Metabolites Shift Equilibria of Biomolecular Condensates

Metabolites are abundant in cells, where condensation of proteins and nucleic acids can organize cellular contents without membranes. Condensates form and dissolve dynamically in response to diverse cellular processes. Understanding condensate phase behavior using full phase diagrams is hampered by the sheer number of distinct molecular species involved. To circumvent this limitation, we introduce susceptibility, a dimensionless descriptor that quantifies dilute-phase responses to solute perturbations and enables direct comparison across condensates. We measure how three model condensates, assembled by distinct cohesive mechanisms, respond to amino acids, nucleotides, and a crowder. We find that these small molecules generically modulate condensate phase equilibria, with susceptibilities spanning over five orders of magnitude. These magnitudes reflect underlying molecular interactions, consistent with theoretical descriptions of condensation including Flory-Huggins and polyphasic linkage theories. We extend susceptibility to multicomponent perturbations by expressing the response as a weighted sum of individual solute perturbations. Applying these principles, we exploit enzymatic activity to induce condensation and modulate material properties. Our work establishes metabolites as generic modulators of biomolecular condensates with relevance to cellular physiology and provides a framework for programming condensates with desired phase and material properties. SIGNIFICANCE STATEMENTCells compartmentalize biochemistry using biomolecular condensates formed by phase separation. Although cells contain thousands of small molecules, little is known about their influence on condensation. In such complex mixtures, mapping full phase diagrams is infeasible because the required measurements scale exponentially with the number of components. Alternatively, we introduce susceptibility to characterize the system response about a working composition. Working with three distinct model condensates, we show that common metabolites generically modulate phase equilibria, consistent with theories of mass action and phase separation. We apply this framework to induce condensation and tune material properties using enzymatic reactions. Our work provides a framework for understanding and harnessing small molecules to regulate biomolecular condensation.