Comprehensive Regulation of Liquid–Liquid Phase Separation of Polypeptides

Abstract

The elucidation of the molecular driving forces responsible for Liquid–liquid Phase Separation (LLPS) of proteins and nucleic acids within living cells is crucial for understanding its biological functions and its role in related diseases. In the present study, we investigated the regulation of LLPS in a series of polypeptides with repetitive proline and arginine (PR) sequences by modifying their length and the salt concentration in the solution. Our findings indicate that higher salt concentrations are necessary for LLPS of repetitive PR peptides longer than eight PRs, which emerges as a threshold value. To pinpoint the molecular forces driving the LLPS in peptides, we sequentially introduced various concentrations of hydrophobic disruptors, such as 1,6-hexanediol, and electrostatic regulators, such as ethyl alcohol and 6-Aminocaproic acid. We further modulated the electrostatic interaction by introducing ethyl alcohol and 6-Aminocaproic acid to alter the dielectric constant of the solution. The inclusion of ethyl alcohol intensified the electrostatic interaction between arginine molecules, facilitating LLPS of PR15, while 6-Aminocaproic acid yielded the reverse effect. We deduced that the phase separation in peptide systems is conjointly driven by hydrophobicity and electrostatic interactions. These insights can guide the regulation of LLPS in other peptide and protein systems, and could be pivotal in addressing abnormal aggregations of proteins and nucleic acids.