Biomineralization Proteins Research Articles

Aspartic Acid Binding on Hydroxyapatite Nanoparticles with Varying Morphologies Investigated by Solid-State NMR Spectroscopy and Molecular Dynamics Simulation.

Hydroxyapatite (HAP) exhibits a highly oriented hierarchical structure in biological hard tissues. The formation and selective crystalline orientation of HAP is a process that involves functional biomineralization proteins abundant in acidic residues. To obtain insights into the process of HAP mineralization and acidic residue binding, synthesized HAP with specific lattice planes including (001), (100), and (011) are structurally characterized following the adsorption of aspartic acid (Asp). The adsorption affinity of Asp on HAP surfaces is evaluated quantitatively and demonstrates a high dependency on the HAP morphological form. Among the synthesized HAP nanoparticles (NPs), Asp exhibits the strongest adsorption affinity to short HAP nanorods, which are composed of (100) and (011) lattice planes, followed by nanosheets with a preferential expression of the (001) facet, to which Asp displays a similar but slightly lower binding affinity. HAP nanowires, with the (100) lattice plane preferentially developed, show significantly lower affinity to Asp and evidence of multilayer adsorption compared to the previous two types of HAP NPs. A combination of solid-state NMR (SSNMR) techniques including 13C and 15N CP-MAS, relaxation measurements and 13C-31P Rotational Echo DOuble Resonance (REDOR) is utilized to characterize the molecular structure and dynamics of Asp-HAP bionano interfaces with 13C- and 15N-enriched Asp. REDOR is used to determine 13C-31P internuclear distances, providing insight into the Asp binding geometry where stronger 13C-31P dipolar couplings correlate with binding affinity determined from Langmuir isotherms. The carboxyl sites are identified as the primary binding groups, facilitated by their interaction with surface calcium sites. The Asp chelation conformations revealed by SSNMR are further refined with molecular dynamics (MD) simulation where specific models strongly agree between the SSNMR and MD models for the various surfaces.

Integrative computational framework to decipher the functions of shell proteins in biomineralization

Integrative computational framework to decipher the functions of shell proteins in biomineralization

Proteolytic profiles of two isoforms of human AMBN expressed in E. coli by MMP-20 and KLK-4 proteases

Ameloblastin is a protein in biomineralization of tooth enamel. However recent results indicate that this is probably not its only role in an organism. Enamel matrix formation represents a complex process enabled via specific crosslinking of two proteins – the most abundant amelogenin and the ameloblastin (AMBN). The human AMBN (hAMBN) gene possesses 13 protein coding exons with alternatively spliced transcripts and the longest isoform about 447 amino acid residues. It has been described that AMBN molecules in vitro assemble into oligomers via a sequence encoded by exon 5. Enamel is formed by the processing of enamel proteins by two specific proteases - enamelysin (MMP-20) and kallikrein 4 (KLK-4). The scaffold made of AMEL and non-amelogenin proteins is cleaved and removed from the developed tooth enamel. The hAMBN is expressed in two isoforms (ISO I and II), which could lead to their different utilization determined by distinct proteolytic profiles. In this study, we compared proteolytic profiles of both isoforms of hAMBN expressed in E. coli after proteolysis by MMP-20, KLK-4, and their 1:2 mixture. Proteolysis products were analysed and cleavage sites were identified by mass spectrometry. The proteolytic profiles of two AMBN isoforms showed different results, although we have to determine that the analysed AMBN was not post-translationally modified as expressed in prokaryotic cells. These results may lead to the suggestion of potentially divergent roles of AMBN isoforms cleavage products in various cell signalling pathways such as calcium buffering or signalling cascades.

