Unraveling the Molecular Pathways of Protein Fibrillation under Thermal Acid Hydrolysis

Abstract Artificial amyloid fibrils formed by globular proteins under thermal acid hydrolysis have drawn extensive attention due to their exceptional mechanical properties and ability to form functional materials. However, the mechanisms underlying their formation, particularly the initiation of fibrillation under heat‐induced acid hydrolysis, are not yet fully understood. By developing a general approach that integrates experiment and theory, the molecular pathways by which β‐lactoglobulin (β‐lg) monomers convert into artificial fibrils under heat‐induced aggregation and acid hydrolysis at concentrations of 0.15–2% w/w are revealed. Despite all mature β‐lg fibrils originating from heat‐induced intermediate aggregates, most aggregates are inactive structures without forming fibrils. Only a minority of aggregates (active structures) convert into fibrils facilitated by heat‐induced acid hydrolysis. Particularly, the peptides with largely consistent protein sequences, exhibiting small variations, are identified as the building blocks for fibril elongation throughout the fibrillation process. Moreover, secondary nucleation is inhibited during fibril formation. The results expand the theoretical framework for understanding amyloid formation induced by thermal acid hydrolysis, which paves the way for precise control of artificial amyloid formation.