Siliplant1 B-domain precipitates silica spheres, aggregates, or gel, depending on Si-precursor to peptide ratios.

Silica is extensively deposited by plants, however, only little is known about the molecular control over this process. Siliplant1 is the only known plant protein to precipitate biosilica. The protein contains seven repeats made of three domains. One of the domains exhibits a conserved sequence, which catalyzes silica precipitation in vitro. Here, silica was synthesized by the activity of a peptide carrying this conserved sequence. Infrared spectroscopy and thermal gravimetric analyses showed that the peptide was bound to the mineral. Scanning electron microscopy showed that silica-peptide particles of 22 ± 4 nm aggregated to spherical structures of 200-300 nm when the ratio of silicic acid to the peptide was below 183:1 molecules. When the ratio was about 183:1, similar particles aggregated into irregular structures, and silica gel formed at higher ratios. Solid-state NMR spectra indicated that the irregular aggregates were richer in Si-O-Si bonds as well as disordered peptide. Our results suggest that the peptide catalyzed the condensation of silicic acid and the formation of ∼20 nm particles, which aggregated into spheres. Excess of the peptide stabilized surface Si-OH groups that prevented spontaneous Si-O-Si bonding between aggregates. Under Si concentrations relevant to plant sap, the peptide and possibly Siliplant1, could catalyze nucleation of silica particles that aggregate into spherical aggregates.

Selective excitation with recoupling pulse schemes uncover properties of disordered mineral phases in bone-like apatite grown with bone proteins.

Bone construction has been under intensive scrutiny for many years using numerous techniques. Solid-state NMR spectroscopy helped unravel key characteristics of the mineral structure in bone owing to its capability of analyzing crystalline and disordered phases at high-resolution. This has invoked new questions regarding the roles of persistent disordered phases in structural integrity and mechanical function of mature bone as well as regarding regulation of early events in formation of apatite by bone proteins which interact intimately with the different mineral phases to exert biological control. Here, spectral editing tethered to standard NMR techniques is employed to analyze bone-like apatite minerals prepared synthetically in the presence and absence of two non-collagenous bone proteins, osteocalcin and osteonectin. A 1H spectral editing block allows excitation of species from the crystalline and disordered phases selectively, facilitating analysis of phosphate or carbon species in each phase by magnetization transfer via cross polarization. Further characterization of phosphate proximities using SEDRA dipolar recoupling, cross-phase magnetization transfer using DARR and T1/T2 relaxation times demonstrate that the mineral phases formed in the presence of bone proteins are more complex than bimodal. They reveal disparities in the physical properties of the mineral layers, indicate the layers in which the proteins reside and highlight the effect that each protein imparts across the mineral layers.

Solvent Dependent Nuclear Magnetic Resonance Molecular Parameters Based on a Polarization Consistent Screened Range Separated Hybrid Density Functional Theory Framework.

Nuclear magnetic resonance (NMR) properties of solvated molecules are significantly affected by the solvent. We, therefore, employ a polarization consistent framework that efficiently addresses the solvent polarizing environment effects. Toward this goal a dielectric screened range separated hybrid (SRSH) functional is invoked with a polarizable continuum model (PCM) to properly represent the orbital gap in the condensed phase. We build on the success of range separated hybrid (RSH) functionals to address the erroneous tendency of traditional density functional theory (DFT) to collapse the orbital gap. Recently, the impact of RSH that properly opens up the orbital gap in gas-phase calculations on NMR properties has been assessed. Here, we report the use of SRSH-PCM that produces properly solute orbital gaps in calculating isotropic nuclear magnetic shielding and chemical shift parameters of molecular systems in the condensed phase. We show that in contrast to simpler DFT-PCM approaches, SRSH-PCM successfully follows expected dielectric constant trends.

Lysozyme is Sterically Trapped Within the Silica Cage in Bioinspired Silica-Lysozyme Composites: A Multi-Technique Understanding of Elusive Protein-Material Interactions.

