collective motions. The amplitudes we used in this method were calculated by the least square method to minimize the deviation to the positions of the recognition helices when bound to the DNA. This prognostic of the target position of the recognition helices was made upon the methodical analysis of 27 structures of homodimeric transcriptional regulators, that present the Helix-Turn-Helix motif, complexed with DNA. This approach allowed us to describe the stereochemical fitting of the recognition helices into the DNA major grooves with new geometrical parameters intimatelly related to the symmetry of the complex, the residue sequence of the recognition helices and the base sequence of the DNA site, providing thus support to model new complexes.

Le transporteur mitochondrial de nucléotides adényliques (Ancp), localisé dans la membrane interne mitochondriale, catalyse l'échange ADP/ATP entre le cytoplasme et la matrice mitochondriale. Il lie deux classes d'inhibiteurs naturels avec une grande spécificité et une haute affinité. Ces deux types d'inhibiteurs, BA et CATR, stabilisent Ancp dans deux conformations distinctes impliquées dans le transport des nucléotides. La compréhension des changements conformationnels subits par Ancp est essentielle pour décrire précisément le mécanisme d'échange des nucléotides. La structure atomique du transporteur de bœuf a montré que les hélices transmembranaires 1, 3 et 5 sont coudées par la présence de prolines qui pourraient donc être impliquées dans les changements conformationnels associés au transport.Dans la première partie de ce manuscrit, ces prolines ont été mutées en alanine ou en leucine et les conséquences de ces mutations ont été étudiées au niveau de la cellule (phénotype, morphologie, contenu en protéines) et des mitochondries en examinant le transport lui-même ainsi que toutes les fonctions mitochondriales (respiration, contenu en protéines, importation des protéines, morphologie…). Il peut-être conclu de ces études que ces prolines jouent un rôle dans le transport mais également dans la biogenèse mitochondriale (import d'Ancp, équilibre fusion/fission mitochondriale).Dans la deuxième partie, ont été étudiées des mutations qui stabilisent Ancp dans la conformation BA ou CATR. L'objectif était d'obtenir des formes stables du transporteur représentant des états intermédiaires du transport pour en étudier la structure atomique par cristallographie. Les résultats préliminaires sont prometteurs. / The mitochondrial ADP/ATP carrier (Ancp), located in the inner mitochondrial membrane, catalyzes the ADP/ATP exchange between the cytoplasm and the mitochondrial membrane. Two classes of natural inhibitors can bind to the carrier with high specificity and affinity. These two families of inhibitors, BA and CATR, stabilize Ancp in two different conformations, which are involved in the nucleotide transport. Understanding the conformational changes undergone by Ancp is essential to describe precisely the nucleotide exchange mechanism. The atomic structure of the Beef Ancp unveiled kinks in transmembrane helices 1, 3 and 5 induced by the prolines, which therefore could be involved in the conformational changes associated with the nucleotide transport. In the first part of this manuscript, the three prolines were mutated into alanine or leucine and the results of these mutations were studied at the level of the cell (phenotype, morphology, protein content) and of the mitochondria by examining the transport itself and various mitochondria functions (respiration, protein content, protein import, morphology...). It can be concluded from these studies that these prolines play a key role in the transport but also in mitochondria biogenesis (Ancp import, mitochondrial fusion/fission balance).In the second part were studied mutations that stabilize Ancp in BA or CATR conformation. The goal was to obtain stable forms of Ancp that would correspond to intermediate steps of the transport to study their atomic structure by crystallography. The preliminary results are promising.

