Bending and branching of calcite laths in the foliated microstructure of pectinoidean bivalves occurs at coherent crystal lattice orientation

Foliated calcite is widely employed by some important pteriomorph bivalve groups as a construction material. It is made from calcite laths, which are inclined at a low angle to the internal shell surface, although their arrangement is different among the different groups. They are strictly ordered into folia in the anomiids, fully independent in scallops, and display an intermediate arrangement in oysters. Pectinids have particularly narrow laths characterized by their ability to change their growth direction by bending or winding, as well as to bifurcate and polyfurcate. Electron backscatter analysis indicates that the c-axes of laths are at a high, though variable, angle to the growth direction, and that the laths grow preferentially along the projection of an intermediate axis between two a-axes, although they can grow in any intermediate direction. Their main surfaces are not particular crystallographic faces. Analyses done directly on the lath surfaces demonstrate that, during the bending/branching events, all crystallographic axes remain invariant. The growth flexibility of pectinid laths makes them an excellent space-filling material, well suited to level off small irregularities of the shell growth surface. We hypothesize that the exceptional ability of laths to change their direction may be promoted by the mode of growth of biogenic calcite, from a precursor liquid phase induced by organic molecules.     

In Vitro Calcite Crystal Morphology Is Modulated by Otoconial Proteins Otolin-1 and Otoconin-90

Otoconia are formed embryonically and are instrumental in detecting linear acceleration and gravity. Degeneration and fragmentation of otoconia in elderly patients leads to imbalance resulting in higher frequency of falls that are positively correlated with the incidence of bone fractures and death. In this work we investigate the roles otoconial proteins Otolin-1 and Otoconin 90 (OC90) perform in the formation of otoconia. We demonstrate by rotary shadowing and atomic force microscopy (AFM) experiments that Otolin-1 forms homomeric protein complexes and self-assembled networks supporting the hypothesis that Otolin-1 serves as a scaffold protein of otoconia. Our calcium carbonate crystal growth data demonstrate that Otolin-1 and OC90 modulate in vitro calcite crystal morphology but neither protein is sufficient to produce the shape of otoconia. Coadministration of these proteins produces synergistic effects on crystal morphology that contribute to morphology resembling otoconia.     

In situ observation of elementary growth processes of protein crystals by advanced optical microscopy

To start systematically investigating the quality improvement of protein crystals, the elementary growth processes of protein crystals must be first clarified comprehensively. Atomic force microscopy (AFM) has made a tremendous contribution toward elucidating the elementary growth processes of protein crystals and has confirmed that protein crystals grow layer by layer utilizing kinks on steps, as in the case of inorganic and low-molecular-weight compound crystals. However, the scanning of the AFM cantilever greatly disturbs the concentration distribution and solution flow in the vicinity of growing protein crystals. AFM also cannot visualize the dynamic behavior of mobile solute and impurity molecules on protein crystal surfaces. To compensate for these disadvantages of AFM, in situ observation by two types of advanced optical microscopy has been recently performed. To observe the elementary steps of protein crystals noninvasively, laser confocal microscopy combined with differential interference contrast microscopy (LCM-DIM) was developed. To visualize individual mobile protein molecules, total internal reflection fluorescent (TIRF) microscopy, which is widely used in the field of biological physics, was applied to the visualization of protein crystal surfaces. In this review, recent progress in the noninvasive in situ observation of elementary steps and individual mobile protein molecules on protein crystal surfaces is outlined.     

Coccolith biomineralisation studied with atomic force microscopy

Biomineralisation can only be understood as an interplay between organic and mineral phases. With this objective, we conducted an investigation of coccoliths using atomic force microscopy (AFM), an ultra-high resolution technique that requires no surface coating and can be used in air or under solution at ambient conditions of temperature and pressure. The detailed morphology, crystal structure, organic scales and organic coating of the coccolith species Coccolithus pelagicus , Helicosphaera carteri and Oolithotus fragilis were investigated. The fine structure of coccoliths is very complex, with the calcite either being smooth, dominated by steps or tuberculate; organic cover can be either granular or fibrous. Behaviour of coccolith surfaces during dissolution is influenced both by mineral and organic material and different surface types show variable resistance to dissolution. The organic coating protects element faces against etching. Through atomic resolution AFM, it is possible to establish the crystallographic structure of the distal shields of C. pelagicus and O. fragilis . Though elements of both species are dominated by stable crystal faces, there are important differences between them, with the external edge of elements being parallel to a cleavage direction in C. pelagicus but parallel to the atomic rows in O. fragilis . Thus, there is evidence that the biomineralisation of each species, and also of select areas of coccoliths of the same species, is markedly different.     