Lysozyme is widely known to promote the formation of condensed silica networks from solutions containing silicic acid, in a reproducible and cost-effective way. However, little is known about the fate of the protein after the formation of the silica particles. Also, the relative arrangement of the different components in the resulting material is a matter of debate. In this study, we investigate the nature of the protein-silica interactions by means of solid-state nuclear magnetic resonance spectroscopy, small-angle X-ray scattering, and electron microscopy. We find that lysozyme and silica are in intimate contact and strongly interacting, but their interaction is neither covalent nor electrostatic: lysozyme is mostly trapped inside the silica by steric effects.

Sourcing Herod the Great's calcite-alabaster bathtubs by a multi-analytic approach.

Herod "the Great", king of Judea in the second half of the first century BC, was known for his building projects, wealth, and political power. Two of his personal calcite-alabaster bathtubs, found in the Kypros fortress and the palace of Herodium, are among the very limited archaeological evidence of his private life. It seemed plausible that they were imported from Egypt, the main source of calcite-alabaster in ancient periods. Yet, the recent identification of a calcite quarry in the Te'omim cave, Israel, challenges this hypothesis. Here, we developed an approach for identification of the source of calcite-alabaster, by combination of four analytical methods: ICP, FTIR, ssNMR and isotope ratio. These methods were then applied to Herod's bathtubs demonstrating that they were indeed quarried in Israel rather than in Egypt.

Molecular differences in collagen organization and in organic-inorganic interfacial structure of bones with and without osteocytes.

Bone is a fascinating biomaterial composed mostly of type-I collagen fibers as an organic phase, apatite as an inorganic phase, and water molecules residing at the interfaces between these phases. They are hierarchically organized with minor constituents such as non-collagenous proteins, citrate ions and glycosaminoglycans into a composite structure that is mechanically durable yet contains enough porosity to accommodate cells and blood vessels. The nanometer scale organization of the collagen fibrous structure and the mineral constituents in bone were recently extensively scrutinized. However, molecular details at the lowest hierarchical level still need to be unraveled to better understand the exact atomic-level arrangement of all these important components in the context of the integral structure of the bone. In this report, we unfold some of the molecular characteristics differentiating between two load-bearing (cleithrum) bones, one from sturgeon fish, where the matrix contains osteocytes and one from pike fish where the bone tissue is devoid of these bone cells. Using enhanced solid-state NMR measurements, we underpin disparities in the collagen fibril structure and dynamics, the mineral phases, the citrate content at the organic-inorganic interface and water penetrability in the two bones. These findings suggest that different strategies are undertaken in the erection of the mineral-organic interfaces in various bones characterized by dissimilar osteogenesis or remodeling pathways and may have implications for the mechanical properties of the particular bone. STATEMENT OF SIGNIFICANCE: Bone boasts unique interactions between collagen fibers and mineral phases through interfaces holding together this bio-composite structure. Over evolution, fish have gone from mineralizing their bones aided by certain bone cells called osteocytes, like tetrapod, to mineralization without these cells. Here, we report atomic level differences in collagen fiber cross linking and organization, porosity of the mineral phases and content of citrate molecules at the bio-mineral interface in bones from modern versus ancient fish. The dissimilar structural features may suggest disparate mechanical properties for the two bones. Fundamental level understanding of the organic and inorganic components in bone and the interfacial interactions holding them together is essential for successful bone repair and for treating better tissue pathologies.

Biogenic Guanine Crystals Are Solid Solutions of Guanine and Other Purine Metabolites.

Highly reflective crystals of the nucleotide base guanine are widely distributed in animal coloration and visual systems. Organisms precisely control the morphology and organization of the crystals to optimize different optical effects, but little is known about how this is achieved. Here we examine a fundamental question that has remained unanswered after over 100 years of research on guanine: what are the crystals made of? Using solution-state and solid-state chemical techniques coupled with structural analysis by powder XRD and solid-state NMR, we compare the purine compositions and the structures of seven biogenic guanine crystals with different crystal morphologies, testing the hypothesis that intracrystalline dopants influence the crystal shape. We find that biogenic "guanine" crystals are not pure crystals but molecular alloys (aka solid solutions and mixed crystals) of guanine, hypoxanthine, and sometimes xanthine. Guanine host crystals occlude homogeneous mixtures of other purines, sometimes in remarkably large amounts (up to 20% of hypoxanthine), without significantly altering the crystal structure of the guanine host. We find no correlation between the biogenic crystal morphology and dopant content and conclude that dopants do not dictate the crystal morphology of the guanine host. The ability of guanine crystals to host other molecules enables animals to build physiologically "cheaper" crystals from mixtures of metabolically available purines, without impeding optical functionality. The exceptional levels of doping in biogenic guanine offer inspiration for the design of mixed molecular crystals that incorporate multiple functionalities in a single material.

