- Nanoscale assembly processes revealed in the nacroprismatic transition zone of Pinna nobilis mollusc shells
Robert Hovden*, Stephan E. Wolf*, Megan E. Holtz, Frédéric Marin, David A. Muller and Lara A. Estroff (* equal contribution) in Nature Communications (2015) , Vol. 6, No. 10097. DOI:10.1038/ncomms10097.
Abstract: Intricate biomineralization processes in molluscs engineer hierarchical structures with meso-, nano- and atomic architectures that give the final composite material exceptional mechanical strength and optical iridescence on the macroscale. This multiscale biological assembly inspires new synthetic routes to complex materials. Our investigation of the prism–nacre interface reveals nanoscale details governing the onset of nacre formation using high-resolution scanning transmission electron microscopy. A wedge-polishing technique provides unprecedented, large-area specimens required to span the entire interface. Within this region, we find a transition from nanofibrillar aggregation to irregular early-nacre layers, to well-ordered mature nacre suggesting the assembly process is driven by aggregation of nanoparticles (~50–80 nm) within an organic matrix that arrange in fibre-like polycrystalline configurations. The particle number increases successively and, when critical packing is reached, they merge into early-nacre platelets. These results give new insights into nacre formation and particle-accretion mechanisms that may be common to many calcareous biominerals.
- Synthesis of calcium carbonate biological materials : how many proteins are needed ?
Frédéric Marin, Nathalie Le Roy, Benjamin Marie, Paula Ramos-Silva, Stephan E. Wolf, Sana Benhamada, Nathalie Guichard, Françoise Immel and Nathalie Le Roy in Key Eng. Mater. (2014) , Vol. 614, pp. 52-61. DOI:10.4028/www.scientific.net/KEM.614.52.
Abstract: In Nature, calcium carbonate biomineralizations are the most abundant mineralized structures of biological origin. Because many exhibit remarkable characteristics, several attempts have been made to use them as substitution materials for bone reconstruction or as models for generating biomimetic composites that exhibit tailored properties. CaCO3 biomineralizations contain small amounts of amalgamate of proteins and polysaccharides that are secreted during the calcification process. They contribute to control the morphology of the crystallites and to spatially organize them in well-defined microstructures. These macromolecules, collectively defined as the skeletal matrix, have been the focus of a large number of studies aiming at synthesizing in vitro biomimetic materials, according to a bottom-up approach. However, recent proteomic investigations performed on the organic matrices associated to mollusc shells or to coral skeletons have quashed our hopes to generate, with only few macromolecular ingredients, biomimetic materials with properties approaching to those of natural biominerals. As a mean value, each matrix comprises a minimum of few tens of different proteins that seem to be strictly associated to calcium carbonate biominerals. Among the proteins that are currently detected, one finds RLCDs-containing proteins (Repetitive-Low-Complexity Domains), enzymes, proteins with protease inhibitors domains and at last, proteins that contains typical ECM (ExtraCellular Matrix) domains. Today, we still do not understand how the skeletal matrix works, and unveiling its complex functioning is one of the challenges for the coming decade, both from fundamental and applied viewpoints. Is it realistic to attempt generating abiotically, in a test tube at room temperature, biomimetic composites that mimic natural biomineralizations in their properties? If so, and by supposing that we know the individual functions of all the components of the matrix, is there a minimal number of proteins required for producing in vitro calcium carbonate biomaterials that approximate natural biominerals? These issues are of importance for the future research directions in biomaterials science.
- 'Shellome': proteins involved in mollusc shell biomineralization: diversity, functions
Frédéric Marin, Benjamin Marie, Sana Ben Hamada, Paula Silva, Nathalie Le Roy, Stephan E. Wolf, Caroline Montagnani, Caroline Joubert and David Piquemal in Recent Adv. Pearl Res. (2013) , Vol. 33, pp. 149-66.