Streptavidin 2D Crystal Substrates for Visualizing Biomolecular Processes by Atomic Force Microscopy

Flat substrate surfaces are a key to successful imaging of biological macromolecules by atomic force microscopy (AFM). Although usable substrate surfaces have been prepared for still imaging of immobilized molecules, surfaces that are more suitable have recently been required for dynamic imaging to accompany the progress of the scan speed of AFM. In fact, the state-of-the-art high-speed AFM has achieved temporal resolution of 30 ms, a capacity allowing us to trace molecular processes played by biological macromolecules. Here, we characterize three types of streptavidin two-dimensional crystals as substrates, concerning their qualities of surface roughness, uniformity, stability, and resistance to nonspecific protein adsorption. These crystal surfaces are commonly resistant to nonspecific protein adsorption, but exhibit differences in other properties to some extent. These differences must be taken into consideration, but these crystal surfaces are still useful for dynamic AFM imaging, as demonstrated by observation of calcium-induced changes in calmodulin, GroES binding to GroEL, and actin polymerization on the surfaces.     

Modification of calcite crystal growth by abalone shell proteins: an atomic force microscope study.

A family of soluble proteins from the shell of Haliotis rufescens was introduced over a growing calcite crystal being scanned in situ by an atomic force microscope (AFM). Atomic step edges on the crystal surface were altered in shape and speed of growth by the proteins. Proteins attached nonuniformly to the surface, indicating different interactions with crystallographically different step edges. The observed changes were consistent with the habit modification induced by this family of proteins, as previously observed by optical microscopy. To facilitate further studies in this area, AFM techniques and certain AFM imaging artifacts are discussed in detail.     

Incorporation of microcrystals by growing protein and virus crystals

In the course of time-lapse video and atomic force microscopy (AFM) investigations of macromolecular crystal growth, we frequently observed the sedimentation of microcrystals and three-dimensional nuclei onto the surfaces of much larger, growing protein or virus crystals. This was followed by the direct incorporation over time of the smaller crystals into the bulk of the larger crystals. In some cases, clear indications were present that upon absorption of the small crystal onto the surface of the larger, there was proper alignment of the respective lattices, and consolidation proceeded without observable defect formation, i.e., the two lattices knitted together without discontinuity. In the case of at least one virus crystal, cubic satellite tobacco mosaic virus (STMV), addition of three-dimensional nuclei and subsequent expansion provided the principal growth mechanism at high supersaturation. This process has not been reported for growth from solution of conventional crystals. In numerous other instances, the lattices of the small and larger crystals were obviously misaligned, and incorporation occurred with the formation of some defect. This phenomenon of small crystals physically embedded in larger crystals could only degrade the overall diffraction and materials properties of macromolecular crystals.     

Atomistic Simulation of Mineral Surfaces

Abstract

Atomistic simulation techniques are now able to model the structure of mineral surfaces at the atomic level. In this paper we begin to address the question of whether surface reactivity can be studied reliably by modelling the surface reactivity of calcite, fluorite and forsterite under aqueous conditions. We first used energy minimisation techniques to investigate the interaction between the minerals calcite and fluorite with water and methanoic acid. The relative adsorption energies suggest that methanoic acid preferentially adsorbs onto fluorite surfaces, while water adsorbs preferentially onto calcite as inferred from experiments on mineral separation. Molecular Dynamics simulations were also used to model the effect of temperature on the adsorption of water on the calcite {1014} and fluorite {111} surfaces. Furthermore we used these techniques to model point defect formation at surfaces. We are also interested in modelling the competition between associative and dissociative adsorption on mineral surfaces. Simulations of adsorption of water on the low-index forsterite surfaces have predicted the adsorption energies and equilibrium morphology. The calculated equilibrium morphology adequately reproduces the experimental morphology of forsterite suggesting that the relative stabilities of the surfaces, both unhydrated and hydroxylated, are calculated correctly.     

