Characterization of two molluscan crystal‐modulating biomineralization proteins and identification of putative mineral binding domains

Abstract

Ethylenediamine-tetraacetic acid extracted water-soluble matrix proteins in molluscan shells secreted from the mantle epithelia are believed to control crystal nucleation, morphology, orientation, and phase of the deposited mineral. Previously, atomic force microscopy demonstrated that abalone nacre proteins bind to growing step edges and to specific crystallographic faces of calcite, suggesting that inhibition of calcite growth may be one of the molecular processes required for growth of the less thermodynamically stable aragonite phase. Previous experiments were done with protein mixtures. To elucidate the role of single proteins, we have characterized two proteins isolated from the aragonitic component of nacre of the red abalone, Haliotis rufescens. These proteins, purified by hydrophobic interaction chromatography, are designated AP7 and AP24 (aragonitic protein of molecular weight 7 kDa and 24 kDa, respectively). Degenerate oligonucleotide primers corresponding to N-terminal and internal peptide sequences were used to amplify cDNA clones by a polymerase chain reaction from a mantle cDNA library; the deduced primary amino acid sequences are presented. Preliminary crystal growth experiments demonstrate that protein fractions enriched in AP7 and AP24 produced CaCO(3) crystals with morphology distinct from crystals grown in the presence of the total mixture of soluble aragonite-specific proteins. Peptides corresponding to the first 30 residues of the N-terminal sequences of both AP7 and AP24 were generated. The synthetic peptides frustrate the progression of step edges of a growing calcite surface, indicating that sequence features within the N-termini of AP7 and AP24 include domains that interact with CaCO(3). CD analyses demonstrate that the N-terminal peptide sequences do not possess significant percentages of alpha-helix or beta-strand secondary structure in solution. Instead, in both the presence and absence of Ca(II), the peptides retain unfolded conformations that may facilitate protein-mineral interaction.

CaCO3Biomineralization: Acidic 8-kDa Proteins Isolated from Aragonitic Abalone Shell Nacre Can Specifically Modify Calcite Crystal Morphology

“…While acetic acid treatment may induce protein hydrolysis, this method of demineralization has been successful for recovering intact the soluble shell proteins AP7 and AP24 from abalone nacre, since the gel electrophoretic mass of the native proteins corresponded well to the calculated protein masses based on cDNA sequences. 13 The total mixture of these solubilized nacre proteins contained several protein species that were separated in SDS-PAGE gels: seven bands were observed to stain intensely with Coomassie Brilliant Blue, and several additional bands stained faintly at higher molecular masses ( Figure 1, lane 1). The previously characterized nacre proteins AP7 and AP24 13 can be identified as bands migrating at the corresponding molecular masses indicated.…”

Expressed sequence tag profiles from calcifying and non-calcifying cultures of Emiliania huxleyi

“…Nineteen of the most abundant ESTs in the f/50 library derived from lith forming cells show no significant match to any sequence present in the f/2 library derived from non-calcifying cells, nor do they show any significant match to any of any GenBank sequences, including biomineralization proteins. Our laboratory intends to obtain full length sequences for these clones that may represent marker sequences for calcification, and characterize their biochemical and biophysical properties with respect to what is known regarding silaffins (Kroger et al 2000;Kroger et al 2001;Kroger et al 2002), lustrin , pearlin (Miyashita et al 2000), nacrein (Miyamoto et al 1996) and other biomineralization proteins (Michenfelder et al 2003;Wustman et al 2002;Zhang et al 2000). Many biomineral-associated proteins tend to have no distinct secondary or tertiary structure but rather feature extended, repeating b-turn, loop, or random coil conformations (Michenfelder et al 2003;Wustman et al 2002;Xu and Evans 1999;Zhang et al 2002;Zhang et al 2000) and contain specific amino acid sequence motifs.…”

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

“…The exact roles of these macromolecules are still unknown while what is known, is that this fraction is necessary for normal mineralized tissue growth and development, as well as its materials properties [19][20][21][22]. 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].…”