Exoskeleton protein repertoires in decapod crustaceans revealed distinct biomineralization evolution with molluscs

Crustaceans are the champions of mineral mobilization and deposition in the animal kingdom due to their unique ability to rapidly and periodically mineralize and demineralize their exoskeletons. They are commonly covered with mineralized exoskeletons for protection and regularly molt throughout their lives. Mineralized crustacean exoskeletons are formed under the control of macromolecules especially matrix proteins but the types of matrix proteins are understudied compared to those in molluscan shells. This gap hinders our understanding of their evolutionary paths compared with those of molluscs. Here, we comprehensively analyzed matrix proteins in the exoskeleton of two crabs, one shrimp, and one crayfish and resulted in a major improvement (∼10-fold) in the identification of biomineralization proteins compared to conventional methods for decapod crustaceans. By a comparison with well-studied molluscan biomineralization proteins, we found that decapod crustaceans evolved novel proteins to form mineralized exoskeletons while sharing some proteins with those of molluscs. Our study sheds light on their evolution and adaption to different environment for exoskeleton formation and provides a foundation for further studies of mineralization in crustaceans under normal and climate-changed conditions. SignificanceMost crustaceans have mineralized exoskeletons as protection. How they form these hierarchical structures is still unclear. This is due partially to the understudied matrix proteins in the minerals. This study filled such a gap by using proteomic analysis of matrix proteins from four decapod crustacean exoskeletons. Many novel proteins were discovered which enabled a solid comparison with those of molluscs. By comparison, we proposed that crustaceans evolved novel proteins to form mineralized exoskeletons while sharing some proteins with those of molluscs. This is useful for us to understand the evolution of two major biomineralized phylum.

Molecular compartmentalization in a syncytium: restricted mobility of proteins within the sea urchin skeletogenic mesenchyme.

Multinucleated cells, or syncytia, are found in diverse taxa. Their biological function is often associated with the compartmentalization of biochemical or cellular activities within the syncytium. How such compartments are generated and maintained is poorly understood. The sea urchin embryonic skeleton is secreted by a syncytium, and local patterns of skeletal growth are associated with distinct sub-domains of gene expression within the syncytium. For such molecular compartments to be maintained and to control local patterns of skeletal growth: (1) the mobility of TFs must be restricted to produce stable differences in the transcriptional states of nuclei within the syncytium; and (2) the mobility of biomineralization proteins must also be restricted to produce regional differences in skeletal growth. To test these predictions, we expressed fluorescently tagged forms of transcription factors and biomineralization proteins in sub-domains of the skeletogenic syncytium. We found that both classes of proteins have restricted mobility within the syncytium and identified motifs that limit their mobility. Our findings have general implications for understanding the functional and molecular compartmentalization of syncytia.

Functional mimicry of sea urchin biomineralization proteins with CaCO3-binding peptides selected by phage display.

The intricate process of biomineralization, e.g. in sea urchins, involves the precise interplay of highly regulated mineralization proteins and the spatiotemporal coordination achieved through compartmentalization. However, the investigation of biomineralization effector molecules, e.g. proteins, is challenging, due to their very low abundance. Therefore, we investigate the functional mimicry in the bioinspired precipitation of calcium carbonate (CaCO3) with artificial peptides selected from a peptide library by phage display based on peptide-binding to calcite and aragonite, respectively. The structure-directing effects of the identified peptides were compared to those of natural protein mixes isolated from skeletal (test) structures of two sea urchin species (Arbacia lixula and Paracentrotus lividus). The calcium carbonate samples deposited in the absence or presence of peptides were analyzed with a set of complementary techniques with regard to morphology, polymorph, and nanostructural motifs. Remarkably, some of the CaCO3-binding peptides induced morphological features in calcite that appeared similar to those obtained in the presence of the natural protein mixes. Many of the peptides identified as most effective in exerting a structure-directing effect on calcium carbonate crystallization were rich in basic amino acid residues. Hence, our in vitro mineralization study further highlights the important, but often neglected, role of positively charged soluble organic matrices associated with biological and bioinspired CaCO3 deposition.

The molecular basis for pore pattern morphogenesis in diatom silica.

