BPS2026 – Mechanistic insights into the liquid-liquid phase separation of Boc-protected amino acids from simulation and experiment

Biomolecular phase separation provides spatial organization within cells to facilitate key cellular processes. The molecular drivers of this liquid-liquid phase separation of proteins and nucleic acids are an active area of study, and the precise interactions and combinations of residues in protein sequences that drive de-mixing are an area of intense research interest. We observed that some Boc-protected amino acids form liquid-like condensates in an ethanol-water solvent system. By using these short peptides as a prototypical system for protein phase separation, we can systematically explore the impact of diverse side chain chemistries on peptide condensation by simply changing the protected amino acid’s identity. Assaying a collection of protected amino acids underscored the importance of hydrophobicity as a driver of the peptides’ de-mixing into a stable dense phase. We combined these experimental observations with molecular dynamics simulations of the peptide-solvent systems to gain mechanistic insights into the interactions driving and maintaining their phase separation. Our atomistic simulations, which reproduce the amino acid-specific trends in phase separation, provide details on the composition of the dense phase and of the interfacial region. The simulations reveal the role of peptide-solvent interactions and of the peptide protonation state in promoting phase separation, which are supported by further experimental observations.