Uncovering the Role of Bicarbonate in Calcium Carbonate Formation at Near-Neutral pH.

Mechanistic pathways relevant to mineralization are not well-understood fundamentally, let alone in the context of their biological and geological environments. Through quantitative analysis of ion association at near-neutral pH, we identify the involvement of HCO3- ions in CaCO3 nucleation. Incorporation of HCO3- ions into the structure of amorphous intermediates is corroborated by solid-state nuclear magnetic resonance spectroscopy, complemented by quantum mechanical calculations and molecular dynamics simulations. We identify the roles of HCO3- ions as being through (i) competition for ion association during the formation of ion pairs and ion clusters prior to nucleation and (ii) incorporation as a significant structural component of amorphous mineral particles. The roles of HCO3- ions as active soluble species and structural constituents in CaCO3 formation are of fundamental importance and provide a basis for a better understanding of physiological and geological mineralization.

New Horizons of Nonclassical Crystallization.

Nonclassical crystallization (NCC) summarizes a number of crystallization pathways, which differ from the classical layer-by-layer growth of crystals involving atomic/molecular building units. Common to NCC is that the building units are larger and include nanoparticles, clusters, or liquid droplets, providing multiple handles for their control at each elementary step. Therefore, many different pathways toward the final single crystals are possible and can be influenced by appropriate experimental parameters or additives at each step of crystal growth. NCC allows for a plethora of crystallization strategies toward complex crystalline (hybrid) materials. In this perspective, we summarize the current state of the art with a focus on the new horizons of NCC with respect to mechanistic understanding, high-performance materials, and new applications. This gives a glimpse on what will be possible in the future using these crystallization approaches: Examples are new electrodes and storage materials, (photo)catalysts, building materials, porous or crystalline materials with complex shape, structural hierarchy, and anisotropic single crystals.

Self-Assembly Toolbox of Tailored Supramolecular Architectures Based on an Amphiphilic Protein Library.

The molecular structuring of complex architectures and the enclosure of space are essential requirements for technical and living systems. Self-assembly of supramolecular structures with desired shape, size, and stability remains challenging since it requires precise regulation of physicochemical and conformational properties of the components. Here a general platform for controlled self-assembly of tailored amphiphilic elastin-like proteins into desired supramolecular protein assemblies ranging from spherical coacervates over molecularly defined twisted fibers to stable unilamellar vesicles is introduced. The described assembly protocols efficiently yield protein membrane-based compartments (PMBC) with adjustable size, stability, and net surface charge. PMBCs demonstrate membrane fusion and phase separation behavior and are able to encapsulate structurally and chemically diverse cargo molecules ranging from small molecules to naturally folded proteins. The ability to engineer tailored supramolecular architectures with defined fusion behavior, tunable properties, and encapsulated cargo paves the road for novel drug delivery systems, the design of artificial cells, and confined catalytic nanofactories.

From Solute, Fluidic and Particulate Precursors to Complex Organizations of Matter.

The organization of matter from its constitutive units recruits intermediate states with distinctive degrees of self-association and molecular order. Existing as clusters, droplets, gels as well as amorphous and crystalline nanoparticles, these precursor forms have fundamental contributions towards the composition and structure of inorganic and organic architectures. In this personal account, we show that the transitions from atoms, molecules or ionic species to superstructures of higher order are intertwined with the interfaces and interactions of precursor and intermediate states. Structural organizations distributed across different length scales are explained by the multistep nature of nucleation and crystallization, which can be guided towards functional hybrid materials by the strategic application of additives, templates and reaction environments. Thus, the non-classical pathways for material formation and growth offer conceptual frameworks for elucidating, inducing and directing fascinating material organizations of biogenic and synthetic origins.

A Model Sea Urchin Spicule Matrix Protein Self-Associates To Form Mineral-Modifying Protein Hydrogels.

