Using an Unstructured Peptide as scaffold to Study a Consensus Iron-binding Motif from CyaY Protein Family

DIEGO SEBASTIAN VAZQUEZ; ANGEL YONE; NORA VIZIOLI; FRANCISCO LUIS GONZÁLEZ FLECHA; MARIANO CAMILO GONZÁLEZ LEBRERO; JAVIER SANTOS

CABA

Congreso; XLI Congreso Anual de la Sociedad Argentina de Biofísica; 2012

Universidad Nacional de Tucuman

Regulation of enzymatic activity by metal ions is crucial in cellular processes. Proteins belonging frataxin family (CyaY, PDB ID: 1EW4), which control intracellular iron homeostasis, have a cluster of acidic residues located between the N-terminal α-helix and β1-strand that are apparently responsible for iron binding and iron exchange1. To understand how frataxin protein works as an iron chaperon, we studied the interaction of metal ions with a putative iron binding site from the bacterial frataxin CyaY. For this purpose, a peptide corresponding to the C-terminal α-helix from E. coli thioredoxin (wt-HCT) was redesigned including the motif (E/D)(E/D)xx(E/D)(E/D) from the bacterial frataxin. Thus, we synthesized and studied an engineered 16 residues-long amphipathic peptide called pIBS, (from putative Iron Binding Site). Preliminary results obtained by circular dichroism, capillary zone electrophoresis, isothermal titration calorimetry and molecular dynamics simulations indicate us that: (i) In the absence of metal ions both, pIBS and wt-HCT, behave as a random-coil peptides in solution; (ii) The interaction pIBS-iron induces the acquisition of helical structure, stabilizing a segment of seven residues consistent with two-turn α-helix conformation; (iii) Peptide pIBS binds iron with an 1:1 stoichiometry and (iv) The interaction between peptide and metal ion seems to be specific for iron since other ions assayed did not produce that effect at similar concentrations. Furthermore, this system will allow us to study one by one, different putative binding sites from CyaY family and how the local context may alter the metal ion affinity.