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PubMed1979

Juvenile hormone modifies larvae and silk gland development in Bombyx mori.

Topical application of a Juvenile Hormone Analogue (ZR 515) during the first half of the first half of the last (fifth) larval instar of Bombyx mori induces a prolongation of this instar and increases larvae weight and silk secretion. Later treatment can lead to an extra larval molt. Moreover, the hormone analogue stops either the development or the secretory activity of the silk gland, depending on the period of treatments. When Juvenile Hormone is administered at the beginning of the fifth instar, silk gland growth as well as RNA and protein syntheses are first inhibited and later resumed and amplified. Finaly silk production increases up to 150 per cent of controls. One of the primarly effects of treatments seems to be RNA synthesis. Thus Juvenile Hormone analogue application could be a new tool for studying RNA transcription and/or processing.

Animals↗
PubMed2002

Fork head alternative binding drives stage-specific gene expression in the silk gland of Bombyx mori.

Here, we identified the main transactivator of fhx, the gene encoding the silk protein fibrohexamerin in posterior silk gland cells (PSG), as the homeotic SGF1/fork head factor. The same factor also stimulates sericin-1, another silk protein encoding gene, in the middle silk gland cells. SGF1/fork head is present in the silk gland nuclei during the whole course of larval life, but its binding to the fhx promoter occurs at intermolt and not during molt, when fhx is respectively turned on and off. The alternative binding of the factor is associated with specific changes in the fhx chromatin topology in PSG cells. Taken together, our results show that stabilization of SGF1/fork head to its target sequence is critical to promote fhx transcription at each intermolt. We also found that fhx is characterized by a PSG-specific DNase I hypersensitive site in the first intron, present during molt and intermolt, i.e. independent of the transcriptional status of the gene. All these data suggest that differential chromatin accessibility and fork head activation are crucial in controlling the spatial and temporal regulation of the fhx gene in the posterior silk gland cells.

Animals↗
PubMed1998

Evolution of repetitive proteins: spider silks from Nephila clavipes (Tetragnathidae) and Araneus bicentenarius (Araneidae).

Spider silks are highly repetitive proteins, characterized by regions of polyalanine and glycine-rich repeating units. We have obtained two variants of the Spidroin 1 (NCF-1) silk gene sequence from Nephila clavipes. One sequence (1726 bp) was from a cloned cDNA, and the other (1951 bp) was from PCR of genomic DNA. When these sequences are compared with each other and the previously published Spidroin 1 sequence, there are differences due to sequence rearrangements, as well as single base substitutions. These variations are similar to those that have been reported from other highly repetitive genes, and probably represent the results of unequal cross-overs. We have also obtained 708 bp of sequence from pCR of genomic DNA from Araneus biocentenarius. This sequence shows considerable similarity to a dragline sequence (ADF-3) from A. diadematus, as well as Spidroin 2 (NCF-2) from N. clavipes. Minor but consistent differences in the repeating unit sequence between A. bicentenarius and A. diadematus suggest that concerted evolution or gene conversion processes are acting to maintain similarity among repeat units within a single gene.

Amino Acid Sequence↗
PubMed2000

Lessons from nature--protein fibers.

Silks are protein fibers with remarkable mechanical properties. The discovery of the structural features that govern these properties is a challenge for biochemistry and structural biology. This review summarizes the results of the biochemistry of silk proteins as well as the knowledge of the molecular biology of the respective genes. In addition, an overview is presented on the efforts to produce recombinant silk proteins by biotechnological techniques.

Animals↗
PubMed2000

Controlling beta-sheet assembly in genetically engineered silk by enzymatic phosphorylation/dephosphorylation.

Enzymatic phosphorylation and dephosphorylation reactions were used to modify a genetically engineered variant of spider dragline silk. The approximately 25 kDa protein was phosphorylated with cyclic AMP-dependent kinase and dephosphorylated with calf intestinal alkaline phosphatase. Phosphorylation inhibited beta-sheet assembly of the protein and enhanced solubility to about 5 mg/mL in water, compared to about 20% of this level upon enzymatic dephosphorylation. The cyclability of the phosphorylation-dephosphorylation system was confirmed by MALDI with a model peptide. Kinetic studies conducted with [gamma-(32)P]ATP illustrate that the phosphorylation reaction proceeds over 6 h. Secondary structure of the phosphorylated and dephosphorylated proteins was determined by CD and FTIR. The results illustrate that an enzymatic phosphorylation event can be used to control the solution structure of a protein like silk, which has a tendency to prematurely precipitate due to the formation of beta-sheets.

Amino Acid Sequence↗
PubMed2002

Conformation transition in silk protein films monitored by time-resolved Fourier transform infrared spectroscopy: effect of potassium ions on Nephila spidroin films.

