AP7 is an extracellular aragonite-associated protein of the nacre layer of the mollusk Haliotis rufescens and possesses a 36-amino acid C-terminal domain that exhibits sequence homology to the C subclass of the RING domain intracellular protein family. We report here novel findings which implicate AP7 as a member of the intrinsically disordered protein class (IDP) and reveal new aspects of AP7 mineralization activity. AP7 is partially disordered but can undergo additional folding in the presence of TFE. AP7 binds Zn(II) ions in a non-tetracoordinate complex but does not require Zn(II) either for folding or for in vitro function. In addition to limiting calcite crystal growth, AP7 is also observed to induce aggregate formation within in vitro mineralization assays, and these aggregates are either amorphous (type A) or crystalline (type B) in appearance. The type A aggregate displays an irregular morphology and round, dark, electron dense deposits that do not give rise to a diffraction pattern. In contrast, the type B aggregates possess either organized parallel crystal clusters or highly dense hexagonal clusters that are confirmed by electron diffraction to be aragonite. This stabilization of aragonite is remarkable in that it occurred in the presence of AP7 alone and did not require typical aragonite stabilization agents such as Mg(II), other nacre proteins, or an organized organic matrix. The ability of a partially disordered C-RING protein to perform inorganic phase stabilization represents a new twist on both the RING domain and IDP stories, and this process of aggregate formation may provide an important clue with regard to the protein-mediated nacre formation process.

The formation of aragonite in the mollusk shell nacre layer is linked to the assembly of framework protein complexes that interact with β-chitin polysaccharide. What is not yet understood is how framework nacre proteins control crystal growth. Recently, a 30 AA intrinsically disordered nacre protein sequence (n16N) derived from the n16 framework nacre protein was found to form aragonite, vaterite, or ACC deposits when adsorbed onto β-chitin. Our present study now establishes that n16N assembles to form amorphous nonmineralized supramolecular complexes that nucleate calcium carbonate polymorphs in vitro. These complexes contain unfolded or disordered (54% random coil, 46% β structures) n16N polypeptide chains that self-assemble in response to alkaline pH shift. The pH-dependent assembly process involves two stages, and it is likely that side chain salt-bridging interactions are a major driving force in n16N self-association. Intriguingly, Ca(II) ions are not required for n16N assembly but do shift the assembly process to higher pH values, and it is likely that Ca(II) plays some role in stabilizing the monomeric form of n16N. Using preassembled fibril-spheroid n16N assemblies on Si wafers or polystyrene supports, we were able to preferentially nucleate vaterite at higher incidence compared to control scenarios, and it is clear that the n16N assemblies are in contact with the nucleating crystals. We conclude that the framework nacre protein sequence n16N assembles to form supramolecular complexes whose surfaces act as nucleation sites for crystal growth. This may represent a general mineralization mechanism employed by framework nacre proteins in general.

Thin tablets and films of calcium carbonate have been grown at the air-water interface via an amorphous precursor route using soluble process-directing agents and a Langmuir monolayer based on resorcarene. By using appropriate concentrations of poly(acrylic acid-sodium salt) in combination with Mg2+ ion, an initially amorphous film is deposited on the monolayer template, which subsequently crystallizes into a mosaic film composed of a mixture of single-crystalline and spherulitic patches of calcite and aragonite. Of particular importance is the synthesis of single-crystalline "tablets" of aragonite (approximately 600 nm thick), because this phase generally forms needle-like polycrystalline aggregates when grown in vitro. To our knowledge, a tabular single-crystalline morphology of aragonite has only been observed in the nacreous layer of mollusk shells. Therefore, this in vitro system may serve as a useful model for examining mechanistic issues pertinent to biomineralization, such as the influence of organic templates on nucleation from an amorphous phase.

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.

The formation of calcite prism architecture in the prismatic layer of the mollusk shell involves the participation of a number of different proteins. One protein family, Asprich, has been identified as a participant in amorphous calcium carbonate stabilization and calcite architecture in the prismatic layer of the mollusk, Atrina rigida . However, the functional role(s) of this protein family are not fully understood due to the fact that insufficient quantities of these proteins are available for experimentation. To overcome this problem, we employed stepwise solid-phase synthesis to recreate one of the 10 members of the Asprich family, the 61 AA single chain protein, Asprich "3". We find that the Asprich "3" protein inhibits the formation of rhombohedral calcite crystals and induces the formation of round calcium carbonate deposits in vitro that contain calcite and amorphous calcium carbonate (ACC). This mineralization behavior does not occur under control conditions, and the formation of ACC and calcite is similar to that reported for the recombinant form of the Asprich "g" protein. Circular dichroism studies reveal that Asprich "3" is an intrinsically disordered protein, predominantly random coil (66%), with 20-30% β-strand content, a small percentage of β-turn, and little if any α-helical content. This protein is not extrinsically stabilized by Ca(II) ions but can be stabilized by 2,2,2-trifluoroethanol to form a structure consisting of turn-like and random coil characteristics. This finding suggests that Asprich "3" may require other extrinsic interactions (i.e., with mineral or ionic clusters or other macromolecules) to achieve folding. In conclusion, Asprich "3" possesses in vitro functional and structural qualities that are similar to other reported for other Asprich protein sequences.

