Size of Biomolecular Condensates Dictates Fate in Liquid-Solid Phase Transitions through Amorphous-Amyloid Competition.

Proteins function not only through intramolecular folding and intermolecular complex formation but also through phase transitions driven by intermolecular interactions. Such phases, including liquid-like condensates, amorphous aggregates (AAs), and amyloid fibrils, are linked to distinct biological functions and pathologies. Although the transition from liquid-like condensates to amyloids has been extensively studied, the kinetic relationships between amyloids and other metastable solid states under cell-sized confinement remain unclear, which may hinder the establishment of effective therapeutic strategies. This knowledge gap arises because bulk-scale experiments inevitably lead to the conversion of metastable phases into the most stable phase. We developed a droplet-based microfluidic system that quantifies amyloid nucleation and metastable AA formation. Using the yeast prion protein Sup35, we found that condensates convert into both amyloids and AAs and that AA formation imposes a kinetic barrier that suppresses amyloid formation in a size-dependent manner at the micrometer scale, highlighting the importance of size effects in condensate-to-amyloid transitions. Furthermore, we demonstrated that the well-known amyloid inhibitor (-)-epigallocatechin-3-gallate paradoxically promoted amyloid formation at low concentrations by modulating the AA and amyloid nucleation kinetics. This phenomenon cannot, in principle, be observed in bulk-scale experiments and became apparent only under micrometer-scale confinement in the present system. These findings provide fundamental insights into protein phase transitions in cellular environments and may guide the development of novel therapeutic strategies targeting the metastable aggregates of amyloidogenic proteins.