A crystal modulating protein from molluscan nacre that limits the growth of calcite in vitro

Protein-mediated biomineral formation and polymorph selection in the mollusk shell involves the participation of a number of different proteins. One of these proteins, AP7, has been identified as a participant in nacre (aragonite) formation in the mollusk Haliotis rufescens. However, the role(s) of this protein in aragonite formation is (are) poorly understood, due to the fact that low quantities of this protein are recoverable from the nacre matrix. To overcome this problem, we employed stepwise solid-phase tBOC synthesis to recreate the 66-AA single-chain protein AP7 and utilized this synthetic form for in vitro mineralization studies. We find that the AP7 protein promotes incomplete or interrupted crystal growth and step edge roundening in a concentration-dependent fashion. Using synthetic peptides which represent the 30-AA N-terminal (AP7N) and 36-AA C-terminal (AP7C) subdomains of AP7, we have identified that the mineral modification activity of AP7 is localized to the unstructured, conformationally labile N-terminal subdomain. Interestingly, the 36-AA C-terminal subdomain has no observable direct effect on in vitro calcium carbonate crystal growth; however, we cannot rule out the possibility that AP7C plays an indirect role in AP7 mineralization activity. Qualitative structural studies reveal that AP7, although possessing Zn(II) fingerlike -His-(X) ₆-His, -Cys-(X)₂-Cys-, and -Cys-(X)₄-Cys- motifs within its C-terminal region, does not possess the structural characteristics of known Zn(II) finger polypeptides, as evidenced by the presence of an ordered, α-helical conformation within the C-terminal subdomain of apo-AP7. Given its associative nature with AP24 and the multifunctional capabilities of the 30-AA N-terminal domain, it is likely that the AP7 protein possesses multifunctional capabilities with regard to nacre formation within the mollusk shell.