Enzyme Active Bath Affects Protein Condensation

TL;DR

This study demonstrates that enzymatic activity in an active bath can enhance protein phase separation by acting as an effective temperature, influencing droplet size, density, and concentration, with implications for cellular organization.

Contribution

It provides the first experimental evidence that enzyme activity can modulate liquid-liquid phase separation by functioning as an effective temperature.

Findings

Active enzyme baths increase droplet size and density.

Enzymatic activity enhances protein condensation.

Results suggest enzyme activity acts as an effective temperature.

Abstract

We investigate how an active bath of enzymes influences the liquid-liquid phase separation (LLPS) of a non-interacting condensing protein. The enzyme we choose to use as the active driver is urease, an enzyme that has been shown by several groups to exhibit enhanced diffusion in the presence of its substrate. The non-interacting LLPS protein is ubiquilin-2, a protein that condenses with increasing temperature and salt. Using a microfluidic device with semipermeable membranes, we create a chemostatic environment to maintain the substrate content to feed the enzymatic bath and remove the products of the chemical reaction. Thus, we isolate the physical enhanced fluctuations from the chemical changes of the enzyme activity. We also compare the results to controls without activity or in the presence of the products of the reaction. We find that the active bath is able to enhance droplet size, density, and concentration, implying that more ubiquilin-2 is in condensed form. This result is consistent with an interpretation that the active bath acts as an effective temperature. Simulations provide an underlying interpretation for our experimental results. Together, these findings provide the first demonstration that physical enzymatic activity can act as an effective temperature to modify LLPS behavior, with implications for intracellular organization in the enzymatically active cellular environment.