Distinct Dynamic Signatures of Amyloidogenic Insulin Revealed by Neutron Spin Echo Spectroscopy

Abstract

Insulin is a well-known model protein for studying the formation of amyloid fibrils. At low pH-values, in the presence of sodium chloride (NaCl), and at elevated temperatures, insulin readily aggregates and forms amyloid fibrils. Without NaCl, but in the presence of ethanol, the lag time of this temperature-induced aggregation is increased drastically. In this study, we have analyzed the dynamical properties of bovine insulin following these two aggregation pathways by using neutron spin echo (NSE) spectroscopy. In addition, small-angle X-ray scattering (SAXS) and thioflavin T (ThT) fluorescence experiments were carried out to track the concomitant structural changes of insulin. Measurements have mainly been performed at 318 K, where amyloid fibrils evolve over 25 h, when the insulin solution contains 0.1 mM of NaCl at pD = 2. In contrast, no amyloid fibrils are formed during 25 h at 318 K, when the insulin solution contains ethanol with a volume fraction of 20 % at pD = 2. Remarkably, the NSE data reveal distinct dynamic signatures of insulin under these two solvent conditions. Collective diffusion of insulin molecules can be inferred from an increased diffusion coefficient at low wavevector transfers in the non-fibrillating sample, whereas self-diffusion is observed in the other case. The SAXS data confirm these dynamic behaviors, because a pronounced correlation peak is only observed under conditions of collective diffusion. The dynamic responses of insulin, as revealed here by NSE spectroscopy, are in agreement with intermolecular interaction potentials derived recently from measurements of the static structure factors of insulin and lysozyme.