Multistep Crystallization via Biomolecular and Ionic Additives : Bidirectional Synergetic Interactions in Protein-Directed Mineralization

Abstract

In living systems, the biologically programmed interplay of organic and inorganic constituents often produces materials with fascinating morphologies and organizations. As a promising model system for elucidating biogenic nucleation and crystallization, sea urchins produce elaborate Mg-calcite spines via the deposition of amorphous precursors. The crystalline phase is deposited with spatiotemporal precision, thereby facilitating a mesocrystalline organization of co-oriented nanocrystals embedded in a matrix of amorphous mineral and biomolecules. Despite the prevalence of C-type lectin-like (CTL) proteins in the spine proteome, the mechanistic roles of this motif towards mineral nucleation and crystallization are not well understood. Therefore, the primary objective of this research is to investigate protein-mineral interactions during the early stages of calcium carbonate (CaCO₃) formation, and how they influence and direct the course of crystallization in near physiological scenarios.<br />Given the pH dependent speciation of (bi)carbonate ions, a methodology to quantitatively investigate CaCO₃ mineralization at near-neutral pH levels (pH 7.5–9.0) is established. In particular, the contributions of HCO₃^- ions as active soluble species and structural constituents in mineral nucleation are revealed. The titration methodology further identifies the specific effects of CTL proteins on mineralization, elucidating the biochemical mechanisms concerning crystal formation and growth. By investigating recombinant forms of three CTL proteins from the spine proteome (SM50, SM30A, LSM34), the proteins are found to inhibit the onset of mineral nucleation and also affect the solubility product of initially formed mineral phases in a pH- and ion-dependent manner. Small angle X-ray scattering (SAXS) and analytical ultracentrifugation (AUC) reveal that these proteins self-associate to distinct supramolecular structures during mineralization in response to the applied ionic and pH conditions. Collectively, these findings suggest that biomineralization encompasses bidirectional processes involving (i) biomolecules that modulate the nucleation and crystallization of inorganics and (ii) distinct mineral precursors that influence the self-association of biomolecules. In addition, elucidating the phase transformation of minerals, the transitions of amorphous mixed metal carbonates towards crystalline superstructures are found to be synergistically regulated by environmental water contents and occluded Mg²⁺ ion contents. In this regard, a novel mechanism for mesocrystal formation via interface-coupled dissolution-re-precipitation of mesoscale amorphous precursors is proposed. Overall biomineralization emerges as a biologically programmed multistep crystallization reaction, wherein ionic and molecular additives influence the nucleation and transformation of mineral phases towards distinct crystalline superstructures.