Cold stimulus during the fibrillation lag phase induces molecularly distinct amyloid strains

Abstract Amyloid polymorphism is important in neurodegenerative and metabolic disease. Understanding fibril molecular polymorphism is critical to understanding why certain protein aggregates made from the same protein are pathogenic while others are benign. Here, we generate two polymorphs of human insulin, as a model system, induced by a thermal change in the nucleation (lag) phase. Both amyloid strains predominantly exhibit β-sheet structures; however, the cold-induced fibrils (formed under a two-h cold (4 °C) shock) display a more heterogeneous structure compared to the more uniform content in the conventional warm fibrils (formed at 37 °C the whole time). These structural variations highlight the complexity of the nucleation and growth mechanisms, with cold fibrils potentially trapped in non-equilibrium states due to a higher nucleation barrier. We find that the functional activity of cold/warm fibrils is unique in in vitro seeding/amyloid propagation, stability against inhibition, and cellular cytotoxicity. Our results show how amyloid polymorphs could differentially modulate pathogenicity. Moreover, the importance of environmental conditions on the molecular features of amyloid nucleation may be critical for design and stability of future protein biologics beyond therapeutic insulin.