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ABSTRACT: Silks are semi-crystalline solids in which protein chains are associated by intermolecular hydrogen bonding within ordered crystallites, and by entanglement within unordered regions. By varying the type of protein secondary structure within crystallites and the overall degree of molecular order within fibers, arthropods produce fibers with a variety of physical properties suited to many purposes. We characterized silk produced as a tactile stimulus during mating by the grey silverfish (Ctenolepisma longicaudata) using Fourier transform infrared spectroscopy, polarized Raman spectroscopy, gel electrophoresis and amino acid analysis. Fibers were proteinaceous-the main component being a 220 kDa protein-and were rich in Gln/Glu, Leu, and Lys. The protein structure present was predominantly random coil, with a lesser amount of beta-structure. Silk fibers could readily be solubilized in aqueous solutions of a mild chaotrope, sodium dodecyl sulfate, indicating protein chains were not cross-linked by disulfide or other covalent bonds. We conclude that entanglement is the major mechanism by which these silk proteins cohere into a solid material. We propose silks used as short-term tactile cues are subject to less stringent requirements for molecular order relative to other silks, allowing the random coil structure to be favored as an adaptation promoting maximal entanglement and adhesion.
Insect biochemistry and molecular biology 04/2013; · 3.25 Impact Factor
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ABSTRACT: While there is growing interest in producing biomaterials containing coiled coils, relatively few studies have made use of naturally occurring fibrous proteins. In this study, we have characterized fibrous proteins used by mother praying mantises to produce an extensive covering for their eggs called an ootheca, and demonstrate production of artificial ootheca using recombinantly produced proteins. Examination of natural oothecae by infrared spectroscopy and solid-state nuclear magnetic resonance revealed the material to consist of proteins organized predominately as coiled coils. Two structural proteins, Mantis Fibroin 1 and Mantis Fibroin 2, were identified in ootheca from each of three species. Between species, the primary sequences of both proteins had diverged considerably but other features were tightly conserved-including low molecular weight, high abundance of Ala, Glu, Lys and Ser, and a triblock-like architecture with extensive central coiled coil domain. Mantis fibroin hydrophobic cores had an unusual composition containing high levels of alanine and aromatic residues. Recombinantly-produced mantis fibroins folded into coiled coils in solution and could be fabricated into solid materials with high coiled coil content. The structural features of mantis fibroins and their straightforward recombinant production make them promising templates for the production of coiled coil biomimetics materials.
Biomacromolecules 11/2012; · 5.48 Impact Factor
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ABSTRACT: Honeybee larvae produce silken cocoons that provide mechanical stability to the hive. The silk proteins are small and non-repetitive and therefore can be produced at large scale by fermentation in E. coli. The recombinant proteins can be fabricated into a range of forms; however the resultant material is soluble in water and requires a post production stabilizing treatment. In this study, we describe the structural and mechanical properties of sponges fabricated from artificial honeybee silk proteins that have been stabilized in aqueous methanol baths or by dry heating. Aqueous methanol treatment induces formation of ß-sheets, with the amount of ß-sheet dictated by methanol concentration. Formation of ß-sheets renders sponges insoluble in water and generates a reversibly compressible material. Dry heat treatments at 190°C produce a water insoluble material, that is stiffer than the methanol treated equivalent but without significant secondary structural changes. Honeybee silk proteins are particularly high in Lys, Ser, Thr, Glu and Asp. The properties of the heat treated material are attributed to generation of lysinoalanine, amide (isopeptide) and/or ester covalent cross-links. The unique ability to stabilize material by controlling secondary structure rearrangement and covalent cross-linking allows us to design recombinant silk materials with a wide range of properties.
PLoS ONE 01/2012; 7(12):e52308. · 4.09 Impact Factor
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ABSTRACT: Raspy crickets (Orthoptera: Gryllacrididae) are unique among the orthopterans in producing silk, which is used to build shelters. This work studied the material composition and the fabrication of cricket silk for the first time. We examined silk-webs produced in captivity, which comprised cylindrical fibers and flat films. Spectra obtained from micro-Raman experiments indicated that the silk is composed of protein, primarily in a beta-sheet conformation, and that fibers and films are almost identical in terms of amino acid composition and secondary structure. The primary sequences of four silk proteins were identified through a mass spectrometry/cDNA library approach. The most abundant silk protein was large in size (300 and 220 kDa variants), rich in alanine, glycine and serine, and contained repetitive sequence motifs; these are features which are shared with several known beta-sheet forming silk proteins. Convergent evolution at the molecular level contrasts with development by crickets of a novel mechanism for silk fabrication. After secretion of cricket silk proteins by the labial glands they are fabricated into mature silk by the labium-hypopharynx, which is modified to allow the controlled formation of either fibers or films. Protein folding into beta-sheet structure during silk fabrication is not driven by shear forces, as is reported for other silks.
