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

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, β2-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.