Physical Phenomena Governing Mineral Morphogenesis in Molluscan Nacre.

Mollusks, as well as many other living organisms, have the ability to shape mineral crystals into unconventional morphologies and to assemble them into complex functional mineral-organic structures, an observation that inspired tremendous research efforts in scientific and technological domains. Despite these, a biochemical toolkit that accounts for the formation of the vast variety of the observed mineral morphologies cannot be identified yet. Herein, phase-field modeling of molluscan nacre formation, an intensively studied biomineralization process, is used to identify key physical parameters that govern mineral morphogenesis. Manipulating such parameters, various nacre properties ranging from the morphology of a single mineral building block to that of the entire nacreous assembly are reproduced. The results support the hypothesis that the control over mineral morphogenesis in mineralized tissues happens via regulating the physico-chemical environment, in which biomineralization occurs: the organic content manipulates the geometric and thermodynamic boundary conditions, which in turn, determine the process of growth and the form of the biomineral phase. The approach developed here has the potential of providing explicit guidelines for the morphogenetic control of synthetically formed composite materials.

Dietary supplementation with nacre reduces cortical bone loss in aged female mice.

Aging is associated with detrimental bone loss leading to fragility fractures in both men and women. Notably, a majority of bone loss with aging is cortical, as well as a large number of fractures are non-vertebral and at the non-hip sites. Nacre is a product of mollusks composed of calcium carbonate embedded in organic components. As our previous study demonstrated the protective effect of nacre supplementation on trabecular bone loss in ovariectomized rats, we sought to evaluate the effect of dietary nacre on bone loss related to aging in female mice which do not suffer true menopause as observed in women. The current study compared the effect of a 90-day long nacre-supplemented diet to that of Standard or CaCO3 diets on both bone mass and strength in 16-month-old C57BL/6 female mice. Multiple approaches were performed to assess the microarchitecture and mechanical properties of long bones, analyze trabecular histomorphometry, and measure bone cell-related gene expressions, and bone turnover markers. In the cortex, dietary nacre improved cortical bone strength in line with lower expression levels of genes reflecting osteoclasts activity compared to Standard or CaCO3 diets (p < 0.05). In the trabeculae, nacre-fed mice were characterized by a bone remodeling process more active than the other groups as shown by greater histomorphometric parameters and osteoblast-related gene expressions (p < 0.05). But these differences were not exhibited at the level of the trabecular microarchitecture at this age. Collectively, these data suggest that dietary nacre should be a potential candidate for reducing aging-associated cortical bone loss in the elderly.

Low-complexity domain-containing protein (LCDP), induced accumulation of calcium carbonate individual crystals to irregular polycrystals in the triangle sail mussel Hyriopsis cumingii.

The nacre layer is composed of sheet-like aragonite crystals, with often loosely arranged polycrystal aragonite sheets which may induce poor mechanical properties in shells. In this study, a full-length low-complexity domain-containing protein (LCDP) cDNA from the triangle sail mussel Hyriopsis cumingii was generated and its role in shell formation investigated. The full-length cDNA was 1058 bp; it had an open reading frame (ORF) of 714 bp encoding 237 amino acids and contained a 20-amino acid signal peptide at the N-terminus and two low-complexity domains. H. cumingii LCDP was not homologous with other species. Tissue expression analyses showed that LCDP was specifically expressed in the mantle. In shell repair assays, significantly higher LCDP expression was observed in the shell repair group from days 12-21 (p < 0.01). After LCDP silencing, aragonite flake shapes in pearl layers became irregular with disordered deposition, while calcium carbonate (CaCO3) crystal surfaces in prismatic layers became rougher and organic matrices between crystals appeared skeletonized, indicating the importance of biomineralization. Our in vitro CaCO3 crystallization assays showed that LCDP induced single crystals to polycrystals, probably via loose arrangement between aragonite flakes. These results provide new insights on freshwater mollusk biomineralization and a theoretical basis for improved pearl quality.

Nacre extract from pearl oyster attenuates amyloid beta-induced memory impairment.