PLoS ONE 01/2012; 7(2):e30408. · 4.09 Impact Factor
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ABSTRACT: Extraction of genome sequences from metagenomic data is crucial for reconstructing the metabolism of microbial communities that cannot be mimicked in the laboratory. A complete Methanococcus maripaludis genome was generated from metagenomic data derived from a thermophilic subsurface oil reservoir. M. maripaludis is a hydrogenotrophic methanogenic species that is common in mesophilic saline environments. Comparison of the genome from the thermophilic, subsurface environment with the genome of the type species will provide insight into the adaptation of a methanogenic genome to an oil reservoir environment.
Journal of bacteriology 10/2011; 193(19):5595. · 3.94 Impact Factor
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ABSTRACT: In this article, we review current knowledge about the silk produced by the larvae of bees, ants, and hornets [Apoidea and Vespoidea: Hymenoptera]. Different species use the silk either alone or in composites for a variety of purposes including mechanical reinforcement, thermal regulation, or humidification. The characteristic molecular structure of this silk is α-helical proteins assembled into tetrameric coiled coils. Gene sequences from seven species are available, and each species possesses a copy of each of four related silk genes that encode proteins predicted to form coiled coils. The proteins are ordered at multiple length scales within the labial gland of the final larval instar before spinning. The insects control the morphology of the silk during spinning to produce either fibers or sheets. The silk proteins are small and non repetitive and have been produced artificially at high levels by fermentation in E. coli. The artificial silk proteins can be fabricated into materials with structural and mechanical properties similar to those of native silks.
Biopolymers 08/2011; 97(6):446-54. · 2.87 Impact Factor
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ABSTRACT: Honeybees produce silken cocoons containing four related fibrous proteins. High levels of each of the honeybee silk proteins can be produced recombinantly by fermentation in Escherichia coli. In this study we have used electrospinning to fabricate a single recombinant honeybee silk protein, AmelF3, into nanofibers of around 200 nm diameter. Infrared spectroscopy found that the molecular structure of the nanofibers was predominantly coiled coil, essentially the same as native honeybee silk. Mats of the honeybee nanofibers were treated with methanol or by water annealing, which increased their β-sheet content and rendered them water insensitive. The insoluble mats were degraded by protease on a time scale of hours to days. The protease gradually released proteins from the solid state and these were subsequently rapidly degraded into small peptides without the accumulation of partial degradation products. Cell culture assays demonstrated that the mats allowed survival, attachment and proliferation of fibroblasts. These results indicate that honeybee silk proteins meet many prerequisites for use as a biomaterial.
Acta biomaterialia 06/2011; 7(10):3789-3795. · 3.98 Impact Factor
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ABSTRACT: Honeybee silk is composed of four fibrous proteins that, unlike other silks, are readily synthesized at full-length and high yield. The four silk genes have been conserved for over 150 million years in all investigated bee, ant and hornet species, implying a distinct functional role for each protein. However, the amino acid composition and molecular architecture of the proteins are similar, suggesting functional redundancy. In this study we compare materials generated from a single honeybee silk protein to materials containing all four recombinant proteins or to natural honeybee silk. We analyse solution conformation by dynamic light scattering and circular dichroism, solid state structure by Fourier Transform Infrared spectroscopy and Raman spectroscopy, and fiber tensile properties by stress-strain analysis. The results demonstrate that fibers artificially generated from a single recombinant silk protein can reproduce the structural and mechanical properties of the natural silk. The importance of the four protein complex found in natural silk may lie in biological silk storage or hierarchical self-assembly. The finding that the functional properties of the mature material can be achieved with a single protein greatly simplifies the route to production for artificial honeybee silk.
