Functional and structural studies of a mini-ferritin protein

Dissertação para obtenção do Grau de Mestre em Bioquímica

DNA-binding protein from starved cells (Dps) are mini-ferritins mainly expressed in bacteria during severe environmental stress. These proteins with a highly conserved structure provide wide protection to cells and function as iron-storage proteins. Some Dps can also bind DNA and the N-terminus has been suggested to be involved in this interaction, due to its positively charged residues. This thesis focused on the functional and structural features of recombinant Dps protein from Pseudomonas nautica 617 (P. nautica). To investigate the iron incorporation mechanism, iron uptake assays using H2O2 and O2 as co-substrate for iron oxidation were performed with Dps WT and F46G variant, using UVVisible spectroscopy. The results showed that Phe46, located close to the ferroxidase center (FOC), does not influence the amount of iron stored. Nevertheless, this residue affectes the iron core formation when O2 was used as co-substrate. Structural characterization of the incorporation of Fe(II) and Cu(II) was performed with X-ray crystallography. High resolution crystal structures of Apo-Dps, Dps incubated with 12 Fe(II)/Dps (12Fe-Dps) and Dps incubated with 12 Cu(II)/Dps (12Cu-Dps) were obtained. It was possible to observe the binding of the metals to the FOCs with different coordination geometry as well as geometrically different FOCs. Additionally Fe(II) and Cu(II) atoms were assigned in a position where hydrophilic pores can be created and serve as entry routes for the metals. For the characterization of the Dps-DNA interaction, electrophoretic mobility shift assays (EMSAs) were carried out with Dps WT and Q14E variant. The results showed that this protein can bind DNA, but the affinity for DNA significantly decreases in the presence of the negative charge in the N-terminus. In this sense, mutations in the N-terminus that may increase the affinity for DNA binding were produced by site-directed mutagenesis.