IQUIFIB   02644
INSTITUTO DE QUIMICA Y FISICOQUIMICA BIOLOGICAS "PROF. ALEJANDRO C. PALADINI"
Unidad Ejecutora - UE
congresos y reuniones científicas
Título:
Function and Stability: Design and Characterization of Human Frataxin Variants
Autor/es:
SEWELL EK; SANTOS J; CASTRO IH
Lugar:
La Plata, Buenos Aires
Reunión:
Congreso; Reunión Anual de la Sociedad Argentina de Biofísica; 2018
Institución organizadora:
SAB
Resumen:
FUNTION AND STABILITY: DESIGN AND CHARACTERIZATION OF HUMAN FRATAXIN VIARIANTSIgnacio Castro1,2, Ellioth Sewell1,2, Javier Santos1,21Instituto de Química y Fisicoquímica Biológicas, Dr. Alejandro Paladini (UBA-CONICET). C.A.B.A.2Departamento de Fisiología y Biología Molecular y Celular. Facultad de Ciencias Exactas y Naturales. Universidad de Buenos Aires. C.A.B.A.Frataxin (FXN) is a mitochondrial protein involved in [Fe-S] cluster formation, it is an allosteric activator of the protein complex NFS1/ACP-ISD11/ISCU. FXN deficient expression results in Friedreich's ataxia. This deficient expression is caused by a decrease in the fxn gene transcription because of the amplification of repetitive sequences in the first intron, or by the presence of mutations that lead to FXN variants with functional deficiency or decreased stability, with more tendency to aggregation or with altered internal mobility. Here, we show results concerning the design and study of a set of human FXN variants. Our goal was to find FXN functional variants with increased thermodynamic stability in vitro and decreased tendency to degradation in the cell. We used the stability predictors FOLDX, Dynamut and PoPMuSiC and we chose eight point-mutants which are predicted as more stable than the wild-type protein. We discarded mutants involving positions that may affect the interaction with the protein complex NFS1/ACP-ISD11/ISCU and the positions located near reported pathological mutations. The recombinant variants were successfully expressed in E. coli and purified to develop the functional and structural characterization. Also, we studied the effect of the combination of a stabilizing point mutation with the mutation K147R. Although the mutation K147R was reported that inhibit its ubiquitination and degradation by the proteasome, it is thermodynamically less stable than the wild-type protein. We observed that double mutant is more stable than the FXN K147R and it is functional.