We used time-resolved Fourier transform infrared spectroscopy (FTIR) to follow a conformation transition in Nephila spidroin film from random coil and/or helical structures to beta-sheet induced by the addition of KCl from 0.01 to 1.0 mol/L in D(2)O. Time series difference spectra showed parallel increases in absorption at 1620 and 1691 cm(-)(1), indicating formation of beta-sheet, together with a coincident loss of intensity of approximately 1650 cm(-)(1), indicating decrease of random coil and/or helical structures. Increase in KCl concentration produced an increased rate of the conformation transition that may attributable to weakening of hydrogen bonds within spidroin macromolecules. The conformation transition was a biphasic process with [KCl] > or = 0.3 mol/L but monophasic with [KCl] < or = 0.1 mol/L. This suggests that, at high KCl concentrations, segments of the molecular chain are adjusted first and then the whole molecule undergoes rearrangement. We discuss the possible significance of these findings to an understanding of the way that spiders spin silk.

Animals↗
PubMed2004

Transition to a beta-sheet-rich structure in spidroin in vitro: the effects of pH and cations.

Unlike man-made fibers, the silks of spiders are spun from aqueous solutions and at atmospheric pressure in a process still poorly understood. The molecular mechanism of this process involves the conversion of a highly concentrated, predominantly disordered silk protein (spidroin) into beta-sheet-rich structures. To help store and transport the spidroins in solution, as well as probably control their conversion, a liquid crystalline arrangement is established in the storage region in the ampulla and persists into the duct. Although it has been suggested that changes in the concentration of hydrogen and metal ions play a role in the formation of the solid thread, there is no reported evidence that these ions influence the secondary structure of native spidroin in solution. Here, we demonstrate that pH values between approximately 3.5 and 4.5 induce a slow change of conformation from the disordered to the beta-sheet-rich form. We also report that Al(3+), K(+), and Na(+) ions induce similar changes in structure, while Ca(2+) and Mg(2+) stabilize the predominantly disorder state of the protein. Cs(+) and Li(+) have no apparent effect. Direct volumetric and spectrophotometric titration showed a pI of 4.22 +/- 0.33 and apparent pK values of 6.74 +/- 0.71 and 9.21 +/- 0.27, suggesting a mechanism for the effect of low pH on the protein and a rationale for the observed reduction in pH in the duct. We discuss the importance of these findings for the spinning process and the active role played by the spider to alter the kinetics of the transition.

Animals↗
PubMed2004

Primary structure elements of spider dragline silks and their contribution to protein solubility.

Spider silk proteins have mainly been investigated with regard to their contribution to mechanical properties of the silk thread. However, little is known about the molecular mechanisms of silk assembly. As a first step toward characterizing this process, we aimed to identify primary structure elements of the garden spider's (Araneus diadematus) major dragline silk proteins ADF-3 and ADF-4 that determine protein solubility. In addition, we investigated the influence of conditions involved in mediating natural thread assembly on protein aggregation. Genes encoding spider silk-like proteins were generated using a cloning strategy, which is based on a combination of synthetic DNA modules and PCR-amplified authentic gene sequences. Comparing secondary structure, solubility, and aggregation properties of the synthesized proteins revealed that single primary structure elements have diverse influences on protein characteristics. Repetitive regions representing the largest part of dragline silk proteins determined the solubility of the synthetic proteins, which differed greatly between constructs derived from ADF-3 and ADF-4. Factors, such as acidification and increases in phosphate concentration, which promote silk assembly in vivo generally decreased silk protein solubility in vitro. Strikingly, this effect was pronounced in engineered proteins comprising the carboxyl-terminal nonrepetitive regions of ADF-3 or ADF-4, indicating that these regions might play an important role in initiating assembly of spider silk proteins.

Amino Acid Motifs↗
PubMed2005

Molecular characterization and evolutionary study of spider tubuliform (eggcase) silk protein.

As a result of hundreds of millions of years of evolution, orb-web-weaving spiders have developed the use of seven different silks produced by different abdominal glands for various functions. Tubuliform silk (eggcase silk) is unique among these spider silks due to its high serine and very low glycine content. In addition, tubuliform silk is the only silk produced just during a short period of time, the reproductive season, in the spider's life. To understand the molecular characteristics of the proteins composing this silk, we constructed tubuliform-gland-specific cDNA libraries from three different spider families, Nephila clavipes, Argiope aurantia, and Araneus gemmoides. Sequencing of tubuliform silk cDNAs reveals the repetitive architecture of its coding sequence and novel amino acid motifs. The inferred protein, tubuliform spidroin 1 (TuSp1), contains highly homogenized repeats in all three spiders. Amino acid composition comparison of the predicted tubuliform silk protein sequence to tubuliform silk indicates that TuSp1 is the major component of tubuliform silk. Repeat unit alignment of TuSp1 among three spider species shows high sequence conservation among tubuliform silk protein orthologue groups. Sequence comparison among TuSp1 repetitive units within species suggests intragenic concerted evolution, presumably through gene conversion and unequal crossover events. Comparative analysis demonstrates that TuSp1 represents a new orthologue in the spider silk gene family.