PLoS ONE 01/2011; 6(2):e16489. · 4.09 Impact Factor
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ABSTRACT: Onychophora are ancient, carnivorous soft-bodied invertebrates which capture their prey in slime that originates from dedicated glands located on either side of the head. While the biochemical composition of the slime is known, its unusual nature and the mechanism of ensnaring thread formation have remained elusive. We have examined gene expression in the slime gland from an Australian onychophoran, Euperipatoides rowelli, and matched expressed sequence tags to separated proteins from the slime. The analysis revealed three categories of protein present: unique high-molecular-weight proline-rich proteins, and smaller concentrations of lectins and small peptides, the latter two likely to act as protease inhibitors and antimicrobial agents. The predominant proline-rich proteins (200 kDa+) are composed of tandem repeated motifs and distinguished by an unusually high proline and charged residue content. Unlike the highly structured proteins such as silks used for prey capture by spiders and insects, these proteins lack ordered secondary structure over their entire length. We propose that on expulsion of slime from the gland onto prey, evaporative water loss triggers a glass transition change in the protein solution, resulting in adhesive and enmeshing thread formation, assisted by cross-linking of complementary charged and hydrophobic regions of the protein. Euperipatoides rowelli has developed an entirely new method of capturing prey by harnessing disordered proteins rather than structured, silk-like proteins.
Proceedings of the Royal Society B: Biological Sciences 11/2010; 277(1698):3255-63. · 5.41 Impact Factor
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ABSTRACT: The cuticle of a Mycterophallus cetoniine scarab species displays both red iridescence due to a multilayer reflector mechanism and rainbow iridescence due to a superimposed diffraction grating mechanism. This is the first reported example of an animal possessing two independent classes of structural colors arising from interference at the wavelengths of visible light. In this work, the Mycterophallus cuticle is characterized by light microscopy, spectrophotometry, scanning electron microscopy, and transmission electron microscopy. We compare the cuticle of the Mycterophallus species to two closely related Lomaptera scarab species, one with only a multilayer reflector and the second with only a diffraction grating. We calculate the correspondence between the nanostructural parameters and the optical properties of the Mycterophallus cuticle to determine the relative optical contributions of the two color mechanisms and the interactions between them.
Journal of Morphology 11/2010; 271(11):1300-5. · 1.54 Impact Factor
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ABSTRACT: Transgenic production of silkworm and spider silks as biomaterials has posed intrinsic problems due to the large size and repetitive nature of the silk proteins. In contrast the silk of honeybees (Apis mellifera) is composed of a family of four small and non-repetitive fibrous proteins. We report recombinant production and purification of the four full-length unmodified honeybee silk proteins in Escherichia coli at substantial yields of 0.2-2.5 g/L. Under the correct conditions the recombinant proteins self-assembled to reproduce the native coiled coil structure. Using a simple biomimetic spinning system we could fabricate recombinant silk fibers that replicated the tensile strength of the native material.
Biomaterials 03/2010; 31(9):2695-700. · 7.40 Impact Factor
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ABSTRACT: Silks play a crucial role in the survival and reproduction of many insects. Labial glands, Malpighian tubules, and a variety of dermal glands have evolved to produce these silks. The glands synthesize silk proteins, which become semicrystalline when formed into fibers. Although each silk contains one dominant crystalline structure, the range of molecular structures that can form silk fibers is greater than any other structural protein group. On the basis of silk gland type, silk protein molecular structure, and the phylogenetic relationship of silk-producing species, we grouped insect silks into 23 distinct categories, each likely to represent an independent evolutionary event. Despite having diverse functions and fundamentally different protein structures, these silks typically have high levels of protein crystallinity and similar amino acid compositions. The substantial crystalline content confers extraordinary mechanical properties and stability to silk and appears to be required for production of fine protein fibers.
Annual Review of Entomology 10/2009; 55:171-88. · 11.45 Impact Factor
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ABSTRACT: In silico substrate docking of both stereoisomers of the pesticide chlorfenvinphos (CVP) in the phosphotriesterase from Agrobacterium radiobacter identified two residues (F131 and W132) that prevent productive substrate binding and cause stereospecificity. A variant (W131H/F132A) was designed that exhibited ca. 480-fold and 8-fold increases in the rate of Z-CVP and E-CVP hydrolysis, respectively, eliminating stereospecificity.
