A Infecção pelo dengue afeta todas as regiões tropicais e subtropicais do globo, registra aproximadamente 390 milhões de casos por ano, e se destaca como um problema emergente de proporções crescentes, especialmente no Brasil, que atualmente responde por 60% dos casos no continente americano. Assim, ações para criar, adaptar e atender as condições para promover avanços na compreensão do processo de infecção pelo vírus em nível molecular pode ser de grande valor, tanto para enfrentar os desafios impostos pela conjuntura atual do dengue, quanto pela possibilidade de ampliar o conhecimento de mecanismos moleculares para outros vírus relacionados. Aqui, focando na proteína E do envelope do vírus do dengue, abordamos o problema da infecção viral sob os aspectos estruturais da proteína do envelope (E) quando enfrenta as condições ambientais distintas ao longo do caminho desde o trato digestivo do vetor exotérmico até a maquinaria celular do hospedeiro homeotérmico. Para este efeito, empregamos simulações de Dinâmica Molecular, avaliando e quantificando os processos de rearranjo conformacional do o domínio III isolado da proteína E dos quatro sorotipos de dengue, os quais são induzidos por alterações nos parâmetros termodinâmicos intensivos (pH 3, 5 e 7, T = 298K e 310K). Além disso, a configuração quaternária pentarradial de cinco domínios III é estudada, correlacionando flexibilidades específicas das regiões de loops a ajuste induzido a eventuais ligantes. Nós também verificamos a estabilidade estrutural do complexo DIII com o fragmento da região ligante de antígeno de um anticorpo monoclonal (Fab 1A1D-2), bem como as forças de interação entre a interface de ligação, identificando resíduos-chave. Além disso, os eventos relacionados com a interação da proteína E (tanto na forma monomérica quanto na dimérica) com o envelope lipídico viral são minuciosamente estudados, abrindo o caminho para um possível estudo centrado na formação de trímeros da proteína E, levando à fusão de membranas e subsequente inoculação do RNA viral no citoplasma. Finalmente, alternativas são propostas para a compreensão do mecanismo de ação da fosfolipase do veneno de Crotalus durissus terrificus - a cascavel sul-americana, que tem se mostrado eficaz na inibição in vitro da infecção pelo vírus do dengue. / Dengue infection burdens all tropical and subtropical regions of the globe, registers approximately 390 million cases annually, and stands out as an emerging problem of increasing proportions, especially in Brazil, which currently accounts for 60% of cases in the American continent. Thus, actions to create, adapt and meet conditions to promote advances in understanding the process of virus infection at the molecular level can be of great value both to meet the challenges posed by current conjuncture of dengue, and for the possibility to extend the knowledge of the molecular mechanisms to related viruses. Here, focusing on the E protein of the dengue virus envelope, we address the problem of viral infection under stereochemical aspects of the envelope (E) protein when it undergoes the distinct environmental conditions along the pathway from the exothermic vector\'s digestive tract to the homeothermic host\'s cell machinery. To this end, we employ molecular dynamics to assess and quantify the processes of conformational rearrangement of isolated domain III of the E protein of the four serotypes of dengue, as induced by changes on intensive thermodynamic parameters (pH 3, 5 and 7; T = 298K and 310K). Further, the 5-fold quaternary configuration of five domains III is studied, correlating specific loop regions flexibilities with induced fit with eventual ligands. We also check the structural stability and interaction forces between the interface of the complex DIII and the antigen binding fragment of a monoclonal antibody (Fab 1A1D-2), identifying key residues. Additionally events related to the interaction of the E protein (both as a monomer and as a dimer) with the viral membrane are thoroughly studied, paving the way for a possible study focused on the formation of trimers of E protein, leading to membrane fusion and subsequent inoculation of viral RNA in the cytoplasm. Finally, alternatives are proposed for understanding the mechanism of action of the phospholipase from the venom of Crotalus durissus terrificus - the South American rattlesnake, which has proven effective in vitro inhibition of Dengue virus infection.