In the purple sea urchin Strongylocentrotus purpuratus, the formation and mineralization of fracture-resistant skeletal elements such as the embryonic spicule require the combinatorial participation of numerous spicule matrix proteins such as the SpSM30A-F isoforms. However, because of limited abundance, it has been difficult to pursue extensive biochemical studies of the SpSM30 proteins and deduce their role in spicule formation and mineralization. To circumvent these problems, we expressed a model recombinant spicule matrix protein, rSpSM30B/C, which possesses the key sequence attributes of isoforms "B" and "C". Our findings indicate that rSpSM30B/C is expressed in insect cells as a single polypeptide containing variations in glycosylation that create microheterogeneity in rSpSM30B/C molecular masses. These post-translational modifications incorporate O- and N-glycans and anionic mono- and bisialylated and mono- and bisulfated monosaccharides on the protein molecules and enhance its aggregation propensity. Bioinformatics and biophysical experiments confirm that rSpSM30B/C is an intrinsically disordered, aggregation-prone protein that forms porous protein hydrogels that control the in vitro mineralization process in three ways: (1) increase the time interval for prenucleation cluster formation and transiently stabilize an ACC polymorph, (2) promote and organize single-crystal calcite nanoparticles, and (3) promote faceted growth and create surface texturing of calcite crystals. These features are also common to mollusk shell nacre proteins, and we conclude that rSpSM30B/C is a spiculogenesis protein that exhibits traits found in other calcium carbonate mineral modification proteins.

Insect Cell Glycosylation and Its Impact on the Functionality of a Recombinant Intracrystalline Nacre Protein, AP24.

The impacts of glycosylation on biomineralization protein function are largely unknown. This is certainly true for the mollusk shell, where glycosylated intracrystalline proteins such as AP24 (Haliotis rufescens) exist but their functions and the role of glycosylation remain elusive. To assess the effect of glycosylation on protein function, we expressed two recombinant variants of AP24: an unglycosylated bacteria-expressed version (rAP24N) and a glycosylated insect cell-expressed version (rAP24G). Our findings indicate that rAP24G is expressed as a single polypeptide containing variations in glycosylation that create microheterogeneity in rAP24G molecular masses. These post-translational modifications incorporate O- and N-glycans and anionic monosialylated and bisialylated, and monosulfated and bisulfated monosaccharides on the protein molecules. AFM and DLS experiments confirm that both rAP24N and rAP24G aggregate to form protein phases, with rAP24N exhibiting a higher degree of aggregation, compared to rAP24G. With regard to functionality, we observe that both recombinant proteins exhibit similar behavior within in vitro calcium carbonate mineralization assays and potentiometric titrations. However, rAP24G modifies crystal growth directions and is a stronger nucleation inhibitor, whereas rAP24N exhibits higher mineral phase stabilization and nanoparticle containment. We believe that the post-translational addition of anionic groups (via sialylation and sulfation), along with modifications to the protein surface topology, may explain the changes in glycosylated rAP24G aggregation and mineralization behavior, relative to rAP24N.

Synergistic Biomineralization Phenomena Created by a Combinatorial Nacre Protein Model System.

In the nacre or aragonite layer of the mollusk shell, proteomes that regulate both the early stages of nucleation and nano-to-mesoscale assembly of nacre tablets from mineral nanoparticle precursors exist. Several approaches have been developed to understand protein-associated mechanisms of nacre formation, yet we still lack insight into how protein ensembles or proteomes manage nucleation and crystal growth. To provide additional insights, we have created a proportionally defined combinatorial model consisting of two nacre-associated proteins, C-RING AP7 (shell nacre, Haliotis rufescens) and pseudo-EF hand PFMG1 (oyster pearl nacre, Pinctada fucata), whose individual in vitro mineralization functionalities are well-documented and distinct from one another. Using scanning electron microscopy, flow cell scanning transmission electron microscopy, atomic force microscopy, Ca(II) potentiometric titrations, and quartz crystal microbalance with dissipation monitoring quantitative analyses, we find that both nacre proteins are functionally active within the same mineralization environments and, at 1:1 molar ratios, synergistically create calcium carbonate mesoscale structures with ordered intracrystalline nanoporosities, extensively prolong nucleation times, and introduce an additional nucleation event. Further, these two proteins jointly create nanoscale protein aggregates or phases that under mineralization conditions further assemble into protein-mineral polymer-induced liquid precursor-like phases with enhanced ACC stabilization capabilities, and there is evidence of intermolecular interactions between AP7 and PFMG1 under these conditions. Thus, a combinatorial model system consisting of more than one defined biomineralization protein dramatically changes the outcome of the in vitro biomineralization process.

On the biophysical regulation of mineral growth: Standing out from the crowd.

