Mechanistic modeling of amyloid oligomer and protofibril formation

Abstract

Early phase of amyloid formation, where prefibrillar aggregates such as oligomers and protofibrils are often observed, is crucial for elucidating pathogenesis. However, since oligomers and protofibrils form transiently and heterogeneously, the detailed mechanisms of their formation remain unclear. Here, we have investigated the early aggregation process of bovine and human insulin by static and dynamic light scattering in combination with thioflavin T (ThT) fluorescence and Fourier transform infrared (FTIR) spectroscopy. The time dependence of light scattering has revealed that oligomers and protofibrils form in bovine insulin, in contrast to no significant aggregation in human insulin. By focusing on bovine insulin for kinetic analysis, it has been revealed that the protofibril formation process was divided into two steps with reference to fractal dimension. When modeled the experimental data of static and dynamic light scattering based on the Smoluchowski aggregation kinetics with fractal aggregation and end-to-end association, we found the initial formation of spherical oligomers and their subsequent uniaxial docking. Furthermore, the analysis of temperature and salt concentration dependence revealed that the end-to-end association is the rate-limiting step, where structure organization occurred with dehydration. The established model for protofibril formation where oligomers are incorporated as a precursor provides insight into the molecular mechanism how protein molecules assemble during the early stage of amyloid formation. SignificanceAmyloid oligomers and protofibrils have attracted attention as critical causes of neurodegenerative diseases; however, detailed formation processes of these aggregates have been poorly understood. In this study, we established a mechanistic model of oligomer and protofibril formation of bovine insulin based on Smoluchowski aggregation kinetics in terms of static and dynamic light scattering. It has been demonstrated that early aggregation proceeds by initial fractal-like aggregation to form oligomers, and subsequent their end-to-end docking to form protofibrils. The latter step is a rate-limiting step, where structural organization occurs with dehydration. The established model is expected to broadly applicable to a variety of proteins, and thus will provides valuable insights for accelerating therapeutic development and anti-neurodegenerative drug design.