Probing the binding modes and dynamics of histidine on fumed silica surfaces by solid-state NMR.

Silica nanoparticles can be designed to exhibit a diverse range of morphologies (e.g. non-porous, mesoporous), physical properties (e.g. hydrophobic, hydrophilic) and a wide range of chemical and biomolecular surface functionalizations. In the present work, the adsorption complex of histidine (His) and fumed silica nanoparticles (FSN) is probed using thermal analysis (TGA/DTG) and a battery of solid-state (SS) NMR methods supported by DFT chemical shift calculations. Multinuclear (1H/13C/15N) one- and two-dimensional magic angle spinning (MAS) SSNMR experiments were applied to determine site-specific interactions between His and FSN surfaces as a function of adsorption solution concentration, pH and hydration state. By directly comparing SSNMR observables (linewidth, chemical shift and relaxation parameters) for His-FSN adsorption complexes to various crystalline, amorphous and aqueous His forms, the His structural and dynamic environment on FSN surfaces could be determined at an atomic level. The observed 13C and 15N MAS NMR chemical shifts, linewidths and relaxation parameters show that the His surface layer on FSN has a significant dependence on pH and hydration state. His is highly dynamic on FSN surfaces under acidic conditions (pH 4) as evidenced by sharp resonances with near isotropic chemical shifts regardless of hydration level indicating a non-specific binding arrangement while, a considerably more rigid His environment with defined protonation states is observed at near neutral pH with subtle variations between hydrated and anhydrous complexes. At near neutral pH, less charge repulsion occurs on the FSN surface and His is more tightly bound as evidenced by considerable line broadening likely due to chemical shift heterogeneity and a distribution in hydrogen-bonding strengths on the FSN surface. Multiple His sites exchange with a tightly bound water layer in hydrated samples while, direct interaction with the FSN surface and significant chemical shift perturbations for imidazole ring nitrogen sites and some carbon resonances are observed after drying. The SSNMR data was used to propose an interfacial molecular binding model between His and FSN surfaces under varying conditions setting the stage for future multinuclear, multidimensional SSNMR studies of His-containing peptides on silica nanoparticles and other nanomaterials of interest.

Correction: Biosynthesis of the oxygenated diterpene nezukol in the medicinal plant Isodon rubescens is catalyzed by a pair of diterpene synthases.

[This corrects the article DOI: 10.1371/journal.pone.0176507.].

Antioxidant Sensing by Spiropyrans: Substituent Effects and NMR Spectroscopic Studies.

The development of stimuli-responsive small molecules for probing biologically active antioxidants such as glutathione (GSH) has important ramifications in the detection of oxidative stress. An ideal sensor for biological applications should exhibit sufficient sensitivity and selectivity for detection at physiological concentrations and be reversible to allow continuous and dynamic monitoring of antioxidant levels. Designing a suitable sensor thus requires a detailed understanding of activation properties and mechanism of action. In this work, we report a new set of GSH-responsive spiropyrans and demonstrate how changes in the electronic structure of spiropyrans influence GSH sensing with high specificity versus other structurally similar and biologically relevant redox-active molecules. The sensitivity, selectivity, kinetics, binding constant, and reversibility of GSH-responsive-substituted spiropyrans were investigated using UV-vis spectroscopy and laser irradiation experiments. Detailed studies of the mechanism of interaction between spiropyrans with GSH were investigated using NMR spectroscopy. Understanding how electronic effects impact the sensing ability of spiropyrans toward antioxidants and elucidating the mechanism of the spiropyran-GSH interaction will facilitate the design of more effective sensors for detection of antioxidants in vivo.

Functional characterization of the cytochrome P450 monooxygenase CYP71AU87 indicates a role in marrubiin biosynthesis in the medicinal plant Marrubium vulgare.

