Aromatic regions and Charges of amino acids Govern the Phase-separating Behavior of Dimeric Peptides

Intrinsically disordered proteins (IDPs) can undergo intracellular liquid-liquid phase separation (LLPS) to form membraneless compartments in cells. However, the existing molecular grammar for LLPS are mainly on IDPs, the sequence features that determine the phase behavior of minimalistic peptide remain elusive. Here, we present a library of simplified cysteine-containing peptide stickers to probe the phase separation behavior of their oxidized dimeric forms. Our findings reveal that LLPS of these simple peptide models are primarily governed by apparent ratio of arginine residues to aromatic residues (Rarg/aro) and the specific type of aromatic residues. The measured saturation concentrations (Csat) for LLPS display a linear relationship with the hydrophobicity of the aromatic residues, highlighting their critical role in phase behavior. Capitalizing on these observations, we incorporated functional residues, such as RGD motifs and catalytic triads (SHD), into the peptide designs to enhance their functionality. Furthermore, redox-active disulfide spacers enabled precise control over condensation and dissolution in response to glutathione, facilitating intracellular delivery and glutathione-mediated release of cargos, including mRNA. These results establish a versatile framework for designing and predicating peptide-based coacervates, advancing their potential applications in intracellular delivery, mRNA vaccines, and understanding the origins of life.