Rational design of a chimeric peptide with iron binding capacity

VAZQUEZ D; YONE A; VIZIOLI, NM; GONZÁLEZ FLECHA, FL; GONZÁLEZ LEBRERO, MC; SANTOS, J

Buenos Aires

Simposio; 18th Symposium on Biomedical, Biopharmaceutical and Industrial Applications of Capillary Electrophoresis and Microchip Technology; 2012

Regulation of enzymatic activity by low molecular weight non-protein ligands (e.g. metallic ions) is a process of crucial importance to understand the principles governing cellular processes. The main role of frataxin protein is to control intracellular iron homeostasis. Frataxin has a patch of acidic residues between the N-terminal helix and β1-strand which would be responsible for iron binding. In this work, we studied the conformational properties, and the capacity and specificity of a putative iron binding site of the CyaY family (E/D)(E/D)xx(E/D) (E/D). A synthetic peptide corresponding to the C-terminal helix of the thioredoxin from E. coli (HCT, peptide wild-type) was redesigned by the insertion of the putative EExxED motif, obtaining a 16 residues-long amphipathic peptide named pIBS (from putative Iron Binding Site). In this study, circular dichroism, light scattering, isothermal titration calorimetry, capillary electrophoresis (CE) techniques and computational calculations were applied. CE analyses were carried out in an uncoated fused silica capillary (32 cm x 50 µm ID) and using two different background electrolytes: one, a 2.0 mM Tris and 5.0 mM sodium chloride solution, pH 7.2, and the other, the same solution with 60 µM Fe (III). Differences in the electrophoretic behavior of the mutant and the wild-type peptides according to the peptide capacity for iron binding were found. Other preliminary results indicated that i) the pIBS-Fe (III) interaction induced helical structure of a 7 residues-long stretch; and ii) such interaction seemed to be specific for iron since other ions such as calcium, magnesium, manganese, cadmium, cobalt and nickel assayed at similar concentration did not produce that effect. The experimental system will allow us to characterize the iron binding site of the frataxin, and to optimize its iron-affinity by means of local amino acid sequence perturbations.