Fig. 3.
Modulation of stability of biomolecular condensates formed by scaffolds (valency of 3) and different types of clients assessed through liquid–liquid phase diagrams. Clients are either low-valency proteins (LV), i.e., valency of 2, or higher-than-scaffold valency proteins (HV), i.e., valency of 4. The lines joining the data points in the phase diagrams are shown as a visual guide to facilitate comparison of the coexistence regions. The values on the vertical axes of the phase diagrams have been normalized (for comparison purposes) by dividing over the critical point of the pure scaffold system (i.e., for the 3-valency pure system). (A) Destabilization of liquid-drop formation by adding LV strongly competing clients to the condensate. Throughout, we define strongly competing clients as those that bind to scaffolds at the same sites, and with the same strength, as in scaffold–scaffold interactions. We compare two cases: 67% scaffold proteins (purple triangles) with 33% high-affinity clients versus 33% scaffolds (orange squares) and 67% clients. The dotted line illustrates the constant value of the inverse interaction strength used in Fig. 4 to evaluate the molecular mechanism of condensate formation. (B) Negligible change in the stability of a biomolecular condensate by addition of LV poorly competing clients. Poorly competing clients are defined as those that bind to scaffolds with one-half of the strength of the scaffold self-interactions, while using the same binding sites as those used for scaffold–scaffold interactions. We compare 67% scaffolds (green triangles) with 33% clients versus 33% scaffolds (black squares) with 67% clients. Note that the diluted-liquid branch exhibits higher densities as the proportion of poorly competing clients increases because clients are predominantly excluded from the condensate and, therefore, concentrated in the diluted phase (SI Appendix, Fig. S3). (C) Increase in condensate stability by addition of clients in two scenarios. First, a moderate increase in condensate stability is observed upon addition of 33 LV noncompeting clients (cyan triangles). These clients bind to scaffolds with the same strength as the scaffolds’ self-interactions but use alternate binding sites in the scaffold exclusively devoted to scaffold–client interactions. Second, a significant increase in stability of the condensate is observed upon addition of 33% strongly competing clients with higher-than-scaffold valencies (magenta diamonds). The black arrows indicate the direction toward which the critical parameters move upon the addition of clients. Error bars in the phase diagrams are of the same size as or smaller than the symbols. Typical statistical uncertainties are provided in SI Appendix, Table S5. Numerical values of the critical points are given in SI Appendix, Table S2.