Abstract: Since the mid-seventies, there has been a considerable emphasis on the key-role played by extracellular organic macromolecules associated to mollusc shell biomineralization. In particular, the proteins occluded within the shell are supposed to fulfill several distinct functions, listed as follows: to provide a gel-like or colloidal micro-environment where crystallization can occur, to compartmentalize this environment in relation to the future microstructure, to promote nucleation and favour crystal growth in privileged crystallographic axes, to stop crystal growth when necessary. Beside these functions related to the interaction with the mineral, some specific shell proteins function as enzymes, while others exert signalling activities towards the calcifying mantle epithelium. The topographic models of shell mineralization, which emerged a decade ago for describing nacre ultrastructure, translate very imperfectly the complexity of the calcification process at the molecular level. For a few years, we have undertaken to obtain and compare the "shellomes" the full shell protein contents of several mollusc models by combining a proteomic approach to available EST data sets. Surprisingly, our findings suggest that, from model to model (pearl oyster versus mussel, for example), the nacre protein contents may exhibit very few similarities. In this review paper, functional and evolutionary implications of our data are briefly discussed.
- Merging models of biomineralisation with concepts of nonclassical crystallisation: is a liquid amorphous precursor involved in the formation of the prismatic layer of the Mediterranean Fan Mussel Pinna nobilis?
Stephan E. Wolf, Ingo Lieberwirth, Filipe Natalio, Jean-Francois Bardeau, Nicolas Delorme, Franziska Emmerling, Raul Barrea, Michael Kappl and Frédéric Marin in Faraday Disc. (2012) , Vol. 159, pp. 433-448. DOI:10.1039/c2fd20045g. This article is part of themed collection: Crystallisation – A Biological Perspective
Abstract: The calcitic prisms of Pinna nobilis (Pinnidae, Linnaeus 1758) are shown to be perfect examples of a mesocrystalline material. Based on their ultrastructure and on the occurrence of an amorphous transient precursor during the early stages of prism formation, we provide evidence for the pathway of mesocrystallisation proposed by Seto et al. (2012), which proceeds not by self-organized oriented attachment of crystalline nano-bricks but by aggregation of initially amorphous nanogranules which later transform by epitaxial nucleation to a three-dimensional array of well aligned nanocrystals. We further fathom the role of a liquid amorphous calcium carbonate in biomineralisation processes and provide strong evidence for the occurrence of PILP-like intermediates during prism formation. We develop a new scenario of prism formation based on the presented findings presented findings and discuss the implications of a speculative liquid amorphous calcium carbonate (LACC) intermediate in vivo.
- Carbonate-coordinated metal complexes precede the formation of liquid amorphous mineral emulsions of divalent metal carbonates
Stephan E.Wolf, Lars Müller, Raul Barrea, Christopher J. Kampf, Jork Leiterer, Ulrich Panne, Thorsten Hoffmann, Franziska Emmerling and Wolfgang Tremel in Nanoscale (2011) , Vol. 3, pp. 1158-65. DOI:10.1039/c0nr00761g.
Abstract: During the mineralisation of metal carbonates MCO3 (M = Ca, Sr, Ba, Mn, Cd, Pb) liquid-like amorphous intermediates emerge. These intermediates that form via a liquid/liquid phase separation behave like a classical emulsion and are stabilized electrostatically. The occurrence of these intermediates is attributed to the formation of highly hydrated networks whose stability is mainly based on weak interactions and the variability of the metal-containing pre-critical clusters. Their existence and compositional freedom are evidenced by electrospray ionization mass spectrometry (ESI-MS). Liquid intermediates in non-classical crystallisation pathways seem to be more common than assumed.
- Strong Stabilization of Amorphous Calcium Carbonate Emulsion by Ovalbumin: Gaining Insight into the Mechanism of 'Polymer-Induced Liquid Precursor' Processes.
Stephan E. Wolf, Jork Leiterer, Vitaliy Pipich, Raul Barrea, Franziska Emmerling and Wolfgang Tremel in J. Am. Chem. Soc. (2011) , Vol. 132, No. 32, pp. 1520-6. DOI:10.1021/ja202622g.
