CONICET | Buscador de Institutos y Recursos Humanos

IFABP is a 15 kDa β-barrel protein whose amyloid-like aggregation is triggered with 25% TFE. Under equilibrium conditions, 10% TFE fosters conformational changes akin to those leading to aggregation-prone species. In the presence of TFE, GdmCl-induced denaturation profiles show a plateau region (at ~ 1.25 M GdmCl) that arises as TFE increases, being most evident at 15%. Turbidity reveals that the latter is due to protein aggregation. GdmCl (up to 1.25M) accelerates the rate of aggregation but higher concentration slows down the process. Far UV-CD of the aggregate uncovers  structure different from that of the native protein, a fact indicative of amyloid aggregation. To further explore the impact of TFE/GdmCl on stability, temperature-induced denaturation profiles were assessed. In all cases, heating leads to a reduction in the dichroic signal at 216 nm. However, a more realistic understanding of the process arises from the comparison of the evolution with temperature of the shape and intensity of the full set of spectra. TFE leads to reduced stability and a concomitant amorphous aggregation. GdmCl also reduces stability but no aggregation is observed. When both are present, amyloid-like aggregation initiates immediately. Moreover, the spectrum remains the same upon cooling, and a simple visual inspection reveals a colloidal appearance that clearly differs from the flocculation observed when thermally induced aggregation occurs. In summary, two parallel aggregation pathways are taking place: one leading to amorphous and the other leading to amyloid aggregation. The presence of TFE at sub-aggregating concentration promotes the first, whereas if GdmCl is also present, the second route takes over. The explanation of these findings is to be found in the complex influence of the additives on the ground states and/or heights of transitions states barriers leading to unfolded or aggregated species.