The thermodynamic hypothesis of protein folding, known as the “Anfinsen’s dogma” states that the native structure of a protein represents a free energy minimum determined by the amino acid sequence. However, inconsistent with the Anfinsen’s dogma, globular proteins can misfold to form amyloid fibrils, which are ordered aggregates associated with diseases such as Alzheimer’s and Parkinson’s diseases. Here, we present a general concept for the link between folding and misfolding. We tested the accessibility of the amyloid state for various proteins upon heating and agitation. Many of them showed Anfinsen-like reversible unfolding upon heating, but formed amyloid fibrils upon agitation at high temperatures. We show that folding and amyloid formation are separated by the supersaturation barrier of a protein. Its breakdown is required to shift the protein to the amyloid pathway. Thus, the breakdown of supersaturation links the Anfinsen’s intramolecular folding universe and the intermolecular misfolding universe.

Amyloid fibrils are supramolecular protein assemblies represented by a cross-β structure and fibrous morphology, whose structural architecture has been previously investigated. While amyloid fibrils are basically a main-chain-dominated structure consisting of a backbone of hydrogen bonds, side-chain interactions also play an important role in determining their detailed structures and physicochemical properties. In amyloid fibrils comprising short peptide segments, a steric zipper where a pair of β-sheets with side chains interdigitate tightly is found as a fundamental motif. In amyloid fibrils comprising longer polypeptides, each polypeptide chain folds into a planar structure composed of several β-strands linked by turns or loops, and the steric zippers are formed locally to stabilize the structure. Multiple segments capable of forming steric zippers are contained within a single protein molecule in many cases, and polymorphism appears as a result of the diverse regions and counterparts of the steric zippers. Furthermore, the β-solenoid structure, where the polypeptide chain folds in a solenoid shape with side chains packed inside, is recognized as another important amyloid motif. While side-chain interactions are primarily achieved by non-polar residues in disease-related amyloid fibrils, the participation of hydrophilic and charged residues is prominent in functional amyloids, which often leads to spatiotemporally controlled fibrillation, high reversibility, and the formation of labile amyloids with kinked backbone topology. Achieving precise control of the side-chain interactions within amyloid structures will open up a new horizon for designing useful amyloid-based nanomaterials.

Structural polymorphism of insulin amyloids in vivo can be recognized using novel amyloid specific fluorescent probes, pFTAA and BTD21.