Biomineralization Controlled by Liquid-Liquid Phase Separation of a Highly Charged Protein

Area of Science:

• Biomineralization
• Materials Science
• Biochemistry

Background:

• Molecular mechanisms of biomineralization are not fully understood.
• Nonclassical crystallization pathways suggest transient liquid phases stabilized by polymers.
• Direct evidence for protein-containing liquid phases in biomineralization is lacking.

Purpose of the Study:

• To demonstrate that acid-rich proteins regulate calcium carbonate nucleation and growth through liquid-liquid phase separation (LLPS).
• To investigate the role of protein-calcium condensates (LPCCs) as crystallization precursors.
• To explore the impact of protein charge and solution crowding on mineral formation.

Main Methods:

• Utilized AGARP, an acid-rich protein from coral (Acropora millepora), as a model system.
• Investigated LLPS under physiologically relevant, crowded conditions.
• Analyzed calcium carbonate nucleation and growth in the presence and absence of AGARP.

Main Results:

• AGARP induced LLPS, forming liquid protein-calcium condensates (LPCCs) that act as crystallization precursors.
• LPCCs exposed to carbonate ions resulted in smooth-edged calcium carbonate morphologies.
• Under low-crowding conditions, AGARP led to amorphous calcium carbonate (ACC) formation.
• AGARP remained intrinsically disordered, indicating charge-mediated interactions are key.

Conclusions:

• LPCCs are biologically relevant intermediates preceding mineralization.
• LLPS provides a new molecular framework bridging phase separation and biomineralization.
• Protein phase behavior can inform bioinspired materials design.