ABSTRACT
Crystalline biominerals do not resemble faceted crystals. Current explanations for this property involve formation via amorphous phases. Using X-ray absorption near-edge structure (XANES) spectroscopy and photoelectron emission microscopy (PEEM), here we examine forming spicules in embryos of Strongylocentrotus purpuratus sea urchins, and observe a sequence of three mineral phases: hydrated amorphous calcium carbonate (ACC · H(2)O) â dehydrated amorphous calcium carbonate (ACC) â calcite. Unexpectedly, we find ACC · H(2)O-rich nanoparticles that persist after the surrounding mineral has dehydrated and crystallized. Protein matrix components occluded within the mineral must inhibit ACC · H(2)O dehydration. We devised an in vitro, also using XANES-PEEM, assay to identify spicule proteins that may play a role in stabilizing various mineral phases, and found that the most abundant occluded matrix protein in the sea urchin spicules, SM50, stabilizes ACC · H(2)O in vitro.
Subject(s)
Biocompatible Materials/chemistry , Calcification, Physiologic , Calcium Carbonate/chemistry , Phase Transition , Animals , Biocompatible Materials/metabolism , Calcium Carbonate/metabolism , Crystallization , Embryo, Nonmammalian/chemistry , Embryo, Nonmammalian/metabolism , Embryo, Nonmammalian/ultrastructure , Extracellular Matrix Proteins/genetics , Extracellular Matrix Proteins/metabolism , Gene Expression Regulation, Developmental , Microscopy, Electron/methods , Minerals/chemistry , Minerals/metabolism , Reverse Transcriptase Polymerase Chain Reaction , Strongylocentrotus purpuratus/chemistry , Strongylocentrotus purpuratus/embryology , Strongylocentrotus purpuratus/metabolism , Water/chemistry , X-Ray Absorption Spectroscopy/methodsABSTRACT
Proteins play a major role in the formation of all biominerals. In mollusk shell nacre, complex mixtures and assemblies of proteins and polysaccharides were shown to induce aragonite formation, rather than the thermodynamically favored calcite (both aragonite and calcite are CaCO(3) polymorphs). Here we used N16N, a single 30 amino acid-protein fragment originally inspired by the mineral binding site of N16, a protein in the nacre layer of the Japanese pearl oysters (Pinctada fucata). In a calcite growth solution this short peptide induces in vitro biomineralization. This model biomineral was analyzed using X-ray PhotoElectron Emission spectroMicroscopy (X-PEEM) and found to be strikingly similar to natural nacre: lamellar aragonite with interspersed N16N layers. This and other findings combined suggest a hypothetical scenario in which in vivo three proteins (N16, Pif80, and Pif97) and a polysaccharide (chitin) work in concert to form lamellar nacre.
