Nacre Protein Sequence Compartmentalizes Mineral Polymorphs in Solution

Abstract

The Japanese pearl oyster (Pinctada fucata) n16 framework matrix protein is an integral part of the growth and formation of the mollusk shell biomineralization mechanism. It is a required component of the extracellular matrix with a dual mineralization role, as an anchor component to synchronize the assembly of the beta-chitin and N-series, Pif-series protein extracellular matrix for aragonite formation and as a regulator of aragonite formation itself. However, the mechanism by which this protein controls aragonite formation is not understood. Here, we investigate the mineralization potential and kinetics of the 30 AA N-terminal portion of the n16 protein, n16N. This sequence has been demonstrated to form either vaterite or aragonite depending upon conditions. Using in situ potentiometric titration methods, we find that n16N is indeed responsible for the self-assembly characteristics found in vivo and in vitro but is not involved with active Ca2+ binding or mineral nucleation processes. Upon the basis of time- and peptide concentration-dependent sampling of mineral deposits that form in solution, we find that n16N is responsible for controlling where mineralization occurs in bulk solution. This protein sequence acts as a molecular spacer that organizes the mineralization space and promotes the formation of mineral constituents that contain ACC, vaterite, and aragonite. Without the concerted action of the n16N assemblage, unregulated calcite formation occurs exclusively. Thus, the n16 protein provides the regulation needed to have the characteristic polymorph, crystalline orientations, and related mechanical properties associated to the microstructure of mollusk shells.

Polymorphs, Proteins, and Nucleation Theory: A Critical Analysis

“…For aragonite, what we must draw from are studies conducted with mollusk shell nacre-associated protein sequences [34][35][36][37][38][39][40][41][42][43][44][45][46][47][48][49][50][51]. For in vitro-based studies, several different carbonate-based assays have been used to achieve supersaturation conditions necessary for calcium carbonate crystal growth, with no control over pH, and the duration of these nucleation experiments were variable (i.e., from several hours up to 7 days) [36,38,39,[41][42][43][44][46][47][48][49][50][51].…”

“…Second, and perhaps most importantly, genomic and proteomic studies of biomineralization proteins have uncovered a vast repertory of proteins [6][7][8]13,14,[26][27][28] that could potentially manage many aspects of proposed nucleation processes, including polymorphism. However, although many protein studies have confirmed that polymorphs do form when proteins are present [34][35][36][37][38][39][40][41][42][43][44][45][46][47][48][49][50][51][52][53], these studies have not provided information that is needed for detailing the mechanisms of polymorph formation, or, have utilized a variety of testing methods that prevent cross-comparisons of datasets necessary for mechanism building [34][35][36][37][38][39][40][41][42][43][44][45][46][47][48][49][50][51]…”

Mineralization and non-ideality: on nature’s foundry

“…For instance, lysozyme (monomer: 14.3 kDa) and ovalbumin (monomer: 45 kDa) have distinct molar mass, surface area and shapes (Table S1), as well as exhibit distinct aggregation propensities in dependence of ionic and pH conditions (Ianeselli et al 2010). For certain proteins, Ca 2+ ions can modulate self-association states via direct interactions (Shearwin and Winzor 1988;Rao et al 2013;Seto et al 2014), suggesting ion-dependent macromolecular conformation bias during mineralization. Further due to distinct density fluctuations and conformations of crowding agents in topologically-frustrated void spaces, crowding induced by different biomacromolecules can have dissimilar consequences (Samiotakis et al 2009).…”

Emergence of order in self-assembly of the intrinsically disordered biomineralisation peptide n16N

“…The 30AA (amino acid chain length) N-terminal region named n16N has been used as an n16 mimic in experimental biomineralisation studies [25][26][27][28][29][30][31][32][33]. Highlights include use of n16N with a Kevlar substrate producing lamellar aragonite [30], and n16N selecting aragonite when bound to β-chitin [29], a macromolecule with a substantial presence in the putative organic biomineralisation matrix of nacre [34].…”

“…Highlights include use of n16N with a Kevlar substrate producing lamellar aragonite [30], and n16N selecting aragonite when bound to β-chitin [29], a macromolecule with a substantial presence in the putative organic biomineralisation matrix of nacre [34]. n16N has been deemed 'the key selfassembly/aragonite forming domain' [33], and is the focus of the current work. Dynamic light scattering has confirmed that n16N oligomerises into μm-scale supramolecular assemblies [32]; evidence from circular dichroism spectra and NMR show that these oligomers are composed of 54% random coil structure and 46% β-structure [22,32,35].…”