A Complicated Relationship: Glycosylation, Ca(II), and Primary Sequence Affect the Interactions and Kinetics between Two Model Mollusk Shell Intracrystalline Nacre Proteins.

The formation of the mollusk shell requires the participation of proteins, many of which may be interactive with one another. We examined a model protein pair system from the mollusk Haliotis rufescens, wherein we probed the interactions between recombinant forms of two major nacre layer proteins, AP7, and the glycoprotein, AP24. Here, the focus was on the impact that the AP24 glycosylation and primary sequence had on AP24-AP7 binding. We find that both the glycosylated and nonglycosylated variants of AP24 bound to AP7 but with different quantities, kinetics, and internal rearrangements. Moreover, the binding of AP7 with nonglycosylated and glycosylated AP24 was found to be Ca(II)-dependent and -independent, respectively. Yet both variants of AP24 combine with AP7 to form hybrid hydrogel particles that are similar in their physical properties. Thus, AP7 and AP24 protein sequences are interactive and form hydrogels, but the interactions are tuned by glycosylation and Ca(II). These features may have an impact on the nacre matrix formation.

Skeletal development in the sea urchin relies upon protein families that contain intrinsic disorder, aggregation-prone, and conserved globular interactive domains.

The formation of the sea urchin spicule skeleton requires the participation of hydrogel-forming protein families that regulate mineral nucleation and nanoparticle assembly processes that give rise to the spicule. However, the structure and molecular behavior of these proteins is not well established, and thus our ability to understand this process is hampered. We embarked on a study of sea urchin spicule proteins using a combination of biophysical and bioinformatics techniques. Our biophysical findings indicate that recombinant variants of the two most studied spicule matrix proteins, SpSM50 and SpSM30B/C (S. purpuratus) have a conformational landscape that include a C-terminal random coil/intrinsically disordered MAPQG sequence coupled to a conserved, folded N-terminal C-type lectin-like (CTLL) domain, with SpSM50 > SpSM30B/C with regard to intrinsic disorder. Both proteins possess solvent-accessible unfolded MAQPG sequence regions where Asn, Gln, and Arg residues may be accessible for protein hydrogel interactions with water molecules. Our bioinformatics study included seven other spicule matrix proteins where we note similarities between these proteins and rare, unusual proteins that possess folded and unfolded traits. Moreover, spicule matrix proteins possess three types of sequences: intrinsically disordered, amyloid-like, and folded protein-protein interactive. Collectively these reactive domains would be capable of driving protein assembly and hydrogel formation. Interestingly, three types of global conformations are predicted for the nine member protein set, wherein we note variations in the arrangement of intrinsically disordered and interactive globular domains. These variations may reflect species-specific requirements for spiculogenesis. We conclude that the molecular landscape of spicule matrix protein families enables them to function as hydrogelators, nucleators, and assemblers of mineral nanoparticles.

The Biomineralization Proteome: Protein Complexity for a Complex Bioceramic Assembly Process.

There are over 62 different biominerals on Earth and a diverse array of organisms that generate these biominerals for survival. This review will introduce the process of biomineralization and the current understanding of the molecular mechanisms of mineral formation, and then comparatively explore the representative secretomes of two well-documented skeletal systems: vertebrate bone (calcium phosphate) and invertebrate mollusk shell (calcium carbonate). It is found that both skeletal secretomes have gross similarities and possess proteins that fall into four functional categories: matrix formers, nucleation assisters, communicators, and remodelers. In many cases the mineral-associated matrix former and nucleation assister sequences in both skeletal systems are unique and possess interactive conserved globular domains, intrinsic disorder, post-translational modifications, sequence redundancy, and amyloid-like aggregation-prone sequences. Together, these molecular features create a protein-based environment that facilitates mineral formation and organization and argue in favor of conserved features that evolve from the mollusk shell to bone. Interestingly, the mollusk shell secretome appears to be more complex compared to that of bone tissue, in that there are numerous protein subcategories that are required for the nucleation and organization of inner (nacre) and outer (prismatic) calcium carbonate regions of the shell. This may reflect the organizational and material requirements of an exoskeletal protective system.

