Supersaturation-Dependent Formation of Amyloid Fibrils

Abstract

The supersaturation of a solution refers to a non-equilibrium phase in which the solution is trapped in a soluble state, even though the solute's concentration is greater than its thermodynamic solubility. Upon breaking supersaturation, crystals form and the concentration of the solute decreases to its thermodynamic solubility. Soon after the discovery of the prion phenomena, it was recognized that prion disease transmission and propagation share some similarities with the process of crystallization. Subsequent studies exploring the structural and functional association between amyloid fibrils and amyloidoses solidified this paradigm. However, recent studies have not necessarily focused on supersaturation, possibly because of marked advancements in structural studies clarifying the atomic structures of amyloid fibrils. On the other hand, there is increasing evidence that supersaturation plays a critical role in the formation of amyloid fibrils and the onset of amyloidosis. Here, we review the recent evidence that supersaturation plays a role in linking unfolding/folding and amyloid fibril formation. We also introduce the HANABI (HANdai Amyloid Burst Inducer) system, which enables high-throughput analysis of amyloid fibril formation by the ultrasonication-triggered breakdown of supersaturation. In addition to structural studies, studies based on solubility and supersaturation are essential both to developing a comprehensive understanding of amyloid fibrils and their roles in amyloidosis, and to developing therapeutic strategies.

Keywords: amorphous aggregation; amyloid fibrils; amyloid β; protein misfolding; solubility; supersaturation; ultrasonication; β2-microglobulin.

Figure 1

Protein and precipitant concentration-dependent phase diagram common to native crystals and aggregates of denatured proteins. Regions I, II, III, and IV represent undersaturation, the metastable region, the labile region, and the amorphous region, respectively. Crystallization and amyloid fibril formation occur from regions II and III. The figure was modified from So et al. [6], with permission. Copyright 2016 Elsevier.

Figure 2

Heating- and agitation-dependent amyloid formation of proteins. According to their aggregation behavior, Type S, A, and B proteins were defined. Left: ThT assays upon heating in the presence (upper) or absence (lower) of stirring. The intensities of ThT fluorescence and LS are indicated by blue and red lines, respectively. n = 3. Middle: TEM images of the samples after heating in the presence (upper) or absence (lower) of stirring. Right: Structures of proteins used with their names and pdb codes. The figure was created based on Noji et al. [2].

Figure 3

Temperature- and NaCl concentration-dependent conformational phase diagrams of β2m before and after the linkage of folding and misfolding transitions. (A) Temperature dependencies of thermodynamic parameters (ΔG(T), ΔH(T), and TΔS(T)) for folding (Mechanism 1, panel (i)) and amyloid formation (Mechanism 2, panel (ii)). Fractions of N, D, and P states for folding (panel (iii)), amyloid formation (panel (iv)), and their linked conditions (panel (v)) are also shown. The plots were made using 0.1 mg/mL β2m, 1.0 M NaCl, and pH 7.0. (B) Phase diagrams for folding/unfolding (Mechanism 1, panel (i)), amyloid misfolding (Mechanism 2, panel (ii)), and their linked conditions (panel (iii)). Lines show the simulated phase boundaries at 0.1 mg/mL β2m and pH 7.0. The figure was modified from Noji et al. [12].

Figure 4

General schematic conformational phase diagram and the three transitions. Three types of amyloidogenic proteins were plotted on a general phase diagram of aggregation (A), and diagrams of average hydrophobicity vs. number of amino acid residues (B) or ∆S_conf (C). In c, ∆S_conf represents an increase upon denaturation of the main chain with (points within the frame) and without (points outside the frame) the contribution of disulfide bonds. The figure was reproduced from Noji et al. [78].

Figure 5

Overview of the HANABI-2000 system. (A) A 3D schematic illustration of the optimized sonoreactor for the amyloid-fibril assays, HANABI-2000. The dimensions of the device are 500 × 550 × 550 mm³. (B) A block chart of the control units of HANABI-2000. The figure is reproduced from Nakajima et al. [98] with permission. (C) The ThT time–course curves (n = 36), which are irradiated with ultrasound with the compensation procedure. The figure was modified from Nakajima et al. [98] with permission. Copyright 2021 Elsevier.

Figure 6

t_half heat maps of aggregation reactions. (A) Under quiescence, (B) under shaking, and (C) under ultrasonication. The yellow dots denote the solubility of acidic β2m monomer at each salt concentration, determined by ultracentrifugation and the ELISA assay. The dotted lines in panels (B,C) indicate the phase boundaries under quiescence, which are varied under agitation as indicated by yellow arrows. The figure is reproduced from Nakajima et al. [104] with permission. Copyright 2021 American Chemical Society.