Fig. 3: Modulation of enzymatic activity in nanoclusters.

A Initial rates of homogeneous NOX solutions at 280 nM and 1 µM (gray), of heterogeneous systems formed by the different fusion proteins at 1 µM protein concentration (“Total (2 phases)”), and of the dilute phase in equilibrium with the dense phase in the heterogeneous systems (“Dilute phase”). n ≥ 3 independent experiments. Data were presented as mean values ± SEM. B Size distribution of the dilute phase after removing micron-sized condensates by centrifugation measured by dynamic light scattering, showing the presence of nanoclusters. C Schematic phase diagram showing the protein concentration at which nanoclusters were formed (280 nM, red square), and the c_sat (300 nM, blue circle). The Y-axis represents temperature normalized by the interaction coefficient (χ). D, E Size distribution of 280 nM protein solutions in 25 mM Tris, 20 mM NaCl, pH 7.5 measured by dynamic light scattering (D) and nanoparticle tracking analysis (E), showing the presence of nanoclusters. F Presence and absence of nanoclusters as detected by dynamic light scattering, indicated by green circles and red cross, respectively. The formation of the clusters is abolished at salt concentrations larger than 300 mM. G Initial rates of the solutions at 280 nM in 25 mM Tris at low salt (20 mM NaCl, where nanoclusters are observed, “Nanoclusters c = 280 nM”) and high salt (500 mM NaCl, where the solution is homogeneous, “1 phase c = 280 nM”). The initial rates of the dilute phase in equilibrium with the dense phase in the heterogeneous system reported in panel (A) (“Dilute phase”) are also shown in this panel (G) for comparison. n ≥ 3 independent experiments. Data were presented as mean values ± SEM. Created with BioRender.com.