Composite Materials Design: Biomineralization Proteins and the Guided Assembly and Organization of Biomineral Nanoparticles.

There has been much discussion of the role of proteins in the calcium carbonate biomineralization process, particularly with regard to nucleation, amorphous stabilization/transformation, and polymorph selection. However, there has been little if any discussion of the potential role that proteins might play in another important process: the guided assembly and organization of mineral nanoparticles into higher-ordered structures such as mesocrystals. This review discusses particle attachment theory and recent evidence of mineral-associated proteins forming hydrogels that assemble and organize mineral clusters into crystalline phase. From this discussion we postulate a mechanism by which biomineralization protein hydrogel aggregation assists in mineral nanoparticle assembly and organization within calcium carbonate skeletal elements and discuss potentials ways for harnessing this process in materials design.

Secrets of the Sea Urchin Spicule Revealed: Protein Cooperativity Is Responsible for ACC Transformation, Intracrystalline Incorporation, and Guided Mineral Particle Assembly in Biocomposite Material Formation.

The formation of the sea urchin spicule involves the stabilization and transformation of amorphous calcium carbonate (ACC) and assembly of ACC nanoparticle precursors into a mesoscale single crystal of fracture-resistant calcite. This process of particle assembly or attachment is under the control of a family of proteins known as the spicule matrix [Strongylocentrotus purpuratus (SpSM)] proteome. Recently, two members of this proteome, SpSM50 and the glycoprotein SpSM30B/C-G (in recombinant forms), were found to interact together via SpSM30B/C-G oligosaccharide-SpSM50 protein interactions to form hybrid protein hydrogels with unique physical properties. In this study, we investigate the mineralization properties of this hybrid hydrogel alongside the hydrogels formed by SpSM50 and SpSM30B/C-G individually. We find that the SpSM50 + SpSM30B/C-G hybrid hydrogel is synergistic with regard to surface modifications and intracrystalline inclusions of existing calcite crystals, the inhibition of ACC formation, and the kinetic destabilization of ACC to form a crystalline phase. Most importantly, the hybrid hydrogel phase assembles and organizes mineral particles into discrete clusters or domains within in vitro mineralization environments. Thus, the interactions of SpSM50 and SpSM30B/C-G, mediated by carbohydrate-protein binding, reflect the need for protein cooperativity for the ACC-to-crystalline transformation, intracrystalline void formation, and guided mineral particle assembly processes that are instrumental in spicule formation.

Glycosylation Fosters Interactions between Model Sea Urchin Spicule Matrix Proteins. Implications for Embryonic Spiculogenesis and Biomineralization.

The formation of embryonic mineralized skeletal elements (spicules) in the sea urchin requires the participation of proteins, many of which may interact with one another and assist in the creation of an extracellular matrix wherein mineral formation takes place. To probe this, we created a sea urchin spicule recombinant model protein pair system wherein we tested the interactions between two major spicule proteins, SpSM50 and the glycoprotein, SpSM30B/C. Both proteins are strong hydrogelators that manipulate early and later events in mineral formation. We discovered that the anionic glycan moieties of SpSM30B/C are required for interaction with the SpSM50 protein and that these interactions are Ca(II)-independent. In addition, when these proteins form a complex, they create hybrid hydrogel particles that are physically distinct from their individual counterparts. Thus, glycan-mediated interactions play an important role in in vitro spicule protein assembly and most likely within the spicule itself.

Selective Synergism Created by Interactive Nacre Framework-Associated Proteins Possessing EGF and vWA Motifs: Implications for Mollusk Shell Formation.

