Interplay of Noncovalent Interactions in Phase Separation Mediated by Tyrosine-Rich and Arginine-Rich Polypeptides.

Liquid-liquid phase separation (LLPS) and other forms of condensed phase formation play important roles in many cellular processes. Elucidating the noncovalent interaction networks that underlie condensate formation is a fundamental challenge. Cation-π interactions between Arg and Tyr side chains have been proposed as a driving force for many forms of protein-mediated condensate formation. Efforts to probe this hypothesis with ribosomally generated proteins are constrained by limits on residue incorporation. To transcend these limits, we developed a two-component system comprising a long Arg-rich protein fragment, generated via heterologous expression, and a short anionic Tyr-rich peptide, generated via chemical synthesis. Phase separation occurred when these components were mixed at low concentrations (5 μM each). Global replacements of Tyr with noncanonical residues were conducted to interrogate side chain contributions to condensate formation. The results suggest that cation-π interactions are not essential for phase separation in our system, and that even when cation-π interactions contribute, their role may not be dominant. H-bond donor properties of the Tyr side chain hydroxyl appear to play a significant role, along with Coulombic forces, in driving condensate formation mediated by the anionic Tyr-rich peptide and the Arg-rich protein fragment.