Biogenic mineralization processes are generally regulated by soluble additives and insoluble matrices. This endows precise control over the different stages of mineralization such as the uptake, transport of mineral precursors as well as the subsequent deposition of the mineral phases with consistent compositions and morphologies. Programmed in the interactions of organic molecules with different precursor species and the fine modulation of the niche environments, a formative elegance is reflected in the biological means for crystal formation in comparison to the synthetic counterparts. In order to spotlight the role of prevalent biophysical environments in the emergence of fascinating materials, we revisit biologically modulated mineralization to describe nucleation and crystallization under physicochemical highly non-ideal conditions on account of macromolecular crowding and the gel-like nature of cellular matrices.

An oligomeric C-RING nacre protein influences prenucleation events and organizes mineral nanoparticles.

The mollusk shell nacre layer integrates mineral phases with macromolecular components such as intracrystalline proteins. However, the roles performed by intracrystalline proteins in calcium carbonate nucleation and subsequent postnucleation events (e.g., organization of mineral deposits) in the nacre layer are not known. We find that AP7, a nacre intracrystalline C-RING protein, self-assembles to form amorphous protein oligomers and films on mica that further assemble into larger aggregates or phases in the presence of Ca2+. Using solution nuclear magnetic resonance spectroscopy, we determine that the protein assemblies are stabilized by interdomain interactions involving the aggregation-prone T31-N66 C-terminal C-RING domain but are destabilized by the labile nature of the intrinsically disordered D1-T19 AA N-terminal sequence. Thus, the dynamic, amorphous nature of the AP7 assemblies can be traced to the molecular behavior of the N-terminal sequence. Using potentiometric methods, we observe that AP7 protein phases prolong the time interval for prenucleation cluster formation but neither stabilize nor destabilize ACC clusters. Time-resolved flow cell scanning transmission electron microscopy mineralization studies confirm that AP7 protein phases delay the onset of nucleation and assemble and organize mineral nanoparticles into ring-shaped branching clusters in solution. These phenomena are not observed in protein-deficient assays. We conclude that C-RING AP7 protein phases modulate the time period for early events in nucleation and form strategic associations with forming mineral nanoparticles that lead to mineral organization.

A nacre protein, n16.3, self-assembles to form protein oligomers that dimensionally limit and organize mineral deposits.

The mollusk shell is a complex biological material that integrates mineral phases with organic macromolecular components such as proteins. The role of proteins in the formation of the nacre layer (aragonite mineral phase) is poorly understood, particularly with regard to the organization of mineral deposits within the protein extracellular matrix and the identification of which proteins are responsible for this task. We report new experiments that provide insight into the role of the framework nacre protein, n16.3 (Pinctada fucata), as an organizer or assembler of calcium carbonate mineral clusters. Using a combination of biophysical techniques, we find that recombinant n16.3 (r-n16.3) oligomerizes to form amorphous protein films and particles that possess regions of disorder and mobility. These supramolecular assemblies possess an intrinsically disordered C-terminal region (T64-W98) and reorganize in the presence of Ca(2+) ions to form clustered protein oligomers. This Ca(2+)-induced reorganization leads to alterations in the molecular environments of Trp residues, the majority of which reside in putative aggregation-prone cross-ß strand regions. Potentiometric Ca(2+) titrations reveal that r-n16.3 does not significantly affect the formation of prenucleation clusters in solution, and this suggests a role for this protein in postnucleation mineralization events. This is verified in subsequent in vitro mineralization assays in which r-n16.3 demonstrates its ability to form gel-like protein phases that organize and cluster nanometer-sized single-crystal calcite relative to protein-deficient controls. We conclude that the n16 nacre framework proteome creates a protein gel matrix that organizes and dimensionally limits mineral deposits. This process is highly relevant to the formation of ordered, nanometer-sized nacre tablets in the mollusk shell.

Roles of larval sea urchin spicule SM50 domains in organic matrix self-assembly and calcium carbonate mineralization.

The larval spicule matrix protein SM50 is the most abundant occluded matrix protein present in the mineralized larval sea urchin spicule. Recent evidence implicates SM50 in the stabilization of amorphous calcium carbonate (ACC). Here, we investigate the molecular interactions of SM50 and CaCO3 by investigating the function of three major domains of SM50 as small ubiquitin-like modifier (SUMO) fusion proteins - a C-type lectin domain (CTL), a glycine rich region (GRR) and a proline rich region (PRR). Under various mineralization conditions, we find that SUMO-CTL is monomeric and influences CaCO3 mineralization, SUMO-GRR aggregates into large protein superstructures and SUMO-PRR modifies the early CaCO3 mineralization stages as well as growth. The combination of these mineralization and self-assembly properties of the major domains synergistically enable the full-length SM50 to fulfill functions of constructing the organic spicule matrix as well as performing necessary mineralization activities such as Ca(2+) ion recruitment and organization to allow for proper growth and development of the mineralized larval sea urchin spicule.