Shells are composed of two types of calcium carbonate polymorphs-the prismatic layer and the nacreous layer. Pearls, composed of the nacreous layer, have been used in Chinese medicine since ancient times. We have previously shown that extracts from the nacreous layer improves scopolamine-induced memory impairment. However, whether pearl ameliorates cognitive disorders induced by amyloid-ß 1-40 (Aß1-40) has not been elucidated. In this study, we investigated whether nacre extract improves memory impairment induced by intracerebroventricular injection of Aß1-40. Administration of nacre extract led to recovery from Aß1-40-induced impairments in object recognition, short-term memory, and spatial memory. Nacre extract reversed the increase in lipid peroxidation caused by Aß1-40 in the cerebral cortex by increasing the expression of catalase and superoxide dismutase. In addition, nacre extract recovered the expression and phosphorylation of cyclic AMP response element-binding protein (CREB), which decreased with Aß1-40 treatment, and increased the expression of brain-derived neurotrophic factor and neuropeptide Y, which are regulated by CREB. Nacre extract also suppressed acetylcholine esterase activity and Aß1-40-induced tau phosphorylation. Histochemical analysis of the hippocampus region showed that the nacre extract protected against Aß1-40-induced neuronal loss in the hippocampus. These results suggest that nacre extract protects against Aß1-40-induced neuronal cell death by suppressing oxidative stress and increasing the expression and phosphorylation of CREB.

Biomimetic galactomannan/bentonite/graphene oxide film with superior mechanical and fire retardant properties by borate cross-linking.

With the great demand for high-strength integrated materials in various industries, products from renewable resources were expected to replace petroleum-based materials. Inspired by the hierarchical structure of nacre, in this work, bentonite and graphene oxide (GO) were incorporated into the galactomannan (GM) matrix to prepare a ternary nanocomposite, which was further cross-linked and strengthened with borate. The chemical structure of the composite was analyzed with SEM, FTIR, XPS and XRD, revealing a co-assembly reaction between GO, bentonite and GM, accompanied by the borate crosslinking. This synergistic strengthen effect resulted in a composite possessing a maximum tensile stress and toughness of 231.16 MPa and 4.53 MJ/m3, respectively, harder than most of the previously reported hemicellulose composites. Moreover, the nanocomposites showed excellent fire retardant property with a limiting oxygen index of 46.8 % due to the introduction of bentonite and GO, which shows potential application in fire-protective insulation, packaging and coating.

Nanoscaled pearl powder accelerates wound repair and regeneration in vitro and in vivo.

Pearl powder has been used to treat many diseases like palpitations, insomnia, and epilepsy for thousands of years in Chinese medicine. It has demonstrated antioxidant, antiaging, antiradiative, and tonic activities. Pearl powder contains multiple active proteins, which are nutritious for skin cells and might be advantageous for wound repair and regeneration. However, its healing effect in vivo was not reported yet. This study aims to investigate the effects and the underlying mechanism of the pearl powders with different particle sizes in wound treatment. Briefly, the pearl powder with different sizes was characterized for their particle sizes and morphology. The protein release profiles of these powders were also studied. The influence of the different size of pearl powder in the proliferation, migration of skin cells was evaluated. Then, with the rat skin excision model, the effect of pearl powder on wound repair and regeneration was investigated. It was demonstrated that, all the micro and nanosized pearl powders could both increase the proliferation and migration of skin cells and accelerate the wound closure, as well as significantly enhanced the biomechanic strength of the healed skins. Moreover, the pearl powder treatment could improve the formation and regular deposition of collagen, and enhance the skin angiogenesis. Among all these in vitro and in vivo investigations, nanoscale pearl powder expressed the highest efficiency for healing. The mechanism might be contributed to the increased release of active proteins, enhanced tissue attachment, and the increased cellular uptake for the nano powder at the topical site.

A novel nacre matrix protein hic24 in Hyriopsis cumingii is essential for calcium carbonate nucleation and involved in pearl formation.

Matrix proteins play important roles in molluscan shell biomineralization, which helps in the understanding of mechanisms associated with pearl formation. In this study, we characterized the gene encoding a novel shell-matrix protein, hic24, in Hyriopsis cumingii and investigated its structure and function. The full cDNA sequence of hic24 is 756 bp, with an open reading frame of 654 bp encoding 217 amino acids, including a signal peptide of 18 amino acids. Sequence analysis revealed that the protein is ∼23.5 kDa, and that Gly accounted for 11.5% of the total amino acid content. Secondary structure prediction indicated a structure comprised predominantly by ß-folds. Quantitative real-time polymerase chain reaction and in situ hybridization indicated that hic24 is expressed in the dorsal epithelial cells of the mantle, indicating hic24 as a nacreous-layer matrix protein. Additionally, hic24 expression patterns during pearl biomineralization showed that hic24 regulates the growth of the later nacreous layer. After attenuating hic24 expression by RNA interference in the mantle, we observed that hic24 plays a role in biomineralization of the shell nacre by inhibiting calcium carbonate nucleation.

