AP7, a Partially Disordered Pseudo C-RING Protein, Is Capable of Forming Stabilized Aragonite in Vitro

Abstract

AP7 is an extracellular aragonite-associated protein of the nacre layer of the mollusk Haliotis rufescens and possesses a 36-amino acid C-terminal domain that exhibits sequence homology to the C subclass of the RING domain intracellular protein family. We report here novel findings which implicate AP7 as a member of the intrinsically disordered protein class (IDP) and reveal new aspects of AP7 mineralization activity. AP7 is partially disordered but can undergo additional folding in the presence of TFE. AP7 binds Zn(II) ions in a non-tetracoordinate complex but does not require Zn(II) either for folding or for in vitro function. In addition to limiting calcite crystal growth, AP7 is also observed to induce aggregate formation within in vitro mineralization assays, and these aggregates are either amorphous (type A) or crystalline (type B) in appearance. The type A aggregate displays an irregular morphology and round, dark, electron dense deposits that do not give rise to a diffraction pattern. In contrast, the type B aggregates possess either organized parallel crystal clusters or highly dense hexagonal clusters that are confirmed by electron diffraction to be aragonite. This stabilization of aragonite is remarkable in that it occurred in the presence of AP7 alone and did not require typical aragonite stabilization agents such as Mg(II), other nacre proteins, or an organized organic matrix. The ability of a partially disordered C-RING protein to perform inorganic phase stabilization represents a new twist on both the RING domain and IDP stories, and this process of aggregate formation may provide an important clue with regard to the protein-mediated nacre formation process.

The multiple effects of amino acids on the early stages of calcium carbonate crystallization

“…There are no consistent compositions or structural homologs found amongst these organic fractions, except that they do have regions which are highly repetitive and have characteristics of "intrinsically disordered protein" (IDP) domains, domains implicated in either mineral binding or the assembly process [23][24][25]. Many have associated these IDPs to being active participants in various biological processes like biomineralization [24,[26][27][28].…”

“…Several groups have examined short peptides containing repeats of proline and glycine on mineralization [29,31]. Others have used short peptides with consensus groups from specific known mineralization proteins [18,26,27,32]. In many of these cases presented, the short peptides are often found to have specific affinity to a predominant crystalline face of the growing mineral [22,33] or preference for a specific mineral polymorph [26,27,34].…”

“…Others have used short peptides with consensus groups from specific known mineralization proteins [18,26,27,32]. In many of these cases presented, the short peptides are often found to have specific affinity to a predominant crystalline face of the growing mineral [22,33] or preference for a specific mineral polymorph [26,27,34]. In finding these short peptide sequences, groups have adapted molecular biology techniques to screen through the millions of sequences that have an affinity to a crystal growth step or face.…”

Polymorphs, Proteins, and Nucleation Theory: A Critical Analysis

“…Second, and perhaps most importantly, genomic and proteomic studies of biomineralization proteins have uncovered a vast repertory of proteins [6][7][8]13,14,[26][27][28] that could potentially manage many aspects of proposed nucleation processes, including polymorphism. However, although many protein studies have confirmed that polymorphs do form when proteins are present [34][35][36][37][38][39][40][41][42][43][44][45][46][47][48][49][50][51][52][53], these studies have not provided information that is needed for detailing the mechanisms of polymorph formation, or, have utilized a variety of testing methods that prevent cross-comparisons of datasets necessary for mechanism building [34][35][36][37][38][39][40][41][42][43][44][45][46][47][48][49][50][51]…”

Single Amino Acids as Additives Modulating CaCO3 Mineralization

“…Furthermore, they showed that a part of Ca 2+ ions forms neutral equilibrium clusters before nucleation of the supersaturated solution arises (18). Several groups have also investigated the influence of different additives on the nucleation of CaCO 3 (19)(20)(21)(22). Amongst others, polycarboxylates which usually serve as inhibitors of the formation of scale in laundry detergents or dishwaters were used to investigation inhibition of nucleation (23).…”