Assessing the impact of conformational perturbants on folding and aggregation pathways of a β-barrel fold

Here we explore the interplay between physical and chemical perturbants to unravel links among native folding, amorphous and ordered aggregation scenarios in IFABP (rat intestinal fatty acid binding protein). This small beta-barrel protein undergoes amyloid-like aggregation above 15 % v/v trifluoroethanol. Our aim was to address the influence of sub-aggregating TFE concentrations on the unfolding transitions of IFABP. The urea-induced unfolding process can bona fide be considered a two-state transition where no aggregation takes place. On the other hand, with GdmCl, the appearance of amyloid-like aggregation becomes evident upon TFE challenge. Temperature-induced denaturation profiles show that both additives, TFE and GdmCl decrease protein stability. Whereas amorphous aggregation occurs upon heating in the presence of TFE, no aggregation takes place with GdmCl. Conversely, when both additives are present, amyloid-like aggregation prevails. The explanation for the choice of amorphous or amyloid-like pathways must reconcile the effects of perturbants on both the protein and solvent structures. Key points include the TFE-promoted desolvation of the polypeptide, a process further enhanced by heat. Although GdmCl might prevent amorphous thermal aggregation by solubilizing non-native states, this effect could also favor amyloid aggregation. In addition, the electrolyte-induced segregation of TFE at high enough GdmCl concentration might contribute to the development and/or stabilization of TFE clusters that could act as nucleation-inducing interfaces, thus leading to the observed amyloid aggregation outcome.