The Intrinsically Disordered C‑RING Biomineralization Protein, AP7, Creates Protein Phases That Introduce Nanopatterning and Nanoporosities into Mineral Crystals

We report an interesting process whereby the formation of nanoparticle assemblies on and nanoporosities within calcite crystals is directed by an intrinsically disordered C-RING mollusk shell nacre protein, AP7. Under mineralization conditions, AP7 forms protein phases that direct the nucleation of ordered calcite nanoparticles via a repetitive protein phase deposition process onto calcite crystals. These organized nanoparticles are separated by gaps or spaces that become incorporated into the forming bulk crystal as nanoporosities. This is an unusual example of organized nanoparticle biosynthesis and mineral modification directed by a C-RING protein phase

Formation and Structure of Calcium Carbonate Thin Films and Nanofibers Precipitated in the Presence of Poly(Allylamine Hydrochloride) and Magnesium Ions

That the cationic polyelectrolyte poly­(allylamine hydrochloride) (PAH) exerts a significant influence on CaCO₃ precipitation challenges the idea that only anionic additives have this effect. Here, we show that in common with anionic polyelectrolytes such as poly­(aspartic acid), PAH supports the growth of calcite thin films and abundant nanofibers. While investigating the formation of these structures, we also perform the first detailed structural analysis of the nanofibers by transmission electron microscopy (TEM) and selected area electron diffraction. The nanofibers are shown to be principally single crystal, with isolated domains of polycrystallinity, and the single crystal structure is even preserved in regions where the nanofibers dramatically change direction. The formation mechanism of the fibers, which are often hundreds of micrometers long, has been the subject of intense speculation. Our results suggest that they form by aggregation of amorphous particles, which are incorporated into the fibers uniquely at their tips, before crystallizing. Extrusion of polymer during crystallization may inhibit particle addition at the fiber walls and result in local variations in the fiber nanostructure. Finally, we investigate the influence of Mg²⁺ on CaCO₃ precipitation in the presence of PAH, which gives thinner and smoother films, together with fibers with more polycrystalline, granular structures

Ultrastructure and Crystallography of Nanoscale Calcite Building Blocks in <i>Rhabdosphaera clavigera</i> Coccolith Spines

Coccolithophores create an intricate exoskeleton from nanoscale calcite platelets. Shape, size, and crystal orientation are controlled to a remarkable degree. In this study, the structure of <i>Rhabdosphaera clavigera</i> is described in detail for the first time through a combination of electron microscopy techniques, including three-dimensional electron tomography. The coccolithophore exhibits several micrometer long 5-fold symmetric spines with diameters of approximately 0.5 μm. The nanorystals constituting the spine are arranged radially along the longitudinal axis, protruding from the almost flat disks that form the coccosphere. The stem of the spine is shown to consist of {104} calcite rhombohedra single crystalline platelets, arranged in five separate spiral “staircases”. The spine tip shows 15 structural elements: five large “panels” protruding outward along the lateral plane and five leaf-shaped smaller units which form the topmost steps of the staircases. The outer tip consists of five long thin platelets protruding along the length of the spine axis. This feature extends downward into the spine-core. This core-feature may serve as a base for crystal nucleation and assembly analogous to the proto-coccolith ring in the <i>V/R</i> growth model (Young J. R. et al. Nature, 1992, 356, 516−518). However, we find significant dissimilarities of the crystal elements constituting the spine in comparison to that model

An Oligomeric C‑RING Nacre Protein Influences Prenucleation Events and Organizes Mineral Nanoparticles

The mollusk shell nacre layer integrates mineral phases with macromolecular components such as intracrystalline proteins. However, the roles performed by intracrystalline proteins in calcium carbonate nucleation and subsequent postnucleation events (e.g., organization of mineral deposits) in the nacre layer are not known. We find that AP7, a nacre intracrystalline C-RING protein, self-assembles to form amorphous protein oligomers and films on mica that further assemble into larger aggregates or phases in the presence of Ca²⁺. Using solution nuclear magnetic resonance spectroscopy, we determine that the protein assemblies are stabilized by interdomain interactions involving the aggregation-prone T31–N66 C-terminal C-RING domain but are destabilized by the labile nature of the intrinsically disordered D1–T19 AA N-terminal sequence. Thus, the dynamic, amorphous nature of the AP7 assemblies can be traced to the molecular behavior of the N-terminal sequence. Using potentiometric methods, we observe that AP7 protein phases prolong the time interval for prenucleation cluster formation but neither stabilize nor destabilize ACC clusters. Time-resolved flow cell scanning transmission electron microscopy mineralization studies confirm that AP7 protein phases delay the onset of nucleation and assemble and organize mineral nanoparticles into ring-shaped branching clusters in solution. These phenomena are not observed in protein-deficient assays. We conclude that C-RING AP7 protein phases modulate the time period for early events in nucleation and form strategic associations with forming mineral nanoparticles that lead to mineral organization