Common crystal nucleation mechanism in shell formation of two morphologically distinct calcite brachiopods

Closely related mineral-producing organisms share common biomineralisation processes. We demonstrate that, in cases of disparate mineral structures where crystal growth mechanisms are necessarily diverse, nucleation processes are the common underlying mechanism during shell formation. Detailed crystallography in the context of shell microstructure in two morphologically distinct calcite brachiopods indicates that, despite differences in shell growth and fabric, at the centre of growth, calcite crystals nucleate with the c-axis 0001 parallel to the shell surface. Such detailed contextual crystallography of biomineralisation using electron backscatter diffraction (EBSD) will have significant applications for future research in biological and medical sciences.     

Perlwapin, an abalone nacre protein with three four-disulfide core (whey acidic protein) domains, inhibits the growth of calcium carbonate crystals

We have isolated a new protein from the nacreous layer of the shell of the sea snail Haliotis laevigata (abalone). Amino acid sequence analysis showed the protein to consist of 134 amino acids and to contain three sequence repeats of approximately 40 amino acids which were very similar to the well-known whey acidic protein domains of other proteins. The new protein was therefore named perlwapin. In addition to the major sequence, we identified several minor variants. Atomic force microscopy was used to explore the interaction of perlwapin with calcite crystals. Monomolecular layers of calcite crystals dissolve very slowly in deionized water and recrystallize in supersaturated calcium carbonate solution. When perlwapin was dissolved in the supersaturated calcium carbonate solution, growth of the crystal was inhibited immediately. Perlwapin molecules bound tightly to distinct step edges, preventing the crystal layers from growing. Using lower concentrations of perlwapin in a saturated calcium carbonate solution, we could distinguish native, active perlwapin molecules from denaturated ones. These observations showed that perlwapin can act as a growth inhibitor for calcium carbonate crystals in saturated calcium carbonate solution. The function of perlwapin in nacre growth may be to inhibit the growth of certain crystallographic planes in the mineral phase of the polymer/mineral composite nacre.     

Annealing and melting behavior of poly(l-lactic acid) single crystals as revealed by in situ atomic force microscopy

In situ annealing and melting of folded-chain single crystals of poly(l-lactic acid) (PLLA) was examined by temperature-controlled atomic force microscopy (AFM). Prominent changes in the crystal appearance during annealing could be followed in real time by the AFM at temperatures above the original crystallization temperature. Thickening of the crystal edges could be occasionally observed, and this indicates that the crystal edges are less perfect than the central, well-ordered regions. At higher annealing temperatures, melting of the unthickened part started. The melting of the unthickened region progressed from the boundaries of the thickened portion normal to the growth face, rather than to the folding surfaces. In addition, it is suggested that melting also initiates at defective or distorted sites in the crystal as revealed by transmission electron microscopy (TEM) and AFM.     

Growth and disorder of macromolecular crystals: insights from atomic force microscopy and X-ray diffraction studies

The growth processes and defect structures of protein and virus crystals have been studied in situ by atomic force microscopy (AFM), X-ray diffraction topography, and high-resolution reciprocal space scanning. Molecular mechanisms of macromolecular crystallization were visualized and fundamental kinetic and thermodynamic parameters, which govern the crystallization process of a number of macromolecular crystals, have been determined. High-resolution AFM imaging of crystal surfaces provides information on the packing of macromolecules within the unit cell and on the structure of large macromolecular assemblies. X-ray diffraction techniques provide a bulk probe with poorer spatial resolution but excellent sensitivity to mosaicity and strain. Defect structures and disorder created in macromolecular crystals during growth, seeding, and post-growth treatments including flash cooling were characterized and their impacts on the diffraction properties of macromolecular crystals have been analyzed. The diverse and dramatic effects of impurities on growth and defect formation have also been studied. Practical implications of these fundamental insights into the improvement of macromolecular crystallization protocols are discussed.     