Nacre formation by epithelial cell cultures from mantle of the black-lip pearl oyster, Pinctada margaritifera.

Mantle tissue from the black-lip pearl oyster, Pinctada margaritifera, was cultured in vitro using sterilized seawater supplemented with 0.1% yeast extract as the culture medium. Granular and agranular epithelial cells, hyalinocytes, and fibroblast-like cells were observed in the initial stages of culture. Epithelial cells later formed pseudopodial cell networks containing clusters of granulated cells, which upon maturation released their colored granules. These granules induced formation of nacre crystal deposits on the bottom of the culture plate. Cultures comprised of only granulated epithelial cells were established through periodic sub-culturing of mantle cells and maintained for over 18 mo in a viable condition. Reverse transcriptase PCR of cultured cells demonstrated gene expression of the shell matrix protein, nacrein. To further evaluate the functional ability of cultured granulated epithelial cells, nuclear shell beads were incubated in culture medium containing these cells to induce nacre formation on the beads. Observation of the bead surface under a stereomicroscope at periodic intervals showed the gradual formation of blackish yellow colored nacre deposits. Examination of the bead surface by scanning electron microscopy and energy dispersive X-ray analysis at periodic intervals revealed a distinct brick and mortar formation characteristic of nacre, comprised of aragonite platelets and matrix proteins. Calcium, carbon, and oxygen were the major elements in all stages examined. Our study shows that mantle epithelial cells in culture retain the ability to secrete nacre and can therefore form the basis for future studies on the biomineralization process and its application in development of sustainable pearl culture.

Hierarchically roughened microplatelets enhance the strength and ductility of nacre-inspired composites.

Rough interfaces featuring nanoscale asperities are known to play a major role in the mechanics of nacre. Transferring this concept to artificial bioinspired composites requires a detailed understanding about the effect of the surface topography of reinforcing elements on the mechanical performance of such materials. To gain further insights into the effect of asperity size, hierarchy and coverage on the mechanics of nacre-inspired composites, we decorate alumina microplatelets with silica nanoparticles of selected sizes and use the resulting roughened platelets as reinforcing elements (15vol%) in a commercial epoxy matrix. For a single layer of silica nanoparticles on the platelet surface, increased ultimate strain and toughness are obtained with a large roughening particle size of 250nm. On the contrary, strength and stiffness are enhanced by decreasing the size of asperities using 22nm silica particles. By combining particles of two different sizes (100nm and 22nm) in a hierarchical fashion, we are able to improve stiffness and strength of platelet-reinforced polymers while maintaining high ultimate strain and toughness. Our results indicate that carefully designed hierarchically roughened interfaces lead to a more homogeneous stress distribution within the polymer matrix between the stiff reinforcing elements. By enabling the deformation of a larger fraction of the polymer matrix, this design concept improves the mechanical response of bioinspired composites and can possibly also be exploited to enhance the performance of conventional fiber-reinforced polymers.

Anticonvulsant and sedative-hypnotic activity screening of pearl and nacre (mother of pearl).

ETHNOPHARMACOLOGICAL RELEVANCE: Pearl and nacre are valuable traditional medicines to treat palpitations, convulsions or epilepsy in China for thousands of years. However, the active ingredients are not clear till now. AIM OF THE STUDY: The main purpose of the current investigation was to assess the anticonvulsant and sedative-hypnotic activity of pearl powder and nacre powder, including their corresponding 6 protein extracts. MATERIAL AND METHODS: Determination of the amino acid composition of the obtained protein was carried out by ultra-performance liquid chromatography (UPLC) combined with 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate (AQC) pre-column derivatisation. The influence of the tested drugs on locomotor activity and convulsions latency was recorded. The contents of 5-Hydroxytryptamine (5-HT) and γ-aminobutyric acid (GABA) in brain were detected by enzyme-linked immunesorbent assay (ELISA) kits. In addition, immunohistochemistry was carried out to evaluate the changes of 5-HT3 and GABAB. In parallel, the expressions of them were demonstrated by western blot. RESULTS: The obtained data suggested that pearl original powder (1.1g/kg), pearl water-soluble protein (0.2g/kg), pearl acid-soluble protein (0.275g/kg), pearl conchiolin protein (1.1g/kg), nacre original powder (1.1g/kg), nacre water-soluble protein (0.2g/kg), nacre acid-soluble protein (0.7g/kg) and nacre conchiolin protein (1.1g/kg) could down-regulate the expression of 5-HT3 and up-regulate the level of GABAB to varying degrees compared with the control group. Besides, drug administration also reduced the locomotor activity and increased convulsions latency with a certain mortality. CONCLUSIONS: These findings correlated with the traditional use of pearl and nacre as sedation and tranquilization agents, thus making them interesting sources for further drug development and also providing critical important evidence for the selection of quality control markers.