Biomineral-forming organisms produce inorganic materials with complex, genetically encoded morphologies that are unmatched by current synthetic chemistry. It is poorly understood which genes are involved in biomineral morphogenesis and how the encoded proteins guide this process. We addressed these questions using diatoms, which are paradigms for the self-assembly of hierarchically meso- and macroporous silica under mild reaction conditions. Proteomics analysis of the intracellular organelle for silica biosynthesis led to the identification of new biomineralization proteins. Three of these, coined dAnk1-3, contain a common protein-protein interaction domain (ankyrin repeats), indicating a role in coordinating assembly of the silica biomineralization machinery. Knocking out individual dank genes led to aberrations in silica biogenesis that are consistent with liquid-liquid phase separation as underlying mechanism for pore pattern morphogenesis. Our work provides an unprecedented path for the synthesis of tailored mesoporous silica materials using synthetic biology.

Bioinformatics Analysis of Protein Homologues of Magnetotactic Bacteria Magnetosome Island Proteins in Human Proteome

Background. The number of biogenic magnetic nanoparticles (BMN), present in human organs and tissues in the form of magnetite (ferrimagnetic iron oxide), increases in oncological and neurodegenerative diseases. Therefore, the study of homologues of BMN biomineralization proteins (mam-proteins) of magnetotaxis bacteria (MTB) in human proteome is relevant task. This concern is due primarily to the expediency of establishing patterns of changes in the expression of these proteins and searching for correlations with oncological and neurodegenerative diseases. Objective. We are aimed to conduct the bioinformatic analysis of homologues of MTB mam-proteins in humans and to determine the patterns of changes in the expression of these proteins, as well as to search for their connections with the specified diseases. This will allow to identify the main candidate proteins (among the known homologues of MTB mam-proteins in humans) for experimental verification of their participation in the genetically programmed mechanism of BMN biosynthesis in humans. Methods. The methods of comparative genomics were used, in particular the BLAST (Basic Local Alignment Search Tool) program of the NCBI database. Database tools were also used: NCBI Conserved Domain Search, The Cancer Genome Atlas database, Ensembl database. Results. The bioinformatic analysis of 16 homologues of MTB mam-proteins in humans was carried out, namely: PEX5, ANAPC7, CDC23, CDC27 and SGTA – homologues of MamA in MTB; SLC30A4, SLC30A9, SLC39A3 and SLC39A4 – homologs of MamB and MamM in MTB; HTRA1, HTRA2, HTRA3 and HTRA4 – MamO and MamE homologues in MTB; SCRIB, PDZK1 and PDZD3 – MamE homologues in MTB. Using pairwise alignments, the degree of homology between the mam-proteins of the MTB magnetosome island and the corresponding human proteins was determined, conserved domains and their functions were determined, changes in their expression levels in cancer and normal conditions were determined by analyzing the relevant databases, and the metabolic pathways to which the data proteins are involved were analysed. The analysis of the obtained data allowed to assume the presence of the main homologues of the MTB mam-proteins of the magnetosome island in humans, which cause an increase in the level of BMN in oncological and neurodegenerative diseases, namely: an increase in the expression level of the proteins PEX5, ANAPC7 (homologs of MamA), SLC39A3, SLC39A4 (homologs of MamB and MamM), HTRA4 (MamO and MamE homolog) and SCRIB (MamE homolog). Conclusions. The obtained data allow us to assume that the proteins PEX5, ANAPC7, SGTA, SLC39A3, SLC39A4, HTRA4 and SCRIB are the main homologues of the MTB mam-proteins in humans and cause an increase in the level of BMN in oncological and neurodegenerative diseases.

Atomic Details of Biomineralization Proteins Inspiring Protein Design and Reengineering for Functional Biominerals

Biominerals are extraordinary materials that provide organisms with a variety of functions to support life. The synthesis of biominerals and organization at the macroscopic level is a consequence of the interactions of these materials with proteins. The association of biominerals and proteins is very ancient and has sparked a wealth of research across biological, medical and material sciences. Calcium carbonate, hydroxyapatite, and silica represent widespread natural biominerals. The atomic details of the interface between macromolecules and these biominerals is very intriguing from a chemical perspective, considering the association of chemical entities that are structurally different. With this review I provide an overview of the available structural studies of biomineralization proteins, explored from the Protein Data Bank (wwPDB) archive and scientific literature, and of how these studies are inspiring the design and engineering of proteins able to synthesize novel biominerals. The progression of this review from classical template proteins to silica polymerization seeks to benefit researchers involved in various interdisciplinary aspects of a biomineralization project, who need background information and a quick update on advances in the field. Lessons learned from structural studies are exemplary and will guide new projects for the imaging of new hybrid biomineral/protein superstructures at the atomic level.

