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Comparative Study
. 2012 Sep 4;109(36):14446-51.
doi: 10.1073/pnas.1208228109. Epub 2012 Aug 20.

Distinguishing crystal-like amyloid fibrils and glass-like amorphous aggregates from their kinetics of formation

Yuichi Yoshimura  1 , Yuxi Lin, Hisashi Yagi, Young-Ho Lee, Hiroki Kitayama, Kazumasa Sakurai, Masatomo So, Hirotsugu Ogi, Hironobu Naiki, Yuji Goto
Affiliations
Comparative Study

Distinguishing crystal-like amyloid fibrils and glass-like amorphous aggregates from their kinetics of formation

Yuichi Yoshimura et al. Proc Natl Acad Sci U S A. .

Abstract

Amyloid fibrils and amorphous aggregates are two types of aberrant aggregates associated with protein misfolding diseases. Although they differ in morphology, the two forms are often treated indiscriminately. β(2)-microglobulin (β2m), a protein responsible for dialysis-related amyloidosis, forms amyloid fibrils or amorphous aggregates depending on the NaCl concentration at pH 2.5. We compared the kinetics of their formation, which was monitored by measuring thioflavin T fluorescence, light scattering, and 8-anilino-1-naphthalenesulfonate fluorescence. Thioflavin T fluorescence specifically monitors amyloid fibrillation, whereas light scattering and 8-anilino-1-naphthalenesulfonate fluorescence monitor both amyloid fibrillation and amorphous aggregation. The amyloid fibrils formed via a nucleation-dependent mechanism in a supersaturated solution, analogous to crystallization. The lag phase of fibrillation was reduced upon agitation with stirring or ultrasonic irradiation, and disappeared by seeding with preformed fibrils. In contrast, the glass-like amorphous aggregates formed rapidly without a lag phase. Neither agitation nor seeding accelerated the amorphous aggregation. Thus, by monitoring the kinetics, we can distinguish between crystal-like amyloid fibrils and glass-like amorphous aggregates. Solubility and supersaturation will be key factors for further understanding the aberrant aggregation of proteins.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
Formation of β2m amyloid fibrils and amorphous aggregates at 0 (gray), 100 (red), and 1000 (blue) mM NaCl monitored by measuring light scattering at 350 nm (A) and ThT fluorescence at 480 nm (B). The solution was subjected to agitation with a stirring magnet. The inset in panel A is a close-up view of the early time course at 1000 mM NaCl.
Fig. 2.
Fig. 2.
Effects of the NaCl concentration on the kinetics and morphology of β2m aggregation. The β2m solutions were subjected to agitation with a stirring magnet at 600 rpm. (A, B) Dependencies on the NaCl concentration of the maximum ThT fluorescence at 480 nm (A) and the lag time of the increase in ThT fluorescence at 480 nm (B). The solid lines are drawn as an eye-guide. In panel B, the lag time at 0, 900, and 1000 mM NaCl was not able to be quantified because ThT fluorescence did not show a significant enhancement (see Fig. S2). (C–F) AFM images of the β2m aggregates at 100 (C), 300 (D), 500 (E), and 1000 (F) mM NaCl. The white bars represent 1 μm. (G–J) TEM images of the β2m aggregates at 100 (G), 300 (H), 500 (I), and 1000 (J) mM NaCl. The black bars represent 250 nm.
Fig. 3.
Fig. 3.
Effects of various forms of agitation on the growth of β2m amyloid fibrils at 100 (A) and 1000 (B) mM NaCl monitored by measuring light scattering at 350 nm. The solutions of β2m were agitated with a stirring magnet or irradiation with ultrasonication pulses. The effects of seeding were also examined. Light scattering intensity at 350 nm was monitored as a function of time. For comparison, the kinetics under quiescent conditions are also shown. When the effects of ultrasonic pulses or seeding were examined, the solutions were stirred.
Fig. 4.
Fig. 4.
Effects of agitation on the growth of β2m amyloid fibrils monitored by measuring ThT fluorescence at 480 nm (A–D) and ANS fluorescence at 480 nm (E–H). The solutions of β2m were agitated with a stirring magnet or irradiation with ultrasonic pulses. The effects of seeding were also examined. The NaCl concentrations were 100 (A, E), 300 (B, F), 500 (C, G), and 1000 (D, H) mM NaCl. When the effects of ultrasonic pulses or seeding were examined, the solutions were stirred.
Fig. 5.
Fig. 5.
Schematic diagrams for the phase transitions producing distinct aggregates. (A) NaCl-dependent conformational transition of a folded protein (e.g. hen egg lysozyme at pH 4.7). As a representative parameter, specific volume is plotted against NaCl concentration. (B) Phase diagrams of β2m at pH 2.5 depending on the NaCl and β2m concentrations. Agitation causes a downward shift of the metastability curve, which is indicated by an arrow. Filled circles indicate the experimental conditions employed in this study, illustrating the agitation-triggered fibrillation at 100 mM NaCl. In panels A and B, Regions 1, 2, 3, and 4 represent thermodynamically stable region with soluble monomers, metastable region without spontaneous nucleation, labile region with spontaneous nucleation, and glass region produced with too many nuclei, respectively. (C) General phase diagram of the conformational states of peptides and proteins dependent on conformational uniqueness and concentration. The representative conformational states are monomers, crystals, amyloid fibrils, and amorphous aggregates. In this phase diagram, amorphous aggregates of unfolded proteins and those of folded proteins are not distinguished. As for crystallization and amyloid fibrillation, supersaturation critically determines the phase transition as shown in panels A and B.
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