Cu2+-Induced self-assembly and amyloid formation of a cyclic D,L-α-peptide: structure and function.

In a wide spectrum of neurodegenerative diseases, self-assembly of pathogenic proteins to cytotoxic intermediates is accelerated by the presence of metal ions such as Cu2+. Only low concentrations of these early transient oligomeric intermediates are present in a mixture of species during fibril formation, and hence information on the extent of structuring of these oligomers is still largely unknown. Here, we investigate dimers as the first intermediates in the Cu2+-driven aggregation of a cyclic D,L-α-peptide architecture. The unique structural and functional properties of this model system recapitulate the self-assembling properties of amyloidogenic proteins including ß-sheet conformation and cross-interaction with pathogenic amyloids. We show that a histidine-rich cyclic D,L-α-octapeptide binds Cu2+ with high affinity and selectivity to generate amyloid-like cross-ß-sheet structures. By taking advantage of backbone amide methylation to arrest the self-assembly at the dimeric stage, we obtain structural information and characterize the degree of local order for the dimer. We found that, while catalytic amounts of Cu2+ promote aggregation of the peptide to fibrillar structures, higher concentrations dose-dependently reduce fibrillization and lead to formation of spherical particles, showing self-assembly to different polymorphs. For the initial self-assembly step to the dimers, we found that Cu2+ is coordinated on average by two histidines, similar to self-assembled peptides, indicating that a similar binding interface is perpetuated during Cu2+-driven oligomerization. The dimer itself is found in heterogeneous conformations that undergo dynamic exchange, leading to the formation of different polymorphs at the initial stage of the aggregation process.

Structure and Dynamics Perturbations in Ubiquitin Adsorbed or Entrapped in Silica Materials Are Related to Disparate Surface Chemistries Resolved by Solid-State NMR Spectroscopy.

Protein immobilization on material surfaces is emerging as a powerful tool in the design of devices and active materials for biomedical and pharmaceutical applications as well as for catalysis. Preservation of the protein's biological functionality is crucial to the design process and is dependent on the ability to maintain its structural and dynamical integrity while removed from the natural surroundings. The scientific techniques to validate the structure of immobilized proteins are scarce and usually provide limited information as a result of poor resolution. In this work, we benchmarked the ability of standard solid-state NMR techniques to resolve the effects of binding to dissimilar silica materials on a model protein. In particular, the interactions between ubiquitin and the surfaces of MCM41, SBA15, and silica formed in situ were tested for their influence on the structure and dynamics of the protein. It is shown that the protein's globular fold in the free state is only slightly perturbed in the three silica materials. Local motions on a residue level that are quenched by immobilization or, conversely, that arise from the process are also detailed. NMR measurements show that these perturbations are unique to each silica material and can serve as reporters of the characteristic surface chemistry.

Osteopontin regulates biomimetic calcium phosphate crystallization from disordered mineral layers covering apatite crystallites.

Details of apatite formation and development in bone below the nanometer scale remain enigmatic. Regulation of mineralization was shown to be governed by the activity of non-collagenous proteins with many bone diseases stemming from improper activity of these proteins. Apatite crystal growth inhibition or enhancement is thought to involve direct interaction of these proteins with exposed faces of apatite crystals. However, experimental evidence of the molecular binding events that occur and that allow these proteins to exert their functions are lacking. Moreover, recent high-resolution measurements of apatite crystallites in bone have shown that individual crystallites are covered by a persistent layer of amorphous calcium phosphate. It is therefore unclear whether non-collagenous proteins can interact with the faces of the mineral crystallites directly and what are the consequences of the presence of a disordered mineral layer to their functionality. In this work, the regulatory effect of recombinant osteopontin on biomimetic apatite is shown to produce platelet-shaped apatite crystallites with disordered layers coating them. The protein is also shown to regulate the content and properties of the disordered mineral phase (and sublayers within it). Through solid-state NMR atomic carbon-phosphorous distance measurements, the protein is shown to be located in the disordered phases, reaching out to interact with the surfaces of the crystals only through very few sidechains. These observations suggest that non-phosphorylated osteopontin acts as regulator of the coating mineral layers and exerts its effect on apatite crystal growth processes mostly from afar with a limited number of contact points with the crystal.

