Prediction and characterization of liquid-liquid phase separation of minimalistic peptides

Liquid-liquid phase separation (LLPS) of proteins mediates the assembly of biomolecular condensates involved in physiological and pathological processes. Identifying the minimalistic building blocks and the sequence determinant of protein phase separation is an urgent need but remains challenging partially due to lack of methodologies to characterize the phase behavior. Here, we demonstrate computational tools to efficiently quantify the microscopic fluidity of liquid condensates and the temperature-dependent phase diagram of peptides. We comprehensively explore the LLPS abilities of all 400 dipeptide combinations of coded amino acids and observe the occurrences of spontaneous LLPS in three categories of dipeptides. Our predictions are validated by turbidity assays and differential interference contrast (DIC) microscopy. We demonstrate that dipeptides, minimal but complete, possess multivalent interactions sufficient for LLPS, suggesting LLPS is a general property of peptides and proteins, independent of their sequence length. This study paves the way for the prediction and characterization of peptide phase behavior at the molecular level.