A Infecção pelo dengue afeta todas as regiões tropicais e subtropicais do globo, registra aproximadamente 390 milhões de casos por ano, e se destaca como um problema emergente de proporções crescentes, especialmente no Brasil, que atualmente responde por 60% dos casos no continente americano. Assim, ações para criar, adaptar e atender as condições para promover avanços na compreensão do processo de infecção pelo vírus em nível molecular pode ser de grande valor, tanto para enfrentar os desafios impostos pela conjuntura atual do dengue, quanto pela possibilidade de ampliar o conhecimento de mecanismos moleculares para outros vírus relacionados. Aqui, focando na proteína E do envelope do vírus do dengue, abordamos o problema da infecção viral sob os aspectos estruturais da proteína do envelope (E) quando enfrenta as condições ambientais distintas ao longo do caminho desde o trato digestivo do vetor exotérmico até a maquinaria celular do hospedeiro homeotérmico. Para este efeito, empregamos simulações de Dinâmica Molecular, avaliando e quantificando os processos de rearranjo conformacional do o domínio III isolado da proteína E dos quatro sorotipos de dengue, os quais são induzidos por alterações nos parâmetros termodinâmicos intensivos (pH 3, 5 e 7, T = 298K e 310K). Além disso, a configuração quaternária pentarradial de cinco domínios III é estudada, correlacionando flexibilidades específicas das regiões de loops a ajuste induzido a eventuais ligantes. Nós também verificamos a estabilidade estrutural do complexo DIII com o fragmento da região ligante de antígeno de um anticorpo monoclonal (Fab 1A1D-2), bem como as forças de interação entre a interface de ligação, identificando resíduos-chave. Além disso, os eventos relacionados com a interação da proteína E (tanto na forma monomérica quanto na dimérica) com o envelope lipídico viral são minuciosamente estudados, abrindo o caminho para um possível estudo centrado na formação de trímeros da proteína E, levando à fusão de membranas e subsequente inoculação do RNA viral no citoplasma. Finalmente, alternativas são propostas para a compreensão do mecanismo de ação da fosfolipase do veneno de Crotalus durissus terrificus - a cascavel sul-americana, que tem se mostrado eficaz na inibição in vitro da infecção pelo vírus do dengue. / Dengue infection burdens all tropical and subtropical regions of the globe, registers approximately 390 million cases annually, and stands out as an emerging problem of increasing proportions, especially in Brazil, which currently accounts for 60% of cases in the American continent. Thus, actions to create, adapt and meet conditions to promote advances in understanding the process of virus infection at the molecular level can be of great value both to meet the challenges posed by current conjuncture of dengue, and for the possibility to extend the knowledge of the molecular mechanisms to related viruses. Here, focusing on the E protein of the dengue virus envelope, we address the problem of viral infection under stereochemical aspects of the envelope (E) protein when it undergoes the distinct environmental conditions along the pathway from the exothermic vector\'s digestive tract to the homeothermic host\'s cell machinery. To this end, we employ molecular dynamics to assess and quantify the processes of conformational rearrangement of isolated domain III of the E protein of the four serotypes of dengue, as induced by changes on intensive thermodynamic parameters (pH 3, 5 and 7; T = 298K and 310K). Further, the 5-fold quaternary configuration of five domains III is studied, correlating specific loop regions flexibilities with induced fit with eventual ligands. We also check the structural stability and interaction forces between the interface of the complex DIII and the antigen binding fragment of a monoclonal antibody (Fab 1A1D-2), identifying key residues. Additionally events related to the interaction of the E protein (both as a monomer and as a dimer) with the viral membrane are thoroughly studied, paving the way for a possible study focused on the formation of trimers of E protein, leading to membrane fusion and subsequent inoculation of viral RNA in the cytoplasm. Finally, alternatives are proposed for understanding the mechanism of action of the phospholipase from the venom of Crotalus durissus terrificus - the South American rattlesnake, which has proven effective in vitro inhibition of Dengue virus infection.

