Role of Pif97 in Nacre Biomineralization: In Vitro Characterization of Recombinant Pif97 as a Framework Protein for the Association of Organic–Inorganic Layers in Nacre

Abstract

Nacre is the inner layer of the mollusc shell and provides exceptional toughness via its highly organized organic−inorganic composite structure. Pif is an organic matrix protein from the nacreous layer of the pearl oyster Pinctada fucata and exhibits regulatory behavior in nacre formation. Here, we investigated features of Pif97, the Nterminal of Pif, using a recombinant form of Pif97 produced in Escherichia coli. We observed that recombinant Pif97 was able to efficiently form a complex with calcium ions. Additionally, recombinant Pif97 showed both in vitro growth inhibition of thermodynamically stable calcite, stabilization of amorphous calcium carbonate, and exclusive binding affinity to metastable aragonite and chitin. These results imply the participation of Pif97 in the calcification of nacre including the association of the inorganic phase and polysaccharide template. We propose that recombinant Pif97 has inherent characteristics of the native form, which are significant for interrelating with organic matrix and inorganic calcium carbonate during nacre biomineralization.

Control of nacre biomineralization by Pif80 in pearl oyster

“…S1, A and B). A similar result was observed for native Pif97 ( 18 ). Native Pif80 in its denatured state has been shown to be functional in in vitro mineralization ( 15 ), indicating that the particular conformation of Pif80 is not critical to its proper function.…”

“…Pif has drawn much attention because of its indispensability in normal nacre formation, its contribution to the crystallization of nacre-like aragonites ( 15 ), and the wide distribution of its homologs from bivalves to gastropods ( 17 ). While the role of Pif97 can be readily inferred by its annotated domain structure ( 18 ), the specific role of Pif80 has not been determined because of the absence of predictable functional domains. Nevertheless, Pif80 has interesting structural features such as a high ratio of charged and repetitive amino acid residues and the presence of a cluster of acidic amino acid residues.…”

Ps19, a novel chitin binding protein from Pteria sterna capable to mineralize aragonite plates in vitro

“…Biochemical assays for Ps19 have shown it has chitin-binding abilities, which is a vital characteristic for interactions between shell matrix proteins and organic biomolecules to create a frame for calcium carbonate mineralization [5,26,46,47]. β-chitin is one of the main organic components of interlamellar nacre plates, and it is also considered as a possible nucleation site for aragonite sheets due to its interaction with other matrix proteins [48][49][50]. According to this evidence, Ps19 may not be only involved in aragonite plate mineralization but also in its nucleation.…”

A bioinspired dual-crosslinked tough silk protein hydrogel as a protective biocatalytic matrix for carbon sequestration

“…In addition, the morphology and polymorphs of the CaCO 3 were analyzed by FE-SEM and X-ray powder diffraction (Rigaku, Japan) as previously described. 46 In vitro CaCO 3 crystallization…”

“…The effect of dc-ngCA-silk hydrogel on in vitro CaCO 3 crystallization was investigated by exploiting the slow diffusion of ammonium carbonate vapor in calcium chloride solution as previously described. 46 The morphology of the precipitated CaCO 3 was analyzed by FE-SEM.…”