Amino Acid Sequence↗
PubMedDate not supplied

Rheological characterization of nephila spidroin solution.

We report the results of an investigation into the rheology of solutions of natural spider silk dope (spinning solution). We demonstrate that dilute dope solutions showed only shear thinning as the shear rate increased while more concentrated solutions showed an initial shear thinning followed by a shear thickening and a subsequent decline in viscosity. The critical shear rate for shear thickening depended on dope concentration and was very low in concentrated solutions. This helps to explain how spiders are able to spin silk at very low draw rates and why they use a very concentrated dope solution. We also show that the optimum shear rate for shear thickening in moderately concentrated solutions occurred at pH 6.3 close to the observed pH at the distal end of the spider's spinning duct. Finally, we report that the addition of K(+) ions to dilute dope solutions produced a spontaneous formation of nanofibrils that subsequently aggregated and precipitated. This change was not seen after the addition of other common cations. Taken together, these observations support the hypothesis that the secretion of H(+) and K(+) by the spider's duct together with moderate strain rates produced during spinning induce a phase separation in the silk dope in which the silk protein (spidroin) molecules are converted into insoluble nanofibrils.

Animals↗
PubMedDate not supplied

Structural conformation of spidroin in solution: a synchrotron radiation circular dichroism study.

Spider silk is made and spun in a complex process that tightly controls the conversion from soluble protein to insoluble fiber. The mechanical properties of the silk fiber are modulated to suit the needs of the spider by various factors in the animal's spinning process. In the major ampullate (MA) gland, the silk proteins are secreted and stored in the lumen of the ampulla. A particular structural fold and functional activity is determined by the spidroins' amino acid sequences as well as the gland's environment. The transition from this liquid stage to the solid fiber is thought to involve the conversion of a predominantly unordered structure to a structure rich in beta-sheet as well as the extraction of water. Circular dichroism provides a quick and versatile method for examining the secondary structure of silk solutions and studying the effects of various conditions. Here we present the relatively novel technique of synchrotron radiation based circular dichroism as a tool for investigating biomolecular structures. Specifically we analyze, in a series of example studies on structural transitions induced in liquid silk, the type of information accessible from this technique and any artifacts that might arise in studying self-assembling systems.

Circular Dichroism↗
PubMedDate not supplied

Conserved C-termini of Spidroins are secreted by the major ampullate glands and retained in the silk thread.

The C-termini of Spidroins produced in the major and minor ampullate glands of spiders are highly conserved. Despite this conservation, no corresponding peptides have been identified in the spinning dopes or the silk filaments so far. To prove their presence or absence, polyclonal antibodies derived against fusion proteins containing the conserved C-terminal regions of both Spidroin 1 and 2 from the spider Nephila clavipes were generated. The antibodies reacted with high molecular weight polypeptides of the corresponding gland extracts and solubilized major ampullate filament and in addition to filament cross-sections. This demonstrates the existence of C-terminal specific peptides in the spinning dope and the mature Spidroins. Both the fusion proteins as well as the proteins contained within the gland lumen showed a reduction in their size under reducing conditions indicating the presence of disulfide bonds. Their high conservation and the biochemical data suggest crucial roles the C-termini play in the formation and/or structure of the corresponding silk filaments.

Animals↗
PubMedDate not supplied

Effects of electrospinning and solution casting protocols on the secondary structure of a genetically engineered dragline spider silk analogue investigated via Fourier transform Raman spectroscopy.

Micrometer and submicrometer diameter fibers of recombinant dragline spider silk analogues, synthesized via protein engineering strategies, have been electrospun from 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP) and compared with cast films via Raman spectroscopy in order to assess changes in protein conformation that may result from the electrospinning process. Although the solvent casting process was shown to result in predominantly beta-sheet conformation similar to that observed in the bulk, the electrospinning process causes a major change in conformation from beta-sheet to alpha-helix. A possible mechanism involving electric field-induced stabilization of alpha-helical segments in HFIP solution during the electrospinning process is discussed.

Amino Acid Sequence↗
PubMedDate not supplied

Further investigation on potassium-induced conformation transition of Nephila spidroin film with two-dimensional infrared correlation spectroscopy.