Abstract: The impact of the ovo proteins ovalbumin and lysozyme—present in the first stage of egg shell formation—on the homogeneous formation of the liquid amorphous calcium carbonate (LACC) precursor, was studied by a combination of complementing methods: in situ WAXS, SANS, XANES, TEM, and immunogold labeling. Lysozyme (pI = 9.3) destabilizes the LACC emulsion whereas the glycoprotein ovalbumin (pI = 4.7) extends the lifespan of the emulsified state remarkably. In the light of the presented data: (a) Ovalbumin is shown to behave commensurable to the 'polymer-induced liquid precursor' (PILP) process proposed by Gower et al. Ovalbumin can be assumed to take a key role during eggshell formation where it serves as an effective stabilization agent for transient precursors and prevents undirected mineralization of the eggshell. (b) It is further shown that the emulsified LACC carries a negative surface charge and is electrostatically stabilized. (c) We propose that the liquid amorphous calcium carbonate is affected by polymers by depletion stabilization and de-emulsification rather than 'induced' by acidic proteins and polymers during a so-called polymer-induced liquid-precursor process. The original PILP coating effect, first reported by Gower et al., appears to be a result of a de-emulsification process of a stabilized LACC phase. The behavior of the liquid amorphous carbonate phase and the polymer-induced liquid-precursor phase itself can be well described by colloid chemical terms: electrostatic and depletion stabilization and de-emulsification by depletion destabilization.
- Formation of silicones mediated by the sponge enzyme silicatein-α
Stephan E. Wolf, Ute Schlossmacher, Anna Pietuch, Bernd Mathiasch, Heinz-Christoph Schröder, Werner E G Müller and Wolfgang Tremel in Dalton Transact. (2010) , Vol. 39, pp. 9245-9. DOI:10.1039/B921640E. This article is part of a themed issue: New Horizon of Organosilicon Chemistry.
Abstract: The sponge-restricted enzyme silicatein-α catalyzes in vivo silica formation from monomeric silicon compounds from sea water (i.e. silicic acid) and plays the pivotal role during synthesis of the siliceous sponge spicules. Recombinant silicatein-α, which was cloned from the demosponge Suberites domuncula (phylum Porifera), is shown to catalyze in vitro condensation of alkoxy silanes during a phase transfer reaction at neutral pH and ambient temperature to yield silicones like the straight-chained polydimethylsiloxane (PDMS). The reported condensation reaction is considered to be the first description of an enzymatically enhanced organometallic condensation reaction.
- Evidence for biogenic processes during formation of ferromanganese crusts from the Pacific Ocean: implications of biologically induced mineralization.
Xiao-Hong Wang, Stephan E. Wolf, Ute Schlossmacher, Filipe Natalio, Heinz-Christoph Schröder, Wolfgang Tremel and Werner E G Müller in Micron (2009) , Vol. 40, No. 5-6, pp. 526-35. DOI:10.1016/j.micron.2009.04.005.
Abstract: Ferromanganese [Fe/Mn] crusts formed on basaltic seamounts, gain considerable economic importance due to their high content of Co, Ni, Cu, Zn and Pt. The deposits are predominantly found in the Pacific Ocean in depths of over 1000m. They are formed in the mixing layer between the upper oxygen-minimum zone and the lower oxygen-rich bottom zone. At present an almost exclusive abiogenic origin of crust formation is considered. We present evidence that the upper layers of the crusts from the Magellan Seamount cluster are very rich in coccoliths/coccolithophores (calcareous phytoplankton) belonging to different taxa. Rarely intact skeletons of these unicellular algae are found, while most of them are disintegrated into their composing prisms or crystals. Studies on the chemical composition of crust samples by high resolution SEM combined with an electron probe microanalyzer (EPMA) revealed that they are built of distinct stacked piles of individual compartments. In the center of such piles Mn is the dominant element, while the rims of the piles are rich in Fe (mineralization aspect). The compartments contain coccospheres usually at the basal part. Energy dispersive X-ray spectroscopy (EDX) analyses showed that those coccospheres contain, as expected, CaCO3 but also Mn-oxide. Detailed analysis displayed on the surface of the coccolithophores a high level of CaCO3 while the concentration of Mn-oxide is relatively small. With increasing distance from the coccolithophores the concentration of Mn-oxide increases on the expense of residual CaCO3. We conclude that coccoliths/coccolithophores are crucial for the seed/nucleation phase of crust formation (biomineralization aspect). Subsequently, after the biologically induced mineralization phase Mn-oxide deposition proceeds "auto"catalytically.
