Intrinsically disordered regions that drive phase separation form a robustly distinct protein class.

Protein phase separation is thought to be a primary driving force for the formation of membrane-less organelles, which control a wide range of biological functions from stress response to ribosome biogenesis. Among phase-separating (PS) proteins, many have intrinsically disordered regions (IDRs) that are needed for phase separation to occur. Accurate identification of IDRs that drive phase separation is important for testing the underlying mechanisms of phase separation, identifying biological processes that rely on phase separation, and designing sequences that modulate phase separation. To identify IDRs that drive phase separation, we first curated datasets of folded, ID, and PS ID sequences. We then used these sequence sets to examine how broadly existing amino acid property scales can be used to distinguish between the three classes of protein regions. We found that there are robust property differences between the classes and, consequently, that numerous combinations of amino acid property scales can be used to make robust predictions of protein phase separation. This result indicates that multiple, redundant mechanisms contribute to the formation of phase-separated droplets from IDRs. The top-performing scales were used to further optimize our previously developed predictor of PS IDRs, ParSe. We then modified ParSe to account for interactions between amino acids and obtained reasonable predictive power for mutations that have been designed to test the role of amino acid interactions in driving protein phase separation. Collectively, our findings provide further insight into the classification of IDRs and the elements involved in protein phase separation.