We used two-dimensional (2D) correlation infrared spectroscopy to study further the potassium-induced conformation transition in Nephila spidroin films. It provided increased resolution and important new information on the sequence of events in the conformation transition process, showing that beta-sheet formed from the helical component before they formed from random coil. It also showed more evidence that formation of the 1691 cm(-1) (turn/bend) peak did not proceed with the same kinetics as the 1620 cm(-1) (antiparallel beta-sheet component) one, so we attribute the 1691 cm(-1) peak to turns which formed with different kinetics as the antiparallel beta-sheets. We present a single coherent and detailed hypothesis for the assembly and secondary structural transition of silk proteins in vivo and in vitro based on our findings and on evidence from other laboratories.

Animals↗
PubMedDate not supplied

Acid extraction and purification of recombinant spider silk proteins.

A procedure has been developed for the isolation of recombinant spider silk proteins based upon their unique stability and solubilization characteristics. Three recombinant silk proteins, (SpI)7, NcDS, and [(SpI)4/(SpII)1]4, were purified by extraction with organic acids followed by affinity or ion exchange chromatography resulting in 90-95% pure silk solutions. The protein yield of NcDS (15 mg/L culture) and (SpI)7 (35 mg/L) increased 4- and 5-fold, respectively, from previously reported values presumably due to a more complete solubilization of the expressed recombinant protein. [(SpI)4/(SpII)1]4, a hybrid protein based on the repeat sequences of spidroin I and spidroin II, had a yield of 12.4 mg/L. This method is an effective, reproducible technique that has broad applicability for a variety of silk proteins as well as other acid stable biopolymers.

Animals↗
PubMedDate not supplied

An investigation of the divergence of major ampullate silk fibers from Nephila clavipes and Argiope aurantia.

The major ampullate fiber of both Nephila clavipes and Argiope aurantia is composed of two different proteins, MaSp1 and MaSp2. Each of these proteins has a highly conserved pattern of silk-associated amino acid motifs. The GPGXX motif is the only source of proline and is unique to MaSp2. On the basis of the percent of proline, Nephila clavipes major ampullate silk was calculated to consist of 19% MaSp2 and 81% MaSp1, while Argiope aurantia was calculated to have a significantly higher MaSp2 content of 59% with MaSp1 comprising the remaining 41%. To investigate the functional implications of the difference in protein composition, major ampullate silk fibers from Nephila clavipes and Argiope aurantia were mechanically tested and compared. Stress-strain curves produced from polynomial regression show that the two significant differences between major ampullate silk fibers from Nephila clavipes and Argiope aurantia are the average peak load stress and Young's modulus, with Argiope higher for both.

Amino Acid Motifs↗
PubMedDate not supplied

Structure of model peptides based on Nephila clavipes dragline silk spidroin (MaSp1) studied by 13C cross polarization/magic angle spinning NMR.

To obtain detailed structural information for spider dragline spidroin (MaSp1), we prepared three versions of the consensus peptide GGLGGQGAGAAAAAAGGAGQGGYGGLGSQGAGR labeled with 13C at six different sites. The 13C CP/MAS NMR spectra were observed after treating the peptides with different reagents known to alter silk protein conformations. The conformation-dependent 13C NMR chemical shifts and peak deconvolution were used to determine the local structure and the fractional compositions of the conformations, respectively. After trifluoroacetic acid (solvent)/diethyl ether (coagulant) treatment, the N-terminal region of poly-Ala (PLA) sequence, Ala8 and Ala10, adopted predominantly the alpha-helix with a substantial amount of beta-sheet. The central region, Ala15, Ala18, and Leu26, and C-terminal region, Ala31, of the peptide were dominated by either 3(1)-helix or alpha-helix. There was no indication of beta-sheet, although peak broadening indicates that the torsion angle distribution is relatively large. After 9 M LiBr/dialysis treatment, three kinds of conformation, beta-sheet, random coil, and 3(1)-helix, appeared, in almost equal amounts of beta-sheet and random coil conformations for Ala8 and Ala10 residues and distorted 3(1)-helix at the central region of the peptide. In contrast, after formic acid/methanol and 8 M urea/acetonitrile treatments, all of the local structure tends to beta-sheet, although small amounts of random coil are also observed. The peak pattern of the Ala Cbeta carbon after 8 M urea/acetonitrile treatment is similar to the corresponding patterns of silk fiber from Bombyx mori and Samia cynthia ricini. We also synthesized a longer 13C-labeled peptide containing two PLA blocks and three Gly-rich blocks. After 8 M urea/acetonitrile treatment, the conformation pattern was closely similar to that of the shorter peptide.

Acetonitriles↗