Supersaturation-limited and Unlimited Phase Transitions Compete to Produce the Pathway Complexity in Amyloid Fibrillation

Masayuki Adachi,Masatomo So,Kazumasa Sakurai,József Kardos,Yuji Goto

doi:10.1074/jbc.m115.648139

Masayuki Adachi, Masatomo So + Show 3 more

Open Access

https://doi.org/10.1074/jbc.m115.648139

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Abstract

Although amyloid fibrils and amorphous aggregates are two types of aggregates formed by denatured proteins, their relationship currently remains unclear. We used β2-microglobulin (β2m), a protein responsible for dialysis-related amyloidosis, to clarify the mechanism by which proteins form either amyloid fibrils or amorphous aggregates. When ultrasonication was used to accelerate the spontaneous fibrillation of β2m at pH 2.0, the effects observed depended on ultrasonic power; although stronger ultrasonic power effectively accelerated fibrillation, excessively strong ultrasonic power decreased the amount of fibrils formed, as monitored by thioflavin T fluorescence. An analysis of the products formed indicated that excessively strong ultrasonic power generated fibrillar aggregates that retained β-structures but without high efficiency as seeds. On the other hand, when the spontaneous fibrillation of β2m was induced at higher concentrations of NaCl at pH 2.0 with stirring, amorphous aggregates became more dominant than amyloid fibrils. These apparent complexities in fibrillation were explained comprehensively by a competitive mechanism in which supersaturation-limited reactions competed with supersaturation-unlimited reactions. We link the kinetics of protein aggregation and a conformational phase diagram, in which supersaturation played important roles.

Highlights

Relationship between amyloid fibrils and amorphous aggregates has not yet been elucidated
We previously suggested that amyloid fibrils and amorphous aggregates were similar to the crystals and glasses of substances, respectively [8], and that they are represented by the same phase diagram as often used for crystallization of substances (Fig. 1A)
Ultrasonication-forced Amyloid Fibrillation—We examined the dependence of ␤2m fibrillation on ultrasonic power by using different positions of the ultrasonicator bath

Summary

Introduction

Relationship between amyloid fibrils and amorphous aggregates has not yet been elucidated. Results: A competitive mechanism of amyloid fibrillation and amorphous aggregation reproduced the observed aggregation kinetics of ␤2-microglobulin. Amyloid fibrils and amorphous aggregates are two types of aggregates formed by denatured proteins, their relationship currently remains unclear. We used ␤2-microglobulin (␤2m), a protein responsible for dialysis-related amyloidosis, to clarify the mechanism by which proteins form either amyloid fibrils or amorphous aggregates. On the other hand, when the spontaneous fibrillation of ␤2m was induced at higher concentrations of NaCl at pH 2.0 with stirring, amorphous aggregates became more dominant than amyloid fibrils. These apparent complexities in fibrillation were explained comprehensively by a competitive mechanism in which supersaturation-limited reactions competed with supersaturation-unlimited reactions. We link the kinetics of protein aggregation and a conformational phase diagram, in which supersaturation played important roles

Results

Discussion

Conclusion