Author(s): Freire, Filipe Miguel dos Santos
Date: 2012
Persistent ID: http://hdl.handle.net/10362/8775
Origin: Repositório Institucional da UNL
Subject(s): Heme-binding proteins; BH3-only proteins; Biomolecular Crystallography; Nuclear magnetic resonance; Fluorescence quenching
Dissertação para obtenção do Grau de Doutor em Bioquímica, Especialidade Bioquímica Estrutural
Heme is essential to all aerobic organisms, as it is involved in several biological processes. Due to its toxicity and high insolubility, several proteins transiently bind this molecule, ensuring the transport and insertion into heme proteins. A structural study of three heme-binding proteins, human SOUL protein and human and murine p22HBP is described in this dissertation. In chapter 1, an introduction to heme and its biological importance is performed and all the information related to the proteins previously mentioned is summarized. The basic principles of X-ray Crystallography and Nuclear Magnetic Resonance (NMR), the techniques used in the structural characterization of these proteins, are described. For the studies described in this thesis large amounts of pure protein are required. For this reason, hSOUL protein was cloned, and the overexpression and purification of hSOUL optimized – chapter 2. The overall structure of hSOUL is very similar to murine p22HBP solution structure. hSOUL protein structure determined by X-ray Crystallography is described in chapter 3 and the possible biological consequences are discussed. Understanding the heme interaction with hSOUL was an important objective of this work. For that, NMR, Fluorescence Quenching and Visible Spectroscopy studies were performed. The results obtained, and shown in chapter 4, indicate that the interaction, if it exists, is non-specific. The several experiments to crystallize human and murine p22HBP, in order to solve their three-dimensional structure in complex with heme and therefore understand heme-p22HBP interaction, are described in chapter 5. The main conclusions from the present work are drawn in chapter 6 together with the future perspectives.
Fundação para a Ciência e a Tecnologia - (SFRH/BD/30239/2006 e PTDC/QUI/64203/2006
