102. Advances in the functional modulation of the NFS1/ACP-ISD11/ISCU/FXN Fe-S cluster mitochondrial supercomplex by molecular intervention through specific Trojan tutors

CORREA A; CASTRO IH; PIGNATARO MF; SANTOS J

La Plata, Buenos Aires

Congreso; Reunión Anual de la Sociedad Argentina de Biofísica; 2018

SAB

The development of molecules that can specifically bind to targets plays a major role in therapeutics and diagnostics, and also in basic and applied research. As an alternative to antibodies, the use of a different kind of specific molecules against various targets has been tested and characterized over the last years (1-2). The possibility of using them as molecular tutors, that promote conformational stabilization, appears as a promising strategy for the treatment of several diseases. In this work, we employed Sac7d variants (affitins), selected by ribosome display technology using Frataxin (FXN) as the target. The lack of FXN, an allosteric activator of NFS1/ACP-ISD11/ISCU Fe-S cluster supercomplex in mitochondria, leads to Friedreich?s ataxia, a rare inherited disease that causes progressive nervous system damage. Sac7d is a DNA binding protein from S. acidocaldarius and has already been extensively used as protein scaffold. This is a very stable protein of 66 residues (7.6kDa) that has proven to be an effective and extremely plastic scaffold for molecular recognition. Libraries have been prepared with up to 10 to 15 replacements, for example, for specific recognition of human immunoglobulin G and against bacterial outer membrane protein PulD, CelD glicosidase, and others. We performed four rounds of Ribosome Display using a Sac7d library and human FXN (wild-type) as the target. To test whether the selection was successful we (a) performed an ELISA to detect the binding of affitins to FXN and BSA (as a negative control) and (b) we investigated individual clones by subcloning the product of the last round of selection into a pFP1001 vector, transformed E. coli DH5αF´IQ cells, induced affitin expression and performed an ELISA. Positive clones were sequenced. We found that the ratio of 450 OD of FXN to BSA was 1.6, when the optimal value should be around 8-10 (2) suggesting the need of at least one more round of selection to discard the BSA binders.