Direct observation of the transition from calcite to aragonite growth as induced by abalone shell proteins

The mixture of EDTA-soluble proteins found in abalone nacre are known to cause the nucleation and growth of aragonite on calcite seed crystals in supersaturated solutions of calcium carbonate. Past atomic force microscope studies of the interaction of these proteins with calcite crystals did not observe this transition because no information about the crystal polymorph on the surface was obtained. Here we have used the atomic force microscope to directly observe changes in the atomic lattice on a calcite seed crystal after the introduction of abalone shell proteins. The observed changes are consistent with a transition to (001) aragonite growth on a (1014) calcite surface.     

Tectorial structures on the inner ear sensory epithelia of Proteus anguinus (Amphibia,caudata)

The tectorial structures of the inner ear of the proteid salamander Proteus anguinus were studied with transmission and scanning electron microscopy in order to analyze the ultrastructure of the otoconial membranes and otoconial masses of the maculae and the tectorial membrane of the papilla amphibiorum. Both otoconial and tectorial membranes consist of two parts: (1) a compact part and (2) a fibrillar part that joins the membrane with the sensory epithelium. Masses of otoconia occupy the lumina above these membranes. There are two types of calcium carbonate crystals in the otoconial masses within the inner ear of Proteus anguinus. The relatively small otoconial mass of the utricular macula occupies an area no greater than the diameter of the sensory epithelium, and it is composed of calcite crystals. On the other hand, the enormous otoconial masses of the saccular macula and the lagenar macula are composed of aragonite crystals. In the sacculus and lagena, globular structures 2–9 m?m in diameter were discovered on the lower surfaces of the otoconial masses above the sensory epithelia. These globules show a progression from smooth-surfaced, small globules to large globules with spongelike, rough surfaces. It is hypothesized that these globules are precursors of the aragonite crystals and that calcite crystals develop similarly in the utriculus. The presence of globular precursors in adult animals suggests that the formation of new crystals in the otoconial membranes of the sacculus and lagena of Proteus is a continuous, ongoing process.     

Origin and expansion of foliated microstructure in pteriomorph bivalves

The ultrastructure of the calcitic prisms of the prismatic shell layers of pteriomorph bivalves was examined by scanning electronic microscopy and diffraction techniques. Results indicate that the internal structure of the prisms is noticeably different among taxa. In species belonging to the families Pinnidae, Pteriidae, and Isognomonidae (Pterioida), prisms are built up with nanometric calcite crystals. On the other hand, Pectinidae, Propeamussliidae, Anomiidae (order Pectinoida) and the Ostreidae (Ostreoida) have prisms constituted by calcitic laths with micrometric size. These laths are indistinguishable from those constituting the foliated microstructure. In almost all cases, there is mineral continuity from the prisms to the underlying foliated layer, as confirmed by X-ray texture analyses. These findings corroborate a previous assumption that the foliated microstructure derived from calcitic prisms, particularly from those with internal foliated structure. The appearance of the foliated microstructure facilitated drastic mineralogical and microstructural changes in pteriomorph shells-for example, the development of rigid shell margins and the production of largely calcitic shells. Such changes have, no doubt, contributed to the evolutionary success of the groups, which have shown a pronounced diversification over time.     

Microstructure and crystallographic-texture of giant barnacle (Austromegabalanus psittacus) shell

Barnacle shell is a very complex and strong composite bioceramic composed of different structural units which consist of calcite 15 microcrystals of very uniform size. In the study reported herein, the microstructural organization of these units has been examinated in detail with optical and scanning electron microscopy, and X-ray diffraction techniques. These analyses showed that the external part of the shell has a massive microstructure consisting of randomly oriented crystals. Toward the interior, the shell became organized in mineral layers separated by thin organic sheets. Each of these mineral layers has a massive microstructure constituted by highly oriented calcite microcrystals with their c-axes aligned [(001) fibre texture] perpendicular to the organic sheets and the shell surface. Interestingly, in another structural unit, the shell shield, the orientation of the c-axis calcite crystals shifts from being perpendicular to being parallel to the shell surface across its thickness. This study provides evidence that the organic matrix is responsible for the organization of the shell mineral and exterts strong a strict control on the polymorphic type, size and orientation of shell-forming crystals.     

