IQUIFIB   02644
INSTITUTO DE QUIMICA Y FISICOQUIMICA BIOLOGICAS "PROF. ALEJANDRO C. PALADINI"
Unidad Ejecutora - UE
congresos y reuniones científicas
Título:
Grafting a Functional Iron-binding Motif onto a Thioredoxin Scaffold
Autor/es:
VAZQUEZ DS; AGUDELO SUAREZ W; ARAN M; GONZÁLEZ FLECHA FL; GONZÁLEZ LEBRERO MC ; SANTOS J
Lugar:
Villa Carlos Paz, Córdoba
Reunión:
Congreso; Reunion Anual de la Sociedad Argentina de Biofísica; 2013
Institución organizadora:
Sociedad Argentina de Biofísica
Resumen:
Proteins belonging to the CyaY family contain acidic residues clusters arranged in tandem, presumably involved in iron exchange and principally located in the N-terminal region. We endeavored to obtain an individual iron-binding motif grafted onto a foreign protein, thioredoxin (TRX) to evaluate the intrinsic affinity and the role of neighbor residues, bypassing limitations associated with the natural scaffold, such as the presence of multiple binding sites and cooperativity between them. In this first work, we present the characterization of internal dynamics and global properties of the apo and holo conformations, carried out by computational (MD simulations) and equilibrium experiments. As judged by circular dichroism analysis and its hydrodynamic behavior, the protein conserves wild-type signatures. More importantly, the global stability is not significantly altered upon mutation but shows a slightly diminished oxidoreductase activity in absence of iron. In addition, MDS analysis of the secondary structure suggests that the binding of the metal ion may stiffen the grafted region. Furthermore, we evaluate the iron affinity of an isolated peptide containing the iron binding motif (KD=100.1μM, pH4.1) and its conformation, in the presence and absence of the metal ion. The results suggest that 5-6 residues of the peptide acquired helical structure upon iron binding. Finally, by the grafting approach, we expect to shed light in the understanding of protein-metal dynamics and the impact on protein function.