Identification of an "acidic" C-terminal mineral modification sequence from the mollusk shell protein asprich

The formation of the mineralized mollusk shell involves the participation specific proteins to control mineral formation and morphology. Recent sequencing studies have identified a subfamily of seven proteins that are specific for the prismatic or calcitic layer of the mollusk Atrina rigida. This subfamily, named Asprich a through g, are polyanionic in sequence and contain conserved sequence regions at the N- and C-termini that are believed to play a role in mineral formation. Using solid-phase peptide synthesis, we have recreated the 42-AA C-terminal domain of Asprich which contains two conserved subdomains, the 17 AA Asp, Glu, Ala repeat (DEAD17) and the Asp, Glu-rich 25 AA acidic-2 region. Using in vitro calcium carbonate mineralization assays, we demonstrate that the 42 AA C-terminal domain exerts effects on the morphology of forming calcite crystals, leading to the formation of porosities and irregular crystal growth. The two subdomains, DEAD17 and acidic-2, exert similar, albeit weaker, effects on crystal growth as well. Interestingly, when DEAD17 and acidic-2 are present as free peptides in a 1:1 mole mixture in parallel mineralization assays, the resulting peptide-mediated effects on calcite morphology are dramatically different from the results obtained for either the 42 AA C-terminal domain or the individual DEAD17 and acidic-2 domains themselves. These results suggest that the functionality of the individual DEAD17 and acidic-2 subdomains can be transformed depending upon the presence of other free peptide species. Structurally, the 42 AA C-terminal domain and the two subdomains are remarkably similar to one another and are conformationally labile, existing in an equilibrium with random-coil and other undefined secondary structures at neutral pH, traits that are also common to calcium carbonate-specific polypeptide sequences.