Identification of Mineral Modulation Sequences within the Nacre-Associated Oyster Shell Protein, n16

Abstract

Nature's use of proteins to direct and control the synthesis of inorganic solids represents an important paradigm for bioinspired materials synthesis. However, the mechanism by which polypeptides direct inorganic synthesis, particularly with regard to selective formation of crystals polymorphs, remains unknown. An important step in understanding polypeptide-directed inorganic synthesis is the identification of sequence regions in biomineralization proteins that can affect crystal growth. In this report, we identify that the 30 AA N-and C-terminal sequence regions (n16-N and n16-C, respectively) of the oyster shell aragonite-associated protein, n16, exhibit control over the morphology of calcium carbonate crystals grown in geologic calcite overgrowth assays and polyimide (Kevlar)-based assays. Here, we find that calcium carbonate crystals, which grow in the presence of model peptides representing the n16-N and n16-C sequences, adopt dendritic or circular overgrowth in geological calcite overgrowth assays and "staircase" structures in Kevlar-based assays, as compared to negative controls and to parallel assays conducted in the presence of AP24-1, the 30 AA N-terminal sequence region of the nacre-associated protein, AP24, which interrupts step edge growth. Synchrotron X-ray diffraction studies reveal that the crystals grown in the presence of n16-N are calcite. Circular dichroism spectrometry studies of n16-N and n16-C model peptides reveal qualitatively similar solution state conformations that consist of either random coil in equilibrium with other secondary structures (e.g., -strand, turn, loop, polyproline type II) or, at higher concentrations, a -strand secondary structure in equilibrium with random coil. We conclude that the N-and C-terminal sequence regions of the nacre-associated n16 protein most likely play a role in n16-mediated effects on calcium carbonate crystal growth in the nacre layer.Nature's use of proteins to regulate the growth of inorganic solids is one of the major hallmarks of the biomineralization process and represents one possible pathway for the construction of unique and useful inorganic materials. A great deal of interest has centered on calcium carbonate-based mineralization processes, where several crystalline polymorphs (calcite, aragonite, and vaterite) and amorphous calcium carbonates are preferentially created within certain organisms. 1 An interesting model system of polymorph selection exists in the mollusk shell, where two polymorphs, calcite (prismatic layer) and aragonite (nacre layer), coexist side by side. 2-7 Recent protein studies have identified a number of unique polypeptides associated with the nacre layer, 5,6,9 and it is believed that these proteins participate in the stabilization of the aragonite polymorph during shell synthesis. 3-8 Thus, if one is interested in exploiting the principles of bioinspired inorganic synthesis and protein-mediated phase stabilization for material science, 10 we need to obtain a better understanding of how these proteins affect mineral f...

Biomimetic and Bioinspired Crystallization with Macromolecular Additives

“…[Samata et al, 1999] The N-and C-terminal sequences of N16 (N16N and N16C, both 30 amino acids) were analyzed in their structural changes along with concentrations, pH, and the presence of calcium ions. [Amos et al 2011;Kim et al, 2004b] They had a mixture of unfolded and -stranded structures, and they appeared to form supramolecular assemblies regulated by pH and calcium ions. This is consistent with the preliminary analysis of the secondary structure of N16 and with the fact that N16 is a matrix protein.…”

Bivalve Characterization Using Synchrotron Micro X-Ray Fluorescence

“…Experiments investigating the mechanisms involved in growth have been investigated at the National Synchrotron Light Source (NSLS) using small angle X-ray scattering and other techniques [7,8]. Two groups applied synchrotron micro X-ray fluorescence (XRF) techniques to make measurements of metals in whole shells thus bridging the gap between the molecular and field scales [9,10].…”

Probing the Organic-Mineral Interface at the Molecular Level in Model Biominerals

“…This approach has been used to examine the participation of a number of polypeptides within biomineral nucleation and crystal growth assay systems. [9][10][11][12][13][14][15][16][17] Although limited in their scope, such in vitro systems provide much needed information on the role of proteins within mineralizing systems, and can serve as a stepping stone towards more complex model systems that possess larger numbers of molecular participants.…”

“…9,11,[13][14][15][16][17] To examine the molecular features present at the OMI in the organic-mineral systems, we used x-ray absorption near-edge structure (XANES) spectroscopy, a tool sensitive to the electronic and molecular structure. Although XANES cannot provide the complete molecular or mineral three-dimensional structure that diffraction yields, it is exquisitely sensitive to the formation or disruption of chemical bonds, does not require the sample to be crystalline, and can analyze both sides of the OMI in these model systems.…”