BACKGROUND: Horehound (Marrubium vulgare) is a medicinal plant whose signature bioactive compounds, marrubiin and related furanoid diterpenoid lactones, have potential applications for the treatment of cardiovascular diseases and type II diabetes. Lack of scalable plant cultivation and the complex metabolite profile of M. vulgare limit access to marrubiin via extraction from plant biomass. Knowledge of the marrubiin-biosynthetic enzymes can enable the development of metabolic engineering platforms for marrubiin production. We previously identified two diterpene synthases, MvCPS1 and MvELS, that act sequentially to form 9,13-epoxy-labd-14-ene. Conversion of 9,13-epoxy-labd-14-ene by cytochrome P450 monooxygenase (P450) enzymes can be hypothesized to facilitate key functional modification reactions in the formation of marrubiin and related compounds. RESULTS: Mining a M. vulgare leaf transcriptome database identified 95 full-length P450 candidates. Cloning and functional analysis of select P450 candidates showing high transcript abundance revealed a member of the CYP71 family, CYP71AU87, that catalyzed the hydroxylation of 9,13-epoxy-labd-14-ene to yield two isomeric products, 9,13-epoxy labd-14-ene-18-ol and 9,13-epoxy labd-14-ene-19-ol, as verified by GC-MS and NMR analysis. Additional transient Nicotiana benthamiana co-expression assays of CYP71AU87 with different diterpene synthase pairs suggested that CYP71AU87 is specific to the sequential MvCPS1 and MvELS product 9,13-epoxy-labd-14-ene. Although the P450 products were not detectable in planta, high levels of CYP71AU87 gene expression in marrubiin-accumulating tissues supported a role in the formation of marrubiin and related diterpenoids in M. vulgare. CONCLUSIONS: In a sequential reaction with the diterpene synthase pair MvCPS1 and MvELS, CYP71AU87 forms the isomeric products 9,13-epoxy labd-14-ene-18/19-ol as probable intermediates in marrubiin biosynthesis. Although its metabolic relevance in planta will necessitate further genetic studies, identification of the CYP71AU87 catalytic activity expands our knowledge of the functional landscape of plant P450 enzymes involved in specialized diterpenoid metabolism and can provide a resource for the formulation of marrubiin and related bioactive natural products.

Hierarchical spidroin micellar nanoparticles as the fundamental precursors of spider silks.

Many natural silks produced by spiders and insects are unique materials in their exceptional toughness and tensile strength, while being lightweight and biodegradable-properties that are currently unparalleled in synthetic materials. Myriad approaches have been attempted to prepare artificial silks from recombinant spider silk spidroins but have each failed to achieve the advantageous properties of the natural material. This is because of an incomplete understanding of the in vivo spidroin-to-fiber spinning process and, particularly, because of a lack of knowledge of the true morphological nature of spidroin nanostructures in the precursor dope solution and the mechanisms by which these nanostructures transform into micrometer-scale silk fibers. Herein we determine the physical form of the natural spidroin precursor nanostructures stored within spider glands that seed the formation of their silks and reveal the fundamental structural transformations that occur during the initial stages of extrusion en route to fiber formation. Using a combination of solution phase diffusion NMR and cryogenic transmission electron microscopy (cryo-TEM), we reveal direct evidence that the concentrated spidroin proteins are stored in the silk glands of black widow spiders as complex, hierarchical nanoassemblies (∼300 nm diameter) that are composed of micellar subdomains, substructures that themselves are engaged in the initial nanoscale transformations that occur in response to shear. We find that the established micelle theory of silk fiber precursor storage is incomplete and that the first steps toward liquid crystalline organization during silk spinning involve the fibrillization of nanoscale hierarchical micelle subdomains.

Functional Diversity of Diterpene Synthases in the Biofuel Crop Switchgrass.

