Self-assembly of misfolded proteins into ordered fibrillar aggregates known as amyloid results in numerous human diseases. Despite an increasing number of proteins and peptide fragments being recognised as amyloidogenic, how these amyloid aggregates assemble remains unclear. In particular, the identity of the nucleating species, an ephemeral entity that defines the rate of fibril formation, remains a key outstanding question. Here, we propose a new strategy for analyzing the self-assembly of amyloid fibrils involving global analysis of a large number of reaction progress curves and the subsequent systematic testing and ranking of a large number of possible assembly mechanisms. Using this approach, we have characterized the mechanism of the nucleationdependent formation of ␤2-microglobulin (␤2m) amyloid fibrils. We show, by defining nucleation in the context of both structural and thermodynamic aspects, that a model involving a structural nucleus size approximately the size of a hexamer is consistent with the relatively small concentration dependence of the rate of fibril formation, contrary to expectations based on simpler theories of nucleated assembly. We also demonstrate that fibril fragmentation is the dominant secondary process that produces higher apparent cooperatively in fibril formation than predicted by nucleated assembly theories alone. The model developed is able to explain and predict the behavior of ␤2m fibril formation and provides a rationale for explaining generic properties observed in other amyloid systems, such as fibril growth acceleration and pathway shifts under agitation.AIC model comparison analysis ͉ amyloid fibril formation ͉ fibril brittleness ͉ global analysis S elf-assembly of misfolded forms of normally soluble and functional proteins or peptides into amyloid fibrils results in numerous human diseases (1). Understanding how amyloid self-assembly occurs, therefore, is of paramount importance for a molecular interpretation of amyloidosis and for the development of therapies against amyloid disease. Over the past decade, advances have been made toward a more complete description of amyoid fibril formation, including the determination of increasingly refined models of fibril structures (reviewed in ref. 1) and the identification of amyloid precursors and oligomeric states reviewed in ref. 2, one or more of which could be the culprits of cytotoxicity associated with several amyloid diseases (e.g., ref.3). However, the molecular events occurring during the self-assembly process itself remain obscure because of the heterogeneity and the complexity of the early association events.Amyloid fibril self-assembly reactions are generally accepted as a form of nucleated polymerization (4). These reactions are characterized by an initial lag phase where little or no change in fibril concentration can be detected. This is followed by an elongation phase where a large mass percentage of the starting protein material is converted into fibrils. A shared feature among amyloid formation and other nuclea...

Although most proteins can assemble into amyloid-like fibrils in vitro under extreme conditions, how proteins form amyloid fibrils in vivo remains unresolved. Identifying rare aggregation-prone species under physiologically relevant conditions and defining their structural properties is therefore an important challenge. By solving the folding mechanism of the naturally amyloidogenic protein beta-2-microglobulin at pH 7.0 and 37 degrees C and correlating the concentrations of different species with the rate of fibril elongation, we identify a specific folding intermediate, containing a non-native trans-proline isomer, as the direct precursor of fibril elongation. Structural analysis using NMR shows that this species is highly native-like but contains perturbation of the edge strands that normally protect beta-sandwich proteins from self-association. The results demonstrate that aggregation pathways can involve self-assembly of highly native-like folding intermediates, and have implications for the prevention of this, and other, amyloid disorders.

Fibrils associated with amyloid disease are molecular assemblies of key biological importance, yet how cells respond to the presence of amyloid remains unclear. Cellular responses may not only depend on the chemical composition or molecular properties of the amyloid fibrils, but their physical attributes such as length, width, or surface area may also play important roles. Here, we report a systematic investigation of the effect of fragmentation on the structural and biological properties of amyloid fibrils. In addition to the expected relationship between fragmentation and the ability to seed, we show a striking finding that fibril length correlates with the ability to disrupt membranes and to reduce cell viability. Thus, despite otherwise unchanged molecular architecture, shorter fibrillar samples show enhanced cytotoxic potential than their longer counterparts. The results highlight the importance of fibril length in amyloid disease, with fragmentation not only providing a mechanism by which fibril load can be rapidly increased but also creating fibrillar species of different dimensions that can endow new or enhanced biological properties such as amyloid cytotoxicity.