Sequential dissociation of insulin amyloids probed by high pressure Fourier transform infrared spectroscopy

High pressure (HP) Fourier transform infrared (FTIR) spectroscopy has been here employed to investigate the thermodynamic stability of bovine pancreatic insulin (BPI) amyloids. Once the aggregation reaction has started, the backbone arrangement of the proteins forming the amyloid is known to reach a stationary phase in a few hours; after this time the infrared absorption of fibrils becomes stable. It is here shown how further stabilization of the structure during the stationary phase can be probed via FTIR spectroscopy, through the observation of the high pressure behaviour of fibrils formed at different maturation stages. We report on the high pressure fragmentation of insulin amyloids, probed on fibrils formed in the early stages of the stationary phase. Moreover, we noticed a sequentiality high pressure dissociation that seems to respect a pre-existing hierarchy of structures: the stabilization of a protofibrillar state is observed at pressures in the order of a few kbar and our results suggest the possible occurrence of a partial refolding, induced by pressures up to 11.4 kbar. Our findings remark the importance of high pressure in stabilizing intermediate structures and in evaluating the driving forces of fibrillation, demonstrating how the control of electrostatic interactions and hydrophobic effects can be used to characterize the factors that modulate amyloids stability.