Applied and environmental microbiology 07/2009; 75(15):5153-6. · 3.69 Impact Factor
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ABSTRACT: The atrazine chlorohydrolase AtzA has evolved within the past 50 years to catalyze the hydrolytic dechlorination of the herbicide atrazine. It is of wide research interest for two reasons: first, catalytic improvement of the enzyme would facilitate its application in bioremediation, and second, because of its recent evolution, it presents a rare opportunity to examine the early stages in the acquisition of new catalytic activities. Using a structural model of the AtzA-atrazine complex, a region of the substrate-binding pocket was targeted for combinatorial randomization. Identification of improved variants through this process informed the construction of a variant AtzA enzyme with 20-fold improvement in its k(cat)/K(m) value compared with that of the wild-type enzyme. The reduction in K(m) observed in the AtzA variants has allowed the full kinetic profile for the AtzA-catalyzed dechlorination of atrazine to be determined for the first time, revealing the hitherto-unreported substrate cooperativity in AtzA. Since substrate cooperativity is common among deaminases, which are the closest structural homologs of AtzA, it is possible that this phenomenon is a remnant of the catalytic activity of the evolutionary progenitor of AtzA. A catalytic mechanism that suggests a plausible mechanistic route for the evolution of dechlorinase activity in AtzA from an ancestral deaminase is proposed.
Applied and environmental microbiology 03/2009; 75(7):2184-91. · 3.69 Impact Factor
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ABSTRACT: Aposthonia gurneyi, an Australian webspinner species, is a primitive insect that constructs and lives in a silken tunnel which screens it from the attentions of predators. The insect spins silk threads from many tiny spines on its forelegs to weave a filmy sheet. We found that the webspinner silk fibers have a mean diameter of only 65 nm, an order of magnitude smaller than any previously reported insect silk. The purpose of such fine silk may be to reduce the metabolic cost of building the extensive tunnels. At the molecular level, the A. gurneyi silk has a predominantly beta-sheet protein structure. The most abundant clone in a cDNA library produced from the webspinner silk glands encoded a protein with extensive glycine-serine repeat regions. The GSGSGS repeat motif of the A. gurneyi silk protein is similar to the well-known GAGAGS repeat motif found in the heavy fibroin of silkworm silk, which also has beta-sheet structure. As the webspinner silk gene is unrelated to the silk gene of the phylogenetically distant silkworm, this is a striking example of convergent evolution.
International Journal of Biological Macromolecules 07/2008; 43(3):271-5. · 2.45 Impact Factor
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ABSTRACT: Organophosphorus (OP) pesticides poison more than 3,000,000 people every year in the developing world, mostly through intentional self-poisoning. Advances in medical therapy for OP poisoning have lagged, and current treatment is not highly effective with mortality of up to 40% in even the most advanced Western medical facilities. Administration of a broadly active bacterial OP hydrolase to patients in order to hydrolyze OPs in circulation might allow current therapies to be more effective. The objective of this work was to evaluate the efficacy of a new recombinant bacterial OP hydrolase (OpdA), cloned from Agrobacterium radiobacter, in rat models of two chemically distinct but highly toxic and rapidly acting OP pesticides: dichlorvos and parathion. Without OpdA treatment, median time to death in rats poisoned with 3x LD(50) of dichlorvos or parathion was 6 min and 25.5 min, respectively. Administration of a single dose of OpdA immediately after dichlorvos resulted in 100% survival at 24h, with no additional antidotal therapy. After parathion poisoning, OpdA alone caused only a delay to death. However, an additional two doses of OpdA resulted in 62.5% survival at 24 h after parathion poisoning. In combination with pralidoxime therapy, a single dose of OpdA increased survival to 75% after parathion poisoning. Our results demonstrate that OpdA is able to improve survival after poisoning by two chemically distinct and highly toxic OP pesticides.
Toxicology 06/2008; 247(2-3):88-92. · 3.68 Impact Factor
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ABSTRACT: Silks are strong protein fibers produced by a broad array of spiders and insects. The vast majority of known silks are large, repetitive proteins assembled into extended beta-sheet structures. Honeybees, however, have found a radically different evolutionary solution to the need for a building material. The 4 fibrous proteins of honeybee silk are small ( approximately 30 kDa each) and nonrepetitive and adopt a coiled coil structure. We examined silks from the 3 superfamilies of the Aculeata (Hymenoptera: Apocrita) by infrared spectroscopy and found coiled coil structure in bees (Apoidea) and in ants (Vespoidea) but not in parasitic wasps of the Chrysidoidea. We subsequently identified and sequenced the silk genes of bumblebees, bulldog ants, and weaver ants and compared these with honeybee silk genes. Each species produced orthologues of the 4 small fibroin proteins identified in honeybee silk. Each fibroin contained a continuous predicted coiled coil region of around 210 residues, flanked by 23-160 residue length N- and C-termini. The cores of the coiled coils were unusually rich in alanine. There was extensive sequence divergence among the bee and ant silk genes (<50% similarity between the alignable regions of bee and ant sequences), consistent with constant and equivalent divergence since the bee/ant split (estimated to be 155 Myr). Despite a high background level of sequence diversity, we have identified conserved design elements that we propose are essential to the assembly and function of coiled coil silks.
