congresos y reuniones científicas
On the Search of a Comprehensive Representation of Human Frataxin.
Sociedad Argentina de Biofísica
Congreso; XLIII reunión anual de la Sociedad Argentina de Biofísica; 2014
Friedreich?s ataxia (FRDA) is a neurodegenerative disease linked to a deficiency of frataxin (FXN), a mitochondrial protein involved in iron-sulfur cluster biosynthesis. The mature form of human FXN (residues 81―210) contains a well folded C―terminal domain starting at residue 90 with / topology followed by a C-terminal region (CTR), comprising the last 17 residues with a non-periodic structure that packs against the protein?s core. We studied a number of pathological and rationally-designed variants to investigate the impact of the alteration of the CTR on the stability, internal dynamics and folding dynamics of FXN. The pathological mutation L198R yields a global destabilization of the structure, as well as a significant and highly localized alteration of dynamics, mainly involving residues that are in contact with L198 in wild-type FXN. Variant FXN90―195, which is closely related to the FRDA―associated mutant FXN81―193, preserves its native-like structure. However, the truncation of the CTR results in an extreme decrease of global stability and alteration of protein dynamics over a vast range of timescales, including regions far from the CTR, as shown by an increase of the amide proton?water exchange rates, 15N relaxation measurements and relaxation dispersion experiments. Moreover, both mutants exhibit an important deficiency in iron-binding, suggesting coupling dynamics between the CTR and the acidic ridge (helix 1, loop1, strand 1) involved in iron binding. The increased sensitivity to proteolysis observed in vitro for both mutants ―which might imply a faster degradation rate in vivo―, the reduced ability to bind iron, and the enhanced tendency to aggregate exhibited by the truncated variant may explain why the alteration of the CTR causes FRDA. Furthermore, the more drastic alteration of the dynamics and stability of FXNL198R mutant with respect to FXN90―210, is in line with the extremely rapid disease progression observed in patients carrying this mutation. As an extension of this study, we will show preliminary experimental data from the study of the effect that the mutation of loop1 has on the CTR?s local stability and dynamics. We have also investigated FXN folding dynamics. Folding and unfolding kinetic experiments indicate that the FXN folding process may include an intermediate state, and firmly suggest that the vast change in global stability observed in CTR mutations is most probably caused by a native-state destabilization than by a change in the stability of transition state ensembles. Thus, CTR―mediated contacts would not significantly contribute to the TSE energetics. Taken as a whole, these results contribute to understanding how stability and activity are linked to protein motions, and might be valuable for the design of target-specific binders to control local protein motions for stability and activity enhancement.Acknowledgements: UBACyT, CONICET and ANPCyT.