A BACKGROUND ENZYMATIC ACTIVE BATH AFFECTS LIQUID-LIQUID PHASE SEPARATION OF PROTEINS

The cell interior has been suggested to be an active bath driven by a myriad of enzymes. It is an open problem as to how this background activity can affect physical processes in the cell. A biologically relevant process in cells is the formation of membraneless organelles, which are biomolecular condensates that phase separate via liquid-liquid phase separation. We seek to experimentally reconstitute a model system for an active bath of enzymes to determine the effects on the liquid phase separation of a model condensate protein. We use urease, an exothermic and kinetically fast enzyme that converts urea to carbon dioxide and ammonia, as the background enzyme. We use ubiquilin-2 (UBQLN2), a protein that phase separates with high salt and high temperature. Using a unique microfluidic chamber that feeds the enzyme while removing the products, we created a gradient of salt to image the complete phase diagram at one time. We turn on the enzyme and see the phase diagram shifts with enzyme activity. In a complementary approach, we used a UV-Vis Spectroscopy to find the temperature of the phase transition, with and without the enzyme activity. Results from both of the experiments are consistent with the concept of effective temperature set by the activity of enzyme in the active bath.