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

Perovic, Iva, Andreas Verch, Eric P. Chang, Ashit Rao, Helmut Cölfen, Roland Kröger, and John Spencer Evans, 2014
AP7 self-assembling to form amorphous protein oligomers and films observed in situ via TEM
Image courtesy of Biochemistry

Abstract

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.

Impact Statement

Biomineralization study of the role of the shellfish nacre protein, AP7 on the formation of protein stabilized calcite nanostructures using LC-STEM. The precursor solutions were simultaneously mixed in situ and the nucleation and growth were observed in situ in real-time.