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Teresa Margarida Pedrosa Cardoso Estabilidade Universidade de Aveiro Departamento de Biologia 2007 Teresa Margarida Estabilidade Conformacional da Proteinase Pedrosa Cardoso Aspártica – Arquétipo, Pepsina A Conformational Stability of the Archetypal Aspartic Proteinase, Pepsin A Universidade de Aveiro Departamento de Biologia 2007 Teresa Margarida Estabilidade Conformacional da Proteinase Pedrosa Cardoso Aspártica – Arquétipo, Pepsina A Conformational Stability of the Archetypal Aspartic Proteinase, Pepsin A Dissertação apresentada à Universidade de Aveiro para cumprimento dos requisitos necessários à obtenção do grau de Doutor em Bioquímica , realizada sob a orientação científica da Professora Doutora Marlene Maria Tourais de Barros, Professora Associada com Agregação do Centro Regional das Beiras da Universidade Católica Portuguesa, e do Professor Doutor Euclides Manuel Vieira Pires, Professor Associado do Departamento de Bioquímica da Faculdade de Ciências e Tecnologia da Universidade de Coimbra. Apoio financeiro da FCT e do FSE no âmbito do III Quadro Comunitário de Apoio. A meus Pais e Irmã o júri presidente Professor Doutor Vasile Staicu Professor Catedrático do Departamento de Matemática da Universidade de Aveiro Professora Doutora Maria Ana Dias Monteiro Santos Professora Catedrática do Departamento de Biologia da Universidade de Aveiro Professor Doutor Eduardo José Xavier Rodrigues de Pinho e Melo Professor Associado com Agregação da Faculdade de Engenharia de Recursos Naturais da Universidade do Algarve Professora Doutora Marlene Maria Tourais de Barros Professora Associada com Agregação da Universidade Católica Portuguesa, Centro Regional das Beiras – Pólo de Viseu Professor Doutor Euclides Manuel Vieira Pires Professor Associado da Faculdade de Ciências e Tecnologia da Universidade de Coimbra Professora Doutora Paula Cristina Veríssimo Pires Professora Auxiliar da Faculdade de Ciências e Tecnologia da Universidade de Coimbra Professora Doutora Ana Cristina de Fraga Esteves Sarmento Professora Auxiliar Convidada do Departamento de Biologia da Universidade de Aveiro agradecimentos À Universidade de Aveiro, por permitir o desenvolvimento deste projecto. À Fundação para a Ciência e a Tecnologia, pelo financiamento concedido. À Professora Doutora Marlene Barros, pelo apoio científico e logístico. Ao Professor Doutor Euclides Pires, pelas pertinentes sugestões. Aos Professores Nicholas Price e Ala n Cooper, pelos ensinamentos e proveitosas discussões. À Doutora Sharon Kelly, a Tommy Jess e a Mrs. Margaret Nutley, pelo indispensável auxilio no planeamento e realização de trabalho experimental. A todos, pelo caloroso acolhimento durante as minhas estadias em Glasgow. À Cláudia Oliveira, Cristina Sarmento e Anabela Pereira, por apoios prestados durante a realização de trabalho experimental. À Sofia Duarte e Sofia Fraga pela disponibilidade. À Isabel Velada, pela compreensão. Ao Professor Doutor Carlos Pires, pelos conselhos. Aos meus três anjos protectores, por tudo. Mas, sobretudo, pelo exemplo de coragem e força do meu Pai, pelo exemplo de optimismo e compreensão da minha Mãe, pelo exemplo de sinceridade e esforço da minha Irmã. Aos meus Avós, por relevarem as minhas ausências. À restante Família, pela alegria em cada reencontro. Errata da Dissertação “Estabilidade Conformacional da Proteinase Aspártica - Arquétipo, Pepsina A” Na primeira página, deveria ter-se incluído: “Apoio financeiro do POCTI no âmbito do III Quadro Comunitário de Apoio através do projecto POCTI/QUI/60791/2004.” palavras-chave Pepsina A; Acetonitrilo; Hidrocloreto de Guanidina; Ureia; Temperatura; Desnaturação de Proteínas; Estados Conformacionais Não-Na tivos; Estabilidade Conformacional. resumo A pepsina A tem sido considerada um protótipo do grupo das proteinases aspárticas, cuja relevância ao nível da patofisiologia humana tem estimulado intensa pesquisa. As propriedades moleculares e catalíticas d a pepsina A começaram a ser exploradas há cerca de um século. Contudo, a informação existente sobre a estabilidade conformacional desta proteína em condições nativas é insuficiente. Elucidação dos factores responsáveis pela manutenção do estado conformacional naturalmente enrolado e activo de proteínas é vital para estabelecer estratégias de modulação da estabilidade. Com o objectivo de obter novos conhecimentos acerca das bases da estabilidade conformacional da pepsina A, foi adoptada uma abordagem que con sistiu na indução e monitorização da desnaturação da proteína no seu estado naturalmente enrolado. A severidade das condições ambientais foi gradualmente agravada por adição de desnaturantes e / ou modificação de propriedades físicas e químicas do sistema proteína-solvente. Após uma fase de incubação nas condições desnaturantes, com a duração típica de 1 