Characterization of the folding of yeast frataxin: comparing hot and cold denaturation and defining the role of protein frustration

Understanding the process leading a polypeptide chain to its native state is one of the unsolved problems of modern Biochemistry. Even if the sequence of a protein contains all the information necessary to guide the folding process, it has been demonstrated that the residual structure eventually present in the denatured state can tune the folding process. For this reason, studying the characteristics of the denatured state has become an useful tool to understand better the folding process. In this work the attention has been focused on yeast frataxin, a mitochondrial protein orthologous of human frataxin, which is involved in several processes required for the cellular regulation of iron homeostasis. Yeast frataxin is an interesting system because it undergoes cold denaturation at physiological conditions and at temperature above the freezing point of water. Therefore, it gives the chance to study the folding process taking into account both the cold and hot denatured states. The first part of this thesis is thus focused on the characterization of the hot and cold denaturation processes. Analysis of thermal denaturation experiments and kinetic experiments revealed a simple two state folding process. Then the transition states of the reaction have been characterized by using a Φ-value analysis at two different temperatures, in combination with molecular dynamics simulations. The differences in the cold and hot folding processes have been established by comparing the structure of the denatured states with the one of the transition states. The second part is focused instead on the characterization of another interesting aspect of yeast frataxin: the competition between folding and function and its consequences for misfolding and aggregation. Kinetic experiments have been performed on yeast frataxin by using urea as a chaotropic agent. By analyzing the chevron plots obtained under different pH conditions, it has been established that frataxin folds via a broad free energy barrier. This feature has been associated with protein frustration and it gives the possibility to analyze both the early and late events of folding. In order to do that, a Φ-value analysis has been performed and the data obtained have been used in molecular dynamics simulations. In this way it has been demonstrated that at the early stage of folding a significative fraction of nonnative contacts can be observed. The prediction of molecular dynamics simulation has been confirmed by a second round of mutagenesis, mutating those residues that are supposed to result in non canonical Φ-values, confirming a high level of frustration in such regions and a propensity of frataxin to misfold at the early stage of the folding process.