Single Amino Acid Substitutions in Stickers, but Not Spacers, Substantially Alter UBQLN2 Phase Transitions and Dense Phase Material Properties

UBQLN2 450-624 oligomerizes and undergoes temperature-responsive liquid-liquid phase transitions following a closed-loop temperature-concentration phase diagram. We recently showed that disease-linked mutations to UBQLN2 450-624 impart highly varying effects to its phase behavior, ranging from little change to significant decrease of saturation concentration and formation of gels and aggregates. However, how single mutations lead to these properties is unknown. Here, we use UBQLN2 450-624 as a model system to study the sequence determinants of phase separation. We hypothesized that UBQLN2 450-624 regions previously identified to promote its oligomerization are the "stickers" that drive interchain interactions and phase separation. We systematically investigated how phase behavior is affected by all 19 possible single amino acid substitutions at three sticker and two "spacer" (sequences separating stickers) positions. Overall, substitutions to stickers, but not spacers, substantially altered the shape of the phase diagram. Within the sticker regions, increasing hydrophobicity decreased saturation concentrations at low temperatures and enhanced oligomerization propensity and viscoelasticity of the dense phase. Conversely, substitutions to acidic residues at all positions greatly increased saturation concentrations. Our data demonstrate that single amino acid substitutions follow a molecular code to tune phase transition behavior of biopolymers.