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

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.

Liquid Cell Transmission Electron Microscopy and the Impact of Confinement on the Precipitation from Supersaturated Solutions

“…From the morphology of the nanoparticles we speculate that the formed particles were calcite rather than aragonite, vaterite or amorphous calcium carbonate. A similar observation could be made when studying calcium carbonate formation in the presence of proteins where the nacre derived protein AP7 was found to lead to calcium binding and enhanced protein induced local ion concentration [10]. These observations do not exclude a possible role of radiolysis in lowering the pH as has been reported elsewhere [11].…”

“…The zone width has been found to be of the order of 4-5 µm [10] under standard conditions. These experiments had been performed using atmospheric SEM observing precipitation in a petri dish through a Si x N y membrane in the bottom of the dish with no restriction of dimensions.…”

Polymorphs, Proteins, and Nucleation Theory: A Critical Analysis

“…These reported studies utilized mole protein quantities, Ca(II) potentiometric methods, and parallel mineralization assay systems to study the early and later events in calcite-based calcium carbonate nucleation and monitor the formation and stabilization of PNCs and ACC in a time-dependent fashion. What was discovered was very informative: these proteins are distinguishable in terms of what mineral species or steps in the non-classical scheme they affect (Figure 4) [58][59][60][61][62][63][64][65][66][67]. Further, using mineralization assays which overlap with the time periods of the potentiometric titrations and utilize similar solution and supersaturation conditions [58][59][60][61][62][63][64][65][66][67] these studies demonstrated that these proteins form hydrogels that can capture, assemble, and organize mineral nanoparticles ( Figure 5) [62,65] consistent with CPA theory [15].…”

“…As an example, recent in vitro studies involving five recombinant mollusk shell nacre-associated proteins [58][59][60][61][62][63][64][65][66] and ACC-stabilizing sea urchin spicule matrix proteins [67,68] have utilized mineralization assay conditions identical to those employed in non-classical nucleation studies [29][30][31][32][33]. What links these different biomineralization proteins together are two common molecular themes [69,70]: intrinsic disorder, or absence of folding protein structure, and amyloid-like aggregation-prone domains, which promote protein-protein association leading to the formation of protein phases or hydrogels [58][59][60][61][62][63][64][65][66][67][68]. These reported studies utilized mole protein quantities, Ca(II) potentiometric methods, and parallel mineralization assay systems to study the early and later events in calcite-based calcium carbonate nucleation and monitor the formation and stabilization of PNCs and ACC in a time-dependent fashion.…”

“…What was discovered was very informative: these proteins are distinguishable in terms of what mineral species or steps in the non-classical scheme they affect (Figure 4) [58][59][60][61][62][63][64][65][66][67]. Further, using mineralization assays which overlap with the time periods of the potentiometric titrations and utilize similar solution and supersaturation conditions [58][59][60][61][62][63][64][65][66][67] these studies demonstrated that these proteins form hydrogels that can capture, assemble, and organize mineral nanoparticles ( Figure 5) [62,65] consistent with CPA theory [15]. The major conclusions one can draw from these studies are the following: (1) nacre [58][59][60][61][62][63][64][65][66] and sea urchin spicule matrix proteins [67,68] perform seminal tasks that would be critical for eventual polymorph selection and stabilization; (2) the functionalities of these six nacre proteins [58][59][60][61][62][63][64]…”