Protein-Independent Liquid-Liquid Phase Separation of Adenosine Triphosphate Under Crowded Conditions.

Adenosine triphosphate (ATP), a common constituent of protein-rich biomolecular condensates, itself undergoes liquid-liquid phase separation (LLPS) even in the absence of polypeptides. Using polyethylene glycol as macromolecular crowding agent, we observed robust ATP droplet formation at pH 2-11. Moderate NaCl concentrations and lower temperatures promote LLPS by lowering critical ATP and crowder concentrations. While very high salt reverses this trend, the droplets nevertheless survive in hypersaline environments. We argue that both ion-mediated bridging and anomalous underscreening likely contribute to the response of ATP droplets to increasing ionic strength. Most importantly, physiological millimolar ATP concentrations are sufficient for phase separation in the presence of millimolar Mg2 + and crowders, mimicking intracellular conditions. pH tunes intermolecular interactions, evidenced by inversion of the adenosine circular dichroism (CD) signal. These results reveal intrinsic, protein-independent LLPS of ATP with potential roles in cellular compartmentalization and pathological phase transitions.