In the nacre layer of the Pinctada fucata oyster shell there exists a multimember proteome, known as the framework family, which regulates the formation of the aragonite mesoscale tablets and participates in the creation of an organic coating around each tablet. 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 recombinant framework proteins, r-Pif97 (containing a von Willebrand Factor Type A domain (vWA)) and r-n16.3 (containing an EGF-like domain), whose individual in vitro mineralization functionalities are distinct from one another. We find that at 1:1 molar ratios r-Pif97 and r-n16.3 exhibit little or no synergistic activity regarding modifying existing calcite crystals. However, during the early stages of nucleation in solution, we note synergistic effects on nucleation kinetics and ACC formation/stability (via dehydration) that are not observed for the individual proteins. This selective synergism is generated by Ca2+-mediated protein-protein interactions (∼4 molecules of r-n16.3 per 1 molecule of r-Pif97) which lead to the formation of nucleation-responsive hybrid hydrogel particles in solution. Interestingly, in the absence of Ca2+ there are no significant interactions occurring between the two proteins. This unique behavior of the framework-associated n16.3 and Pif97 proteins suggests that the Asp/Glu-containing regions of the vWA and EGF-like domains may play a role in both nacre matrix formation and mineralization.

Intracrystalline incorporation of nacre protein hydrogels modifies the mechanical properties of calcite crystals: a microcompression study.

The fracture toughness of mollusk shell nacre has been attributed to many factors, one of which is the intracrystalline incorporation of nacre-specific proteins. Although mechanical force measurements have been made on the nacre layer and on individual calcium carbonate crystals containing occluded organic molecules and macromolecules, there are few if any studies which examine the impact of occluded proteins on the mechanical properties of calcium carbonate crystals. To remedy this, we performed microcompression studies of calcite crystals grown in the presence and absence of two recombinant nacre proteins, r-AP7 (H. rufescens, intracrystalline proteome) and r-n16.3 (P. fucata, framework proteome), both of which are known aggregators that form hydrogel nanoinclusions within in vitro calcite. We find that, relative to protein-free calcite, the intracrystalline inclusion of either r-AP7 or r-n16.3 nacre protein hydrogels within the calcite crystals leads to a reduction in strength. However, nacre protein-modified crystals were found to exhibit elastic deformation under force compared to control scenarios, with no discernable differences noted between intracrystalline or framework protein-modified crystals. We conclude from our in vitro microcompression studies that the intracrystalline incorporation of nacre proteins can contribute to fracture-resistance of the crystalline phase by significantly reducing both modulus AND critical strength.

Insights into Mollusk Shell Formation: Interlamellar and Lamellar-Specific Nacre Protein Hydrogels Differ in Ion Interaction Signatures.

In the mollusk shell nacre layer, there exist hydrogelator proteomes that play important roles in the formation of the mineral phase. Two of these proteomes, the intracrystalline and the framework, reside in the interior and exterior, respectively, of the nacre tablets. To date there is no clear evidence of what distinguishes an intracrystalline protein from a framework protein regarding the nucleation process. Using Eu(III), phosphate anions, and recombinant versions of the intracrystalline protein, AP7 and the framework protein, n16.3 we probed each protein hydrogel for its interactions with these model ions. Fluorescence spectroscopy of Eu(III) interactions with both protein hydrogels revealed that r-AP7 exhibited enhanced effects on Eu(III) fluorescence compared to r-n16.3, and, 31P NMR experiments demonstrated that r-AP7 had a more significant impact on phosphate anions compared to r-n16.3. Thus, r-AP7 was found to be more of an ion "disruptor" than r-n16.3. Interestingly, these findings correlate with the particle size distributions and internal structure of the hydrogel particles themselves, suggesting that the physical and chemical properties of the hydrogels dictate hydrogel-ion interactions. In conclusion, we confirm that hydrogelator proteomes possess distinguishable ion interaction properties that may impact the nucleation processes in these regions and control the overall formation of mesoscale nacre tablets.

Functional Prioritization and Hydrogel Regulation Phenomena Created by a Combinatorial Pearl-Associated Two-Protein Biomineralization Model System.

