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Universidade De São Paulo Departamento De UNIVERSIDADE DE SÃO PAULO DEPARTAMENTO DE FÍSICA – FACULDADE DE FILOSOFIA, CIÊNCIAS E LETRAS DE RIBEIRÃO PRETO LUIS FELIPE SANTOS MENDES Caracterização estrutural e dinâmica da proteína de estruturação e compactação do complexo de Golgi (GRASP) em solução Ribeirão Preto 2018 Luis Felipe Santos Mendes Caracterização estrutural e dinâmica da proteína de estruturação e compactação do complexo de Golgi (GRASP) em solução Tese apresentada ao Departamento de Física da FFCLRP-USP como parte das exigências para a obtenção do título de doutor em ciências Área de concentração: Física Aplicada à Medicina e Biologia Orientador: Prof. Dr. Antonio Jose da Costa Filho Ribeirão Preto 2018 Autorizo a reprodução total ou parcial deste documento, por meio convencional ou eletrônico, para fins de estudo e pesquisa, desde que citada a fonte. Ficha Catalográfica Mendes, Luis Felipe Santos Caracterização estrutural e dinâmica da proteína de estruturação e compactação do complexo de Golgi (GRASP) em solução, Ribeirão Preto, 2018. 154 p. Tese de Doutorado apresentada à Faculdade de Filosofia Ciências e Letras de Ribeirão Preto/USP – Área de concentração: Física Aplicada à Medicina e Biologia Orientador: Costa-Filho, Antonio José. 1. GRASP 2. Secreção não convencional de proteínas 3. Espectroscopia 4. Proteínas Intrinsicamente Desordenadas 5. Molten Globule Nome: MENDES, Luis Felipe Santos Título: Caracterização estrutural e dinâmica da proteína de estruturação e compactação do complexo de Golgi (GRASP) em solução Tese apresentada ao Departamento de Física da FFCLRP-USP como parte das exigências para obtenção do título de Doutor em Ciências (Física Aplicada à Medicina e Biologia). Área de concentração: Física Aplicada à Medicina e Biologia Aprovado em: ___/___/___ Banca Examinadora Prof. Dr. __________________________ Instituição: __________________________ Julgamento:________________________ Assinatura: __________________________ Prof. Dr. __________________________ Instituição: __________________________ Julgamento:________________________ Assinatura: __________________________ Prof. Dr. __________________________ Instituição: __________________________ Julgamento:________________________ Assinatura: __________________________ Prof. Dr. __________________________ Instituição: __________________________ Julgamento:________________________ Assinatura: __________________________ Prof. Dr. __________________________ Instituição: __________________________ Julgamento:________________________ Assinatura: __________________________ This thesis is dedicated to my family, for all their love, incentive and support. There is no bigger love than this that I feel for you. Acknowledgments To do my PhD in the Physics Department of FFCLRP-USP was undoubtedly one of the best and unique experience I could have ever had in my life. I would like to thank God for giving me the strength to go through all the hard and easy challenges in the process and helped me to get many insights out of nowhere. As a scientist, I should not believe in something or someone I cannot prove the existence but what I feel is much stronger and clearer for me than even the sensation of cold and heat. I will never deny my faith and this has only made me stronger with time. I am especially grateful for all the love, dedication, incentive, support and several other things that my parents have (done) for me since ever. I would never be in this position without your unquestionable love, support, and patience. Hope that this thesis and all the things that I have conquered so far might make you proud of me, despite all the hard work, nights without sleeping (in particular my father working overnight in Petrobras) and headaches that I often give you. I feel that I am getting really far in my career and just like Newton once said, I truly believe that I could only see and go this far because during my entire life I was standing on the shoulders of two giants. Thanks for being my giants, love you both. I am also especially grateful for the friendship, patience and love of my little sister Lidiane. Thanks for helping me not getting crazy during my infinite crises. You were a great roommate and friend. Hope to be a person as kind and good as you one day. Still working on this, it’s hard than do science. “I lived a million miles of memories on that road; with every step I take I know that I'm not alone. You take the home from the boy, but not the boy from his home… Who says you can't go home? (John Bon Jovi, 2005)” I am extremely thankful to my supervisor, Antonio Jose da Costa Filho (Jabah), for his guidance, support, advices and suggestions in all moments during my entire PhD time. Thanks for trusting me and for letting me have the unique opportunity of working under your supervision over the last 8 years of my scientific life. Thanks also for the several beers, whiskies and the friendship. Your support was essential for my growing as a person, scientist, and, of course, for the success of this PhD thesis. I would also like to thank all the actual and former members of the LBM. All of you have an especial part in this thesis. I am thankful to Prof. Dr. Anthony Watts for giving me the unique opportunity of working with you and his group at the University of Oxford-UK. It was such an outstanding experience that I cannot measure or simply define using ordinary words. I would also like to give an especial thank to the Watts group for the help and friendship during this amazing year. I am thankful to Prof. Dr. Christina Redfield for guiding me into the solution NMR world. Thanks for being so patient, coming to the NMR machine with me and sharing a little of your infinite knowledge. I am thankful to Gunnar Jeschke and Alex Smirnov for my short internships in the ETH Zurich and North Caroline State University, respectively. For sure, these times in Switzerland and USA contributed enormously to my scientific maturation. Thanks also for all the members of both groups. I am also thankful to Mariana (Gatinha) for the company, friendship and for being on and by my side from the beginning until the very end of this Ph.D thesis. Our relationship had several stages and I am thankful for each Mariana that you were in my life (the colleague, the friend and the girlfriend). You made and make me a better person and professional. Hope to have you still by my side making me better for as long as you can handle. “If you are lucky enough to find love, remember it is there and don't throw it away (Stephen Hawking)” I am especially grateful to Dr. Luis Guilherme Mansor Basso and Dr. Assuero Faria Garcia for being my friends and indirect co-supervisors during these years. Without you, guys, this thesis would be nothing more than a stamp collection. I believe I have now several good qualities as a professional that I copied-and-pasted from you both. Hope to be as good scientist as you are. I am also thankful to Dra. Patricia Suemy Kumagai for the SRCD data collection presented in chapter 2. It is always an honor to work and have some beer with you. Coming to people that had a special importance for me during these years outside the lab. You all had a huge contribution!!! Science is much more than the work inside the wet lab or on the computer. Without all of you, I would have got crazy in the very beginning. I know that there are several names and I don’t want to forget anyone, so I will avoid a list. If you were by my side during my PhD (and if you are one of these people you will know that you are a part of this), just know that you are important and I am thankful. I would like to thank Tequila for being by my side during this time. You did not choose it, I did, but you still look okay with this hehehehehe There is nothing more pure and truthful than the friendship of an animal. Jurema was not direct by my side but soon she will be in the water castle she deserves and Tequila will have his entire garden to eat without any limits. I am extremely thankful to FAPESP for my grants during my PhD and the BEPE internship. CNPq, BBSRC and CAPES are also acknowledge. “Science knows no country, because knowledge belongs to humanity, and is the torch which illuminates the world.” (Louis Pasteur, Free Lance of Science (1960) by René Jules Dubos, Ch. 3 "Pasteur in Action") “Imagine there's no countries... No need for greed or hunger, a brotherhood of man” (John Lennon, 1971) Let’s connect ourselves through knowledge Resumo 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.
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