- Nucleation and Growth of CaCO3 Mediated by the Egg-White Protein Ovalbumin: A Time-Resolved in situ Study Using Small-Angle Neutron Scattering
Vitaliy Pipich, Mathias Balz, Stephan E. Wolf, Wolfgang Tremel and Dietmar Schwahn in J. Am. Chem. Soc. (2008) , Vol. 130, No. 21, pp. 6879-92. DOI:10.1021/ja801798h.
Abstract: Mineralization of calcium carbonate in aqueous solutions starting from its initiation was studied by time-resolved small-angle neutron scattering (SANS). SANS revealed that homogeneous crystallization of CaCO3 involves an initial formation of thin plate-shaped nuclei which subsequently reassemble to 3-dimensional particles, first of fractal and finally of compact structure. The presence of the egg-white protein ovalbumin leads to a different progression of mineralization through several stages; the first step represents amorphous CaCO3, whereas the other phases are crystalline. The formation and dissolution of the amorphous phase is accompanied by Ca2+-mediated unfolding and cross-linking of about 50 protein monomers showing the characteristic scattering of linear chains with a large statistical segment length. The protein complexes act as nucleation centers for the amorphous phase because of their enrichment by Ca2+ ions. SANS revealed the sequential formation of CaCO3 starting from the amorphous phase and the subsequent formation of the crystalline polymorphs vaterite and aragonite. This formation from less dense to more dense polymorphs follows the Ostwald-Volmer rule.
- Poly(silicate)-metabolizing silicatein in siliceous spicules and silicasomes of demosponges comprises dual enzymatic activities (silica polymerase and silica esterase)
Werner E. G. Müller, Ute Schloßmacher, Xiaohong Wang, Alexandra Boreiko, David Brandt, Stephan E. Wolf, Wolfgang Tremel and Heinz C. Schröder in FEBS J. (2008) , Vol. 275, No. 2, pp. 362-70. DOI:10.1111/j.1742-4658.2007.06206.x.
Abstract: Siliceous sponges can synthesize poly(silicate) for their spicules enzymatically using silicatein. We found that silicatein exists in silica-filled cell organelles (silicasomes) that transport the enzyme to the spicules. We show for the first time that recombinant silicatein acts as a silica polymerase and also as a silica esterase. The enzymatic polymerization/polycondensation of silicic acid follows a distinct course. In addition, we show that silicatein cleaves the ester-like bond in bis(p-aminophenoxy)-dimethylsilane. Enzymatic parameters for silica esterase activity are given. The reaction is completely blocked by sodium hexafluorosilicate and E-64. We consider that the dual function of silicatein (silica polymerase and silica esterase) will be useful for the rational synthesis of structured new silica biomaterials.
- Reply to Mirror Symmetry Breaking of the Centrosymmetric CaCO3 Crystals with Amino Acids
Niklas Loges, Stephan E. Wolf, Martin Panthöfer, Lars Müller, Marc-Christopher Reinnig, Thorsten Hoffmann and Wolfgang Tremel in Angew. Chem. Int. Ed. (2008) , Vol. 47, No. 20, pp. 3683-6. DOI:10.1002/anie.200800120.
Abstract: Like chalk and cheese: Although the model put forward by Lahav and Leiserowitz may be quite reasonable for the crystallization of glycine in the presence of amino acids, it is not the case for the crystallization of CaCO3 (see picture). In the latter case, the amino acids present cannot be considered to be innocent molecules as they are involved as ligands in the coordination chemistry of Ca2+. New results indicate that homochiral impurities are not the driving force for the phase selection: When added intentionally, they annihilate the observed phase selectivity rather than promote it.
- New Insights into the Crystallization Process of Calcium Carbonate by a Contact-Free in situ Scattering Technique using a Levitating Drop Method
Stephan E. Wolf, Jork Leiterer, Michael Kappl, Franziska Emmerling and Wolfgang Tremel in Z. Anorg. Allg. Chem. (2008) , Vol. 634, No. 11, pp. 2015. DOI:10.1002/zaac.200870015.
- Early homogenous amorphous precursor stages of calcium carbonate and subsequent crystal growth in levitated droplets.
