(169ah) Uncovering Residue-Level Driving Forces Underlying the Formation of Biomolecular Condensates

The self-assembly of intrinsically disordered proteins (IDPs) into membraneless compartments, termed membraneless organelles (MLOs) or biomolecular condensates, has implications in understanding cellular function, as well as the design novel biomaterials. Despite the interest in MLOs, a fundamental understanding of the thermodynamic driving forces linking amino acid sequence to phase behavior remains elusive. It has been hypothesized that certain sequence features, specifically, the presence of aromatic residues, and charged residues particularly Arg, encode the phase behavior of prion-like IDPs. Here,¹ we target previously overlooked sequence features through the design of sequence variants of a model polypeptide to investigate if, and how, amino acids and sequence features that fall outside of the existing molecular grammar alter phase behavior. We find that all but one of the sequences designed undergo phase separation, pointing to a model of all residues collectively contributing to the driving forces for phase separation, rather than just a handful. Further, we find that the impact of residue substitutions on the driving forces for phase separation are not explained by their solvation thermodynamics. To identify the cause for the deviations from bulk-phase hydrophobicity measures, we perform alchemical free energy calculations to quantify the free energy of transfer of amino acid sidechain analogs from the water-rich dilute phase to the protein-rich dense phase. We observe a complex interplay between protein-sidechain interactions within the condensate, and sidechain-solvent interactions within the dilute and condensed phases in determining the net favorability or unfavorability of transfer free energies of all amino acids. To summarize, in this work we uncover the contributions of residues previously overlooked in the molecular grammar of phase separation, as well as provide a molecular picture of the forces underlying their contribution to formation and stabilization of biomolecular condensates.

References

1. Rekhi, Shiv, et al. "Expanding the molecular language of protein liquid–liquid phase separation." Nature Chemistry (2024): 1-12.