Nacre-like materials using a simple doctor blading technique: Fabrication, testing and modeling.

The remarkable mechanical performance of biological materials such as bone, nacre, and spider silk stems from their staggered microstructure in which stiff and strong reinforcements are elongated in the direction of loading, separated by softer interfaces, and shifted relative to each other. This structure results in useful combinations of modulus, strength and toughness and therefore is increasingly mimicked in bio-inspired engineering composites. Here, we report the use of a simple and versatile technique based on doctor-blading to fabricate staggered composites of microscopic alumina tablets with high alignment in a chitosan matrix. Tensile tests on these nacre-like materials show that the modulus and strength of the composite films are enhanced by the incorporation of ceramic tablets, but only up to 15vol% after which all properties degrade. This phenomenon, also reported in the past for most of nacre-like materials, composed of micro/nano tablets, obtained from different techniques, has been limiting our ability to produce large volumes of high-performance nacre-like materials. Examination of the structure of the films revealed that at lower tablet concentrations the tablets are well-aligned and well dispersed thorough the volume of the film. At 15vol% and beyond, we observed tablet misalignment and clustering. In order to investigate the impact of these imperfections on material performance we developed large scale finite element models representative of the structure of the composite films. These models show that the mechanical performance significantly degrades with tablet misalignment, and especially at high tablet concentrations. The simulations along with the SEM images therefore quantitatively explain the experimental trends, e.g. the degradation of mechanical properties at high tablet contents.

Nacre extract restores the mineralization capacity of subchondral osteoarthritis osteoblasts.

Osteoarthritis (OA) is the most common cause of joint chronic pain and involves the entire joints. Subchondral osteoarthritic osteoblasts present a mineralization defect and, to date, only a few molecules (Vitamin D3 and Bone Morphogenetic Protein2) could improve the mineralization potential of this cell type. In this context, we have tested for the first time the effect of nacre extract on the mineralization capacity of osteoblasts from OA patients. Nacre extract is known to contain osteogenic molecules which have demonstrated their activities notably on the MC3T3 pre-osteoblastic cell line. For this goal, molecules were extracted from nacre (ESM, Ethanol Soluble Matrix) and tested on osteoblasts of the subchondral bone from OA patients undergoing total knee replacement and on MC3T3 cells for comparison. We chose to investigate the mineralization with Alizarin Red staining and with the study of extracellular matrix (ECM) structure and composition. In a complementary way the structure of the ECM secreted during the mineralization phase was investigated using second harmonic generation (SHG). Nacre extract was able to induce the early presence (after 7 days) of precipitated calcium in cells. Raman spectroscopy and electron microscopy showed the presence of nanograins of an early crystalline form of calcium phosphate in OA osteoblasts ECM and hydroxyapatite in MC3T3 ECM. SHG collagen fibers signal was present in both cell types but lower for OA osteoblasts. In conclusion, nacre extract was able to rapidly restore the mineralization capacity of osteoarthritis osteoblasts, therefore confirming the potential of nacre as a source of osteogenic compounds.

Bioinspired Hierarchical Alumina-Graphene Oxide-Poly(vinyl alcohol) Artificial Nacre with Optimized Strength and Toughness.

Due to hierarchical organization of micro- and nanostructures, natural nacre exhibits extraordinary strength and toughness, and thus provides a superior model for the design and fabrication of high-performance artificial composite materials. Although great progress has been made in constructing layered composites by alternately stacking hard inorganic platelets and soft polymers, the real issue is that the excellent strength of these composites was obtained at the sacrifice of toughness. In this work, inspired by the layered aragonite microplatelets/chitin nanofibers-protein structure of natural nacre, alumina microplatelets-graphene oxide nanosheets-poly(vinyl alcohol) (Al2O3/GO-PVA) artificial nacre is successfully constructed through layer-by-layer bottom-up assembly, in which Al2O3 and GO-PVA act as "bricks" and "mortar", respectively. The artificial nacre has hierarchical "brick-and-mortar" structure and exhibits excellent strength (143 ± 13 MPa) and toughness (9.2 ± 2.7 MJ/m(3)), which are superior to those of natural nacre (80-135 MPa, 1.8 MJ/m(3)). It was demonstrated that the multiscale hierarchical structure of ultrathin GO nanosheets and submicrometer-thick Al2O3 platelets can deal with the conflict between strength and toughness, thus leading to the excellent mechanical properties that cannot be obtained using only one size of platelet. We strongly believe that the work presented here provides a creative strategy for designing and developing new composites with excellent strength and toughness.