Stephan E. Wolf, Jork Leiterer and Michael Kappl, Franziska Emmerling and Wolfgang Tremel in J. Am. Chem. Soc. (2008) , Vol. 130, No. 37, pp. 12342-7. DOI:10.1021/ja800984y. Highlighted in Nachr. Chemie (2009).
Abstract: An in situ study of the contact-free crystallization of calcium carbonate in acoustic levitated droplets is reported. The levitated droplet technique allows an in situ monitoring of the crystallization while avoiding any foreign phase boundaries that may influence the precipitation process by heterogeneous nucleation. The diffusion-controlled precipitation of CaCO3 at neutral pH starts in the initial step with the homogeneous formation of a stable, nanosized liquid-like amorphous calcium carbonate phase that undergoes in a subsequent step a solution-assisted transformation to calcite. Cryogenic scanning electron microscopy studies indicate that precipitation is not induced at the solution/air interface. Our findings demonstrate that a liquid-liquid phase separation occurs at the outset of the precipitation under diffusion-controlled conditions (typical for biomineral formation) with a slow increase of the supersaturation at neutral pH.
- Bioencapsulation of living bacteria (Escherichia coli) with poly(silicate) after transformation with silicatein-alpha gene.
Werner E.G. Müller, Sylvia Engel, Xiaohong Wang, Stephan E. Wolf, Wolfgang Tremel, Narsinh L. Thakurd, Anatoli Krasko, Mugdha Divekar and Heinz C. Schröder in Biomaterials (2008) , Vol. 29, No. 7, pp. 771-9. DOI:10.1016/j.biomaterials.2007.10.038.
Abstract: Bioencapsulation is an intriguing way to immobilize biological materials, including cells, in silica, metal-oxides or hybrid sol-gel polymers. Until now only the sol-gel precursor technology was utilized to immobilize bacteria or yeast cells in silica. With the discovery of silicatein, an enzyme from demosponges that catalyzes the formation of poly(silicate), it became possible to synthesize poly(silicate) under physiological (ambient) conditions. Here we show that Escherichia coli can be transformed with the silicatein gene, its expression level in the presence of isopropyl beta-D-thiogalactopyranoside (IPTG) can be efficiently intensified by co-incubation with silicic acid. This effect could be demonstrated on the level of recombinant protein synthesis as well as by immunostaining analysis. The heterologously produced silicatein is enzymatically active, as confirmed by staining with Rhodamine 123 (formation for poly[silicate] from silicic acid) and by reacting free silicic acid with the beta-silicomolybdato color system. Electron microscopic analysis revealed that the bacteria that express silicatein form a viscous cover around them when growing in the presence of silicic acid. Finally, we demonstrate that the growth kinetics of E. coli remains unaffected whether or not the bacteria had been transformed with silicatein or grown in medium, supplemented with silicic acid. It is concluded that silicatein-mediated encapsulation of bacteria with silica might improve, extend and optimize the range of application of bacteria for the production of recombinant protein.
- Analysis of the axial filament in spicules of the demosponge Geodia cydonium: different silicatein composition in microscleres (asters) and megascleres (oxeas and triaenes).
Werner E G Müller, Ute Schlossmacher, Carsten Eckert, Anatoli Krasko, Alexandra Boreiko, Hiroshi Ushijima, Stephan E. Wolf, Wolfgang Tremel, Isabel M Müller and Heinz-Christoph Schröder in Eur. J. Cell Biol. (2007) , Vol. 86, No. 8, pp. 473-87. DOI:10.1016/j.ejcb.2007.06.002.
