On the Folding Mechanism of Human Frataxin

FARAJ SE; GONZÁLEZ LEBRERO R; ROMAN EA; SANTOS J

Congreso; Reunión anual de la SAB; 2014

SAB

The deficient activity of Frataxin (FXN), a mitochondrial iron-binding protein, is related to Friedreich?s ataxia, a neurodegenerative disease that severely affects limb motricity and cardiac function. FXN is an α/β globular protein, which consists of 130 residues in its mature wild-type variant. The C-terminal region (CTR) of FXN lacks periodic structure and is packed against the protein?s core. We have previously studied a series of naturally occurring (pathogenic) and rationally-designed mutants of the CTR, and found that this stretch is crucial for the consolidation of tertiary structure, since mutations affect thermodynamic stability and molecular dynamics over different timescales. In order to elucidate the role that the CTR plays in the folding mechanism, we performed folding and unfolding kinetics studies of several CTR mutants. At low urea concentrations the refolding branch of the ln(kobs) vs. [urea] plot slightly deviates from linearity, and kinetic traces show a ?burst phase?; both these facts support the hypothesis of the existence of an intermediate state. In addition, we found that the refolding reaction is unaffected in the different variants?even in one that has had the CTR completely removed, and both at 15 and 25 °C?while the unfolding mechanism is altered and correlated with the thermodynamic stability observed in equilibrium unfolding experiments, including its dependence with the buffer?s ionic strength. These results imply that the CTR contributes to the stabilization of the native state, although it may not take a major part in the transition state for the rate-determining step in the folding reaction. All in all, our findings allow us to gain further evidence on the role of the CTR in the stabilization of FXN, as well as to understand the determinants of kinetic stabilization, which may help to explain the pathogenicity of some mutations and to develop stabilizing compounds to be used as drugs to treat the disease.