Expressão, cristalização e determinação estrutural de macromoléculas biológicas por difração de raios X por monocristal

Abstract

Proteins¿ shape and function are intimately related. Hence, the comprehension of their tridimensional structures is necessary for the detailed elucidation of the biological processes in which they participate. Crystallography is a powerful technique applied on protein structure determination. Great knowledge of Physics and Biochemistry is needed when capacitating a professional to be able to work on every stage of the process of structure determination by protein crystallography. The present work aimed at visiting every one of those stages, including recombinant protein expression, protein purification and crystallization, X-ray diffraction experiments (XRD), data processing, structure solution and analyses. To cover the whole process, three proteins were studied: a sphingomyelinase D (SMase D), the bovine papillomavirus-1 E2 DNA binding domain (BPV-1 E2) and the NahK/NahL complex. Different parts of the process of structure determination were covered working with each of the proteins. SMase D was expressed, purified and crystallized and XRD experiments were performed with its crystals; the structure of BPV-1 E2 was elucidated from XRD data and the proposed model was properly analyzed; XRD data from a crystal of the NahK/NahL complex, which indicated an unordered region on the experimental electron density map, were reprocessed and analyzed, resulting on the proposition of a new structural model of the complex.