Biophysical Characterization of Human Frataxin Precursor

FARAJ SE; CASTRO I; SANTOS, J; FERRARI A

Tucumán

Congreso; IIILAFeBS, IX IberoAmerican congress of Biophysics, XLV SAB Annual Meeting; 2016

SAB

Biophysical characterization of human frataxin precursor Friedreich ataxia is a disease caused by reduced expression of a small mitochondrial protein, frataxin. Our laboratory have a large experience studying themature human frataxin (FXNm) (frataxin from residue 90 to 210). The aim of this study is to investigate the secondary, tertiary and quaternary structure of the human frataxin precursor (FXNp), and how the long N-terminal (from residue 1-89) affect its stability. For this purpose, we produced the human frataxin precursor protein in E. Coli and it was purified by Ni-NTA affinity resin. We studied the protein by circular dicrhoism (characterization of native state and thermal unfolding) , static light scattering, fluorescence (chemical unfolding and interaction with ANS) and its protease sensibility by gel electrophoresis. As expected, the extreme N-terminal of FXNp is unstructured, and it seems to affect a little the protein stability to different concentration of chaotrope. Its secondary structure is much similar to the FXNm, but its tertiary structure is very different, so its N-terminal is likely to interact with the rest of the protein. The FXNp is mainly monomeric. The thermal stability of FXNp is less than FXNm, FXNp has a bigger tendency to form aggregates at high temperatures. On the other hand, the FXNp has a TAT peptide (translocation peptide) in the N-terminal position which allows FXNp to enter living cells (B104 and n2a cell lines). In this work, we checked the FXNp ability to enter living cells and its sorting to mitochondria by fluorescence microscopy.