Conformation and stability of a recombinant variant of human frataxin precursor

NOGUERA MARTÍN EZEQUIEL; HERRERA MARIA GEORGINA; CASTRO IGNACIO HUGO; SANTOS JAVIER

Congreso; Reunión Conjunta de Sociedades de Biociencias; 2017

Friedreich´s Ataxia is a disease caused by a decrease in the expression frataxin (FXN) and loss of FXN functionality. The outcome of the lack of FXN function is an inefficient iron-sulfur cluster assembly, with widespread enzymatic deficit and oxidative damage. FXN is translated in the cytoplasm as a precursor of 210 residues (FXN1-210). Then, this protein is imported into the mitochondrial matrix and processed to the mature form (FXN81-210). One outstanding strategy to increase the concentration of active FXN inside mitochondria involves the production of recombinant variants with the capability of crossing cell membranes, thus yielding an active and stable form of the protein.We investigated the conformation and folding of the recombinant precursor variant His6-TAT-FXN1-210 that includes a cell-penetrating peptide (TAT) in the N-terminal region (NTR) and a His tag. We studied the thermal unfolding process by circular dichroism (CD). Results showed unfolding is not reversible unless a given GmdCl concentration is added to the protein sample. In this case, theprocess is completely reversible. Neither urea nor NaCl exhibit the complete effect observed for GdmCl, but a combination of both does it. These results suggest that (i) the NTR establishes interactions favoring FXN aggregation; (ii) aggregation is enhanced at high temperatures but may be attenuated by the presence of additives; and (iii) NTR-mediated interactions are of both sorts: ionic and apolar. His6-TAT-FXN1-210 was studied by light scattering. It is mainly monomeric and, at subdenaturing concentration of GdmCl, it exhibits a Rh larger than the expected for fully-folded protein. According to this, Far-UV CD spectra and secondary structure predictions suggest that the NTR is disordered. To know whether His6-TATFXN1-210 is active, we performed a NFS1 activation assays. Results suggest that FXN function is conserved in this variant.