Quantitative analysis of local dynamics of the C-terminal region of human frataxin

FARAJ SE; ARÁN M; GALLO M; SANTOS J

Cordoba

Congreso; Reunion Anual de la Sociedad Argentina de Biofísica; 2013

Sociedad Argentina de Biofísica

Friedreich´s ataxia is a progressive neurological hereditary disease, caused by the deficiency in the activity of frataxin (FXN), a mitochondrial iron chaperone that regulates the transference of metal ions to other proteins. Previous studies from our laboratory found that the C-terminal region (CTR) is a crucial element in the stabilization of FXN. The local unfolding of the CTR may enable the protein to smoothly modulate its dynamics and stability, acting as a conformational ?lock?. In an attempt to quantify the stability of the interaction between the CTR and the rest of the domain, we replaced, one at a time, three Leu residues from the CTR ?which establish hydrophobic interactions with core residues? by Cys residues. Cys mutants are useful tools to analyze local dynamics through the reactivity of their free thiol, allowing us to infer the dynamics at particular positions (in which Cys are placed). We followed the reaction of Cys modification by DTNB (Ellman?s reagent), and found that different positions possess varying rates of modification. Our results indicate that the difference in free energy of local unfolding of the CTR is half the global stabilization energy. Besides, molecular dynamics simulations results are consistent with our experimental observations, and allow us to estimate average accessibility of Cys residues for each variant, to assess the effective contribution to observed reactivity from local unfolding events. Altogether, these results were analyzed side by side with wild-type protein and L198R mutant NMR relaxation dispersion experiments.