Secondary Nucleation Progressively Clear Kinetically Trapped Species in Multigeneration Seeded Fibril Growth

Amyloid fibrils are supramolecular protein assemblies characterized by their highly ordered structure and remarkable mechanical and chemical properties. Amyloid formation is a very complex process that involves an interplay of various mechanisms-primary nucleation, secondary nucleation, elongation, and fragmentation. Despite its pivotal role, particularly in fibril polymorphism, our understanding of the aggregation process remains incomplete. In this work we employ an in situ approach based on Vibrational Circular Dichroism (VCD), complemented by spectroscopic and microscopic techniques, to investigate fibril formation and accuracy of structural replication in a seeded, multi-generational model. Our results demonstrate that the morphological diversity of amyloid fibrils is governed by the competition between elongation, fragmentation and secondary nucleation pathways. While the high-fidelity elongation promotes the preservation of kinetically stabilized morphologies and fragmentation amplifies the most fragile fibril populations, it is the secondary nucleation that is the dominant mechanism enabling the structural reorganization. These findings suggest that amyloid proliferation involves escaping the kinetically trapped states toward energetically favored polymorphs, representing in overall a process of progressive refinement of amyloid structure perpetuated by secondary nucleation.