<p>Proteins with highly specific binding properties constitute the basis for many important applications in biotechnology and medicine. Immunoglobulins have so far been the obvious choice but recent advances in protein engineering have provided several novel constructs that indeed challenge antibodies. One class of such binding proteins is based on the 58 residues three-helix bundle Z domain from staphylococcal protein A (SPA). These so-called affibodies are selected from libraries containing Z domain variants with 13 randomised positions at the immunoglobulin Fc-binding surface. This thesis aims to describe the principles for molecular recognition in two protein-protein complexes involving affibody proteins. The first complex is formed by the Z_SPA-1 affibody binding to its own ancestor, the Z domain (Kd ~1 μM). The second complex consists of two affibodies: Z_Taq, originally selected to bind Taq DNA polymerase, and anti-Z_Taq, an anti-idiotypic binder to Z_Taq with a Kd ~0.1 μM. The basis for the study is the determination of the three-dimensional structures using NMR spectroscopy supported by biophysical characterization of the uncomplexed proteins and investigation of binding thermodynamics using isothermal titration calorimetry. The free Z_SPA-1 affibody is a molten globule-like protein with reduced stability compared to the original scaffold. However, upon target binding it folds into a well-defined structure with an interface topology resembling that displayed by the immunoglobulin Fc fragment when bound to the Z domain. At the same time, structural rearrangements occur in the Z domain in a similar way as in the Fc-binding process. The complex interface buries 1632 Ų total surface area and 10 out of 13 varied residues in Z_SPA-1 are directly involved in inter-molecular contacts. Further characterization of the molten globule state of Z_SPA-1 revealed a native-like overall structure with increased dynamics in the randomised regions (helices 1 and 2). These features were reduced when replacing some of the mutated residues with the corresponding wild-type Z domain residues. The nature of the free Z_SPA-1 affects the thermodynamics of the complex formation. The contribution from the unfolding equilibrium of the molten globule was successfully separated from the binding thermodynamics. Further decomposition of the binding entropy suggests that the conformational entropy penalty associated with stabilizing the molten globule state of Z_SPA-1 upon binding seriously reduces the binding affinity. The Z_Taq:anti-Z_Taq complex buries in total 1672 Ų surface area and all varied positions in anti-Z_Taq are directly involved in binding. The main differences between the Z:Z_SPA-1 and the Z_Taq:anti-Z_Taq complexes are the relative subunit orientation and certain specific interactions. However, there are also similarities, such as the hydrophobic interface character and the role of certain key residues, which are also found in the SPA:Fc interaction. Structural rearrangements upon binding are also common features of these complexes. Even though neither Z_Taq nor anti-Z_Taq shows the molten globule behaviour seen for Z_SPA-1, there are indications of dynamic events that might affect the binding affinity. This study provides not only a molecular basis for affibody-target recognition, but also contributions to the understanding of the mechanisms regulating protein stability and protein-protein interactions in general.</p>

Proteins with highly specific binding properties constitute the basis for many important applications in biotechnology and medicine. Immunoglobulins have so far been the obvious choice but recent advances in protein engineering have provided several novel constructs that indeed challenge antibodies. One class of such binding proteins is based on the 58 residues three-helix bundle Z domain from staphylococcal protein A (SPA). These so-called affibodies are selected from libraries containing Z domain variants with 13 randomised positions at the immunoglobulin Fc-binding surface. This thesis aims to describe the principles for molecular recognition in two protein-protein complexes involving affibody proteins. The first complex is formed by the ZSPA-1 affibody binding to its own ancestor, the Z domain (Kd ~1 μM). The second complex consists of two affibodies: ZTaq, originally selected to bind Taq DNA polymerase, and anti-ZTaq, an anti-idiotypic binder to ZTaq with a Kd ~0.1 μM. The basis for the study is the determination of the three-dimensional structures using NMR spectroscopy supported by biophysical characterization of the uncomplexed proteins and investigation of binding thermodynamics using isothermal titration calorimetry. The free ZSPA-1 affibody is a molten globule-like protein with reduced stability compared to the original scaffold. However, upon target binding it folds into a well-defined structure with an interface topology resembling that displayed by the immunoglobulin Fc fragment when bound to the Z domain. At the same time, structural rearrangements occur in the Z domain in a similar way as in the Fc-binding process. The complex interface buries 1632 Å2 total surface area and 10 out of 13 varied residues in ZSPA-1 are directly involved in inter-molecular contacts. Further characterization of the molten globule state of ZSPA-1 revealed a native-like overall structure with increased dynamics in the randomised regions (helices 1 and 2). These features were reduced when replacing some of the mutated residues with the corresponding wild-type Z domain residues. The nature of the free ZSPA-1 affects the thermodynamics of the complex formation. The contribution from the unfolding equilibrium of the molten globule was successfully separated from the binding thermodynamics. Further decomposition of the binding entropy suggests that the conformational entropy penalty associated with stabilizing the molten globule state of ZSPA-1 upon binding seriously reduces the binding affinity. The ZTaq:anti-ZTaq complex buries in total 1672 Å2 surface area and all varied positions in anti-ZTaq are directly involved in binding. The main differences between the Z:ZSPA-1 and the ZTaq:anti-ZTaq complexes are the relative subunit orientation and certain specific interactions. However, there are also similarities, such as the hydrophobic interface character and the role of certain key residues, which are also found in the SPA:Fc interaction. Structural rearrangements upon binding are also common features of these complexes. Even though neither ZTaq nor anti-ZTaq shows the molten globule behaviour seen for ZSPA-1, there are indications of dynamic events that might affect the binding affinity. This study provides not only a molecular basis for affibody-target recognition, but also contributions to the understanding of the mechanisms regulating protein stability and protein-protein interactions in general. / QC 20101025