Crystallization, stability, and enzymatic degradation of poly(L-lactide) thin film

Poly(L-lactide) (PLLA) thin film with 100 nm thickness was crystallized at 160 degreesC for 20 min from the melt obtained at 220 degreesC. Hexagonal crystals with three types of growth (derivative growth lamellae, overgrowth multistacked lamellae, and undergrowth multistacked lamellae) were simultaneously observed by atomic force microscopy (AFM). These phenomena are due to the differences of the formative points of secondary crystal nuclei against the basal lamella. Enzymatic degradation of PLLA thin film revealed two types of amorphous regions. These regions were identified as the free amorphous region around the crystals and the restricted amorphous region between the crystal and glass substrate. In situ observation of thermal behavior of lamellar crystals was performed to understand the correlation between the chain folding and stability of the crystal by using temperature-controlled AFM. The morphology of the sectors with [100] growth plane had changed to a comblike morphology despite the fact that the [110] growth plane remained unchanged, suggesting that the stability of the chain folding and the chain-packing state affected the thermal behavior.     

Perlinhibin, a cysteine-, histidine-, and arginine-rich miniprotein from abalone (Haliotis laevigata) nacre, inhibits in vitro calcium carbonate crystallization

We have isolated a 4.785 Da protein from the nacreous layer of the sea snail Haliotis laevigata (greenlip abalone) shell after demineralization with acetic acid. The sequence of 41 amino acids was determined by Edman degradation supported by mass spectrometry. The most abundant amino acids were cysteine (19.5%), histidine (17%), and arginine (14.6%). The positively charged amino acids were almost counterbalanced by negatively charged ones resulting in a calculated isoelectric point of 7.86. Atomic-force microscopy studies of the interaction of the protein with calcite surfaces in supersaturated calcium carbonate solution or calcium chloride solution showed that the protein bound specifically to calcite steps, inhibiting further crystal growth at these sites in carbonate solution and preventing crystal dissolution when carbonate was substituted with chloride. Therefore this protein was named perlinhibin. X-ray diffraction investigation of the crystal after atomic-force microscopy growth experiments showed that the formation of aragonite was induced on the calcite substrate around holes caused by perlinhibin crystal-growth inhibition. The strong interaction of the protein with calcium carbonate was also shown by vapor diffusion crystallization. In the presence of the protein, the crystal surfaces were covered with holes due to protein binding and local inhibition of crystal growth. In addition to perlinhibin, we isolated and sequenced a perlinhibin-related protein, indicating that perlinhibin may be a member of a family of closely related proteins.     

Macromolecular crystal growth as revealed by atomic force microscopy

Direct visualization of macromolecular crystal growth using atomic force microscopy (AFM) has provided a powerful tool in the delineation of mechanisms and the kinetics of the growth process. It has further allowed us to evaluate the wide variety of impurities that are incorporated into crystals of proteins, nucleic acids, and viruses. We can, using AFM, image the defects and imperfections that afflict these crystals, the impurity layers that poison their surfaces, and the consequences of various factors on morphological development. All of these can be recorded under normal growth conditions, in native mother liquors, over time intervals ranging from minutes to days, and at the molecular level.     

IN VITRO RECALCIFICATION OF ORGANIC MATRIX OF SCALLOP SHELL AND SERPULID TUBES

Organic matrix was isolated from the shell of the bivalve Argopectenirradians by decalcification. The capacity of the matrix toinitiate formation of crystals similar in form and orientationto the crystals of normal shell was investigated. Decalcifiedshell matrix placed in an inorganic recalcification solutioninitiated the formation of elongate crystals in parallel arrangementcorresponding to the parallel orientation observed in the matrixfibers and similar to the orientation of the long crystals innormal shell. The detailed form of the crystals deposited invitro was different from that of the normal shell crystals.Electron diffraction analysis of remineralized matrix demonstratedthat the material was calcite, the mineral of normal shell. In contrast, the calcareous tube of the serpulid Hydroides dianthushas crystals lacking uniform arrangement and a matrix whichdoes not have a well-oriented structure. The decalcified tubematrix was recalcified and the mineral posited showed some evidenceof normal orientation. The results demonstrate that matrices of Argopecten shell andHydroides tube can induce crystal formation in vitro. Sincethe soluble matrix would be expected to be removed during decalcification,the observed in vitro effects apparently involve the insolublematrix. (Received 19 June 1984;