Aggregation-phase diagrams of β2-microglobulin reveal temperature and salt effects on competitive formation of amyloids versus amorphous aggregates

Abstract

Several serious diseases are associated with crystal-like amyloid fibrils or glass-like amorphous aggregates of denatured proteins. However, protein aggregation involving both types of aggregates has not yet been elucidated in much detail. Using a protein associated with dialysis-related amyloidosis, β-microglobulin (β2m), we previously demonstrated that amyloid fibrils and amorphous aggregates form competitively depending on salt (NaCl) concentration. To examine the generality of the underlying competitive mechanisms, we herein investigated the effects of heat on acid-denatured β2m at pH 2. Using thioflavin fluorescence, CD, and light scattering analysis along with atomic force microscopy imaging, we found that the temperature-dependent aggregation of β2m markedly depends on NaCl concentration. Stepwise transitions from monomers to amyloids and then back to monomers were observed at low NaCl concentrations. Amorphous aggregates formed rapidly at ambient temperatures at high NaCl concentrations, but the transition from amorphous aggregates to amyloids occurred only as the temperature increased. Combining the data from the temperature- and NaCl-dependent transitions, we constructed a unified phase diagram of conformational states, indicating a parabolic solubility curve with a minimum NaCl concentration at ambient temperatures. Although amyloid fibrils formed above this solubility boundary, amorphous aggregates dominated in regions distant from this boundary. Kinetic competition between supersaturation-limited slow amyloid fibrillation and supersaturation-unlimited fast amorphous aggregation deformed the phase diagram, with amyloid regions disappearing with fast titration rates. We conclude that phase diagrams combining thermodynamics and kinetics data provide a comprehensive view of β2m aggregation exhibiting severe hysteresis depending on the heat- or salt-titration rates.

Possible mechanisms of polyphosphate-induced amyloid fibril formation of β 2 -microglobulin

“…2 and SI Appendix, Fig. S1 B and C), we observed a rapid increase in LS followed by a delayed increase in ThT fluorescence after a lag time, indicating kinetic competition between rapid amorphous aggregation or oligomer formation and the slow conversion to amyloid fibrils, as predicted by the competitive mechanism of amorphous aggregation and amyloid formation (1,2,5,11). However, we have no structural or morphological evidence for transient amorphous aggregation, and further studies are required to verify this.…”

“…LS intensity gradually increased and exceeded the detection limit at 2 M orthoP, at which amorphous aggregates dominated. Previous studies showed that although low concentrations of NaCl produced amyloid fibrils, high concentrations of NaCl (e.g., 1.0 M) produced amorphous aggregates, making an optimum for amyloid formation at ∼0.2 M NaCl (1,2,5,12). Thus, the effects of orthoP on amyloid formation are similar to those of NaCl, although amyloid fibrils remained even at 1 M orthoP.…”

“…Orthophosphate (orthoP), diphosphate (diP), tetraphosphate (tetraP), short polyphosphates with 10 to 15 phosphate groups (polyP-S), and long polyphosphates with 60 to 70 phosphate groups (polyP-L) were used to systematically examine the amyloid formation of β2m under acidic and neutral pH conditions. To accelerate supersaturationlimited amyloid formation, we performed experiments under ultrasonic irradiation (1,2,5). We found that, under both acidic and neutral pH conditions, the amyloid formation of β2m was significantly accelerated by polyPs.…”

“…amyloid fibrillation | amorphous aggregation | dialysis-related amyloidosis | polyphosphates | supersaturation A ggregates of denatured proteins may be classified into two types: amyloid fibrils and amorphous aggregates (1)(2)(3)(4)(5). Amyloid fibrils, in which β-strands are aligned perpendicularly to the fibril's axis (6,7), are associated with a wide range of human diseases, such as Alzheimer's disease, Parkinson's disease, and dialysis-related amyloidosis (8,9).…”

“…Amorphous aggregates without an ordered β-sheet structure are common to denatured proteins and are also associated with diseases such cataracts caused by α-crystallin aggregation (10). We proposed that amyloid fibrils and amorphous aggregates are similar to the crystals and amorphous aggregates (or glass) of solutes or substances (1,2,5,11). We suggested a competitive mechanism of protein aggregation in which supersaturation-limited amyloid formation competes with supersaturation-unlimited amorphous aggregation, to obtain a more comprehensive understanding of protein aggregation (1,2,5,11).…”