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 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.