Fibrils emerging from droplets: Molecular guiding principles behind phase transitions of short peptide-based condensates

Biochemical reactions occurring in highly crowded cellular environments require different means of control to ensure productivity and specificity. Compartmentalization of reagents by liquid-liquid phase separation is one of these means. However, extremely high local protein concentrations of up to 400 mg/ml can result in pathological aggregation into fibrillar amyloid structures, a phenomenon that has been linked to various neurodegenerative diseases. Despite its relevance, the process of liquid-to-solid transition inside condensates is still not well understood at the molecular level. In this work, we use small peptide derivatives that can undergo both liquid-liquid and subsequent liquid-to-solid phase transition as model systems to study both processes at the molecular level. Using solid-state nuclear magnetic resonance (NMR) and transmission electron microscopy, we compare the structure of condensed states of leucine, tryptophan and phenylalanine containing derivatives, distinguishing between liquid-like condensates, amorphous aggregates and fibrils, respectively. A structural model for the fibrils formed by the phenylalanine derivative was obtained by a structure calculation based on NMR distance restraints. Our results show that the fibrils are stabilised by hydrogen bonds and side-chain π-π interactions, which are likely much less pronounced or absent in the liquid and amorphous state. Such noncovalent interactions are equally important for the liquid-to-solid phase transition of proteins, particularly those related to neurodegenerative diseases and our results suggest that aged condensates of these proteins may have partial amyloid-like characteristics.