Active droplets through enzyme-free, dynamic phosphorylation

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 do not exist. We present an enzyme-free reaction cycle that consumes phosphorylating agents by transiently phosphorylating amino acids. The phosphorylated amino acids 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. Our new reaction cycle will be valuable as a model for biological phosphorylation but can also offer insights into protocell formation.