Active droplets through enzyme-free, dynamic phosphorylation

Abstract

Life continuously transduces energy to perform critical functions using energy stored in reactive molecules like ATP or NADH. ATP dynamically phosphorylates active sites on proteins and thereby regulates their function. Inspired by such machinery, regulating supramolecular functions using energy stored in reactive molecules has gained traction. Enzyme-free, synthetic systems that use dynamic phosphorylation to regulate supramolecular processes have not yet been reported, to our knowledge. Here, we show an enzyme-free reaction cycle that consumes the phosphorylating agent monoamidophosphate by transiently phosphorylating histidine and histidine-containing peptides. The phosphorylated species are labile and deactivate through hydrolysis. The cycle exhibits versatility and tunability, allowing for the dynamic phosphorylation of multiple precursors with a tunable half-life. Notably, we show the resulting phosphorylated products can regulate the peptide's phase separation, leading to active droplets that require the continuous conversion of fuel to sustain. The reaction cycle will be valuable as a model for biological phosphorylation but can also offer insights into protocell formation.

Fig. 1. Active coacervate-based compartments by the dynamic phosphorylation of peptides at the expense of a simple phosphorylating agent.

A schematic representation of the chemical reaction cycle coupled to the formation of complex coacervate-based droplets. The chemical reaction cycle converts a deactivated, histidine-containing precursor peptide (blue) to an activated, phosphohistidine-containing product (red) at the expense of the chemical fuel monoamidophosphate (MAP). The precursor acts as a catalyst for the hydrolysis of MAP. The activated product can interact with poly-arginine (purple) to form liquid-liquid phase-separated complex coacervate droplets. The droplets dissolve upon deactivation (dephosphorylation) of the product and the precursor can be recycled.

Fig. 2. Dynamic phosphorylation of amino acids using simple fuels.

a The hydrolysis of the phosphorylating agent MAP. b The amino acid-based catalysts were tested to convert the phosphorylating agent MAP. *pH 10.5 of Tyr containing sample due to low solubility at pH <pK_a. c The phosphorylating agents tested to phosphorylate histidine. **Not suitable due to many side reactions. d The concentration profiles as a function of time. The markers represent data determined by NMR, and the solid line represents a kinetic model. e The reaction network of the cycle with 1-, 3-pHis, and 1,3-bpHis as observed phosphorylated species. All experiments were performed in triplicate, and the conditions used were 75 mM amino acid, 80 mM of MAP, 50 mM DAP or 100 mM TMP in a 500 mM MES buffered solution at pH 6.5.

Fig. 3. Parameters affecting the dynamic phosphorylation of peptides.

a The evolution of the concentration of 3-pHis for Ac-GHG-OH (purple) and His (red). b Addition of pyridine and pH change the observed half-life of 3-pHis. It is increasing with increasing pH and decreasing amount of pyridine. c The structure of the peptide Ac-GHG-OH used in this study. d Representative concentration profiles of 3-pHis at pH 6.5 with increasing concentrations of pyridine. The smaller the squares, the shorter the observed half-life. e Sankey diagram to visualize the phosphate flow in the system (5 mM pyridine, pH 6.5). f, g The percentage of fuel converted to phosphate directly and through pyridine (black) or phosphorylated histidine species (magenta) depends on the pH and the amount of pyridine added. All experiments were performed in triplicate, and the conditions used were 75 mM precursor, 80 mM MAP and pyridine in a 500 mM MES or MOPS buffered solution at pH 5.5, 6.5 or 7.5.

Fig. 4. Dynamic phosphorylation of a peptide forms active compartments.

a A scheme of the chemical reaction cycle combined with complex coacervation-based compartment formation with poly-arginine. b The dynamic phosphorylation-based chemical reaction cycle. c Representative micrographs at various times after adding MAP as fuel. The dotted line represents the water droplet. The micrographs are a maximum z-projection of a z-stack with a pseudocolor-coding. d Image sequence of a FRAP experiment. 500 nM Cy5-R₃₀ was used as a fluorescently labeled peptide. e Normalized intensity of the FRAP experiment over time. The fitting function is shown in red. f The sum of the concentration of 1- and 3-pHis as a function of time predicted by the kinetic model (left Y axis) overlaid on the relative total droplet volume against time measured from the confocal microscopy data. The experiments were carried out in triplicate. The critical coacervation concentration (0.26 mM) is highlighted with the dotted line. g, h Lag time and maximum intensity as a function of MAP concentration. 2.5% PEG₈₀₀₀ was added to the reaction solutions as a crowding agent. All experiments were performed in triplicate, and all data is shown. i Micrographs of the active droplets in the presence of 200 nM polyanions of various structures (Cy3 labeled ssDNA, dsDNA, RNA, or pSS). Unless mentioned otherwise, the conditions used were 20 mM peptide 1, 12.5 mM MAP, 50 mM R₃₀ (charges) and 200 nM sulforhodamine B in a 100 mM MOPS buffered solution at pH 7.5.