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The thin sheets of calcite, termed folia, that make up much of the shell of an oyster are covered by a layer of discrete globules that has been proposed to consist of agglomerations of protein and mineral. Foliar fragments, treated at 475 degrees C for 36 h to remove organic matter, were imaged by atomic force microscopy (AFM) as crystals grew on the foliar surfaces in artificial seawater at calcite supersaturations up to 52-fold. Crystals were also viewed later by scanning electron microscopy. After pyrolysis, the foliar globules persisted only as fragile remnants that were quickly washed away during AFM imaging, revealing an underlying morphology on the foliar laths of a tightly packed continuum of nanometer-scale protrusions. At intermediate supersaturations, crystal formation was seen immediately almost everywhere on these surfaces, each crystal having the same distinctive shape and orientation, even at the outset with crystals as small as a few nanometers. In contrast, nucleation did not occur readily on non-pyrolyzed foliar surfaces, and the crystals that did grow, although slowly at intermediate supersaturations, had irregular shapes. Possible crystallographic features of foliar laths are considered on the basis of the morphology of ectopic crystals and the atomic patterns of various surfaces. A model for foliar lath formation is presented that includes cycles of pulsed secretion of shell protein, removal of the protein from the mineralizing solution upon binding to mineral, and mineral growth at relatively high supersaturation over a time frame of about 1 h for each turn of the cycle.

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Recently antifreeze proteins (AFP) have been the subject of many structure-function relationship studies regarding their antifreeze activity. Attempts have been made to elucidate the structure-function relationship by various amino acid substitutions, but to our knowledge there has been no successful from first principles design of a polypeptide that would bind to designated ice planes along a specific direction. In this paper we show the results of our first attempt on an entirely de novo design of an alanine-lysine-rich antifreeze polypeptide. This 43 residue alanine-lysine peptide exhibits characteristic nonequilibrium freezing point depression and binds to the designated (210) planes of ice along the [122] vector. The structural and thermodynamic properties of this polypeptide were determined using circular dichroism spectroscopy and its nonequilibrium antifreeze properties were investigated using an ice-etching method and nanoliter osmometry.

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The organic layers within biominerals often are viewed as sheets that may function in part to limit and define the underlying crystal structure, as well as to promote formation of the next mineral layer. Some insights into the nature of the sheets were revealed by atomic force microscopy (AFM) of surfaces of freshly cleaved fragments of oyster shell folia. Visible in the micrographs were arrays of globular structures that resembled the globules seen in isolated oyster shell protein bound to calcite, mica, and glass. The results of chemical treatment showed that the foliar globules slowly dissolved in 5.25% NaOCl or 1 N NaOH, reacted with an antibody prepared against an isolated oyster shell protein, and were hydrolyzed by several proteolytic enzymes. These morphological and chemical observations suggested that protein was a significant component of the foliar globules. Although they might also have a significant mineral content, the foliar globules were not effective as nucleators of CaCO3 crystal formation at low levels of supersaturation in artificial seawater. Overall, the results suggested that molecules of oyster shell protein may agglomerate and combine with mineral to form a surface of complex topography that coats the calcite laths but exhibits no obvious correspondence to any specific crystallographic plane.

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The inhibition by phosphocitrate of struvite crystal formation and growth has been examined in the present study. Crystal growth in a gel matrix was controlled by phosphocitrate in a dose-dependent manner. The effects of inhibition were followed using scanning electron microscopy, optical microscopy, and single crystal X-ray analysis. The presence of phosphocitrate induced very strong, crystal face specific inhibition of struvite, leading to total cessation of crystal growth when sufficient concentration of the inhibitor was made available. Crystal growth studies and results from molecular modeling indicated strong affinity of phosphocitrate to (101) faces of struvite. This in turn led to an alteration in the expression of these faces and the development of a characteristic arrowhead struvite morphology. Similar changes were not observed in the presence of identical concentrations of citrate, acetohydroxamic acid, and N-sulfo-2 amino tricarballylate (an analog of phosphocitrate), emphasizing the unique interaction of phosphocitrate with the struvite crystal lattice.

