Amino Acid Transfer Free Energies Reveal Thermodynamic Driving Forces in Biomolecular Condensate Formation.

The self-assembly of intrinsically disordered proteins into biomolecular condensates shows a dependence on the primary sequence of the protein, leading to sequence-dependent phase separation. Methods to investigate this sequence-dependent phase separation rely on effective residue-level interaction potentials that quantify the propensity for the residues to remain in the dilute phase versus the dense phase. The most direct measure of these effective potentials are the distribution coefficients of the different amino acids between the two phases, but due to the lack of availability of these coefficients, proxies, most notably hydropathy, have been used. However, recent work has demonstrated the limitations of the assumption of hydropathy-driven phase separation. In this work, we address this fundamental gap by calculating the transfer free energies associated with transferring each amino acid side chain analog from the dilute phase to the dense phase of a model biomolecular condensate. We uncover an interplay between favorable protein-mediated and unfavorable water-mediated contributions to the overall free energies of transfer. We further uncover an asymmetry between the contributions of positive and negative charges in the driving forces for condensate formation. The results presented in this work provide an explanation for several non-trivial trends observed in the literature and will aid in the interpretation of experiments aimed at elucidating the sequence-dependent driving forces underlying the formation of biomolecular condensates.