On the Use of Double Mutant Cycles to Probe the Molecular Interactions in Biomolecular Condensates

Disordered proteins can form biomolecular condensates by demixing from their environment, enabling reversible compartmentalisation of cellular components in the form of membraneless organelles. Multivalent interactions are essential for this type of phase separation behaviour, and for disordered proteins, the potential for multivalent interactions is encoded in the sequence composition and patterning. Mutational studies have been instrumental in helping elucidate this sequence grammar by perturbing the amino acid sequence and quantifying the resulting changes in the driving force for phase separation. While such studies have provided a detailed and predictive understanding of the driving forces for phase separation, they strictly do not inform on the nature of the interactions that drive phase separation. Here, we propose using double mutant cycles to explore molecular interactions and their contributions to condensate properties more directly. We explore the applicability of double mutant cycles for different types of interactions in condensates formed by the low-complexity domain of hnRNPA1 using coarse-grained molecular dynamics simulations. We find that the interactions between arginine and tyrosine residues, as well as between aromatic residues, contribute mostly additively to the propensity for phase separation. However, for the interactions between charged residues, we find that--in an interplay with the net charge of the protein--there is a measurable non-additive contribution to the phase separation propensity. Based on our results, we envisage that double mutant cycles could provide additional insights into protein phase separation, thus expanding the understanding of the sequence grammar and the underlying molecular interactions.