In the nacre or aragonitic layer of an oyster pearl, there exists a 12-member proteome that regulates both the early stages of nucleation and nanoscale-to-mesoscale assembly of nacre tablets and calcitic crystals from mineral nanoparticle precursors. Several approaches to understanding protein-associated mechanisms of pearl nacre formation have been developed, 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 pearl nacre-associated proteins, PFMG1 and PFMG2 (shell oyster pearl nacre, Pinctada fucata) whose individual in vitro mineralization functionalities are distinct from one another. Using scanning electron microscopy, atomic force microscopy, Ca(II) potentiometric titrations, and quartz crystal microbalance with dissipation monitoring quantitative analyses, we find that at 1:1 molar ratios, rPFMG2 and rPFMG1 co-aggregate in specific molecular ratios to form hybrid hydrogels that affect both the early and later stages of in vitro calcium carbonate nucleation. Within these hybrid hydrogels, rPFMG2 plays a role in defining protein co-aggregation and hydrogel dimension, whereas rPFMG1 defines participation in nonclassical nucleation processes; both proteins exhibit synergy with regard to surface and subsurface modifications to existing crystals. The interactions between both proteins are enhanced by Ca(II) ions and may involve Ca(II)-induced conformational events within the EF-hand rPFMG1 protein, as well as putative interactions between the EF-hand domain of rPFMG1 and the calponin-like domain of rPFMG2. Thus, the pearl-associated PFMG1 and PFMG2 proteins interact and exhibit mineralization functionalities in specific ways, which may be relevant for pearl formation.

A Model Sea Urchin Spicule Matrix Protein, rSpSM50, Is a Hydrogelator That Modifies and Organizes the Mineralization Process.

In the purple sea urchin Strongylocentrotus purpuratus, the formation and mineralization of fracture-resistant skeletal elements such as the embryonic spicule require the combinatorial participation of numerous spicule matrix proteins such as SpSM50. However, because of its limited abundance and solubility issues, it has been difficult to pursue extensive in vitro biochemical studies of SpSM50 protein and deduce its role in spicule formation and mineralization. To circumvent these problems, we expressed a tag-free bacterial model recombinant spicule matrix protein, rSpSM50. Bioinformatics and biophysical experiments confirm that rSpSM50 is an intrinsically disordered, aggregation-prone C-type lectin-like domain-containing protein that forms dimensionally and internally heterogeneous protein hydrogels that control the in vitro mineralization process in three ways. The hydrogels (1) kinetically stabilize the aqueous calcium carbonate system against nucleation and thermodynamically destabilize the initially formed ACC in bulk solution, (2) promote and organize faceted single-crystal calcite and polycrystalline vaterite nanoparticles, and (3) promote surface texturing of calcite crystals and induce subsurface nanoporosities and channels within both calcite and vaterite crystals. Many of these features are also common to mollusk shell nacre proteins and the sea urchin spicule matrix glycoprotein, SpSM30B/C, and we conclude that rSpSM50 is a spiculogenesis hydrogelator protein that exhibits traits found in other calcium carbonate mineral-modification proteins.

Sea Urchin Spicule Matrix Proteins Form Mesoscale "Smart" Hydrogels That Exhibit Selective Ion Interactions.

In the sea urchin embryo spicule, there exists a proteome of >200 proteins that are responsible for controlling the mineralization of the spicule and the formation of a fracture-resistant composite. In this report, using recombinant proteins, we identify that two protein components of the spicule, SM30B/C and SM50, are hydrogelators. Because of the presence of intrinsic disorder and aggregation-prone regions, these proteins assemble to form porous mesoscale hydrogel particles in solution. These hydrogel particles change their size, organization, and internal structure in response to pH and ions, particularly Ca(II), which indicates that these behave as ion-responsive or "smart" hydrogels. Using diffusion-ordered spectroscopy NMR, we find that both hydrogels affect the diffusion of water, but only SM50 affects the diffusion of an anionic solute. Thus, the extracellular matrix of the spicule consists of several hydrogelator proteins which are responsive to solution conditions and can control the diffusion of water and solutes, and these proteins will serve as a model system for designing ion-responsive, composite, and smart hydrogels.

