Synergistic Biomineralization Phenomena Created by a Combinatorial Nacre Protein Model System

Abstract

In the nacre or aragonite layer of the mollusk shell there exist proteomes which regulate both the early stages of nucleation and nano-to-mesoscale assembly of nacre tablets from mineral nanoparticle precursors. Several approaches have been developed to understand protein-associated mechanisms of nacre formation, yet we still lack insight into how protein ensembles or proteomes manage nucleation and crystal growth. To provide additional insights we have created a proportionally-defined combinatorial model consisting of two nacre-associated proteins, C-RING AP7 (shell nacre, H. rufescens) and pseudo-EF hand PFMG1 (oyster pearl nacre, P. fucata) whose individual in vitro mineralization functionalities are well-documented and distinct from one another. Using SEM, flow cell STEM, AFM, Ca(II) potentiometric titrations and QCM-D quantitative analyses, we find that both nacre proteins are functionally active within the same mineralization environments, and at 1:1 mole ratios, synergistically create calcium carbonate mesoscale structures with ordered intracrystalline nanoporosities, extensively prolong nucleation times and introduce an additional nucleation event. Further, these two proteins jointly create nanoscale protein aggregates or phases that under mineralization conditions further assemble into protein-mineral PILP-like phases with enhanced ACC stabilization capabilities, and there is evidence for intermolecular interactions between AP7 and PFMG1 under these conditions. Thus, a combinatorial model system consisting of more than one defined biomineralization protein dramatically changes the outcome of the in vitro biomineralization process.

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.…”

“…As we are starting to see, a common theme that emerges from proteome studies is that these proteins share common traits, such as intrinsic disorder or unfolded structure [69] and aggregation propensity [69] that leads to protein phase or hydrogel formation that provides volume confinement and a pathway to particle assembly and organization [58][59][60][61][62][63][64][65][66][67]. Yet, the sequences of these proteins are unalike [58][59][60][61][62][63][64][65][66][67] and so genetic variation is permissible and even welcomed in biological organisms so long as specific nucleation goals are being met. AP7 is a hydrogelator that forms aggregating gels that contain mineral nanoparticles.…”