Research objectives and content
Iron availability controls growth and proliferation of many microorganisms with clinical or ecological interest. The Fur (Ferric uptake regulation) protein is a bacterial regulator which represses the expression of essential proteins in the presence of iron as corepressor. Previous work in the host laboratory indicates the presence of two sequences with high homology with E. coli fur DNA binding sequences in the species .Anabaena. Those sequences are placed upstream the iron-regulated gene encoding flavodoxin, protein which replaces the iron-containing protein ferredoxin in iron deficient conditions.
The first part of the project consist of the cloning and
overexpression of the Fur protein from Anabaena (preliminary work on the cloning is in progress). The recombinant protein will be purified using either FPLC, HPLC or traditional chromatography. The next step will be the crystallization of Fur and Fur-metal complexes, initially Mn2+, followed by the determination of their 3D structures. The experiments outlined in th project will draw strongly on the experience and expertise of the applicant in the areas of protein purification and protein chemistry, and also the skills obtained while working in protein crystallography during her post-doctoral training.
Training content (objective, benefit and expected impact)
Fur is the only repressor characterized to date that is responsive to the concentration of a nutrious metal ion. It also lacks significant sequence homology to any known DNA-binding protein. So far, no ferric uptake regulation proteins have been crystallized. It is expected that understanding the mechanism of DNA sequence recognition and ligand activation of Fur will give valuable information.