Siliplant1 protein precipitates silica in sorghum silica cells.

Silicon is absorbed by plant roots as silicic acid. The acid moves with the transpiration stream to the shoot, and mineralizes as silica. In grasses, leaf epidermal cells called silica cells deposit silica in most of their volume using an unknown biological factor. Using bioinformatics tools, we identified a previously uncharacterized protein in Sorghum bicolor, which we named Siliplant1 (Slp1). Slp1 is a basic protein with seven repeat units rich in proline, lysine, and glutamic acid. We found Slp1 RNA in sorghum immature leaf and immature inflorescence. In leaves, transcription was highest just before the active silicification zone (ASZ). There, Slp1 was localized specifically to developing silica cells, packed inside vesicles and scattered throughout the cytoplasm or near the cell boundary. These vesicles fused with the membrane, releasing their content in the apoplastic space. A short peptide that is repeated five times in Slp1 precipitated silica in vitro at a biologically relevant silicic acid concentration. Transient overexpression of Slp1 in sorghum resulted in ectopic silica deposition in all leaf epidermal cell types. Our results show that Slp1 precipitates silica in sorghum silica cells.

Peptides from diatoms and grasses harness phosphate ion binding to silica to help regulate biomaterial structure.

Many life forms generate intricate submicron biosilica structures with various important biological functions. The formation of such structures, from the silicic acid in the waters and in the soil, is thought to be regulated by unique proteins with high repeats of specific amino acids and unusual sidechain modifications. Some silicifying proteins are characterized by high prevalence of basic amino acids in their primary structures. Lysine-rich domains are found, for instance, in diatom silaffin proteins and in the sorghum grass siliplant1 protein. These domains exhibit catalytic activity in silica chain condensation, owing to molecular interactions of the lysine amine groups with the forming mineral. The use of amine chemistry by two very remote organisms has motivated us to seek other molecular biosilicification processes that may be common to the two life forms. In diatom silaffins, domains rich in phosphoserine residues are thought to assist the assembly of silaffin molecules into an organic supra-structure which serves as a template for the silica to precipitate on. This mold, held by salt bridges between serine phosphates and lysine amines, dictates the shape of the silica particles formed. Yet, silica synthesized with the dephosphorylated silaffin in phosphate buffer showed similar morphology to the one prepared with the native protein, suggesting that a defined spatial arrangement of serine phosphates is not required to generate silica with the desired shape. Concurrently, free phosphates enhanced the activity of siliplant1 in silica formation. It is therefore beneficial to characterize the involvement of these anions as co-factors in regulated silicification by functional peptides from the two proteins and to understand whether they play similar molecular role in the mechanism of mineralization. Here we analyze the molecular interactions of free phosphate ions with silica and the silaffin peptide PL12 and separately with silica and siliplant1 peptide SLP1 in the two biomimetic silica products generated by the two peptides. MAS NMR measurements show that the phosphate ions interact with the peptides and at the same time may be forming bonds with the silica mineral. This bridging capability may add another avenue by which the structure of the silica material is influenced. A model for the molecular/ionic interactions at the bio-inorganic interface is described, which may have bearings for the role of phosphorylated residues beyond the function as intermolecular cross linkers or free phosphate ions as co-factors in regulation of silicification. STATEMENT OF SIGNIFICANCE: The manuscript addresses the question how proteins in diatoms and plants regulate the biosilica materials that are produced for various purposes in organisms. It uses preparation of silica in vitro with functional peptide derivatives from a sorghum grass protein and from a diatom silaffin protein separately to show that phosphate ions are important for the control that is achieved by these proteins on the final shape of the silica material produced. It portrays via magnetic resonance spectroscopic measurements, in atomic detail, the interface between atoms in the peptide, atoms on the surface of the silica formed and the phosphate ions that form chemical bonds with atoms on the silica as part of the mechanism of action of these peptides.