Equilibrium conformational ensemble of the intrinsically disordered peptide n16N: linking subdomain structures and function in nacre.

n16 is a framework protein family associated with biogenic mineral stabilization, thought to operate at three key interfaces in nacre: protein/ß-chitin, protein/protein, and protein/CaCO3. The N-terminal half of this protein, n16N, is known to be active in conferring this mineral stabilization and organization. While some details relating to the stabilization and organization of the mineral are known, the molecular mechanisms that underpin these processes are not yet established. To provide these molecular-scale details, here we explore current hypotheses regarding the possible subdomain organization of n16N, as related to these three interfaces in nacre, by combining outcomes of Replica Exchange with Solute Tempering molecular dynamics simulations with NMR experiments, to investigate the conformational ensemble of n16N in solution. We verify that n16N lacks a well-defined secondary structure, both with and without the presence of Ca(2+) ions, as identified from previous experiments. Our data support the presence of three different, functional subdomains within n16N. Our results reveal that tyrosine, chiefly located in the center of the peptide, plays a multifunctional role in stabilizing conformations of n16N, for intrapeptide and possibly interpeptide interactions. Complementary NMR spectroscopy data confirm the participation of tyrosine in this stabilization. The C-terminal half of n16N, lacking in tyrosine and highly charged, shows substantive conformational diversity and is proposed as a likely site for nucleation of calcium carbonate. Finally, dominant structures from our predicted conformational ensemble suggest the presentation of key residues thought to be critical to the selective binding to ß-chitin surfaces.

Splice variants of perlucin from Haliotis laevigata modulate the crystallisation of CaCO3.

Perlucin is one of the proteins of the organic matrix of nacre (mother of pearl) playing an important role in biomineralisation. This nacreous layer can be predominately found in the mollusc lineages and is most intensively studied as a compound of the shell of the marine Australian abalone Haliotis laevigata. A more detailed analysis of Perlucin will elucidate some of the still unknown processes in the complex interplay of the organic/inorganic compounds involved in the formation of nacre as a very interesting composite material not only from a life science-based point of view. Within this study we discovered three unknown Perlucin splice variants of the Australian abalone H. laevigata. The amplified cDNAs vary from 562 to 815 base pairs and the resulting translation products differ predominantly in the absence or presence of a varying number of a 10 mer peptide C-terminal repeat. The splice variants could further be confirmed by matrix-assisted laser desorption ionisation time of flight mass spectrometry (MALDI-ToF MS) analysis as endogenous Perlucin, purified from decalcified abalone shell. Interestingly, we observed that the different variants expressed as maltose-binding protein (MBP) fusion proteins in E. coli showed strong differences in their influence on precipitating CaCO3 and that these differences might be due to a splice variant-specific formation of large protein aggregates influenced by the number of the 10 mer peptide repeats. Our results are evidence for a more complex situation with respect to Perlucin functional regulation by demonstrating that Perlucin splice variants modulate the crystallisation of calcium carbonate. The identification of differentially behaving Perlucin variants may open a completely new perspective for the field of nacre biomineralisation.

Effect of reinforcement surface functionalization on the mechanical properties of nacre-like bulk lamellar composites processed by a hybrid conventional method.

Alumina platelet reinforced epoxy matrix composites with an architecture resembling to natural nacre were fabricated by a hybrid conventional method called Hot-press Assisted Slip Casting process (HASC). Correlation between processing parameters, platelet content, platelet orientation and mechanical property enhancement of the fabricated composites was examined. In order to investigate the effect of interfacial compatibility and bonding on the mechanical properties of the fabricated inorganic-organic composites, platelet surfaces were modified with both epoxy- and amino-functional silanes. As received and functionalized platelet surfaces were studied by X-Ray Photoelectron Spectroscopy (XPS) to confirm the success of surface modification. Fabricated bio-inspired bulk lamellar composite materials were characterized in terms of their microstructural architecture and mechanical properties. The results obtained indicated that HASC processed composites exhibit enhanced flexural strength, stiffness and hardness, as compared to neat epoxy and composites fabricated by simple mixing, as a result of their nacre-like architecture with well aligned platelets. It has been also observed that functionalization by both type of silanes improves interfacial adhesion between platelets and epoxy matrix resulting in further enhancement of the mechanical properties of bulk lamellar composites fabricated by HASC.