La reconnaissance moléculaire joue un rôle central dans tous les processus biologiques ainsi que dans le développement de nouvelles biotechnologies. Depuis les 60 dernières années, deux mécanismes de reconnaissance moléculaire ont permis de décrire le couplage entre la liaison et le changement conformationnel observé chez les biomolécules. Le mécanisme par ajustement induit a lieu lorsque le ligand se lie à l'état inactif de la biomolécule et induit un changement de conformation vers la forme active. Le mécanisme par sélection conformationnelle, quant à lui, a lieu lorsque le ligand se lie directement à l'état spontanément actif de plus faible population et le stabilise, déplaçant ainsi la population de biomolécule vers cet état. Bien que nous connaissons des exemples de protéines qui fonctionnent selon chacun de ces mécanismes, nous ne comprenons pas encore les différences entre les performances de ces mécanismes ni les déterminants moléculaires qui leur donnent lieu. Une compréhension approfondie de ces mécanismes nous permettrait de mieux comprendre pourquoi certaines protéines ont évolué selon un mécanisme en particulier ainsi que de s'inspirer de ces mécanismes pour le développement de biotechnologies finement régulées. Jusqu'à aujourd'hui, ces deux mécanismes ont exclusivement été étudiés dans le contexte des biomolécules naturelles, principalement des protéines, dont la complexité dynamique et structurale rend difficile la comparaison et la manipulation individuelle des différents paramètres thermodynamiques. Il est donc particulièrement ardu de caractériser le rôle de chacun de ces paramètres quant à la sélection et la performance de ces mécanismes. Pour contourner ces limitations expérimentales, nos travaux de recherche se sont intéressés à recréer ces mécanismes à l'aide d'interrupteurs d'ADN fluorescents pour lesquels il est possible de prédire et de modifier la structure et les propriétés thermodynamiques ainsi que d'en mesurer l'activation en temps réel. Ce faisant, il a été possible d'observer que le mécanisme par ajustement induit est obtenu lorsque le site de liaison est partiellement accessible dans l'état inactif. Nous avons aussi observé que ce mécanisme permet une activation et une désactivation jusqu'à 10 000 fois plus rapide que la sélection conformationnelle, qui par contraste, donne lieu à une activation plus lente ainsi qu'à un maintien prolongé de l'activation. Ces différences cinétiques suggèrent ainsi un rôle évolutif distinct pour chacun et laissent envisager des applications en biotechnologies pour l'optimisation de la cinétique. / Molecular recognition plays a central role in almost every biological and biotechnological process. Over the last 60 years, two molecular recognition mechanisms have been used to appropriately describe the coupling between binding and conformational change in biomolecules. The induced fit mechanism takes place when ligand binding to the inactive state of the biomolecule induces the conformational change leading to the active state. On the other hand, in the conformational selection mechanism, where active and inactive states exist in equilibrium, the ligand binds selectively to the active state of the biomolecule and shifts the equilibrium towards this state by stabilizing it. Even though these mechanisms have been widely studied, it is still unclear if they differ in performance or how each mechanism can be modulated. Such a fundamental understanding of the differences between these mechanisms would shed light on the reasons for an apparent selective pressure driving the use of a specific mechanism for a given biomolecule and would also allow us to engineer new biomolecules which would benefit from the strengths of these mechanisms. To date, both mechanisms have been exclusively studied in the context of naturally occurring biomolecules, mainly proteins, whose structural and dynamic complexity as well as diversity seem to prevent comparison and manipulation of specific and individual thermodynamic parameters. Consequently, only little progress has been made towards characterizing the role of certain key thermodynamic parameters on the selection and performance of the mechanism. To circumvent this limitation, we have reproduced these mechanisms using simple fluorescent DNA constructs allowing for reliable prediction and variation of both structure and thermodynamics as well as real time monitoring of the activation process in presence of a DNA target. These DNA "switches" allowed us to determine that an induced fit mechanism occurs when the binding site is partially available in the inactive state and that this mechanism allows for a faster activation and deactivation (up to four orders of magnitude) compared to a conformational selection mechanism, which in contrast corresponds to a slower activation and deactivation, leading to a longer activation period. The observed kinetic differences between these mechanisms points towards potential uses for both in different areas of biotechnology as well as some rationale behind evolution favoring one mechanism over the other for a given protein.