Structural evolution of fibril polymorphs during amyloid assembly.

Cryoelectron microscopy (cryo-EM) has provided unprecedented insights into amyloid fibril structures, including those associated with disease. However, these structures represent the endpoints of long assembly processes, and their relationship to fibrils formed early in assembly is unknown. Consequently, whether different fibril architectures, with potentially different pathological properties, form during assembly remains unknown. Here, we used cryo-EM to determine structures of amyloid fibrils at different times during in vitro fibrillation of a disease-related variant of human islet amyloid polypeptide (IAPP-S20G). Strikingly, the fibrils formed in the lag, growth, and plateau phases have different structures, with new forms appearing and others disappearing as fibrillation proceeds. A time course with wild-type hIAPP also shows fibrils changing with time, suggesting that this is a general property of IAPP amyloid assembly. The observation of transiently populated fibril structures has implications for understanding amyloid assembly mechanisms with potential new insights into amyloid progression in disease.

A two-step biopolymer nucleation model shows a nonequilibrium critical point.

Biopolymer self-assembly pathways are complicated by the ability of their monomeric subunits to adopt different conformational states. This means nucleation often involves a two-step mechanism where the monomers first condense to form a metastable intermediate, which then converts to a stable polymer by conformational rearrangement of constituent monomers. Nucleation intermediates play a causative role in amyloid diseases such as Alzheimer's and Parkinson's. While existing mathematical models neglect the conversion dynamics, experiments show that conversion events frequently occur on comparable timescales to the condensation of intermediates and growth of mature polymers and thus cannot be ignored. We present a model that explicitly accounts for simultaneous assembly and conversion. To describe conversion, we propose an experimentally motivated initiation-propagation mechanism in which the stable phase arises locally within the intermediate and then spreads by nearest-neighbor interactions, in a manner analogous to one-dimensional Glauber dynamics. Our analysis shows that the competing timescales of assembly and conversion result in a nonequilibrium critical point, separating a regime where intermediates are kinetically unstable from one where conformationally mixed intermediates accumulate. This strongly affects the accumulation rate of the stable biopolymer phase. Our model is uniquely able to explain experimental phenomena such as the formation of mixed intermediates and abrupt changes in the scaling exponent γ, which relates the total monomer concentration to the accumulation rate of the stable phase. This provides a first step toward a general model of two-step biopolymer nucleation, which can quantitatively predict the concentration and composition of biologically crucial intermediates.

Simple, Reliable Protocol for High-Yield Solubilization of Seedless Amyloid-ß Monomer.

Self-assembly of the amyloid-ß (Aß) peptide to form toxic oligomers and fibrils is a key causal event in the onset of Alzheimer's disease, and Aß is the focus of intense research in neuroscience, biophysics, and structural biology aimed at therapeutic development. Due to its rapid self-assembly and extreme sensitivity to aggregation conditions, preparation of seedless, reproducible Aß solutions is highly challenging, and there are serious ongoing issues with consistency in the literature. In this paper, we use a liquid-phase separation technique, asymmetric flow field-flow fractionation with multiangle light scattering (AF4-MALS), to develop and validate a simple, effective, economical method for re-solubilization and quality control of purified, lyophilized Aß samples. Our findings were obtained with recombinant peptide but are physicochemical in nature and thus highly relevant to synthetic peptide. We show that much of the variability in the literature stems from the inability of overly mild solvent treatments to produce consistently monomeric preparations and is rectified by a protocol involving high-pH (>12) dissolution, sonication, and rapid freezing to prevent modification. Aß treated in this manner is chemically stable, can be stored over long timescales at -80 °C, and exhibits remarkably consistent self-assembly behavior when returned to near-neutral pH. These preparations are highly monomeric, seedless, and do not require additional rounds of size exclusion, eliminating the need for this costly procedure and increasing the flexibility of use. We propose that our improved protocol is the simplest, fastest, and most effective way to solubilize Aß from diverse sources for sensitive self-assembly and toxicity assays.

General Principles Underpinning Amyloid Structure.

Amyloid fibrils are a pathologically and functionally relevant state of protein folding, which is generally accessible to polypeptide chains and differs fundamentally from the globular state in terms of molecular symmetry, long-range conformational order, and supramolecular scale. Although amyloid structures are challenging to study, recent developments in techniques such as cryo-EM, solid-state NMR, and AFM have led to an explosion of information about the molecular and supramolecular organization of these assemblies. With these rapid advances, it is now possible to assess the prevalence and significance of proposed general structural features in the context of a diverse body of high-resolution models, and develop a unified view of the principles that control amyloid formation and give rise to their unique properties. Here, we show that, despite system-specific differences, there is a remarkable degree of commonality in both the structural motifs that amyloids adopt and the underlying principles responsible for them. We argue that the inherent geometric differences between amyloids and globular proteins shift the balance of stabilizing forces, predisposing amyloids to distinct molecular interaction motifs with a particular tendency for massive, lattice-like networks of mutually supporting interactions. This general property unites previously characterized structural features such as steric and polar zippers, and contributes to the long-range molecular order that gives amyloids many of their unique properties. The shared features of amyloid structures support the existence of shared structure-activity principles that explain their self-assembly, function, and pathogenesis, and instill hope in efforts to develop broad-spectrum modifiers of amyloid function and pathology.