Loop insertions in human frataxin to study protein stability and dynamics

MARTÍN EZEQUIEL NOGUERA; ERNESTO A. ROMAN; SANTIAGO E. FARAJ; JAVIER SANTOS

Villa Carlos Paz, Córdoba

Congreso; XLII Reunión Anual de la Sociedad Argentina de Biofísica; 2013

Sociedad Argentina de BIofísica

A complete description of the protein structure requires, in addition to the average localization of all of its atoms, the changes in time of these positions, i.e. its dynamic behavior, and ultimately the characterization of the complete native ensemble, including low-populated conformations. The study of protein dynamics is crucial to understand biological function. Throughout the structural study of the human frataxin, we found that there is a relationship between the dynamics of the C-terminal region (CTR) and loop-1, the segment linking the helix-α1 with the strand-β1 of this single domain protein: sequence mutations that affect the dynamics of CTR, also alter the dynamics of loop-1. In this work, we explored these relationships through the manipulation of protein sequence: introducing mutations in the loop-1 region to alter its local dynamics, and assessing its effects in the dynamics of other regions of the protein. First of all, we investigated whether the loop-1 is tolerant to the sequence changes, without severe alterations in the stability of the native state. For this purpose, we designed two variants: in one of them, the only two residues which establish tertiary contacts with the rest of the protein were mutated to alanine, whereas in the second variant, substitutions and insertions were performed resulting in a loop extension with six new glycine residues. Both variants were characterized by spectroscopic and hydrodynamic methods, and both were shown to be well folded, stable, and monomeric. These results will be presented along a preliminary assessment of the influence of these mutations in conformational dynamics.