The molecular evolution of the pif family proteins in various species of mollusks.

Various novel proteins have been identified from many kinds of mollusk shells. Although such matrix proteins are believed to play important roles in the calcium carbonate crystal formation of shells, no common proteins that interact with calcium carbonate or that are involved in the molecular mechanisms behind shell formation have been identified. Pif consists of two proteins, Pif 80 and Pif 97, which are encoded by a single mRNA. Pif 80 was identified as a key acidic protein that regulates the formation of the nacreous layer in Pinctada fucata, while Pif 97 has von Willebrand factor type A (VWA) and chitin-binding domains. In this study, we identified Pif homologues from Pinctada margaritifera, Pinctada maxima, Pteria penguin, Mytilus galloprovincialis, and in the genome database of Lottia gigantea in order to compare their primary protein sequences. The VWA and chitin-binding domains are conserved in all Pif 97 homologues, whereas the amino acid sequences of the Pif 80 regions differ markedly among the species. Sequence alignment revealed the presence of a novel significantly conserved sequence between the chitin-binding domain and the C-terminus of Pif 97. Further examination of the Pif 80 regions suggested that they share a sequence that is similar to the laminin G domain. These results indicate that all Pif molecules in bivalves and gastropods may be derived from a common ancestral gene. These comparisons may shed light on the correlation between molecular evolution and morphology in mollusk shell microstructure.

An improved failure criterion for biological and engineered staggered composites.

High-performance biological materials such as nacre, spider silk or bone have evolved a staggered microstructure consisting of stiff and strong elongated inclusions aligned with the direction of loading. This structure leads to useful combinations of stiffness, strength and toughness, and it is therefore increasingly mimicked in bio-inspired composites. The performance of staggered composites can be tuned; for example, their mechanical properties increase when the overlap between the inclusions is increased. However, larger overlaps may lead to excessive tensile stress and fracture of the inclusions themselves, a highly detrimental failure mode. Fracture of the inclusions has so far only been predicted using highly simplified models, which hinder our ability to properly design and optimize engineered staggered composites. In this work, we develop a new failure criterion that takes into account the complex stress field within the inclusions as well as initial defects. The model leads to an 'optimum criterion' for cases where the shear tractions on the inclusions is uniform, and a 'conservative' criterion for which the tractions are modelled as point forces at the ends of the overlap regions. The criterion can therefore be applied for a wide array of material behaviour at the interface, even if the details of the shear load transfer is not known. The new criterion is validated with experiments on staggered structures made of millimetre-thick alumina tablets, and by comparison with data on nacre. Formulated in a non-dimensional form, our new criterion can be applied on a wide variety of engineered staggered composites at any length scale. It also reveals new design guidelines, for example high aspect ratio inclusions with weak interfaces are preferable over inclusions with low aspect ratio and stronger interfaces. Together with existing models, this new criterion will lead to optimal designs that harness the full potential of bio-inspired staggered composites.

A C-RING-like domain participates in protein self-assembly and mineral nucleation.

AP7 is a nacre-associated protein of the mollusk shell that forms supramolecular assemblies that nucleate single-crystal aragonite in vitro. AP7 possesses two major sequence regions: a random coil 30-amino acid N-terminal domain (AP7N) and a partially disordered 36-amino acid C-terminal domain (AP7C) that exhibits imperfect sequence homology to the C subclass of the intracellular RING domain family. We report here new findings that implicate the C-RING domain in AP7-mediated supramolecular assembly and single-crystal mineral formation. AP7 protein spontaneously self-assembles over a pH range of 4-9 and is monomeric at pH >9.5. AP7N and AP7C both oligomerize over the pH range of 4-9, with the AP7C sequence closely resembling AP7 in terms of particle morphology and size. In vitro mineralization experiments demonstrate that both AP7N and AP7C form supramolecular assemblies that nucleate single-crystal calcium carbonates. Comparison of previously published nuclear magnetic resonance-based structures of AP7C and AP7N reveals the significant presence of complementary anionic-cationic electrostatic molecular surfaces on AP7C that are not found on AP7N, and this may explain the noted discrepancies between the two domains in terms of self-assembly and single-crystal nucleation. We conclude that the C-RING-like sequence is an important site for AP7 self-association and mineral nucleation, and this represents the first known instance of a RING-like sequence performing these functions within an extracellular protein.