CONICET | Buscador de Institutos y Recursos Humanos

The added complexity to protein folding landscapes due to the incidence of aggregation deserves close experimental scrutiny, given the mounting importance to further the understanding of this phenomenon in biotechnology and biomedical sciences. In this study, we explore the interplay between physical (temperature) and chemical perturbants (trifluoroethanol TFE, guanidinium chloride GdmCl) to unravel links among native folding, amorphous and ordered (amyloid) aggregation scenarios in IFABP (rat intestinal fatty acid binding protein). This small beta barrel protein undergoes amyloid-like aggregation above 15 % v/v TFE, a possible outcome of the ability of this co-solvent to form micelle-like clusters at moderate concentration. As an inverse correlation has generally been postulated between thermodynamic stability of the native state and propensity to amyloid aggregation, our aim here was to address the influence of sub-aggregating TFE concentrations on the urea or GdmCl induced unfolding transitions of IFABP, as monitored by intrinsic fluorescence emission and circular dichroism (CD) spectroscopies. Insights on the process arise from comparing the evolution of the shape and intensity of bands across the full set of CD spectra collected along thermal ramps. The urea-induced unfolding transition of IFABP can be assimilated to a 2-stage system where the stability of the native state is not compromised and no aggregation takes place by influence of TFE. At variance, with GdmCl, the appearance of amyloid-like aggregation becomes evident upon TFEchallenge. Temperature-induced denaturation profiles show that both additives, TFE and GdmCl reduce protein stability. Whereas a concomitant increase of 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, by the same token this effect could 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 -upon reaching a large enough size- could act as nucleation-inducing interfaces, thus leading to the observed amyloid aggregation outcome.

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