The proteomics of the freshwater pearl powder: Insights from biomineralization to biomedical application

The freshwater pearl is one kind of valuable organic jewelry and traditional Chinese medicine (TCM). However, the molecular basis of matrix protein in pearl biomineralization and biomedical applications are largely unknown to date. In this study, the matrix proteins of water-soluble matrix, acid-soluble matrix and acid-insoluble matrix from the freshwater seedless pearl powder were detected using liquid chromatography-tandem mass spectrometry (LC-MS/MS) respectively, and identified against the transcriptomic database of the pearl sac. The results showed that a total of 190 proteins were identified in pearl proteomics, which was divided into eight categories by their potential biomineralization functions. The composition of pearl matrix proteins and the high frequency conserved domains like carbonic anhydrase, von Willebrand factor type A, tyrosinase and chitin binding 2 in protein sequences, implying that the “chitin-silk fibroin gel proteins–acidic macromolecules” model was suitable for description the pearl biomineralization process. Meanwhile, ninety-one of pearl matrix proteins could be classified into seven categories by their potential medical functions including wound healing, osteogenic property, antioxidant activity, neuro-regulation effects, skin lightening effect, anti-inflammatory and anti-apoptotic effects and other immunomodulatory property. In general, these results provided valuable new insights into not only the diversity of pearl matrix protein for mollusc biomineralization, but the molecular basis of pearl matrix proteins responsible for their diverse biological properties in TCM application. SignificanceThe significance of this study included the following points:1.A total of 190 proteins were identified in the freshwater pearl powder, providing valuable new insights into the complexity and diversity of pearl matrix protein in biomineralization.2.Pearl matrix proteins was divided into seven categories by their potential medical functions. This study, for the first time, attempted to unravel the molecular basis responsible for diverse biological properties of pearl powder in TCM application. We believe that this work will provide key basement and open a window for investigating the biological properties of matrix proteins and further illuminating the functional mechanism of pearl powder as TCM.

Shedding light on silica biomineralization by comparative analysis of the silica-associated proteomes from three diatom species.

Morphogenesis of the intricate patterns of diatom silica cell walls is a protein-guided process, yet to date only very few such silica biomineralization proteins have been identified. Therefore, it is currently unknown whether all diatoms share conserved proteins of a basal silica forming machinery, and whether unique proteins are responsible for the morphogenesis of species-specific silica patterns. To answer these questions, we extracted proteins from the silica of three diatom species (Thalassiosira pseudonana, Thalassiosira oceanica, and Cyclotella cryptica) by complete demineralization of the cell walls. Liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) analysis of the extracts identified 92 proteins that we name 'soluble silicome proteins' (SSPs). Surprisingly, no SSPs are common to all three species, and most SSPs showed very low similarity to one another in sequence alignments. In-depth bioinformatics analyses revealed that SSPs could be grouped into distinct classes based on short unconventional sequence motifs whose functions are yet unknown. The results from the in vivo localization of selected SSPs indicates that proteins, which lack sequence homology but share unconventional sequence motifs may exert similar functions in the morphogenesis of the diatom silica cell wall.

A Bivalve Biomineralization Toolbox.

Mollusc shells are a result of the deposition of crystalline and amorphous calcite catalyzed by enzymes and shell matrix proteins (SMP). Developing a detailed understanding of bivalve mollusc biomineralization pathways is complicated not only by the multiplicity of shell forms and microstructures in this class, but also by the evolution of associated proteins by domain co-option and domain shuffling. In spite of this, a minimal biomineralization toolbox comprising proteins and protein domains critical for shell production across species has been identified. Using a matched pair design to reduce experimental noise from inter-individual variation, combined with damage-repair experiments and a database of biomineralization SMPs derived from published works, proteins were identified that are likely to be involved in shell calcification. Eighteen new, shared proteins likely to be involved in the processes related to the calcification of shells were identified by the analysis of genes expressed during repair in Crassostrea gigas, Mytilus edulis, and Pecten maximus. Genes involved in ion transport were also identified as potentially involved in calcification either via the maintenance of cell acid–base balance or transport of critical ions to the extrapallial space, the site of shell assembly. These data expand the number of candidate biomineralization proteins in bivalve molluscs for future functional studies and define a minimal functional protein domain set required to produce solid microstructures from soluble calcium carbonate. This is important for understanding molluscan shell evolution, the likely impacts of environmental change on biomineralization processes, materials science, and biomimicry research.

