Phase Behavior of an Fc-Fusion Protein Reveals Generic Patterns of Ion-Specific Perturbation on Protein-Protein Interactions

Modulation of phase separation temperature (Tph) for liquid-liquid phase separation of an Fc-fusion protein was studied at pH values below, near, and above its pI where the net charge of the protein was positive, neutral, and negative, respectively, in KF, KCl, KSCN, and MgCl2 solutions. At the pH value near the pI, the monotonic drop in Tph for all the salt solutions suggests that both the anion and cation can disrupt attractive protein-protein interactions, effectively salting-in the protein. At the pH below or above the pI, the counter-ion neutralizes the net charge on the protein. The neutralization reduces repulsive protein-protein interactions while the co-ion effectively strengthens them. Then, salting-in behavior appears after the completion of charge neutralization. Last, the complex ion-specific modulation on Tph could be rationalized through the rankings of SCN− > Cl− > F− and Mg2+ > K+ for their interactions with the protein throughout the pH conditions.