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In this paper we report the results of our studies on the stereospecific binding of shorthorn sculpin antifreeze protein (AFP) to (2 -1 0) secondary prism faces of ice. Using ice crystal growth and etching techniques together with molecular modeling, molecular dynamics, and energy minimization, we explain the nature of preferential binding of shorthorn sculpin AFP along the [1 2 2] direction on (2- 1 0) planes. In agreement with ice etching studies, the mechanism of preferential binding suggested by molecular modeling explains why the binding of shorthorn sculpin AFP occurs along [1 2 2] and not along its mirror symmetry-related direction [-1 -2 2] on (2 -1 0). This binding mechanism is based on the protein-crystal surface enantioselective recognition that utilizes both alpha-helical protein backbone matching to the (2 -1 0) surface topography and matching of side chains of polar/charged residues with specific water molecule positions in the ice surface. The mechanisms of winter flounder and shorthorn sculpin antifreeze binding to ice are compared.

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Binding of citrate and phosphocitrate to calcium oxalate monohydrate crystals has been studied using scanning electron microscopy (SEM) and molecular modeling. Phosphocitrate structure has been resolved using low temperature X-ray analysis and ab initio computational methods. The (-1 0 1) crystal surface of calcium oxalate monohydrate is involved in binding of citrate and phosphocitrate, as shown by SEM and molecular modeling. Citrate and phosphocitrate conformations and binding energies to (-1 0 1) faces have been obtained and compared to binding to another set of calcium-rich planes (0 1 0). Difference in inhibitory properties of these compounds has been attributed to better coordination of functional groups of phosphocitrate with calcium ions in (-1 0 1). Relevance of this study to design of new calcium oxalate monohydrate inhibitors is discussed.

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Oyster shell protein and polyaspartate bound to calcite have been visualized at the atomic and molecular levels by atomic force microscopy. The identities of potential binding sites have been suggested from atomic force microscopy (AFM) images and have been evaluated by molecular modeling. Energies and conformations of binding to (110) and (110) prism faces, (001) basal calcium planes, and (104) cleavage planes are considered. The interaction with the basal plane is strongest and is essentially irreversible. Binding to (110) prism surfaces is also energetically favored and selective for orientations parallel or perpendicular to the c-axis. Binding to (110) faces is significantly weaker and orientation nonspecific. If carboxyl groups of the protein or peptide replace select carbonate ions of the (110) face, the binding energy increases significantly, favoring binding in the parallel direction. Binding to (104) cleavage surfaces is weak and probably reversible. Specific alignment of oyster shell protein molecules on calcite surfaces is shown by AFM, and the relevance to the binding model is discussed.

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Synthetic polyanions, including peptide analogs of naturally occurring proteins, have been shown to inhibit the nucleation and growth of calcium salt crystals. The binding characteristics of polyaspartate and aspartate-serine copolymers to calcium carbonate (calcite) and hydroxyapatite (HAP) are presented here. The binding is related to dose-dependent inhibition of crystal growth measured by constant composition assay. Peptide phosphorylation had little effect on binding affinity or crystal growth inhibition with either calcium salt. Spermine was able to reduce hydroxyapatite crystal growth but with lower efficacy than the polyanionic peptides. Spermine reversed some of the HAP growth inhibition produced by an anionic peptide. Binding of a labeled polyanion was reduced by a similar anionic peptide at all concentrations of the label, however, spermine reduced binding only at higher concentrations of the labeled polyanion. The data support the presence of multiple binding site classes on HAP surfaces, some inaccessible to polycations and some at which both polyanions and polycations can bind.

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Soluble organic matrix isolated from dorsal carapaces of the blue crab, Callinectes sapidus, inhibited CaCO3 crystallization when free in solution. Immobilized matrix complexes, prepared by crosslinking soluble matrix to decalcified crab carapace, promoted CaCO3 formation in that crystallization in the presence of the immobilized soluble matrix complexes began sooner than in solution controls. In the experimental treatments, deposition of crystals occured only within the complexes and not in the crystallization solutions. Chitin, a polymer of N-acetyl-D-glucosaminc, and chitosan, a deacetylated chitin, which are both insoluble products of the organic matrix of the crab carapace containing little to no matrix protein, did not promote CaCO3 crystallization. Complexes of immobilized polyanionic synthetic peptides on chitosan also promoted CaCO3 crystallization. Addition of a hydrophobic tail (Ala8) to the polyanionic peptide (Asp20) reduced the rate of promotion, possibly because the hydrophobic tail formed a diffusion barrier around crystal nuclei growth sites, suppressing interactions of nascent crystal nuclei with ions in the bulk solution.

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