Evolution of the Avian Eggshell Biomineralization Protein Toolkit - New Insights From Multi-Omics.

The avian eggshell is a remarkable biomineral, which is essential for avian reproduction; its properties permit embryonic development in the desiccating terrestrial environment, and moreover, are critically important to preserve unfertilized egg quality for human consumption. This calcium carbonate (CaCO3) bioceramic is made of 95% calcite and 3.5% organic matrix; it protects the egg contents against microbial penetration and mechanical damage, allows gaseous exchange, and provides calcium for development of the embryonic skeleton. In vertebrates, eggshell occurs in the Sauropsida and in a lesser extent in Mammalia taxa; avian eggshell calcification is one of the fastest known CaCO3 biomineralization processes, and results in a material with excellent mechanical properties. Thus, its study has triggered a strong interest from the researcher community. The investigation of eggshell biomineralization in birds over the past decades has led to detailed characterization of its protein and mineral constituents. Recently, our understanding of this process has been significantly improved using high-throughput technologies (i.e., proteomics, transcriptomics, genomics, and bioinformatics). Presently, more or less complete eggshell proteomes are available for nine birds, and therefore, key proteins that comprise the eggshell biomineralization toolkit are beginning to be identified. In this article, we review current knowledge on organic matrix components from calcified eggshell. We use these data to analyze the evolution of selected matrix proteins and underline their role in the biological toolkit required for eggshell calcification in avian species. Amongst the panel of eggshell-associated proteins, key functional domains are present such as calcium-binding, vesicle-binding and protein-binding. These technical advances, combined with progress in mineral ultrastructure analyses, have opened the way for new hypotheses of mineral nucleation and crystal growth in formation of the avian eggshell, including transfer of amorphous CaCO3 in vesicles from uterine cells to the eggshell mineralization site. The enrichment of multi-omics datasets for bird species is critical to understand the evolutionary context for development of CaCO3 biomineralization in metazoans, leading to the acquisition of the robust eggshell in birds (and formerly dinosaurs).

Role of Protein in Fungal Biomineralization of Copper Carbonate Nanoparticles

Biomineralization processes are of key importance in the biogeochemical cycling of metals and other elements by microorganisms, and several studies have highlighted the potential applications of nanoparticle synthesis via biomineralization. The roles played by proteins in the transformation and biologically induced biomineralization of metals by microorganisms is not well understood, despite the interactions of protein and nanoparticles at mineral interfaces attracting much interest in various emerging fields for novel biomaterial synthesis. Here, we have elucidated the association and involvement of fungal proteins in the formation of biogenic copper carbonate nanoparticles (CuNPs) using a carbonate-enriched biomass-free ureolytic fungal culture supernatant. Proteomic analysis was conducted that identified the major proteins present in the culture supernatant. Of the proteins identified, triosephosphate isomerase (TPI) exhibited a strong affinity to the CuNPs, and the impact of purified TPI on CuNP formation was studied in detail. The combined use of scanning electron microscopy (SEM) and confocal laser scanning microscopy (CLSM) confirmed that TPI played an important role in controlling the morphology and structure of the nanomaterials. Fourier transform infrared spectroscopy (FTIR) was applied to examine conformational changes of the proteins to further clarity the interaction mechanisms with CuNPs during biomineralization. Such analyses revealed unfolding of proteins on the mineral surface and an increase in β sheets within the protein structure. These results extend understanding of how microbial systems can influence biomineral formation through protein secretion, the mechanisms involved in formation of complex protein/inorganic systems, and provide useful guidelines for the synthesis of inorganic-protein based nanomaterials.

