Pif97, a von Willebrand and Peritrophin Biomineralization Protein, Organizes Mineral Nanoparticles and Creates Intracrystalline Nanochambers

Abstract

The formation of the mollusk nacre layer involves the assembly and organization of mineral nanoparticles into fracture-toughened mesoscale-sized aragonite tablets that possess intracrystalline nanoporosities. At least one nacre protein family, known as the framework proteome, is strategically located as part of a macromolecular coating around each nacre tablet and is believed to participate in tablet formation. Here, we report new studies of a recombinant form (rPif97) of a unique Japanese pearl oyster (Pinctada fucata) nacre framework biomineralization protein, Pif97. This unique protein possesses both a von Willlebrand factor type A domain (vWA, F23-Y161) and a Peritrophin A chitin-binding domain (PAC, E234-D298). rPif97 self-associates or aggregates to form amorphous protein phases that organize both amorphous and single-crystal calcium carbonate nanoparticles in vitro. Further, in the presence of nucleating calcite crystals, rPif97 protein phases deposit onto these crystals and become occluded over time, forming nanochambers within the crystal interior. The formation of these mineral-modifying amorphous protein phases is linked to the presence of intrinsic disorder and amyloid-like cross-β-strand aggregation-prone regions, and three-dimensional modeling indicates that both the vWA and PAC domains are accessible for intermolecular interactions. Thus, the vWA- and PAC-containing Pif97 protein exhibits key functionalities that would allow its participation in mollusk nacre layer tablet assembly and porosity formation.

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]…”

“…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][65] are consistent with either non-classical [29][30][31][32][33] or CPA [16] theories [15]. Note that since these protein assay studies were calcite-based, there were no opportunities to study metastable aragonite or vaterite formation and stabilization.…”

Understanding nanoparticle porosity via nanoimpacts and XPS: electro-oxidation of platinum nanoparticle aggregates

“…This feature gives the nanoparticles additional surface area reflecting the porosity of the individual aggregates and this in turn may lead to changes in environmental adsorption, optical properties and pseudocapacitor performance. [9][10][11][12][13][14][15][16] Nanoporosities can be probed, traditionally, via ex situ gas adsorption isotherm measurements such as the BrunauerEmmett-Teller (BET) method; molecules, such as nitrogen or argon, are used. Such measurements are invaluable for the application of porous nanoparticles for gas-solid studies, for example in catalysis or sensing, and a careful analysis of the measured absorption isotherm may give insight into the shape and size of the particle pores as well as information about the total surface area covered by the adsorbate probe.…”