h, mudanças nas propriedades espectrais, no comportamento hidrodinâmico, na estabilidade térmica e na actividade peptidolítica da pepsina A de suíno foram avaliadas. O solvente orgânico, acetonitrilo, induziu destruição não-cooperativa dos arranjos secundário e terciário desta proteína. Sugere-se que a drástica debilitação das estruturas de hidratação pelo acetonitrilo terá impelido o desenrolamento global. Adicionalmente, a pepsina A revelou-se particularmente resistente à acção desordenante de dois agentes caotrópicos clássicos, o hidrocloreto de guanidina (GdnHCl) e a ureia. Um estado intermediário estável foi identificado durante a reacção de desenrolamen to da proteína nativa induzida pelo GdnHCl. A adição de acetonitrilo a uma molaridade intermédia permitiu a identificação de um diferente estado parcialmente desenrolado na presença de GdnHCl a concentrações pré- transicionais. Dados espectroscópicos indici am promoção de ordem local a nível do esqueleto polipeptídico pela ureia a baixas e altas molaridades. Indução de extenso desenrolamento da proteína pela ureia requereu concentrações quase saturantes deste desnaturante e períodos de exposição de duração su perior a 1 h. Foram detectados fenómenos de aparente estabilização, quando a fase de pré-incubação da proteína em ambos os agentes caotrópicos a baixas molaridades foi prolongada para 3 dias. A pepsina A no seu estado nativo revelou-se bastante termoestável. Contudo, 1 h de exposição da proteína, antes do seu aquecimento, a qualquer dos três desnaturantes a concentrações pré-transicionais foi suficiente para aumentar a termolabilidade da proteína. Contribuições de interacções hidrofóbicas e estruturas de solvatação para a estabilidade conformacional da pepsina A no seu estado biologicamente activo são discutidas nesta dissertação. keywords Pepsin A; Acetonitrile; Guanidine Hydrochloride; Urea; Temperature; Protein Denaturation; Non-Native Conformational States; Conformational Stability. abstract Pepsin A has been considered a proto type of the group of aspartic proteinases, whose significance in human pathophysiology has galvanized intensive research. Molecular and catalytic prope rties of pepsin A have been explored for about a century. However, there is only fragmentary data on the conformational stability of this protein under native conditions. Elucidation of the factors accountable for the maintenance of the natively ordered an d active conformational state of a protein is vital for engendering strategies for tailoring stability. In order to gain new insights into the scaffold of the conformational stability of pepsin A, an approach based on induction and monitoring of denaturati on of the natively folded protein was adopted throughout the investigation reported herein. Environmental stringency was gradually increased by adding denaturants and / or modifying chemical and physical properties of the protein-solvent system. After a pe riod of incubation in the denaturing conditions, which lasted typically 1 h, changes in spectral properties, hydrodynamic behaviour, thermal stability and peptidolytic activity of porcine pepsin A were appraised. The organic solvent, acetonitrile, was foun d to induce non-cooperative destruction of secondary and tertiary arrangements of this protein . Drastic debilitation of hydration structures by acetonitrile is suggested to have triggered global unfolding. In addition, pepsin A revealed to be particularly resistant to the disordering action of two classic chaotropes, guanidine hydrochloride (GdnHCl) and urea. A stable, intermediary state could be identified in the pathway of GdnHCl-induced unfolding of native pepsin A . Upon addition of acetonitrile at an intermediary molarity , a different partially unfolded state could be identified in the presence of GdnHCl at pre-transitional concentrations. It is apparent from spectroscopic data that urea promoted local backbone order at low and high molarities. And, peri ods of exposure longer than 1 h to urea at near-saturating concentrations were required to onset gross unfolding by this denaturant . Phenomena of apparent stabilization were detected when submission of the protein to both chaotropes at low molarities was prolonged to 3 days. Furthermore, pepsin A in its native state was noted to be rather thermostable. However, 1 h - incubation at pre - transitional concentrations of any denaturant prior to heating caused a raise in the protein thermolability. Contributions from hydrophobic interactions and solvation structures to the conformational stability of pepsin A in its biologically active state are discussed in this dissertation. Cover Illustration: Transparent molecular surface of porcine pepsin A (PDB code 4pep ) with the backbone rendered as a ribbon. This figure was generated by GRASS (Graphical
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