Publications (14) View all
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Article: A C-RING-like domain participates in protein self-assembly and mineral nucleation.
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ABSTRACT: 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.Biochemistry 09/2011; 50(41):8880-7. · 3.42 Impact Factor -
Article: Formation of framework nacre polypeptide supramolecular assemblies that nucleate polymorphs.
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ABSTRACT: 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.Biomacromolecules 05/2011; 12(5):1883-90. · 5.48 Impact Factor -
Article: Intrinsically disordered mollusk shell prismatic protein that modulates calcium carbonate crystal growth.
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ABSTRACT: 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.Biomacromolecules 10/2010; 11(10):2539-44. · 5.48 Impact Factor -
Article: The N- and C-Terminal Regions of the Pearl-Associated EF Hand Protein, PFMG1, Promote the Formation of the Aragonite Polymorph in Vitro
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ABSTRACT: 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 (α 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.08/2010; -
Article: Mineralogical signatures of stone formation mechanisms.
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ABSTRACT: 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.Urological Research 08/2010; 38(4):281-92. · 1.23 Impact Factor