Abstract: The skeleton of the siliceous sponges (Porifera: Hexactinellida and Demospongiae) is supported by spicules composed of bio-silica. In the axial canals of megascleres, harboring the axial filaments, three isoforms of the enzyme silicatein (-alpha, -beta and -gamma) have been identified until now, using the demosponges Tethya aurantium and Suberites domuncula. Here we describe the composition of the proteinaceous components of the axial filament from small spicules, the microscleres, in the demosponge Geodia cydonium that possesses megascleres and microscleres. The morphology of the different spicule types is described. Also in G. cydonium the synthesis of the spicules starts intracellularly and they are subsequently extruded to the extracellular space. In contrast to the composition of the silicateins in the megascleres (isoforms: -alpha, -beta and -gamma), the axial filaments of the microscleres contain only one form of silicatein, termed silicatein-alpha/beta, with a size of 25kDa. Silicatein-alpha/beta undergoes three phosphorylation steps. The gene encoding silicatein-alpha/beta was identified and found to comprise the same characteristic sites, described previously for silicateins-alpha or -beta. It is hypothesized, that the different composition of the axial filaments, with respect to silicateins, contributes to the morphology of the different types of spicules.
- Formation of giant spicules in the deep-sea hexactinellid Monorhaphis chuni (Schulze 1904): electron-microscopic and biochemical studies.
Werner E. G. Müller, Carsten Eckert, Klaus Kropf, Xiaohong Wang, Ute Schloßmacher, Christopf Seckert, Stephan E. Wolf, Wolfgang Tremel and Heinz C. Schröder in Cell Tissue Res (2007) , Vol. 329, No. 2, pp. 363-78. DOI:10.1007/s00441-007-0402-x.
Abstract: The siliceous sponge Monorhaphis chuni (Hexactinellida) synthesizes the largest biosilica structures on earth (3 m). Scanning electron microscopy has shown that these spicules are regularly composed of concentrically arranged lamellae (width: 3–10 µm). Between 400 and 600 lamellae have been counted in one giant basal spicule. An axial canal (diameter: ~2 µm) is located in the center of the spicules; it harbors the axial filament and is surrounded by an axial cylinder (100–150 µm) of electron-dense homogeneous silica. During dissolution of the spicules with hydrofluoric acid, the axial filament is first released followed by the release of a proteinaceous tubule. Two major proteins (150 kDa and 35 kDa) have been visualized, together with a 24-kDa protein that cross-reacts with antibodies against silicatein. The spicules are surrounded by a collagen net, and the existence of a hexactinellidan collagen gene has been demonstrated by cloning it from Aphrocallistes vastus. During the axial growth of the spicules, silicatein or the silicatein-related protein is proposed to become associated with the surface of the spicules and to be finally internalized through the apical opening to associate with the axial filament. Based on the data gathered here, we suggest that, in the Hexactinellida, the growth of the spicules is mediated by silicatein or by a silicatein-related protein, with the orientation of biosilica deposition being controlled by lectin and collagen.
- Phasenselektion von Calciumcarbonat durch die Chiralität adsorbierter Aminosäuren
Stephan E. Wolf, Niklas Loges, Bernd Mathiasch, Martin Panthöfer, Ingo Mey, Andreas Janshoff and Wolfgang Tremel in Angew. Chem. (2007) , Vol. 119, No. 29, pp. 5716-21. DOI:10.1002/ange.200700010. This is featured as a VIP paper.
Abstract: Auch Aminosäuren haben Lieblingsplätze: Die Phasenselektion von Calciumcarbonat ist bestimmt durch chirale Aminosäuren, die während der Kristallisation anwesend sind. Das Wechselspiel zwischen Kristalloberfläche und Additiven führt zu einer enantiospezifischen Adsorption der D- und L-Aminosäuren auf chiralen Stufen des Kristallwachstums. Die resultierende Oberflächenpassivierung erzeugt eine kinetische Barriere, welche die Phasenselektion steuert.
- Phase selection of calcium carbonate through the chirality of adsorbed amino acids.
Stephan E. Wolf, Niklas Loges, Bernd Mathiasch, Martin Panthöfer, Ingo Mey, Andreas Janshoff and Wolfgang Tremel in Angew. Chem. Int. Ed. (2007) , Vol. 46, No. 29, pp. 5618-23. DOI:10.1002/anie.200700010. This is featured as a VIP paper.
Abstract: On the phase of it: The phase selection of calcium carbonate (spheres: C gray, Ca green, O red) is determined by chiral amino acids (stick models) present during the crystallization. The interplay of composition and chirality of the crystal surfaces and additives leads to enantiospecific adsorption of the D and L amino acids on chiral surface steps. The resulting surface passivation creates a kinetic barrier, which controls the phase selection.