(126l) Bridging Residue-Level Thermodynamics with Sequence-Dependent Phase Behavior of Intrinsically Disordered Proteins

Like the sequence-structure-function relationship in structured proteins, the primary structure of intrinsically disordered proteins (IDPs) has been shown to impact the formation of biomolecular condensates. This sequence-dependence can be rationalized as a consequence of alterations in the balance of protein-water and protein-protein interactions upon mutation. However, a precise understanding of how specific amino acids alter this balance is still lacking. Previous work has identified a number of key residues and sequence features that underlie the formation and stabilization of biomolecular condensates of IDPs. The heuristics proposed place an emphasis on certain classes of amino acids, namely aromatic and cationic, as primary drivers of phase separation through the formation of favorable protein-protein interactions. In this work, we examine the contributions of amino acids outside of the proposed heuristics in determining the sequence-phase behavior relationship. Through sequence manipulations of a representative model peptide, we find that rather than just a few key residues, all residues present within the protein sequence determine the phase behavior of the protein, pointing to a model of all residues partaking in favorable protein-protein interactions providing the driving forces for phase separation. To better understand the role of solvent in determining the favorability of phase separation, we considered whether solvation thermodynamics could account for the observed sequence-dependent phase behavior of the model polypeptide and found that hydrophobicity alone could not describe the underlying thermodynamics of phase separation. Through alchemical free energy calculations, we uncover the roles of protein and water within the condensed phase, and bulk-like water in the dilute phase in determining the favorability of transfer of amino acid side chain analogs from the water rich dilute phase to the protein rich dense phase. We find that the complex interior within the condensate, consisting of protein and water which is structurally distinct from bulk water, leads to residue level driving forces that cannot adequately be explained by solvation thermodynamics alone. To conclude, in this work we highlight the role of multiple diverse interactions encoded by all residues in a sequence in determining its phase behavior, and provide a molecular picture of the forces underlying to formation and stabilization of biomolecular condensates at the amino acid level.