Understanding regulation of CaCO₃crystallization by de novo designed proteins through in situ imaging and spectroscopy

We investigate CaCO₃ nucleation in the presence of designed proteins presenting arrays of carboxylate groups in patterns expected to mimic the patterns of calcium sites on various faces of the CaCO₃ polymorphs. We combine liquid-phase TEM, liquid and solid state NMR, in situ ATR-FTIR, and molecular modeling to follow the structural and chemical evolution. At moderate supersaturations, the proteins form supramolecular complexes with Ca that drive nucleation of ~3.3 nm calcite, by-passing the amorphous phase observed in pure solution. The initially formed nanocrystals then undergo oriented attachment (OA) to form larger crystals with (110) facets, stabilized by the dissolved proteins. At high supersaturations, the proteins stabilize a viscous dense liquid phase (DLP) that forms by spinodal decomposition and is comprised of 1Ca²⁺:2HCO₃^-:7±2H₂O. The DLP transforms to hollow particles of hydrated amorphous CaCO₃ with the release of CO₂ and H₂O. NMR shows that the same pathway also occurs in pure solution, but the time scales of formation and transformation differ significantly. Molecular simulations indicate that DLP forms via direct condensation of solvated Ca²⁺•(HCO₃^-)₂ complexes that react due to proximity effects in the confinement of the DLP droplets to form CaCO₃, CO₂ and H₂O.