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

Biomineralization plays a critical role in many organisms, yet the molecular mechanisms controlling mineral formation remain incompletely understood. Nonclassical crystallization pathways are proposed to involve transient liquid phases of calcium carbonate stabilized by polymers, such as acid-rich proteins secreted into the skeletal organic matrix. However, direct evidence for protein-containing liquid phases is lacking. Here, it is demonstrated that highly charged acid-rich proteins regulate calcium carbonate nucleation and growth through liquid-liquid phase separation (LLPS). Using AGARP, the first acid-rich protein cloned from the coral Acropora millepora, as a model, it is shown that LLPS occurs under physiologically relevant, crowded conditions, forming liquid protein-calcium condensates (LPCC) that act as crystallization precursors. Exposure of these condensates to carbonate ions triggers crystallization, resulting in complex, smooth-edged morphologies distinct from the sharp-edged structures formed in the absence of the protein. Under low-crowded conditions, the protein-calcium aggregation leads to amorphous calcium carbonate (ACC) formation. AGARP remains intrinsically disordered upon counterion binding, highlighting charge-mediated interactions as key drivers. These findings introduce LPCCs as biologically relevant intermediates preceding mineralization. This mechanism offers a new molecular-level conceptual framework, bridging the fields of phase separation and biomineralization, and suggests strategies for bioinspired materials design leveraging protein phase behavior.