Nacre protein sequence compartmentalizes mineral polymorphs in solution

The Japanese pearl oyster (Pinctada fucata) n16 framework matrix protein is an integral part of the growth and formation of the mollusk shell biomineralization mechanism. It is a required component of the extracellular matrix with a dual mineralization role, as an anchor component to synchronize the assembly of the beta-chitin and N-series, Pif-series protein extracellular matrix for aragonite formation and as a regulator of aragonite formation itself. However, the mechanism by which this protein controls aragonite formation is not understood. Here, we investigate the mineralization potential and kinetics of the 30 AA N-terminal portion of the n16 protein, n16N. This sequence has been demonstrated to form either vaterite or aragonite depending upon conditions. Using in situ potentiometric titration methods, we find that n16N is indeed responsible for the self-assembly characteristics found in vivo and in vitro but is not involved with active Ca²⁺ binding or mineral nucleation processes. Upon the basis of time- and peptide concentration-dependent sampling of mineral deposits that form in solution, we find that n16N is responsible for controlling where mineralization occurs in bulk solution. This protein sequence acts as a molecular spacer that organizes the mineralization space and promotes the formation of mineral constituents that contain ACC, vaterite, and aragonite. Without the concerted action of the n16N assemblage, unregulated calcite formation occurs exclusively. Thus, the n16 protein provides the regulation needed to have the characteristic polymorph, crystalline orientations, and related mechanical properties associated to the microstructure of mollusk shells.