The kinetics of biomimetic mineralization at a fatty acid monolayer interface have been measured in situ by synchrotron x-ray reflectivity. The formation of biologically relevant amorphous calcium carbonate films is affected by soluble macromolecules, supersaturation rate of change, and Mg cations. We find that these solution conditions influence mineral film formation in a complementary fashion. Poly(sodium acrylate) extends the lifetime of metastable amorphous calcium carbonate, solution saturation controls the mineral film growth rate, and Mg cations create a longer induction time. This is the first quantification of potentially competitive biomineralization mechanisms that addresses nucleation and growth of the amorphous mineral phases, which are important in biomineralization.

We employ principles derived from biomineralization to assemble functional nanoscale inorganic materials onto flexible polymer substrates under low-temperature, benign chemical conditions. Our biomimetic approach involves the selective functionalization of polymer surfaces to create regions of differing surface energy which can promote heterogeneous nucleation in designated regions while suppressing it in others. Specifically, ZnO is grown into ordered microarrays on flexible polycarbonate films and polyester filaments directly out of aqueous solutions. The resulting block, checkerboard, and grid shaped microarrays consist of either ZnO nanorods or microplates with feature sizes down to 4 μm. The approach reported is general, does not require traditional lithography, uses no seed layers or self-assembled monolayers, and can be easily applied to a variety of planar or three-dimensional polymer substrates enabling numerous applications in flexible macroelectronics and smart textiles.

Recent studies indicate that the ability of mollusk shell nacre protein sequences to form the calcium carbonate polymorph, aragonite, are linked to the presence of intrinsically disordered sequences within these proteins. Although the exact relationship between protein structural disorder and polymorph formation is not clear, there is a definite interest in discovering other examples of intrinsically disordered nacre protein sequences that can induce aragonite formation. In this report, we extend the relationship between intrinsic disorder and aragonite formation to another set of nacre protein sequences. This protein, known as PFMG1, is associated with pearl formation in the Japanese pearl oyster, Pinctada fucata. We demonstrate that synthetic peptides representing the 30 AA N-and C-terminal sequence regions of PFMG1 nucleate nanoscale-sized aragonite in solution without the need for additional additives. Compared to controls containing no peptide or bovine serum albumin, the PFMG1 terminal sequences appear to form a matrix-like environment around the forming biominerals, and this process will be defined in more detail in later reports. Furthermore, we establish that these PFMG1 terminal sequences possess disordered structures in solution that can be stabilized into partially folded structures (R helix, beta structures) using the structure-stabilizing solvent, 2,2,2-trifluoroethanol. Although we do not know the mechanism by which these peptides promote aragonite nucleation in vitro, we believe that these terminal sequences are participants in PFMG1-mediated aragonite polymorph formation within the oyster pearl and that the intrinsic disorder and folding propensities of these sequences are crucial for this activity.The mollusk shell is a biocomposite that is comprised of two distinct layers of calcium carbonate that coexist with a series of biomacromolecules. [1][2][3][4][5][6][7][8][9][10][11] One of these layers, known as the nacre layer, is comprised of the calcium carbonate polymorph, aragonite, which is thermodynamically unstable. 4 Moreover, the nacre layer is fracture-resistant (3000Â greater than pure aragonite) 5 and under force stress exhibits crack deflection and energy dissipation. [6][7][8] These material properties arise from the presence of proteins which inhabit the aragonite crystals (intercalation or occlusion) 9 and the space surrounding these crystals. [1][2][3] Interestingly, these same proteins may play a role in the nucleation of aragonite [1][2][3]9 and the formation of an amorphous precursor, amorphous calcium carbonate (ACC). 1,12 Hence, the nacre layer represents an important model system for extracting molecular principles (nucleation, protein occlusion, fracture toughness) that Nature uses to create composite materials under ambient conditions.One molecular principle that appears to emerge is the link between disordered protein structure and biomineralization protein function. Recently, a nacre protein, AP7 (Haliotis rufescens), was found to stabilize single and polycrystalline aragon...