First sequencing of ancient coral skeletal proteins

Here we report the first recovery, sequencing, and identification of fossil biomineral proteins from a Pleistocene fossil invertebrate, the stony coral Orbicella annularis. This fossil retains total hydrolysable amino acids of a roughly similar composition to extracts from modern O. annularis skeletons, with the amino acid data rich in Asx (Asp + Asn) and Glx (Glu + Gln) typical of invertebrate skeletal proteins. It also retains several proteins, including a highly acidic protein, also known from modern coral skeletal proteomes that we sequenced by LC–MS/MS over multiple trials in the best-preserved fossil coral specimen. A combination of degradation or amino acid racemization inhibition of trypsin digestion appears to limit greater recovery. Nevertheless, our workflow determines optimal samples for effective sequencing of fossil coral proteins, allowing comparison of modern and fossil invertebrate protein sequences, and will likely lead to further improvements of the methods. Sequencing of endogenous organic molecules in fossil invertebrate biominerals provides an ancient record of composition, potentially clarifying evolutionary changes and biotic responses to paleoenvironments.

Folding of an Intrinsically Disordered Iron-Binding Peptide in Response to Sedimentation Revealed by Cryo-EM.

Biomineralization is mediated by specialized proteins that guide and control mineral sedimentation. In many cases, the active regions of these biomineralization proteins are intrinsically disordered. High-resolution structures of these proteins while they interact with minerals are essential for understanding biomineralization processes and the function of intrinsically disordered proteins (IDPs). Here we used the cavity of ferritin as a nanoreactor where the interaction between M6A, an intrinsically disordered iron-binding domain, and an iron oxide particle was visualized at high resolution by cryo-EM. Taking advantage of the differences in the electron-dose sensitivity of the protein and the iron oxide particles, we developed a method to determine the irregular shape of the particles found in our density maps. We found that the folding of M6A correlates with the detection of mineral particles in its vicinity. M6A interacts with the iron oxide particles through its C-terminal side, resulting in the stabilization of a helix at its N-terminal side. The stabilization of the helix at a region that is not in direct contact with the iron oxide particle demonstrates the ability of IDPs to respond to signals from their surroundings by conformational changes. These findings provide the first glimpse toward the long-suspected mechanism for biomineralization protein control over mineral microstructure, where unstructured regions of these proteins become more ordered in response to their interaction with the nascent mineral particles.

The Influence of Different Classes of Amino Acids on Calcium Phosphates Seeded Growth

Amino acids (AAs) attract attention for elucidating the role of proteins in biomineralization and the preparation of functionalized biomaterials. The influence that AAs exert on calcium phosphate (CaP) mineralization is still not completely understood, as contradictory results have been reported. In this paper, the influence of the addition of different classes of AAs, charged (L-aspartic acid, Asp; L-lysine, Lys), polar (L-asparagine, Asn; L-serine, Ser; L-tyrosine, Tyr), and non-polar (L-phenylalanine, Phe), on CaP growth in the presence of octacalcium phosphate (OCP) and calcium hydrogenphosphate dihydrate (DCPD) seeds was investigated. In control systems (without AAs), a calcium-deficient apatite (CaDHA) layer was formed on the surface of OCP, while a mixture of CaDHA and OCP in the form of spherical aggregates was formed on the surface of DCPD crystals. Charged and non-polar promoted, while polar AAs inhibited CaDHA formation on the OCP seeds. In the case of DCPD, Lys, Asp, and Phe promoted CaP formation, while the influence of other AAs was negligible. The most efficient promotor of precipitation in both cases was non-polar Phe. No significant influence of AAs on the composition and morphology of precipitates was observed. The obtained results are of interest for understanding biomineralization processes and additive controlled material synthesis.