Diterpenoids constitute a diverse class of metabolites with critical functions in plant development, defense, and ecological adaptation. Major monocot crops, such as maize (Zea mays) and rice (Oryza sativa), deploy diverse blends of specialized diterpenoids as core components of biotic and abiotic stress resilience. Here, we describe the genome-wide identification and functional characterization of stress-related diterpene synthases (diTPSs) in the dedicated bioenergy crop switchgrass (Panicum virgatum). Mining of the allotetraploid switchgrass genome identified an expansive diTPS family of 31 members, and biochemical analysis of 11 diTPSs revealed a modular metabolic network producing a diverse array of diterpenoid metabolites. In addition to ent-copalyl diphosphate (CPP) and ent-kaurene synthases predictably involved in gibberellin biosynthesis, we identified syn-CPP and ent-labda-13-en-8-ol diphosphate (LPP) synthases as well as two diTPSs forming (+)-labda-8,13E-dienyl diphosphate (8,13-CPP) and ent-neo-cis-trans-clerodienyl diphosphate (CT-CLPP) scaffolds not observed previously in plants. Structure-guided mutagenesis of the (+)-8,13-CPP and ent-neo-CT-CLPP synthases revealed residue substitutions in the active sites that altered product outcome, representing potential neofunctionalization events that occurred during diversification of the switchgrass diTPS family. The conversion of ent-CPP, ent-LPP, syn-CPP, and ent-neo-CT-CLPP by promiscuous diTPSs further yielded distinct labdane-type diterpene olefins and alcohols. Of these metabolites, the formation of 9ß-hydroxy-syn-pimar-15-ene and the expression of the corresponding genes were induced in roots and leaves in response to oxidative stress and ultraviolet irradiation, indicating their possible roles in abiotic stress adaptation. Together, these findings expand the known chemical space of diterpenoid metabolism in monocot crops toward systematically investigating and ultimately improving stress resilience traits in crop species.

Discovery, Biosynthesis and Stress-Related Accumulation of Dolabradiene-Derived Defenses in Maize.

Terpenoids are a major component of maize (Zea mays) chemical defenses that mediate responses to herbivores, pathogens, and other environmental challenges. Here, we describe the biosynthesis and elicited production of a class of maize diterpenoids, named dolabralexins. Dolabralexin biosynthesis involves the sequential activity of two diterpene synthases, ENT-COPALYL DIPHOSPHATE SYNTHASE (ZmAN2) and KAURENE SYNTHASE-LIKE4 (ZmKSL4). Together, ZmAN2 and ZmKSL4 form the diterpene hydrocarbon dolabradiene. In addition, we biochemically characterized a cytochrome P450 monooxygenase, ZmCYP71Z16, which catalyzes the oxygenation of dolabradiene to yield the epoxides 15,16-epoxydolabrene (epoxydolabrene) and 3ß-hydroxy-15,16-epoxydolabrene (epoxydolabranol). The absence of dolabradiene and epoxydolabranol in Zman2 mutants under elicited conditions confirmed the in vivo biosynthetic requirement of ZmAN2. Combined mass spectrometry and NMR experiments demonstrated that much of the epoxydolabranol is further converted into 3ß,15,16-trihydroxydolabrene (trihydroxydolabrene). Metabolite profiling of field-grown maize root tissues indicated that dolabralexin biosynthesis is widespread across common maize cultivars, with trihydroxydolabrene as the predominant diterpenoid. Oxidative stress induced dolabralexin accumulation and transcript expression of ZmAN2 and ZmKSL4 in root tissues, and metabolite and transcript accumulation were up-regulated in response to elicitation with the fungal pathogens Fusarium verticillioides and Fusarium graminearum Consistently, epoxydolabranol significantly inhibited the growth of both pathogens in vitro at 10 µg mL-1, while trihydroxydolabrene-mediated inhibition was specific to Fverticillioides These findings suggest that dolabralexins have defense-related roles in maize stress interactions and expand the known chemical space of diterpenoid defenses as genetic targets for understanding and ultimately improving maize resilience.

Synthesis of Benzodihydrofurans by Asymmetric C-H Insertion Reactions of Donor/Donor Rhodium Carbenes.

