Análise in situ dos efeitos da temperatura no crescimento de cristais de proteína

• Ano de defesa: 2014
• Autor(a) principal: Thiago Martins Francisco
• Tipo de documento: Dissertação
• Tipo de acesso: Acesso aberto
• Idioma: por
• Instituição de defesa: Universidade Federal de Minas Gerais; UFMG
• Palavras-chave em Português: Temperatura; Difração de raios X; Cristalização de proteínas; Espalhamento de luz; Física
• Link de acesso: http://hdl.handle.net/1843/BUOS-9NBGC3

Studies on crystallization in biological extracts made in the mid-nineteenth century contributed to the discovery of proteins. In the second half of the twentieth century Molecular Biology techniques enabled large-scale production of proteins and X-ray diffraction became the main tool for the investigation of protein structure. From the earliest experiments to the present day, the crystallization process has been the major bottleneck to structural studies of proteins. The current crystallization protocols consist of mixing the precipitating agents, buffers, and additives to the protein solution to promote crystallization discarding various physical parameters. Crystalline samples are obtained by trial and error and with a great rate of non-reproducibility. The aim of this work was to study the effects of the temperature on the protein crystallization process. Crystallization trials with the protein lysozyme at different temperatures showed change of the growth regime, with a remarkable change in the number and in the average size of crystals obtained above and below 12oC. In order to study the influence of temperature on the dynamics of the formation of crystals of lysozyme, crystallization trials with the in situ monitoring by Dynamic Light Scattering technique (DLS) were performed. This technique allowed the determination of the average size of the protein aggregates in solution during crystallization. The results indicated that the temporal evolution of the radii of the aggregates is not affected by temperature. However the amount of aggregates in solution is higher for lower temperatures. Crystals obtained at different temperatures were finally analyzed by X-ray diffraction techniques and the structures obtained did not show any significant change. There are still gaps in the theories that describe the dynamics of the protein crystallization process that justify further studies of this phenomenon, targeting above all, more assertive crystallization protocols.