A família proteína quinases C (PKC) é composta por dez isoenzimas, as quais são capazes de fosforilar resíduos de serina e treonina. A ativação dessas quinases envolve mudanças conformacionais, como a remoção do pseudo-substrato do sítio ativo e associação dessas enzimas com lipídeos em membranas biológicas. Além disso, três fosforilações são importantes para a maturação/ enovelamento da enzima e não estão associadas com o estado de ativação das cPKCs. Apesar dessas quinases estarem envolvidas em vários processos patológicos, como carcinogênese e doenças cardiovasculares, ainda não se estabeleceu a relação entre estado de ativação das PKCs com essas doenças. Isso se deve, em parte, à ausência de ferramentas que possibilitam a distinção das formas ativas e inativas das PKCs. Na presente tese, baseando-se em mudanças conformacionais sofridas pelas PKCs durante o processo de ativação, dois anticorpos contra cPKCs ativas foram racionalmente desenvolvidos, sendo um anticorpo policlonal (anti-C2Cat) e outro monoclonal (4.8E). O anticorpo anti-C2Cat foi desenvolvido a partir de imunização de coelhos com um peptídeo localizado na região de interação entre os domínios C2 e catalítico na PKC inativa. Já o anticorpo monoclonal 4.8E foi produzido após a imunização de camundongos Balb/ C com extrato de proteínas proveniente de células HEK293T superexpressando formas constitutivamente ativas da PKCβI. A seletividade de anti-C2Cat e 4.8E por cPKCs ativas foi demonstrada por ensaios de ELISA e de imunoprecipitação, sendo que os anticorpos sempre apresentaram maior afinidade por cPKCs ativas purificadas, superexpressas ou mesmo as endógenas. O anticorpo anti-C2Cat foi capaz de monitorar a dinâmica espaço-temporal da ativação das cPKCs em linhagens de neuroblastoma (Neuro-2A e SK-N-SH) estimuladas com PMA, morfina, ATP ou glutamato por diferentes tempos. Ainda, um maior conteúdo de cPKCs ativas foi detectado por anti-C2Cat na linhagem de câncer de mama MDA-MB-231 (triplo- negativa) do que em células MCF-7 (ER+). Em acordo com esses dados, anti-C2Cat identificou uma maior ativação de cPKCs em tumores mais agressivos de câncer de mama (subtipo triplo-negativo) do que em tumores menos agressivos (ER+, subtipo luminal). Os anticorpos conformacionais anti-C2Cat e 4.8E foram aplicados para elucidar vias de sinalização que levam à carcinogênese em células MDA-MB-231, por meio da realização de ensaios de co-imunoprecipitação, seguida pela identificação das proteínas por espectrometria de massas. Usando essa abordagem, os resultados sugerem que as cPKCs ativas possam estar envolvidas com a tradução de proteínas envolvidas na migração celular, como actina. Em conjunto, os resultado obtidos na presente tese demonstram duas formas racionais de desenvolver anticorpos contra cPKCs ativas, sendo que algumas aplicações para estas ferramentas foram demonstradas. Estratégias baseadas em mudanças conformacionais, similares às apresentadas aqui, poderão ser utilizadas para a produção racional de anticorpos contra outras quinases ou proteínas / The protein kinase C family (PKC) is composed of ten isoenzymes, which are capable of phosphorylating serine and threonine amino acid residues. PKC activation involves conformational changes, such as removing the pseudo-substrate from the active site and binding of the enzyme to lipids in biological membranes. In addition, PKC undergoes three phosphorylations that are important for the maturation/ folding of the enzyme and are not linked with activation status. Despite the fact that these kinases are involved in various pathological processes, such as carcinogenesis and cardiovascular disease, a relationship between PKC activation status with these diseases has not yet been established. This is partly due to the lack of tools to detect active PKC in tissue samples. In this thesis, based on conformational changes suffered by PKC during its activation, two antibodies against active cPKCs were rationally developed; a polyclonal antibody (anti-C2Cat) and a monoclonal (4.8E). Anti-C2Cat was produced after immunization of rabbits with a peptide located at the interface between the C2 and catalytic domains of cPKCs in an inactive PKC. The monoclonal antibody 4.8E was produced after immunization of Balb/C mice with total lysates from HEK293T cells overexpressing constitutively active forms of PKCβI. The anti-C2Cat and 4.8E specificity by active cPKCs was demonstrated by ELISA and immunoprecipitation assays, where the antibodies always showed higher affinity to active cPKCs. Anti-C2Cat was able to detect the temporal and spatial dynamics of cPKC activation upon receptor (morphine, ATP or glutamate) or phorbol ester stimulation in neuroblastoma lines (Neuro-2A and SK-N-SH). Futhermore, anti-C2Cat is able to detect active PKC in human tissues. Higher levels of active cPKC were observed in the more aggressive triple negative breast cancer tumors as compared to the less aggressive estrogen receptor positive tumors. Also, both antibodies were applied to study signaling pathways that lead to carcinogenesis in MDA-MB-231 cells by performing co-immunoprecipitation and mass spectrometry. Using this approach, the results suggest that active cPKCs may be involved in translation of proteins involved in cell migration, such as actin. Taken together, the results obtained in this thesis showed two rational ways to develop antibodies against active cPKCs and some applications for these tools were demonstrated. Strategies based on conformational changes, similar to those presented herein may be used for rational production of antibodies against other kinases and proteins.

Molecular dynamics (MD) simulations were used to investigate the structural basis for the functions of three proteins: Fc(gamma) receptor III (CD16), von Willebrand factor (VWF), and integrin. CD16, a heavily glycosylated protein expressed on human immune cells, plays a crucial role in immune defense by linking antibody-antigen complexes with cellular effector functions. Glycosylation of CD16 decreases its affinity for IgG. MD simulations were run for CD16-IgG Fc complexes with or without an N-glycan on CD16. The two simulated complexes show different conformations. Molecular Mechanics-Poisson Boltzmann Surface Area (MM-PBSA) approach was used to calculate the binding free energy of the CD16-IgG Fc complexes. The calculated binding free energy helped to identify critical residues. VWF, a multimeric multidomain glycoprotein, initiates platelet adhesion at the sites of vascular injury. A specific VWF metalloprotease, A Disintegrin And Metalloprotease with ThromboSpondin motifs member 13 (ADAMTS-13), cleaves the Tyr1605-Met1606 bond in the VWF A2 domain to generate the full spectrum of plasma VWF species. Shear stress or denaturants assist VWF cleavage by ADAMTS-13 due to the unfolding of A2. MD was used to simulate the unfolding processes of A2 under force or high temperature. The beta-strands of A2 were pulled out sequentially by force, during which the cleavage site changed in steps from the fully buried state to the fully exposed state. Thermal unfolding follows a very different pathway. Integrins are adhesion molecules mediating cell-cell, cell-extracellular matrix, and cell-pathogen interactions. Experiments suggest that integrins can undergo a large-scale change from a bent to an extended conformation, associating with a transition from low to high affinity states, i.e., integrin activation. Steered MD was utilized to simulate the bent-to-extended conformational transition in time of aVb3 integrin. The integrin was observed to change smoothly from the bent to the extended conformation. One major energy barrier was overcome, corresponding to the disruption of the interactions at Hybrid/EGF4/bTD interfaces. A partially extended conformation tends to bend back while a fully extended conformation is stabilized by the coordination of Asp457 with Ca2+ at alpha-genu. Unbending with separated legs overcomes more energy barriers.