Preliminary structural characterization of an iron-binder engineered thioredoxin

DIEGO SEBASTIAN VAZQUEZ; ARÁN, MARTÍN; JAVIER SANTOS

Sierra de la Ventana

Congreso; XLIII Reunión Anual de la Sociedad Argentina de Biofı́sica; 2014

Sociedad Argentina de Biofísica

Frataxin (FXN), a member of the CyaY protein family, is characterized by the presence of a large cluster of acidic residues on its surface principally involved in iron-homeostasis. We have designed and characterized, a short peptide (GRAP) that includes the EExxED motif from the CyaY protein family, using the sequence of the C t helix of E. coli thioredoxin (TRX) as scaffold. GRAP shows certain specificity for Fe 2+ and Fe 3+ characterized by an 1:1 stoichiometry and a K D of 1.9±0.2μM.Iron binding is an entropically driven process that is guided more likely by changes in hydration. Both binding and peptide folding processes take place upon metal interaction. To uncouple the coil-to-helix transition and to study both the iron-binding affinity and the effect on intrinsic dynamics, we grafted the motif onto the full-length TRX (TRXgrap). TRXgrap protein is structured and showed a marginal impact on global stability. Noteworthy, the apo form exhibits a diminished activity. To characterize at the residue level the TRXgrap/Fe 3+ interaction, a preliminary set of NMR and theoretical experiments were carried out. The evaluation of 1 H 15 N-HSQC spectra of TRXgrap in the presence or absence of iron shows that the interaction causes the disappearance of signals of some residues located in the surrounding of the iron-binding motif, compatible with theparamagnetic properties of this metal ion. On the other hand, molecular dynamic simulations of the apo and holo forms shows significant perturbations at the active site in the reduced state suggestingeffects of mutation and metal binding at medium-large distances and explaining the observed reduction of enzimatic activity.