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...

Recently, phage and cell-surface display libraries have been adapted for genetically selecting short peptides for a variety of inorganic materials. Despite the enormous number of inorganic-binding peptides reported and their bionanotechnological utility as synthesizers and molecular linkers, there is still a limited understanding of molecular mechanisms of peptide recognition of and binding to solid materials. As part of our goal of genetically designing these peptides, understanding the binding kinetics and thermodynamics, and using the peptides as molecular erectors, in this report we discuss molecular structural constraints imposed upon the quantitative binding characteristics of peptides with an affinity for inorganics. Specifically, we use a high-affinity seven amino acid Pt-binding sequence, PTSTGQA, as we reported in earlier studies and build two constructs: one is a Cys-Cys constrained "loop" sequence (CPTSTGQAC) that mimics the domain used in the pIII tail sequence of the phage library construction, and the second is the linear form, a septapeptide, without the loop. Both sequences were analyzed for their adsorption behavior on Pt thin films by surface plasmon resonance (SPR) spectroscopy and for their conformational properties by circular dichroism (CD). We find that the cyclic peptide of the integral Pt-binding sequence possesses single or 1:1 Langmuir adsorption behavior and displays equilibrium and adsorption rate constants that are significantly larger than those obtained for the linear form. Conversely, the linear form exhibits biexponential Langmuir isotherm behavior with slower and weaker binding. Furthermore, the structure of the cyclic version was found to adopt a random coil molecular conformation, whereas the linear version adopts a polyproline type II conformation in equilibrium with the random coil. The 2,2,2-trifluoroethanol titration experiments indicate that TFE has a different effect on the secondary structures of the linear and cyclic versions of the Pt binding sequence. We conclude that the presence of the Cys-Cys restraint affects both the conformation and binding behavior of the integral Pt-binding septapeptide sequence and that the presence or absence of constraints could be used to tune the adsorption and structural features of inorganic binding peptide sequences.