Amyloid Gels: Formation of an Insulin Fibrillar Network

Abstract

The formation of insulin amyloid fibrils is important not only for the development of reliable drugs and drug delivery systems, but also for modeling the basic properties of protein self-assembly. Fibrillation kinetics are typically characterized by an initial apparent lag-phase, related to the formation of oligomer, protofibrils and aggregation nuclei. Afterwards, aggregation proceeds over a wide range of length scales via fibril elongation, thickening and/or flocculation, and eventual gelation. Here, we focus on the formation of such a gel, made of insulin amyloid fibrils, upon incubation at high temperature and low pH. By light scattering and rheological techniques, we monitor the development of the structural, dynamical and mechanical properties of fibrillar aggregates, up to the dynamic arrest of the sample and the appearance of a non-ergodic behavior, which marks the occurrence of gelation. Atomic force microscopy imaging on incubated samples highlight the existence of a fibrillar network , as well as a complex hierarchy of different morphologies. Also, small and large angle dynamic light scattering experiments clearly show a non-diffusional dynamic behavior. Our experiments were able to reveal the structural details hidden in the apparent lag-phase, displaying the slow fibril nucleation and elongation. We confirm that this initial stage is followed by an exponential growth of structures of different sizes. These two kinetic stages of structural growth are mirrored by the kinetics of the viscoelastic properties and, in particular, by the growth of the elastic modulus. Our results show that the appearance of a noteworthy elastic network is associated with the initial fibril nucleation and elongation rather than with the formation of larger structures which cause the eventual gelation.