Mechanistic Modeling of Amyloid Oligomer and Protofibril Formation in Bovine Insulin

Early phase of amyloid formation, where prefibrillar aggregates such as oligomers and protofibrils are often observed, is crucial for understanding pathogenesis. However, the detailed mechanisms of their formation have been difficult to elucidate because they tend to form transiently and heterogeneously. Here, we found that bovine insulin protofibril formation proceeds in a monodisperse manner, which allowed us to characterize the detailed early aggregation process by light scattering in combination with thioflavin T fluorescence and Fourier transform infrared spectroscopy. The protofibril formation was specific to bovine insulin, whereas no significant aggregation was observed in human insulin. The kinetic analysis combining static and dynamic light scattering data revealed that the protofibril formation process in bovine insulin can be divided into two steps based on fractal dimension. When modeling the experimental data based on Smoluchowski aggregation kinetics, an aggregation scheme consisting of initial fractal aggregation forming spherical oligomers and their subsequent end-to-end association forming protofibrils was clarified. Furthermore, the analysis of temperature and salt concentration dependencies showed that the end-to-end association is the rate-limiting step, involving dehydration. The established model for protofibril formation, wherein oligomers are incorporated as a precursor, provides insight into the molecular mechanism by which protein molecules assemble during the early stage of amyloid formation.