Metal carbenes appended with two electron-donating groups, known as "donor/donor" carbenes, undergo diastereo- and enantioselective rhodium-catalyzed C-H insertion reactions with ether substrates to form benzodihydrofurans. Unlike the reactions of metal carbenes with electron-withdrawing groups attached, the attenuated electrophilicity enables these reactions to be conducted in Lewis basic solvents (e.g., acetonitrile) and in the presence of water. The diazo precursors for these species are prepared in situ from hydrazone using a mild and chemoselective oxidant (MnO2 ). Although this sequence often can be performed in one-pot, control experiments have elucidated why a "two-pot" process is often more efficient. A thorough screening of achiral catalysts demonstrated that sterically encumbered catalysts are optimal for diastereoselective reactions. Although efficient insertion into allylic and propargylic C-H bonds is observed, competing dipolar cycloaddition processes are noted for some substrates. The full substrate scope of this useful method of benzodihydrofuran synthesis, mechanisms of side reactions, and computational support for the origins of stereoselectivity are described.

Biosynthesis of the oxygenated diterpene nezukol in the medicinal plant Isodon rubescens is catalyzed by a pair of diterpene synthases.

Plants produce an immense diversity of natural products (i.e. secondary or specialized metabolites) that offer a rich source of known and potentially new pharmaceuticals and other desirable bioproducts. The Traditional Chinese Medicinal plant Isodon rubescens (Lamiaceae) contains an array of bioactive labdane-related diterpenoid natural products. Of these, the ent-kauranoid oridonin is the most prominent specialized metabolite that has been extensively studied for its potent antimicrobial and anticancer efficacy. Mining of a previously established transcriptome of I. rubescens leaf tissue identified seven diterpene synthase (diTPSs) candidates. Here we report the functional characterization of four I. rubescens diTPSs. IrTPS5 and IrTPS3 were identified as an ent-copalyl diphosphate (CPP) synthase and a (+)-CPP synthase, respectively. Distinct transcript abundance of IrTPS5 and the predicted ent-CPP synthase IrTPS1 suggested a role of IrTPS5 in specialized ent-kaurene metabolism possibly en route to oridonin. Nicotiana benthamiana co-expression assays demonstrated that IrTPS4 functions sequentially with IrTPS3 to form miltiradiene. In addition, IrTPS2 converted the IrTPS3 product (+)-CPP into the hydroxylated tricyclic diterpene nezukol not previously identified in I. rubescens. Metabolite profiling verified the presence of nezukol in I. rubescens leaf tissue. The proposed IrTPS2-catalyzed reaction mechanism proceeds via the common ionization of the diphosphate group of (+)-CPP, followed by formation of an intermediary pimar-15-en-8-yl+ carbocation and neutralization of the carbocation by water capture at C-8 to yield nezukol, as confirmed by nuclear magnetic resonance (NMR) analysis. Oxygenation activity is rare for the family of class I diTPSs and offers new catalysts for developing metabolic engineering platforms to produce a broader spectrum of bioactive diterpenoid natural products.

Mechanically induced pyrogallol[4]arene hexamer assembly in the solid state extends the scope of molecular encapsulation.

Pyrogallol[4]arene hexamers are hydrogen-bonded molecular capsules of exceptional kinetic stability that can entrap small molecule guests indefinitely, without exchange, at ambient temperatures. Here, we report on the use of a ball mill to induce self-assembly of the capsule components and the guests in the solid state. Stoichiometric amounts of pyrogallol[4]arene and a guest, which can be an arene, alkane, amine, or carboxylic acid, were milled at 30 Hz for fixed durations, dissolved, and characterization by NMR. Most of the resulting encapsulation complexes were kinetically stable but thermodynamically unstable in solution, and the yield of their formation correlates with the duration of the milling and is related to the structures of guest and host. This method extends the scope of molecular encapsulation, as demonstrated by the preparation of kinetically trapped encapsulation complexes of [2.2]paracyclophane, for which we could find no other method of preparation. To gain mechanistic insights into the solid-state assembly process, we characterized the milled powders using 13C CP-MAS NMR, we studied the effects of changing the alkane domain of the host, and we examined how dissolution conditions impact on the distribution of observed encapsulation complexes once in solution. The results support a mechanism comprising mechanically induced solid-state reorganization to produce a mixture rich in nearly or fully assembled guest-filled capsules.

Substitution of Two Active-Site Residues Alters C9-Hydroxylation in a Class†II Diterpene Synthase.