Molecular Biology and Evolution 11/2007; 24(11):2424-32. · 5.55 Impact Factor
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ABSTRACT: Male hilarine flies (Diptera: Empididae: Empidinae) present prospective mates with silk-wrapped gifts. The silk is produced by specialised cells located in the foreleg basitarsus of the fly. In this report, we describe 2.3 kbp of the silk gene from a hilarine fly (Hilara spp.) that was identified from highly expressed mRNA extracted from the prothoracic basitarsus of males. Using specific primers, we found that the silk gene is expressed in the basitarsi and not in any other part of the male fly. The silk gene from the basitarsi cDNA library matched an approximately 220 kDa protein from the silk-producing basitarsus. Although the predicted silk protein sequence was unlike any other protein sequence in available databases, the architecture and composition of the predicted protein had features in common with previously described silks. The convergent evolution of these features in the Hilarini silk and other silks emphasises their importance in the functional requirements of silk proteins.
Insect Biochemistry and Molecular Biology 11/2007; 37(10):1036-43. · 3.25 Impact Factor
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ABSTRACT: Previously we identified juvenile hormone esterase (JHE) from Drosophila melanogaster by the criteria that it showed both appropriate developmental expression and kinetics for juvenile hormone (JH). We also noted three further esterases of D. melanogaster with some JHE-like characteristics, such as a GQSAG active site motif, a particular amphipathic helix, or close phylogenetic relationship with other JHEs. In this study, these JHE-like enzymes were expressed in vitro and their kinetic parameters compared with those of the previously identified JHE. Despite considerable phylogenetic distance between some of the esterases, they could all hydrolyse racemic JHIII. However, only the previously identified JHE had kinetic parameters (K(M) and k(cat)) towards various forms of JH (racemic or individual isomers of JHIII, JHII, JHI, and methyl farnesoate) consistent with a physiological role in JH regulation. Furthermore, only this JHE showed a preference for artificial substrates with acyl chain lengths similar to that of JH. This suggests that there is probably only one physiologically functional JHE in D. melanogaster but multiple esterases with JH esterase activity. Genomic comparisons of the selective JHE across 11 other Drosophila species showed a single orthologue in 10 of them but Drosophila willistoni has 16 full-length copies, five of them with the GQSAG motif and amphipathic helix.
Insect Biochemistry and Molecular Biology 07/2007; 37(6):540-9. · 3.25 Impact Factor
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ABSTRACT: Sulfur is essential for life on Earth, but its availability is limited in many environments. Here the sulfur-starvation response of the model soil bacterium Pseudomonas putida KT2440 is shown to be associated with an approximately fivefold reduction in the total soluble thiol content of the cell. A bioinformatic survey of the P. putida KT2440 genome identified 646 genes encoding proteins with a significantly lower than average sulfur content (low sulfur-content proteins, LSPs), the expression of which may have a role in the global reduction of cellular thiol content during sulfur starvation. Analysis of the genetic organization of the LSP-encoding genes showed that 31% were potentially transcriptionally associated with at least one other gene encoding a protein defined as an LSP. In particular, 55 LSP genes were located in three large clusters, termed low-sulfur islands (LSIs) here. The predicted identities of the proteins encoded by the LSIs strongly suggest that the LSIs have a role in acquiring sulfur from organic sulfur sources during sulfur starvation. This hypothesis was supported by transcription fusion studies on a limited number of LSP promoters under low-sulfur conditions. In a wider survey of bacterial species, LSIs were found to be more prevalent in free-living, Gram-negative bacteria than in Gram-positive or obligately intracellular bacteria.
FEMS Microbiology Letters 03/2007; 267(2):184-93. · 2.04 Impact Factor
