Biophysical characterization of human frataxin precursor

IGNACIO H. CASTRO; SANTIAGO E. FARAJ; JAVIER SANTOS; ALEJANDRO FERRARI

Tucumán

Congreso; III Latin American Federation of Biophysical Societies (LAFeBS) ? IX IberoAmerican Congress of Biophysics ? XLV Reunion Anual SAB; 2016

Sociedad Argentina de Biofísica y Latin American Federation of Biophysical Societies

Friedreich?s ataxia is a disease caused by a reduced expression of frataxin (FXN), a small mitochondrial protein. FXN is translated in the cytoplasm as a precursor of 210 residues. Our laboratory has studied in detail the human mature form. Here we investigate the conformation, and the stability of a precursor variant (thFXN1-210) that includes in the N-terminal a His tag and a TAT peptide to simplify its purification and to be translocated across the cytoplasmic membranes, in cell cultures. FXN1-210 was produced in E. coli and purified by Ni-NTA affinity. The translocation of the fluorescent labeled protein (FITC-thFXN1-210) was checked by fluorescence microscopy using B104 and N2a cell lines. thFXN1-210 was studied by circular dichroism, static light scattering and Trp and ANS fluorescence. The sensitivity to proteolysis was investigated by SDS-PAGE. Protein stability was explored by ureainduced and thermal unfolding experiments. The N-terminal region (NTR) of thFXN1-210 showed to be mostly unstructured, as judged by the analysis of Far-UV CD spectra. Although thFXN1-210 is mainly monomeric, an increase in the aggregation of this variant was evident at high temperatures. Near-UV CD spectra suggest subtle changes in the local environment of aromatic residues. However, neither the presence of the NTR alters significantly the stability of the C-terminal domain in urea unfolding experiments, nor the fluorescent probe ANS binds to the purified precursor, in principle excluding the possibility that the N-terminal keeps the conformation of C-terminal domain in a partially folded state.