Diterpenes form a vast and diverse class of natural products of both ecological and economic importance. Class II diterpene synthase (diTPS) enzymes control the committed biosynthetic reactions underlying diterpene chemical diversity. Homology modelling with site-directed mutagenesis identified two active-site residues in the horehound (Marrubium vulgare) class II diTPS peregrinol diphosphate synthase (MvCPS1); residue substitutions abolished the unique MvCPS1-catalysed water-capture reaction at C9 and redirected enzyme activity toward formation of an alternative product, halima-5(10),13-dienyl diphosphate. These findings contributed new insight into the steric interactions that govern diTPS-catalysed regiospecific oxygenation reactions and highlight the feasibility of diTPS engineering to provide a broader spectrum of bioactive diterpene natural products.

Surface and Wetting Properties of Embiopteran (Webspinner) Nanofiber Silk.

Insects of the order Embioptera, known as embiopterans, embiids, or webspinners, weave silk fibers together into sheets to make shelters called galleries. In this study, we show that silk galleries produced by the embiopteran Antipaluria urichi exhibit a highly hydrophobic wetting state with high water adhesion macroscopically equivalent to the rose petal effect. Specifically, the silk sheets have advancing contact angles above 150°, but receding contact angle approaching 0°. The silk sheets consist of layered fiber bundles with single strands spaced by microscale gaps. Scanning and transmission electron microscopy (SEM, TEM) images of silk treated with organic solvent and gas chromatography mass spectrometry (GC-MS) of the organic extract support the presence of a lipid outer layer on the silk fibers. We use cryogenic SEM to demonstrate that water drops reside on only the first layer of the silk fibers. The area fraction of this sparse outer silk layers is 0.1 to 0.3, which according to the Cassie-Baxter equation yields an effective static contact angle of ∼130° even for a mildly hydrophobic lipid coating. Using high magnification optical imaging of the three phase contact line of a water droplet receding from the silk sheet, we show that the high adhesion of the drop stems from water pinning along bundles of multiple silk fibers. The bundles likely form when the drop contact line is pinned on individual fibers and pulls them together as it recedes. The dynamic reorganization of the silk sheets during the droplet movement leads to formation of "super-pinning sites" that give embiopteran silk one of the strongest adhesions to water of any natural hydrophobic surface.

Structural characterization of nanofiber silk produced by embiopterans (webspinners).

Embiopterans produce silken galleries and sheets using exceptionally fine silk fibers in which they live and breed. In this study, we use electron microscopy (EM), Fourier-transform infrared (FT-IR) spectroscopy, wide angle X-ray diffraction (WAXD) and solid-state nuclear magnetic resonance (ssNMR) techniques to elucidate the molecular level protein structure of webspinner (embiid) silks. Silks from two species Antipaluria urichi and Aposthonia ceylonica are studied in this work. Electron microscopy images show that the fibers are about 90-100 nm in diameter, making webspinner silks among the finest of all known animal silks. Structural studies reveal that the silk protein core is dominated by ß-sheet structures, and that the protein core is coated with a hydrophobic alkane-rich surface coating. FTIR spectra of native embiid silk shows characteristic alkane CH2 stretchings near 2800-2900 cm-1, which decrease approximately 50% after washing the silk with 2 : 1 CHCl3 : MeOH. Furthermore, 13C ssNMR data shows a significant CH2 resonance that is strongly affected by the presence of water, supporting the idea that the silk fibers are coated with a hydrocarbon-rich layer. Such a layer is likely used to protect the colonies from rain. FTIR data also suggests that embiid silks are dominated by ß-sheet secondary structures similar to spider and silkworm silk fibers. NMR data confirms the presence of ß-sheet nanostructures dominated by serine-rich repetitive regions. A deconvolution of the serine Cß NMR resonance reveals that approximately 70% of all seryl residues exist in a ß-sheet structure. This is consistent with WAXD results that suggest webspinner silks are 70% crystalline, which is the highest crystalline fraction reported for any animal silks. The work presented here provides a molecular level structural picture of silk fibers produced by webspinners.

