Enzyme Active Bath Affects Protein Condensation

TL;DR

The study investigates whether an enzymatically active bath can modulate liquid-liquid phase separation (LLPS) of a non-interacting condensate protein, isolating physical activity from chemistry via a chemostatic microfluidic chamber. It combines experimental observations of UBQLN2-450C condensates under a urease-driven active bath with coarse-grained sticker–spacer polymer simulations that are parametrized by the bath Peclèt number $Pe$. The results show that enzymatic activity enhances droplet size, density, and the protein partitioning into the condensed phase, while urease partitioning into droplets is only weakly affected by activity. This supports the interpretation that the active bath acts as an effective temperature, providing tunable control over nanoscale LLPS with potential implications for intracellular organization and active materials design.

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.

Figures (10)

• Figure 1: Model systems for protein phase separation and enzyme activity. (A) Ubiquilin-2 is a model phase separating protein. (i) Schematic of full length ubiquilin-2 (top), and the truncated form from 450-624 aa used in this study (bottom). (ii) Disordered propensity of UBQLN2-450C. (iii) Charge of UBQLN2-450C, at pH 6.8. (B) Urease is the enzyme used to create an active bath. (i) Crystal structure of the urease hexamer (PDB # 3LA4 visualized with VMD HUMP96). (ii) Chemical reaction of urea hydrolysis catalyzed by urease Kot2003. (iii) Schematic of active bath driven by urease enhanced diffusion. Without urea, the system is thermally-driven with no activity (left). With urea, the enzyme activity enhances the diffusion of urease to create an active bath (right).
• Figure 2: Microfluidic device for chemostatic control. (A) Microfluidic chamber with semipermeable membrane walls. (i) Schematic of chamber with three lanes: two outer lanes allow flow to deliver small molecules and remove enzyme catalysis products. The center lane has the experimental system of proteins. (ii) Photograph of the microfluidic chamber. (B) Surface treatment of the chamber. (i) Triblock co-polymer, Pluronic F127 is used to block the surface to inhibit adhesion of UBQN2-450C. (ii) X-Z image of a UBQN2-450C droplet shows no adhesion to the surface. The red, dashed line denotes the surface of the chamber. (C) Microfluidic control of UBQN2-450C phase transition driven with NaCl. Time series showing the formation of condensed UBQN2-450C droplets when 300 mM NaCl is flowed in the outer lanes (top). Condensation is reversed when 0 NaCl is flowed in the outer lanes (bottom). Salt concentration is estimated via 1D diffusion equation with D=$1.958 \times 10^{-9}$$m^2/s$Ghaffari2013.
• Figure 3: Droplet size, number density, and area fraction. (A) Fluorescence image of droplets shows a single slice of the z-scan. (B) Droplet detection using automated particle analysis. (C) CDFs of maximum cross-sectional area of droplets for active bath (orange-filled circles), no activity control (blue-filled circles), and product control (green-filled circles). The CDF is fit with a log-normal CDF function (dash lines). (D) Average droplet area obtained from CDF fit for active condition (orange bar), no activity control (blue bar), and product control (green bar). Error bars represent SEM. Reported p-values calculated using the KS test. (E) Droplet density of active condition (orange bar), no activity control (blue bar) and reaction product control (green bar). (F) Area fraction of droplets calculated with density and average droplet sizes (Supp. Eqn. S2) for active condition (orange bar), the no activity control (blue bar), and the product control (green bar). For the density and area fraction, error bars represent the systematic error due to under-counting.
• Figure 4: UBQL2-450C partition coefficient (PC) quantified for all conditions. (A) Fluorescence image of droplets comparing the active condition (left), the no activity control (middle), and the product control (right) displayed at the same size (scale bar is 10 $\mu m$ or all images) and the same look-up table (AU 61-11321). (B) Quantification method to determine the partition coefficient. (i) Detection and selection of the background region around the droplets to quantify the background intensity with (ii) overlay on the original droplet image. (iii) Detection and selection of the droplets to quantify the droplet intensity with (iv) overlay on the original droplet image. For all images, the green line represents the selection boundary. (C) The average partition coefficient (PC) obtained from CDF fits (Supp. Fig. S1A). (D) Bulk measurement of the dilute phase using spectrophotometry after centrifugation to remove the condensed phase (N = 15 for each condition). Higher absorbance (A.U.) reports higher UBQLN2-450C concentration.
• Figure 5: Active bath controls condensation of sticker-spacer polymers. (A) Snapshots of sticker–spacer polymers in (i) a passive bath ($Pe = 0$) and (ii) an active bath ($Pe = 0.5$). (B) Average size (fraction of particles) of the droplets. (C) Average partition coefficient for polymers and bath particles as a function of $Pe$, at fixed polymer binding affinity $\epsilon_{AB} = 5$.