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

Area of Science:

• Biochemistry
• Cell Biology
• Biophysics

Background:

• Adenosine triphosphate (ATP) is a key molecule in cellular energy transfer.
• ATP is often found within protein-rich biomolecular condensates.
• Biomolecular condensates form via liquid-liquid phase separation (LLPS).

Purpose of the Study:

• To investigate the intrinsic phase separation behavior of ATP independent of proteins.
• To determine the conditions under which ATP undergoes LLPS.
• To explore the potential cellular and pathological implications of ATP's phase separation.

Main Methods:

• Utilized polyethylene glycol as a macromolecular crowding agent to induce ATP droplet formation.
• Varied pH, sodium chloride (NaCl) concentration, and temperature to study LLPS.
• Employed circular dichroism (CD) spectroscopy to analyze pH-dependent interactions.

Main Results:

• Observed robust ATP droplet formation across a wide pH range (2-11).
• Found that moderate NaCl concentrations and lower temperatures promote ATP LLPS.
• Demonstrated ATP phase separation at physiological concentrations with millimolar Mg²⁺ and crowders.
• Noted that high salt concentrations initially reverse the trend but droplets remain stable in hypersaline conditions.
• Observed pH-dependent changes in intermolecular interactions via CD spectroscopy.

Conclusions:

• ATP exhibits intrinsic, protein-independent liquid-liquid phase separation (LLPS).
• ATP LLPS is influenced by pH, ionic strength, temperature, and crowding.
• Physiological conditions can support ATP phase separation, suggesting roles in cellular compartmentalization.
• ATP's phase separation may be relevant to pathological processes.