Molecular “Tuning” of Crystal Growth by Nacre-Associated Polypeptides

Abstract

The formation and stabilization of the aragonite polymorph in the nacre layer of mollusks is an intriguing process, yet very little is known with regard to the participation of proteins in this process. Previously, we identified the 30 AA N-terminal mineral binding domains (AP7-N, AP24-N, n16-N) of three different nacre-specific proteins (AP7, AP24, n16). These three domains differ in primary sequence and induce morphological changes in CaCO3 crystals in vitro. Using AFM microscopy, we investigated the adsorption of AP7-N, AP24-N, and n16-N onto calcite dislocation hillocks. We observe that both AP7-N and AP24-N are multifunctional; they not only inhibit obtuse step advance but also induce rounded, amorphous-appearing deposits on hillock terraces. In contrast, n16-N pins corner sites at the junction of acute and obtuse steps and promote the emergence of a new set of steps approximately oriented along a line joining the obtuse−obtuse and the acute−acute corners. Random scrambling of the n16-N and AP7-N sequences resulted in substantially reduced mineral modification activities, indicating that the primary sequence of both polypeptides is crucial for correct recognition of surface features. These findings indicate that nacre proteins evolved specialized mineral interaction domains that either recognize different surface features (AP7-N, AP24-N versus n16-N), or recognize the same features, but with different binding and catalytic activities (AP24-N versus AP7-N). These functional differences may arise from the differences in primary and secondary structure specific to each N-terminal domain. Hypothetically, this molecular diversity would allow complementary and simultaneous protein control (“molecular tuning”) of different features of the crystal growth process.

Role of molecular charge and hydrophilicity in regulating the kinetics of crystal growth

“…It should be noted that step velocity data in these studies were obtained at slightly different supersaturation and ionic strength conditions (6,7). The most potent additive was AP8-␣, an 8-kDa, highly acidic protein extracted from the mineralized tissue of abalone (10).…”

“…The widely accepted view of the latter mechanism is that macromolecular modifiers in solution are capable of directing growth morphology, but have only neutral or inhibitory effects on growth rate. Recently, this dogma was challenged by showing that low nanomolar levels of proteins isolated from abalone nacre actually increased rates of calcite growth by as much as 5-fold by accelerating the kinetics of molecular step propagation across mineral surfaces (6,7).…”

Modulation of Calcium Oxalate Dihydrate Growth by Selective Crystal-face Binding of Phosphorylated Osteopontin and Polyaspartate Peptide Showing Occlusion by Sectoral (Compositional) Zoning

“…Conserved among many mammalian and avian species, our selected poly-Asp 86 -93 peptide is a linear, contiguous stretch of amino acids rich in aspartic acid, as found in the primary sequence of OPN (68) and proposed to be one of the potent mineral binding motifs of this protein (34,69). It has been shown that small, linear acidic polypeptides, some artificially constructed and others reflecting real protein sequence, can potently regulate the kinetics of biomineral growth (inhibition (34,70,71) or acceleration (72,73)) and impurity-mineral interactions (74).…”

Biomimetic and Bioinspired Crystallization with Macromolecular Additives

“…For example, in situ AFM studies were performed for AP7N, AP8, and AP24N. [Fu et al, 2005b;Kim et al, 2006b] These studies revealed the surprising effects of growth acceleration that had not been observed before, and the underlying mechanism appeared to be through lowering the activation barriers for moving ions from the solution phase to the crystal growth sites [Elhadj et al, 2006;Kim et al, 2006b]. These results indicate the ability of the biomacromolecules to modulate the mineral/solution interfaces to control the kinetics of crystallization.…”