The N- and C-Terminal Regions of the Pearl-Associated EF Hand Protein, PFMG1, Promote the Formation of the Aragonite Polymorph in Vitro

Abstract

Recent studies indicate that the ability of mollusk shell nacre protein sequences to form the calcium carbonate polymorph, aragonite, are linked to the presence of intrinsically disordered sequences within these proteins. Although the exact relationship between protein structural disorder and polymorph formation is not clear, there is a definite interest in discovering other examples of intrinsically disordered nacre protein sequences that can induce aragonite formation. In this report, we extend the relationship between intrinsic disorder and aragonite formation to another set of nacre protein sequences. This protein, known as PFMG1, is associated with pearl formation in the Japanese pearl oyster, Pinctada fucata. We demonstrate that synthetic peptides representing the 30 AA N-and C-terminal sequence regions of PFMG1 nucleate nanoscale-sized aragonite in solution without the need for additional additives. Compared to controls containing no peptide or bovine serum albumin, the PFMG1 terminal sequences appear to form a matrix-like environment around the forming biominerals, and this process will be defined in more detail in later reports. Furthermore, we establish that these PFMG1 terminal sequences possess disordered structures in solution that can be stabilized into partially folded structures (R helix, beta structures) using the structure-stabilizing solvent, 2,2,2-trifluoroethanol. Although we do not know the mechanism by which these peptides promote aragonite nucleation in vitro, we believe that these terminal sequences are participants in PFMG1-mediated aragonite polymorph formation within the oyster pearl and that the intrinsic disorder and folding propensities of these sequences are crucial for this activity.The mollusk shell is a biocomposite that is comprised of two distinct layers of calcium carbonate that coexist with a series of biomacromolecules. [1][2][3][4][5][6][7][8][9][10][11] One of these layers, known as the nacre layer, is comprised of the calcium carbonate polymorph, aragonite, which is thermodynamically unstable. 4 Moreover, the nacre layer is fracture-resistant (3000Â greater than pure aragonite) 5 and under force stress exhibits crack deflection and energy dissipation. [6][7][8] These material properties arise from the presence of proteins which inhabit the aragonite crystals (intercalation or occlusion) 9 and the space surrounding these crystals. [1][2][3] Interestingly, these same proteins may play a role in the nucleation of aragonite [1][2][3]9 and the formation of an amorphous precursor, amorphous calcium carbonate (ACC). 1,12 Hence, the nacre layer represents an important model system for extracting molecular principles (nucleation, protein occlusion, fracture toughness) that Nature uses to create composite materials under ambient conditions.One molecular principle that appears to emerge is the link between disordered protein structure and biomineralization protein function. Recently, a nacre protein, AP7 (Haliotis rufescens), was found to stabilize single and polycrystalline aragon...

Calcite Formation in Soft Coral Sclerites Is Determined by a Single Reactive Extracellular Protein

“…This ratio is apparently driven by changes in spreading rates along midocean ridges (1). The calcification process of aragonite and calcite mineralogy in mollusk shells (3)(4)(5)(6)(7)(8) and some information about calcite (9, 10) have been reported, but our knowledge of the mechanism of the direct biological formation of calcite in marine organisms, especially in corals, remains incomplete. To develop a more complete understanding of calcite formation, detailed information concerning how biomolecules contribute to the kinetics of crystal formation, as well as the structural details of both the surface and the interior of single crystals in the submicrometer to nanometer scale must be analyzed.…”

Biomimetic and Bioinspired Crystallization with Macromolecular Additives

“…Note that the critical concentration of the protein required for aragonite polymorph was much higher than that for aragonitic shell proteins. [Amos et al, 2010] Similar behavior was also seen with soy globulins, but it was unclear if the proteins were solely responsible for this result because the aragonite was prepared at slightly elevated temperature (50ºC) that could induce some aragonite formation. [Liu et al, 2009] Initially formed vaterite transformed into mesocrystal-like calcite.…”

“…[Liu et al, 2007;Skelton et al, 1994] The secondary structure of the PFMG1 was studied for the terminal domains, and they possessed the so-called intrinsically disordered protein sequences (IDP) similar to AP7 and N16. [Amos et al, 2010] It is interesting that the terminal fragment peptides were able to generate aragonite, although the occurrence was not exclusive probably due to the lack of other components that usually participate in the biomineral generation. Clearly, much more multidisciplinary work is expected to be done until we fully understand the principles of mineralization in the biological species.…”

Polymorphs, Proteins, and Nucleation Theory: A Critical Analysis

“…For aragonite, what we must draw from are studies conducted with mollusk shell nacre-associated protein sequences [34][35][36][37][38][39][40][41][42][43][44][45][46][47][48][49][50][51]. For in vitro-based studies, several different carbonate-based assays have been used to achieve supersaturation conditions necessary for calcium carbonate crystal growth, with no control over pH, and the duration of these nucleation experiments were variable (i.e., from several hours up to 7 days) [36,38,39,[41][42][43][44][46][47][48][49][50][51].…”

“…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]…”