Mechanical and physical properties of recombinant spider silk films using organic and aqueous solvents.

Spider silk has exceptional mechanical and biocompatibility properties. The goal of this study was optimization of the mechanical properties of synthetic spider silk thin films made from synthetic forms of MaSp1 and MaSp2, which compose the dragline silk of Nephila clavipes. We increased the mechanical stress of MaSp1 and 2 films solubilized in both HFIP and water by adding glutaraldehyde and then stretching them in an alcohol based stretch bath. This resulted in stresses as high as 206 MPa and elongations up to 35%, which is 4× higher than the as-poured controls. Films were analyzed using NMR, XRD, and Raman, which showed that the secondary structure after solubilization and film formation in as-poured films is mainly a helical conformation. After the post-pour stretch in a methanol/water bath, the MaSp proteins in both the HFIP and water-based films formed aligned ß-sheets similar to those in spider silk fibers.

Reversible assembly of ß-sheet nanocrystals within caddisfly silk.

Nuclear magnetic resonance (NMR) and X-ray diffraction (XRD) experiments reveal the structural importance of divalent cation-phosphate complexes in the formation of ß-sheet nanocrystals from phosphorylated serine-rich regions within aquatic silk from caddisfly larvae of the species Hesperophyla consimilis. Wide angle XRD data on native caddisfly silk show that the silk contains a significant crystalline component with a repetitive orthorhombic unit cell aligned along the fiber axis with dimensions of 5.9 Å × 23.2 Å × 17.3 Å. These nanocrystalline domains depend on multivalent cations, which can be removed through chelation with ethylenediaminetetraacetic acid (EDTA). A comparison of wide angle X-ray diffraction data before and after EDTA treatment reveals that the integrated peak area of reflections corresponding to the nanocrystalline regions decreases by 15-25% while that of the amorphous background reflections increases by 20%, indicating a partial loss of crystallinity. (31)P solid-state NMR data on native caddisfly silk also show that the phosphorylated serine-rich motifs transform from a rigid environment to one that is highly mobile and water-solvated after treatment with EDTA. The removal of divalent cations through exchange and chelation has therefore caused a collapse of the ß-sheet structure. However, NMR results show that the rigid phosphorus environment is mostly recovered after the silk is re-treated with calcium. The (31)P spin-lattice (T1) relaxation times were measured at 7.6 ± 3.1 and 1 ± 0.5 s for this calcium-recovered sample and the native silk sample, respectively. The shorter (31)P T1 relaxation times measured for the native silk sample are attributed to the presence of paramagnetic iron that is stripped away during EDTA chelation treatment and replaced with diamagnetic calcium.

ß-Sheet nanocrystalline domains formed from phosphorylated serine-rich motifs in caddisfly larval silk: a solid state NMR and XRD study.

Adhesive silks spun by aquatic caddisfly (order Trichoptera) larvae are used to build both intricate protective shelters and food harvesting nets underwater. In this study, we use (13)C and (31)P solid-state NMR and wide angle X-ray diffraction (WAXD) as tools to elucidate molecular protein structure of caddisfly larval silk from the species Hesperophylax consimilis . Caddisfly larval silk is a fibroin protein based biopolymer containing mostly repetitive amino acid motifs. NMR and X-ray results provide strong supporting evidence for a structural model in which phosphorylated serine repeats (pSX)4 complex with divalent cations Ca(2+) and Mg(2+) to form rigid nanocrystalline ß-sheet structures in caddisfly silk. (13)C NMR data suggests that both phosphorylated serine and neighboring valine residues exist in a ß-sheet conformation while glycine and leucine residues common in GGX repeats likely reside in random coil conformations. Additionally, (31)P chemical shift anisotropy (CSA) analysis indicates that the phosphates on phosphoserine residues are doubly ionized, and are charge-stabilized by divalent cations. Positively charged arginine side chains also likely play a role in charge stabilization. Finally, WAXD results finds that the silk is at least 7-8% crystalline, with ß-sheet interplane spacings of 3.7 and 4.5 Å.