Crystallization and preliminary X-ray analysis of the C-type lectin domain of the spicule matrix protein SM50 from Strongylocentrotus purpuratus. Corrigendum.

The identity of the crystallized protein in the article by Juneja et al. [(2014), Acta Cryst. F70, 260-262] is corrected.

Oil Displacement in Calcite-Coated Microfluidic Chips via Waterflooding at Elevated Temperatures and Long Times.

In microfluidic studies of improved oil recovery, mostly pore networks with uniform depth and surface chemistry are used. To better mimic the multiple porosity length scales and surface heterogeneity of carbonate reservoirs, we coated a 2.5D glass microchannel with calcite particles. After aging with formation water and crude oil (CRO), high-salinity Water (HSW) was flooded at varying temperatures and durations. Time-resolved microscopy revealed the CRO displacements. Precise quantification of residual oil presented some challenges due to calcite-induced optical heterogeneity and brine-oil coexistence at (sub)micron length scales. Both issues were addressed using pixel-wise intensity calibration. During waterflooding, most of the ultimately produced oil gets liberated within the first pore volume (similar to glass micromodels). Increasing temperature from 22 °C to 60 °C and 90 °C produced some more oil. Waterflooding initiated directly at 90 °C produced significantly more oil than at 22 °C. Continuing HSW exposure at 90 °C for 8 days does not release additional oil; although, a spectacular growth of aqueous droplets is observed. The effect of calcite particles on CRO retention is weak on flat surfaces, where the coverage is ~20%. The calcite-rich pore edges retain significantly more oil suggesting that, in our micromodel wall roughness is a stronger determinant for oil retention than surface chemistry.

Cloning and expression of a tyrosinase from Aspergillus oryzae in Yarrowia lipolytica: application in L-DOPA biotransformation.

Amplification of the tyrosinase gene (melO) from the genomic DNA of Aspergillus oryzae NCIM 1212 yielded a 1.6-kb product. This gene was cloned into pYLEX1, and the resulting pTyro-YLEX1 vector was transformed in Yarrowia lipolytica strain Po 1 g. A clone displaying the highest specific activity for tyrosinase (10.94 U/mg) was used for obtaining the complementary DNA (cDNA) and for protein expression studies. cDNA sequence analysis indicated the splicing of an intron present in the melO gene by Po 1 g. Native polyacrylamide gel electrophoresis, acidification at pH 3.0 followed by activity staining with L-DOPA indicated the expression of an active tyrosinase. The clone over-expressing the tyrosinase transformed L-tyrosine to L-DOPA. On optimization of conditions for the biotransformation (pH 4.0, temperature 60° C and with 3.5 mg of biomass), 0.4 mg/ml of L-DOPA was obtained.

On Biomineralization: Enzymes Switch on Mesocrystal Assembly.

Cellular machineries guide the bottom-up pathways toward crystal superstructures based on the transport of inorganic precursors and their precise integration with organic frameworks. The biosynthesis of mesocrystalline spines entails concerted interactions between biomolecules and inorganic precursors; however, the bioinorganic interactions and interfaces that regulate material form and growth as well as the selective emergence of structural complexity in the form of nanostructured crystals are not clear. By investigating mineral nucleation under the regulation of recombinant proteins, we show that SpSM50, a matrix protein of the sea urchin spine, stabilizes mineral precursors via vesicle-confinement, a function conferred by a low-complexity, disordered region. Site-specific proteolysis of this domain by a collagenase initiates phase transformation of the confined mineral phase. The residual C-type lectin domain molds the fluidic mineral precursor into hierarchical mesocrystals identical to structural crystal modules constituting the biogenic mineral. Thus, the regulatory functions of proteolytic enzymes can guide biomacromolecular domain constitutions and interfaces, in turn determining inorganic phase transformations toward hybrid materials as well as integrating organic and inorganic components across hierarchical length scales. Bearing striking resemblance to biogenic mineralization, these hybrid materials recruit bioinorganic interactions which elegantly intertwine nucleation and crystallization phenomena with biomolecular structural dynamics, hence elucidating a long-sought key of how nature can orchestrate complex biomineralization processes.