A Model Sea Urchin Spicule Matrix Protein Self-Associates To Form Mineral-Modifying Protein Hydrogels.

In the purple sea urchin Strongylocentrotus purpuratus, the formation and mineralization of fracture-resistant skeletal elements such as the embryonic spicule require the combinatorial participation of numerous spicule matrix proteins such as the SpSM30A-F isoforms. However, because of limited abundance, it has been difficult to pursue extensive biochemical studies of the SpSM30 proteins and deduce their role in spicule formation and mineralization. To circumvent these problems, we expressed a model recombinant spicule matrix protein, rSpSM30B/C, which possesses the key sequence attributes of isoforms "B" and "C". Our findings indicate that rSpSM30B/C is expressed in insect cells as a single polypeptide containing variations in glycosylation that create microheterogeneity in rSpSM30B/C molecular masses. These post-translational modifications incorporate O- and N-glycans and anionic mono- and bisialylated and mono- and bisulfated monosaccharides on the protein molecules and enhance its aggregation propensity. Bioinformatics and biophysical experiments confirm that rSpSM30B/C is an intrinsically disordered, aggregation-prone protein that forms porous protein hydrogels that control the in vitro mineralization process in three ways: (1) increase the time interval for prenucleation cluster formation and transiently stabilize an ACC polymorph, (2) promote and organize single-crystal calcite nanoparticles, and (3) promote faceted growth and create surface texturing of calcite crystals. These features are also common to mollusk shell nacre proteins, and we conclude that rSpSM30B/C is a spiculogenesis protein that exhibits traits found in other calcium carbonate mineral modification proteins.

Synergistic Biomineralization Phenomena Created by a Combinatorial Nacre Protein Model System.

In the nacre or aragonite layer of the mollusk shell, proteomes that regulate both the early stages of nucleation and nano-to-mesoscale assembly of nacre tablets from mineral nanoparticle precursors exist. 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, Haliotis rufescens) and pseudo-EF hand PFMG1 (oyster pearl nacre, Pinctada fucata), whose individual in vitro mineralization functionalities are well-documented and distinct from one another. Using scanning electron microscopy, flow cell scanning transmission electron microscopy, atomic force microscopy, Ca(II) potentiometric titrations, and quartz crystal microbalance with dissipation monitoring quantitative analyses, we find that both nacre proteins are functionally active within the same mineralization environments and, at 1:1 molar 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 polymer-induced liquid precursor-like phases with enhanced ACC stabilization capabilities, and there is evidence of 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.

Insect Cell Glycosylation and Its Impact on the Functionality of a Recombinant Intracrystalline Nacre Protein, AP24.

The impacts of glycosylation on biomineralization protein function are largely unknown. This is certainly true for the mollusk shell, where glycosylated intracrystalline proteins such as AP24 (Haliotis rufescens) exist but their functions and the role of glycosylation remain elusive. To assess the effect of glycosylation on protein function, we expressed two recombinant variants of AP24: an unglycosylated bacteria-expressed version (rAP24N) and a glycosylated insect cell-expressed version (rAP24G). Our findings indicate that rAP24G is expressed as a single polypeptide containing variations in glycosylation that create microheterogeneity in rAP24G molecular masses. These post-translational modifications incorporate O- and N-glycans and anionic monosialylated and bisialylated, and monosulfated and bisulfated monosaccharides on the protein molecules. AFM and DLS experiments confirm that both rAP24N and rAP24G aggregate to form protein phases, with rAP24N exhibiting a higher degree of aggregation, compared to rAP24G. With regard to functionality, we observe that both recombinant proteins exhibit similar behavior within in vitro calcium carbonate mineralization assays and potentiometric titrations. However, rAP24G modifies crystal growth directions and is a stronger nucleation inhibitor, whereas rAP24N exhibits higher mineral phase stabilization and nanoparticle containment. We believe that the post-translational addition of anionic groups (via sialylation and sulfation), along with modifications to the protein surface topology, may explain the changes in glycosylated rAP24G aggregation and mineralization behavior, relative to rAP24N.

