Regulation of enzymatic reactions by chemical composition of peptide biomolecular condensates

Abstract

Biomolecular condensates are condensed intracellular phases that are formed by liquid-liquid phase separation (LLPS) of proteins, either in the absence or presence of nucleic acids. These condensed phases regulate various biochemical reactions by recruitment of enzymes and substrates. Developments in the field of LLPS facilitated new insights on the regulation of compartmentalized enzymatic reactions. Yet, the influence of condensate chemical composition on enzymatic reactions is still poorly understood. Here, by using peptides as minimalistic condensate building blocks and β-galactosidase as a simple enzymatic model we show that the reaction is restricted in homotypic peptide condensates, while product formation is enhanced in peptide-RNA condensates. Our findings also show that condensate composition affects the recruitment of substrate, the spatial distribution, and the kinetics of the reaction. Thus, these findings can be further employed for the development of microreactors for biotechnological applications.

Fig. 1. Regulation of enzymatic reactions in homotypic vs. heterotypic condensates.

Schematic illustration showing the three condensate model systems (from left to right): homotypic condensate formed by peptide LLPS, heterotypic condensate formed by peptide-peptide LLPS, and heterotypic condensate formed by peptide-RNA LLPS. β-gal-catalyzed hydrolysis of 4-MUG to the fluorogenic product 4-MU is performed in each of the condensate systems.

Fig. 2. Hydrolysis of 4-MUG in designed biomolecular condensates.

Fluorescence spectroscopy analysis of product formation over time (λ_ex = 320 nm and λ_em = 450 nm). a–c Kinetics of β-gal-catalyzed 4-MUG hydrolysis in the absence of condensates (black line) or in homotypic (a, gray line), heterotypic peptide-peptide (b, blue line) or peptide-RNA (c, red line) condensates. d–f Initial reaction rate of d the free enzyme, e enzymatic reaction in peptide-peptide condensates, and f enzymatic reaction in peptide-RNA condensates as a function of substrate concentration. Values represent an average of three independent measurements; error bars represent SD. All measurements represent the total contribution of the dilute and condensed phases.

Fig. 3. Condensate composition affects recruitment of substrate and enzyme.

a, b Encapsulation efficiency (EE) of Atto633-labeled β-gal and 4-MUG in either homotypic (gray), heterotypic peptide-peptide (blue) or peptide-RNA (red) condensates. EE of Atto633-β-gal was analyzed using confocal microscopy analysis at λ_ex = 640 nm (a), and the EE of 4-MUG was analyzed by fluorescence spectroscopy at λ_ex = 315 nm and λ_em = 370 nm (b). Values represent an average of 3 independent measurements; error bars represent SD. c Confocal microscopy images of encapsulated Atto633-β-gal in the different condensate systems obtained using λ_ex = 640 nm. Scale bars = 50 µm.

Fig. 4. Spatial regulation of 4-MU formation in homotypic and heterotypic condensates.

a, b, c Confocal microscopy images of 4-MU formation over time in a homotypic, b heterotypic peptide-peptide or c peptide-RNA condensates. Images were acquired using a λ_ex = 405 nm laser and using z-stacking analysis. All images represent the middle section of a z-stack. The transmitted light images of condensates (left) were taken at t = 0 min. Scale bars = 50 µm d. Fluorescence intensity of 4-MU in homotypic (gray), peptide-peptide (blue), or peptide-RNA (red) condensates over time, obtained by confocal microscopy analysis. Data represent the average of N = 30 condensates from 3 independent experiments for the heterotypic systems (10 droplets from each experiment), and N = 20 condensates from 2 independent experiments for the homotypic condensates. Error bars represent SD.

Fig. 5. Peptide diffusivity in homotypic vs. heterotypic peptide-peptide and peptide-RNA condensates.

Fluorescence recovery after photobleaching (FRAP) analysis of homotypic, heterotypic peptide-peptide and peptide-RNA condensates, performed using FITC-labeled peptides. Homotypic, peptide-peptide and peptide-RNA condensates were formed by 100 μM/20 mM, 125 μM/5 mM, and 50 μM/2 mM unlabeled/labeled peptides, respectively. a Confocal fluorescence images of condensates before, immediately after, and 35 sec after photobleaching. Analysis was performed using λ_ex = 488 nm laser. Scale bars = 5 µm. b–d Recovery plots from FRAP analysis of b homotypic, c heterotypic peptide-peptide and d peptide-RNA condensates. e t_1/2 values calculated from recovery plots. Values of recovery plots and t_1/2 represent averages of N = 7, 6, and 8 for homotypic, peptide-peptide, and peptide-RNA condensates, respectively. Error bars represent SD.

Fig. 6. Kinetics parameters of enzymatic reaction inside condensates with varying hydrophobicity.

a, b Maximum velocity (a) and catalytic coefficient (K_cat) (b) of enzymatic reaction in peptide-peptide condensates formed by cationic peptides with varying hydrophobicity. According to a one-way ANOVA test, the differences between the parameters are significant. c, d Maximum velocity (c) and catalytic coefficient (K_cat) (d) in peptide-RNA condensates formed by cationic peptides with varying hydrophobicity. According to a one-way ANOVA test, the differences between the parameters are not significant.