Electrostatic-Hydrophobic Competition Induces Re-Entrant Phase Separation in Designer Polypeptides

The liquid-liquid phase separation (LLPS) of intrinsically disordered proteins is governed by a delicate balance among electrostatic, hydrophobic, and aromatic interactions, yet how residue identity reshapes this balance remains incompletely understood. Although significant progress has been made toward establishing a molecular grammar linking sequence to phase behavior, the specific roles of individual hydrophobic residues in modulating the electrostatic-hydrophobic competition have not been systematically dissected. Here, we use a residue-level coarse-grained model to examine the phase behavior of artificially designed peptides with controlled charge patterning and amino acid composition. We find that even when the arrangement of charged and neutral residues is kept fixed, the identity of the neutral residue leads to qualitatively distinct phase behaviors. Phenylalanine monotonically enhances dense-phase formation, whereas alanine progressively suppresses it. In contrast, isoleucine produces a striking re-entrant behavior: increasing its fraction first weakens condensate formation by disrupting electrostatically stabilized associations, but beyond an intermediate composition, effective hydrophobic interactions become dominant and drive the re-emergence of a dense yet relatively loose and dynamic phase. These results illustrate that hydrophobic residues do not merely modulate LLPS strength, but can fundamentally alter the competition between interaction modes and thereby change the phase-separation regime. Our findings are consistent with a picture in which sequence composition modulates condensate formation via a balance among electrostatic, hydrophobic, and aromatic-type interactions, as captured by our coarse-grained model. Preliminary tests on LAF-1-RGG mutants further suggest that these trends may be transferable to biologically relevant IDRs. Together, this work provides additional insights into sequence-dependent LLPS and may inform the rational design of condensates with tunable material properties.