Aragonite-Associated Mollusk Shell Protein Aggregates To Form Mesoscale "Smart" Hydrogels.

In the mollusk shell there exists a framework silk fibroin-polysaccharide hydrogel coating around nacre aragonite tablets, and this coating facilitates the synthesis and organization of mineral nanoparticles into mesocrystals. In this report, we identify that a protein component of this coating, n16.3, is a hydrogelator. Due to the presence of intrinsic disorder, aggregation-prone regions, and nearly equal balance of anionic and cationic side chains, this protein assembles to form porous mesoscale hydrogel particles in solution and on mica surfaces. These hydrogel particles change their dimensionality, organization, and internal structure in response to pH and ions, particularly Ca(II), which indicates that these behave as ion-responsive or "smart" hydrogels. Thus, in addition to silk fibroins, the gel phase of the mollusk shell nacre framework layer may actually consist of several framework hydrogelator proteins, such as n16.3, which can promote mineral nanoparticle organization and assembly during the nacre biomineralization process and also serve as a model system for designing ion-responsive, composite, and smart hydrogels.

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

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-ß-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.

Interactions of amelogenin with phospholipids.

Amelogenin protein has the potential to interact with other enamel matrix proteins, mineral, and cell surfaces. We investigated the interactions of recombinant amelogenin rP172 with small unilamellar vesicles as model membranes, toward the goal of understanding the mechanisms of amelogenin-cell interactions during amelogenesis. Dynamic light scattering (DLS), fluorescence spectroscopy, circular dichroism (CD), and nuclear magnetic resonance (NMR) were used. In the presence of phospholipid vesicles, a blue shift in the Trp fluorescence emission maxima of rP172 was observed (∼334 nm) and the Trp residues of rP172 were inaccessible to the aqueous quencher acrylamide. DLS studies indicated complexation of rP172 and phospholipids, although the possibility of fusion of phospholipids following amelogenin addition cannot be ruled out. NMR and CD studies revealed a disorder-order transition of rP172 in a model membrane environment. Strong fluorescence resonance energy transfer from Trp in rP172 to DNS-bound-phospholipid was observed, and fluorescence polarization studies indicated that rP172 interacted with the hydrophobic core region of model membranes. Our data suggest that amelogenin has ability to interact with phospholipids and that such interactions may play key roles in enamel biomineralization as well as reported amelogenin signaling activities.

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.

An oligomeric C-RING nacre protein influences prenucleation events and organizes mineral nanoparticles.

The mollusk shell nacre layer integrates mineral phases with macromolecular components such as intracrystalline proteins. However, the roles performed by intracrystalline proteins in calcium carbonate nucleation and subsequent postnucleation events (e.g., organization of mineral deposits) in the nacre layer are not known. We find that AP7, a nacre intracrystalline C-RING protein, self-assembles to form amorphous protein oligomers and films on mica that further assemble into larger aggregates or phases in the presence of Ca2+. Using solution nuclear magnetic resonance spectroscopy, we determine that the protein assemblies are stabilized by interdomain interactions involving the aggregation-prone T31-N66 C-terminal C-RING domain but are destabilized by the labile nature of the intrinsically disordered D1-T19 AA N-terminal sequence. Thus, the dynamic, amorphous nature of the AP7 assemblies can be traced to the molecular behavior of the N-terminal sequence. Using potentiometric methods, we observe that AP7 protein phases prolong the time interval for prenucleation cluster formation but neither stabilize nor destabilize ACC clusters. Time-resolved flow cell scanning transmission electron microscopy mineralization studies confirm that AP7 protein phases delay the onset of nucleation and assemble and organize mineral nanoparticles into ring-shaped branching clusters in solution. These phenomena are not observed in protein-deficient assays. We conclude that C-RING AP7 protein phases modulate the time period for early events in nucleation and form strategic associations with forming mineral nanoparticles that lead to mineral organization.