Mineralization and non-ideality: on nature's foundry.

Understanding how ions, ion-clusters and particles behave in non-ideal environments is a fundamental question concerning planetary to atomic scales. For biomineralization phenomena wherein diverse inorganic and organic ingredients are present in biological media, attributing biomaterial composition and structure to the chemistry of singular additives may not provide a holistic view of the underlying mechanisms. Therefore, in this review, we specifically address the consequences of physico-chemical non-ideality on mineral formation. Influences of different forms of non-ideality such as macromolecular crowding, confinement and liquid-like organic phases on mineral nucleation and crystallization in biological environments are presented. Novel prospects for the additive-controlled nucleation and crystallization are accessible from this biophysical view. In this manner, we show that non-ideal conditions significantly affect the form, structure and composition of biogenic and biomimetic minerals.

Crystallization and preliminary X-ray analysis of the C-type lectin domain of the spicule matrix protein SM50 from Strongylocentrotus purpuratus.

Sea urchin spicules have a calcitic mesocrystalline architecture that is closely associated with a matrix of proteins and amorphous minerals. The mechanism underlying spicule formation involves complex processes encompassing spatio-temporally regulated organic-inorganic interactions. C-type lectin domains are present in several spicule matrix proteins in Strongylocentrotus purpuratus, implying their role in spiculogenesis. In this study, the C-type lectin domain of SM50 was overexpressed, purified and crystallized using a vapour-diffusion method. The crystal diffracted to a resolution of 2.85 Å and belonged to space group P212121, with unit-cell parameters a = 100.6, b = 115.4, c = 130.6 Å, α = ß = γ = 90°. Assuming 50% solvent content, six chains are expected to be present in the asymmetric unit.

Removal of hexavalent chromium ions by Yarrowia lipolytica cells modified with phyto-inspired Fe0/Fe3O4 nanoparticles.

The removal of hexavalent chromium [Cr (VI)], an important ground water pollutant by phyto-inspired Fe(0)/Fe(3)O(4) nanocomposite-modified cells of Yarrowia lipolytica (NCIM 3589 and NCIM 3590), was investigated. Electron microscopy and magnetometer studies indicated an effective modification of yeast cell surfaces by the nanocomposites. The effect of pH, temperature, agitation speed, contact time and initial metal ion concentration on the removal of Cr (VI) was determined. The specific uptake values at pH 2.0 were 186.32±3.17 and 137.31±4.53 mg g(-1) for NCIM 3589 and NCIM 3590, respectively, when 1000 mg L(-1) of metal ion concentrations were used. The equilibrium data fitted to Scatchard, Langmuir and linearized Freundlich models suggesting that adsorption played a role in the removal of Cr (VI) ions. The surface modified yeast cells displayed higher values of Langmuir and Scatchard coefficients than the unmodified cells indicating that the former were more efficient in Cr (VI) removal. The enhanced detoxification of Cr (VI) ions by this composite material could be attributed to the reductive power of the Fe(0)/Fe(3)O(4) nanocomposites as well the yeast cell surface functional groups.

Phyto-inspired silica nanowires: characterization and application in lipase immobilization.

Silica nanostructures were phyto-fabricated on different surfaces by using pomegranate (Punica granatum) leaf extracts. On zinc films, nanowires were obtained. On other surfaces such as silica, alumina, zinc oxide, and glass, spherical aggregates, cubic assemblies, microflakes, and acicular structures, respectively, were observed. The nanowires developed on Zn surfaces were characterized by scanning electron microscope-energy dispersive spectrometer (SEM-EDS), X-ray diffraction (XRD), photoluminescence, and Fourier transform infrared (FTIR) spectroscopic analysis. XRD profiles displayed peaks at 2.4, 4.9, and 12.1° indicating the presence of silica nanostructures. When excited at 340 nm, the reaction mixtures displayed a characteristic blue luminescence at 404 nm. FTIR spectra showed the existence of Si-OH and Si-O-Si bonds. The nanowires were functionalized with amine groups and used for the covalent immobilization of Candida rugosa lipase. The immobilized enzyme displayed better pH and temperature stability and retained 80% activity after 20 cycles. This paper highlights a novel route for the phyto-mediated growth of silica nanowires on Zn surfaces, their characterization and effective use as a matrix for enzyme immobilization.