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.

Significance: Although amyloid fibrils are associated with numerous pathologies, the factors triggering amyloid formation remain largely unknown. Polyphosphates were recently reported to induce amyloid fibrils of several amyloidogenic proteins. We found that, under both acidic and neutral pH conditions, the amyloid formation of β ₂ -microglobulin was significantly accelerated by polyphosphates. We elucidated the underlying pH-dependent mechanisms, which will be important for a more comprehensive understanding of the amyloid formation of proteins. Due to the marked ability of polyphosphates to induce the amyloid formation of β ₂ -microglobulin, even at neutral pH, polyphosphates may play an important role in the development of dialysis-related amyloidosis.

Proteins in either a native or denatured conformation often aggregate at an isoelectric point (pI), a phenomenon known as pI precipitation. However, only a few studies have addressed the role of pI precipitation in amyloid formation, the crystal-like aggregation of denatured proteins. We found that α-synuclein, an intrinsically disordered protein of 140 amino acid residues associated with Parkinson's disease, formed amyloid fibrils at pI (= 4.7) under the low-sodium phosphate conditions. Although α-synuclein also formed amyloid fibrils at a wide pH range under high concentrations of sodium phosphate, the pI-amyloid formation was characterized by marked amyloid-specific thioflavin T fluorescence and clear fibrillar morphology, indicating highly ordered structures. Analysis by heteronuclear NMR in combination with principal component analysis suggested that amyloid formation under low and high phosphate conditions occurred by distinct mechanisms. The former was likely to be caused by the intermolecular attractive charge-charge interactions, where α-synuclein has +17 and −17 charges even with the zero net charge. On the other hand, the latter was caused by the phosphate-dependent salting-out effects. pI-amyloid formation may play a role in the membrane-dependent amyloid formation of α-synuclein, where the negatively charged membrane surface reduces the local pH to pI and the membrane hydrophobic environment enhances electrostatic interactions. The results extend the supersaturation-limited mechanism of amyloid formation: Amyloid fibrils are formed under a variety of conditions of decreased solubility of denatured proteins triggered by the breakdown of supersaturation.

β₂-microglobulin (β₂m) and its truncated variant ΔΝ6 are co-deposited in amyloid fibrils in the joints, causing the disorder dialysis-related amyloidosis (DRA). Point mutations of β₂m result in diseases with distinct pathologies. β₂m-D76N causes a rare systemic amyloidosis with protein deposited in the viscera in the absence of renal failure, whilst β₂m-V27M is associated with renal failure, with amyloid deposits forming predominantly in the tongue. Here we use cryoEM to determine the structures of fibrils formed from these variants under identical conditions in vitro. We show that each fibril sample is polymorphic, with diversity arising from a ‘lego-like’ assembly of a common amyloid building block. These results suggest a ‘many sequences, one amyloid fold’ paradigm in contrast with the recently reported ‘one sequence, many amyloid folds’ behaviour of intrinsically disordered proteins such as tau and Aβ.

Amyloidosis-associated amyloid fibrils are formed by denatured proteins when supersaturation of denatured proteins is broken. β₂-Microglobulin (β2m) forms amyloid fibrils and causes dialysis-related amyloidosis in patients receiving long-term hemodialysis. Although amyloid fibrils of β2m in patients are observed at neutral pH, formation of β2m amyloids in vitro has been difficult to discern at neutral pH because of the amyloid-resistant native structure. Here, to further understand the mechanism underlying in vivo amyloid formation, we investigated the relationship between protein folding/unfolding and misfolding leading to amyloid formation. Using thioflavin T assays, CD spectroscopy, and transmission EM analyses, we found that β2m efficiently forms amyloid fibrils even at neutral pH by heating with agitation at high-salt conditions. We constructed temperature- and NaCl concentration–dependent conformational phase diagrams in the presence or absence of agitation, revealing how amyloid formation under neutral pH conditions is related to thermal unfolding and breakdown of supersaturation. Of note, after supersaturation breakdown and following the law of mass action, the β2m monomer equilibrium shifted to the unfolded state, destabilizing the native state and thereby enabling amyloid formation even under physiological conditions with a low amount of unfolded precursor. The amyloid fibrils depolymerized at both lower and higher temperatures, resembling cold- or heat-induced denaturation of globular proteins. Our results suggest an important role for heating in the onset of dialysis-related amyloidosis and related amyloidoses.