Interplay between Local Diffusion, Concentration, and Inter-Protein Alignment Promotes Cross-β-Sheet Transitions at Condensate Interfaces

Biomolecular condensates govern cellular organization through dynamic, membraneless compartments whose material properties are dictated by intermolecular interactions. While liquid–liquid phase separation enables reversible condensate self-assembly, its dysregulation can trigger the formation of pathological solid-like states, often associated with neurodegenerative disorders. Here, we investigate the molecular mechanisms underlying condensate solidification, focusing on the role of inter-protein β-sheet transitions. Using a minimal protein coarse-grained model, we reveal that the condensate’s interface acts as a hotspot for inter-protein β-sheet nucleation and growth. Residue- and segment-resolved analyses show that enhanced interfacial mobility, particularly of terminal domains, coupled with locally high protein concentrations, creates optimal conditions for inter-protein β-sheet-sheet formation. Moreover, favourable inter-protein orientational alignment of terminal domains emerges at the interface. We find that asymmetric sequences—e.g. those with defined hydrophobic vs. more hydrophilic regions—further amplify this effect, resulting in local density fluctuations which concentrate aggregation-prone domains at the interface, and collectively promote inter-protein β-sheet transitions. Such amphiphilic interfacial organization is driven by surface-tension minimization, and stabilizes protein structural rearrangements accentuating the spatial bias of inter-protein structural transitions. Altogether, our results demonstrate that general protein polymer-physics models, capturing only essential features such as flexibility and sequence patterning, inherently display condensate solidification at the interface. These findings identify the interface as a central regulator of condensate hardening, linking molecular-scale interactions to mesoscale phase behaviour, and providing mechanistic insight into the spatiotemporal onset of protein dysregulation in membraneless organelles.