Polymer Theory for Sequence-Specific Phase Separation Behaviors of Charged Intrinsically Disordered Proteins

Abstract

Liquid-liquid phase separation in biology underpins the formation of mesoscopic, liquid-like assemblies of DNA, RNA, and proteins, termed “membraneless organelles” or “biomolecular condensates”. Without a membrane, these assemblies can respond to environmental stimuli rapidly and play a critical role in many cellular regulation processes. Intrinsically disordered proteins (IDPs), proteins that do not fold when isolation because of the depletion of hydrophobicity and the abundance of polar, charged, and aromatic residues, have been observed to form membraneless organelles in vivo with other biomolecules as well as undergo phase separation in vitro by themselves. The phase separation propensity of an IDP is determined by its amino acid sequence, not only the composition but also the sequence pattern of its amino acids. To investigate this “sequence-specific” phenomenon, we develop a polymer physics theory, the “random-phase approximation (RPA)” theory, for the phase separation of charged IDPs. The theory predicts that the charge sequence pattern and the number of aromatic residues are critical for the phase separation propensity of IDP. We have applied this theory to the RNA helicase IDP Ddx4 and its two mutants with scrambled charge sequence and mutations reducing its aromaticity. Our predictions are consistent with experiments. We also applied the theory to 30 model IDP sequences with the same positively and negatively charged residues but different sequence patterns, and discovered a connection between the sequence-specific single-chain compactness and multi-chain phase separation propensity. Aqueous solutions with two IDP species with different charge sequences have also been addressed by our formulation, whereby a “fuzzy” molecular recognition mechanism supported by the similarity/dissimilarity of the two IDP sequences is indicated. Taken together, our RPA theory establishes a framework for systematically investigating and rationalizing sequence-specific phase separation behaviors of charged biomolecules.