We describe herein the characterization of the equilibrium point for A beta(1-40) amyloid fibril elongation in phosphate-buffered saline at 37 degrees C. Seeded fibril elongation progresses rapidly to a reproducible end point of 0.7-1.0 microM unpolymerized monomeric peptide. This remaining monomeric material is functional, since after concentration it supports fibril elongation. Incubation of fibrils in the same buffer results in dissociation to a final monomer concentration in the same range. This robust C(r) value is equivalent to the A beta(1-40) fibril dissociation constant, Kd. The fact that a similar value for Kd is obtained from a ratio of dissociation and elongation rate constants further supports the view that these values are associated with a position of dynamic equilibrium and therefore are related to free energies of amyloid fibril elongation. The C(r) value reported here for wild-type A beta(1-40) fibrils corresponds to a free energy of fibril elongation of about -9 kcal/mol, a value similar to free energies of folding for small globular proteins. Elongation and dissociation of amyloid fibrils from point mutants of A beta(1-40) also yield C(r) values, different for different mutants, that reflect stabilizing/destabilizing effects. Interestingly, assembly of A beta(1-40) fibrils in the presence of a saturating concentration of the amyloid dye thioflavin T does not measurably affect fibril stability, in contrast to the commonly observed stabilization of globular proteins by ligand binding. The ability to quantify and compare amyloid fibril thermodynamic stabilities makes it possible to include fibrils, and potentially other aggregates, in quantitative descriptions of protein folding landscapes.

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In experiments designed to characterize the basis of amyloid fibril stability through mutational analysis of the Aβ(1-40) molecule, fibrils exhibit consistent, significant structural malleability. In these results, and in other properties, amyloid fibrils appear to more resemble plastic materials generated from synthetic polymers than they do globular proteins. Thus, like synthetic polymers and plastics, amyloid fibrils exhibit both polymorphism, the ability of one polypeptide to form aggregates of different morphologies, and isomorphism, the ability of different polypeptides to grow into a fibrillar amyloid morphology. This view links amyloid with the prehistorical and 20 th Century use of proteins as starting materials to make films, fibers, and plastics, and with the classic protein fiber stretching experiments of the Astbury group. Viewing amyloid from the point of view of the polymer chemist may shed new light on issues such as the role of protofibrils in the mechanism of amyloid formation, the biological potency of fibrils, and the prospects for discovering inhibitors of amyloid fibril formation.Amyloid fibrils and amyloid-related protein aggregates are associated with a variety of human diseases of the brain and the periphery (1,2). The past decade has seen significant progress in our understanding of this alternatively folded protein structural motif. At the same time, much remains to be learned about details of amyloid structure, the basis of fibrillogenesis, and the nature of protein aggregate cytotoxicity.Experimental work over the past 10-15 years has revealed the amyloid folding motif to be a nearly ubiquitous alternative folded state that can be accessed by many polypeptides. For globular proteins, the route to amyloid often depends on initial weakening of the native state; in addition, for all proteins and peptides, amyloid formation also depends on packing constraints within the fibril (3). Reviewed here are mutagenesis experiments designed to focus on understanding the nature of the packing constraints within the amyloid motif.The results show that amyloid fibrils, like globular proteins, achieve stability through a mixture of hydrophobic and electrostatic forces. At the same time, the response of amyloid to mutagenesis differs in a number of ways from the typical response of globular proteins, and suggests instead a significant resemblance to synthetic polymers and plastics. A relationship to synthetic polymers provides a comfortable context for a number important features of amyloid fibrils, including: (a) the ability of so many naturally occurring proteins to form amyloid, (b) the observation of multiple conformational states of amyloid fibrils from the same polypeptide sequence, (c) the role of protofibrillar intermediates on the amyloid assembly pathway, (d) the biological potency of amyloid prions, and (e) the mixed success of attempts to identify inhibitors of the amyloid formation process. † Supported by NIH grant R01 AG018416 (RW). ‡ Present address: