Self-organization pathways and spatial heterogeneity in insulin amyloid fibril formation.

At high temperature and low pH, the protein hormone insulin is highly prone to form amyloid fibrils, and for this reason it is widely used as a model system to study fibril formation mechanisms. In this work, we focused on insulin aggregation mechanisms occurring in HCl solutions (pH 1.6) at 60 degrees C. By means of in situ Thioflavin T (ThT) staining, the kinetics profiles were characterized as a function of the protein concentration, and two concurrent aggregation pathways were pointed out, being concentration dependent. In correspondence to these pathways, different morphologies of self-assembled protein molecules were detected by atomic force microscopy images also evidencing the presence of secondary nucleation processes as a peculiar mechanism for insulin fibrillation. Moreover, combining ThT fluorescence and light scattering, the early stages of the process were analyzed in the low concentration regime, pointing out a pronounced spatial heterogeneity in the formation of the first stable fibrils in solution and the onset of the secondary nucleation pathways.