How does osteocalcin lacking γ-glutamic groups affect biomimetic apatite formation and what can we say about its structure in mineral-bound form?

Non-collagenous proteins such as osteocalcin function as regulators of the mineralization process in bone. Osteocalcin undergoes post-translational modification adding an extra carboxylate group on three of its glutamate residues to enhance interaction with bone mineral. In this work, we examine regulation of biomimetic apatite formation by osteocalcin that was not modified after translation. We analyze the structural features in the protein and mineral-protein interfaces to elicit the unmodified protein's fold inside the mineral and to unveil the species that interact with the mineral surface. The results presented here give clues on the protein's active role in controlling the mineral phases that are formed on hydroxyapatite crystals and its ability to influence the extent of order in these crystals.

Dynamics in hydrophilic and hydrophobic molecular chains tethered to MCM41-type mesoporous silica upon wetting and dehydration processes.

Surface modified mesoporous silica materials are important materials for heterogeneous catalysis and are attracting attention as potential drug carriers. The functionality of these materials relies on the physical and chemical properties of the tethers attached to MCM41 silica surface. These chemically linked tails act as molecular brushes, that can capture pollutant molecules, anchor points for catalysts and can host drug molecules. To utilize the full potential of the tailored silica surfaces, one should infer their properties at different levels of solvation. Here, 1H MAS NMR spectroscopy is used to monitor the dynamic properties of two modified MCM41 materials, an aminopropyl tethered MCM41 and an octyl tethered MCM41, through the process of controlled hydration. The surface site resolved measurements demonstrate how the chemical nature of the two tethers governs the way water molecules are directed to the different sites in the porous materials.

Spectroscopic Discrimination of Sorghum Silica Phytoliths.

Grasses accumulate silicon in the form of silicic acid, which is precipitated as amorphous silica in microscopic particles termed phytoliths. These particles comprise a variety of morphologies according to the cell type in which the silica was deposited. Despite the evident morphological differences, phytolith chemistry has mostly been analysed in bulk samples, neglecting differences between the varied types formed in the same species. In this work, we extracted leaf phytoliths from mature plants of Sorghum bicolor (L.) Moench. Using solid state NMR and thermogravimetric analysis, we show that the extraction methods alter greatly the silica molecular structure, its condensation degree and the trapped organic matter. Measurements of individual phytoliths by Raman and synchrotron FTIR microspectroscopies in combination with multivariate analysis separated bilobate silica cells from prickles and long cells, based on the silica molecular structures and the fraction and composition of occluded organic matter. The variations in structure and composition of sorghum phytoliths suggest that the biological pathways leading to silica deposition vary between these cell types.

Minerals in the pre-settled coral Stylophora pistillata crystallize via protein and ion changes.

Aragonite skeletons in corals are key contributors to the storage of atmospheric CO2 worldwide. Hence, understanding coral biomineralization/calcification processes is crucial for evaluating and predicting the effect of environmental factors on this process. While coral biomineralization studies have focused on adult corals, the exact stage at which corals initiate mineralization remains enigmatic. Here, we show that minerals are first precipitated as amorphous calcium carbonate and small aragonite crystallites, in the pre-settled larva, which then evolve into the more mature aragonitic fibers characteristic of the stony coral skeleton. The process is accompanied by modulation of proteins and ions within these minerals. These findings may indicate an underlying bimodal regulation tactic adopted by the animal, with important ramification to its resilience or vulnerability toward a changing environment.

Engineering l-asparaginase for spontaneous formation of calcium phosphate bioinspired microreactors.

Active bioinspired materials are appealing biotechnological targets, and their study is gaining momentum. These materials, which comprise of an inorganic matrix and one or more biomolecules, are extremely variable and therefore may result difficult to characterize in their intimate structure. In this work we have prepared a hydroxyapatite-l-asparaginase composite, with the perspective of using it in acute leukemia treatment. We demonstrate that the use of electron microscopy and powder X-ray diffraction, combined with the atomic-resolution information coming from solid-state NMR, allows us to understand the topology of the material and how the different components interplay to obtain an active composite.