CONICET | Buscador de Institutos y Recursos Humanos

Protein structures experience internal motions which are fundamental for their biological function, also known as conformational dynamics. The aim of this study is to investigate how the perturbation of flexibility in a specific region of a model protein impacts on backbone motions of the rest of the protein and in thermodynamic stability. To this end, we produced a number of variants of human frataxin, where residues corresponding to loop-1 were modified by means of substitutions, insertions or deletions, in order to alter the backbone flexibility of this segment, e.g. we replaced all of the 7 residues of this loop by the same number of glycine amino acids. All of the studied variants were well folded, stable and monomeric, as evaluated by circular dichroism, thermal and chemical unfolding, and light-scattering measurements. We studied the impact of modifications in protein dynamics using NMR methods, including relaxation T1/T2, heteronuclear NOE and water-NOE experiments, and relaxation dispersion measurements. This set of NMR methods allowed us to characterize dynamics in the different timescales. As expected, modifications of backbone dynamics were mostly localized in the loop-1 and neighboring residues in the tertiary structure, however the dynamics of some residues far from the modification point were also perturbed. This phenomenon was mainly evidenced by water-NOE experiments as a reduction in the amide protection, and by relaxation dispersion that reveals subtle perturbations of the H-bond network. These results are presented here in combination with coarse grained and accelerated molecular dynamic simulations to infer relations of local motions between different parts of the protein and to investigate the atomistic details of these processes.