Titanium mineralization in ferritin: a room temperature nonphotochemical preparation and biophysical characterization.

The incremental addition of titanium(III) citrate to H-chain homopolymers of human ferritin results in the formation of 1.5-6.5-nm particles of amorphous TiO(2) within the nanocage of the protein. The mineralization conditions are mild, featuring ambient temperature and no need for photochemical activation. Low ratios of titanium to protein favor intraprotein mineralization, and the products are characterized by stained and unstained transmission electron microscopy, UV-vis spectroscopy, dynamic light scattering, analytical ultracentrifugation, and metal analysis. With up to 1,000 equiv of metal, there is no change to the protein hydrodynamic radius or diffusion constant. There is, however, a systematic shift in the sedimentation coefficient, which confirms mineralization within the protein core.

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.

Formation of framework nacre polypeptide supramolecular assemblies that nucleate polymorphs.

The formation of aragonite in the mollusk shell nacre layer is linked to the assembly of framework protein complexes that interact with ß-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 ß-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% ß 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.

Intrinsically disordered mollusk shell prismatic protein that modulates calcium carbonate crystal growth.

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% ß-strand content, a small percentage of ß-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.

Mineralogical signatures of stone formation mechanisms.

The mechanisms involved in biomineralization are modulated through interactions with organic matrix. In the case of stone formation, the role of the organic macromolecules in the complex urinary environment is not clear, but the presence of mineralogical 'signatures' suggests that some aspects of stone formation may result from a non-classical crystallization process that is induced by acidic proteins. An amorphous precursor has been detected in many biologically controlled mineralization reactions, which is thought to be regulated by non-specific interactions between soluble acidic proteins and mineral ions. Using in vitro model systems, we find that a liquid-phase amorphous mineral precursor induced by acidic polypeptides can lead to crystal textures that resemble those found in Randall's plaque and kidney stones. This polymer-induced liquid-precursor process leads to agglomerates of coalesced mineral spherules, dense-packed spherulites with concentric laminations, mineral coatings and 'cements', and collagen-associated mineralization. Through the use of in vitro model systems, the mechanisms involved in the formation of these crystallographic features may be resolved, enhancing our understanding of the potential role(s) that proteins play in stone formation.

AP7, a partially disordered pseudo C-RING protein, is capable of forming stabilized aragonite in vitro.

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.

Evidence of mineralization activity and supramolecular assembly by the N-terminal sequence of ACCBP, a biomineralization protein that is homologous to the acetylcholine binding protein family.

Several biomineralization proteins that exhibit intrinsic disorder also possess sequence regions that are homologous to nonmineral associated folded proteins. One such protein is the amorphous calcium carbonate binding protein (ACCBP), one of several proteins that regulate the formation of the oyster shell and exhibit 30% conserved sequence identity to the acetylcholine binding protein sequences. To gain a better understanding of the ACCBP protein, we utilized bioinformatic approaches to identify the location of disordered and folded regions within this protein. In addition, we synthesized a 50 AA polypeptide, ACCN, representing the N-terminal domain of the mature processed ACCBP protein. We then utilized this polypeptide to determine the mineralization activity and qualitative structure of the N-terminal region of ACCBP. Our bioinformatic studies indicate that ACCBP consists of a ten-stranded beta-sandwich structure that includes short disordered sequence blocks, two of which reside within the primarily helical and surface-accessible ACCN sequence. Circular dichroism studies reveal that ACCN is partially disordered in solution; however, ACCN can be induced to fold into an alpha helix in the presence of TFE. Furthermore, we confirm that the ACCN sequence is multifunctional; this sequence promotes radial calcite polycrystal growth on Kevlar threads and forms supramolecular assemblies in solution that contain amorphous-appearing deposits. We conclude that the partially disordered ACCN sequence is a putative site for mineralization activity within the ACCBP protein and that the presence of short disordered sequence regions within the ACCBP fold are essential for function.

Mechanism of formation of concentrically laminated spherules: implication to Randall's plaque and stone formation.

We report on the formation of calcium phosphate multi-laminated spherules via a polymer-induced liquid-like precursor (PILP) process. In this non-classical crystallization route, the precipitation of liquid-like amorphous calcium phosphate (ACP) particles is promoted using anionic polypeptide additives, and these droplets coalesce to form globules that later crystallize into spherulites. During crystallization of the amorphous globules, the polymer additive, as well as the waters of hydration, is excluded ahead of the crystallization front, but some polymer becomes entrapped within diffusion-limited zones. This results in the formation of concentric laminations with layers of variable density from organic-rich inclusions. The striking resemblance of these spherules with the crystals of the Randall's plaque and other laminated stones suggests that such biological structures may form via an amorphous precursor process as well. Given the organic-rich environment present in the urinary tract, one might expect a large amount of organic materials to become entrapped within the stratified zones of a forming stone during this type of solidification and transformation process.

Formation of single-crystalline aragonite tablets/films via an amorphous precursor.

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.

Photodetector arrays directly assembled onto polymer substrates from aqueous solution.

We report a simple and inexpensive approach to directly assemble arrays of cadmium sulfide (CdS) semiconductors onto transparent flexible poly(ethylene terephthalate) (PET) sheets via a polymer-mediated selective nucleation and growth process from an aqueous solution. This strategy of assembling functional materials onto plastics utilizes the surface functional molecules of the UV photooxidation patterned polymer to direct the nucleation and growth of CdS. We demonstrated that such assembled structures are viable for flexible macroelectronics, as manifested by the fabrication of CdS photodetector arrays on PET that can withstand bending. The best devices exhibited a specific detectivity of 3 x 10(11) cm Hz(1/2) W(-1) at 514-nm excitation wavelength at a modulation frequency of 90 Hz at room temperature. This direct assembly strategy eliminates additional lithography and etching steps during the deposition of the active inorganic semiconductor layer, is general to other inorganic materials and plastic substrates, and can enable low-cost, wearable, and/or disposable flexible electronics.

Complementary control by additives of the kinetics of amorphous CaCO3 mineralization at an organic interface: in-situ synchrotron x-ray observations.

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.