Assessing the intra-crystalline approach to amino acid geochronology of Neogloboquadrina pachyderma (sinistral)

Abstract While amino acid (AA) geochronology has been widely applied to foraminiferal biomineral proteins, there has been limited assessment of the potential of isolating an ‘intra-crystalline’ fraction of proteins to improve the reliability of AA geochronology for foraminifera. In this study, bleaching experiments were carried out on the foraminifer Neogloboquadrina pachyderma (sinistral) from an independently dated core from the south eastern Norwegian Sea spanning the last 120 ka. Results show that this species contains a bleach-resistant fraction of biomineral proteins, and that this intra-crystalline fraction may behave as a closed system. Racemisation in both the intra-crystalline and whole-protein fractions was found to systematically increase with time. Isoleucine epimerisation showed reliable trends with depth when analysed by ion exchange chromatography (IEC); analysis by reverse-phase liquid chromatography (RP-HPLC) resulted in reliable age-D/L relationships for aspartic acid, glutamic acid and alanine. Isolating the intra-crystalline fraction with a 48 h bleach treatment reduced the influence of contamination in younger material. Therefore, this paper recommends that the intra-crystalline approach is used when analysing Neogloboquadrina pachyderma (sinistral) for AA geochronology, especially for younger samples. Material-specific regression equations developed in this study successfully convert IEC A/I to RP-HPLC D/L values for aspartic acid, glutamic acid and alanine, enabling integration of geochronologies developed using different methods for this class of biomineral.

Molecular Characterization of a Novel Shell Matrix Protein With PDZ Domain From Mytilus coruscus.

Mollusk shells are products of biomineralization and possess excellent mechanical properties, and shell matrix proteins (SMPs) have important functions in shell formation. A novel SMP with a PDZ domain (PDZ-domain-containing-protein-1, PDCP-1) was identified from the shell matrices of Mytilus coruscus. In this study, the gene expression, function, and location of PDCP-1 were analyzed. PDCP-1 was characterized as an ∼70 kDa protein with a PDZ (postsynaptic density/discs large/zonula occludes) domain and a ZM (ZASP-like motif) domain. The PDCP-1 gene has a high expression level and specific location in the foot, mantle and adductor muscle. Recombinantly expressed PDCP-1 (rPDCP-1) altered the morphology of calcite crystals, the polymorph of calcite crystals, binding with both calcite and aragonite crystals, and inhibition of the crystallization rate of calcite crystals. In addition, anti-rPDCP-1 antibody was prepared, and immunohistochemistry and immunofluorescence analyses revealed the specific location of PDCP-1 in the mantle, the adductor muscle, and the aragonite (nacre and myostracum) layer of the shell, suggesting multiple functions of PDCP-1 in biomineralization, muscle-shell attachment, and muscle attraction. Furthermore, pull-down analysis revealed 19 protein partners of PDCP-1 from the shell matrices, which accordingly provided a possible interaction network of PDCP-1 in the shell. These results expand the understanding of the functions of PDZ-domain-containing proteins (PDCPs) in biomineralization and the supramolecular chemistry that contributes to shell formation.

Controls of nature: Secondary, tertiary, and quaternary structure of the enamel protein amelogenin in solution and on hydroxyapatite

Amelogenin, a protein critical to enamel formation, is presented as a model for understanding how the structure of biomineralization proteins orchestrate biomineral formation. Amelogenin is the predominant biomineralization protein in the early stages of enamel formation and contributes to the controlled formation of hydroxyapatite (HAP) enamel crystals. The resulting enamel mineral is one of the hardest tissues in the human body and one of the hardest biominerals in nature. Structural studies have been hindered by the lack of techniques to evaluate surface adsorbed proteins and by amelogenin’s disposition to self-assemble. Recent advancements in solution and solid state nuclear magnetic resonance (NMR) spectroscopy, atomic force microscopy (AFM), and recombinant isotope labeling strategies are now enabling detailed structural studies. These recent studies, coupled with insights from techniques such as CD and IR spectroscopy and computational methodologies, are contributing to important advancements in our structural understanding of amelogenesis. In this review we focus on recent advances in solution and solid state NMR spectroscopy and in situ AFM that reveal new insights into the secondary, tertiary, and quaternary structure of amelogenin by itself and in contact with HAP. These studies have increased our understanding of the interface between amelogenin and HAP and how amelogenin controls enamel formation.