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PubMed2004

Structural role of tyrosine in Bombyx mori silk fibroin, studied by solid-state NMR and molecular mechanics on a model peptide prepared as silk I and II.

The influence of the bulky and H-bonding Tyr side-chain on its Ala- and Gly-rich environment in Bombyx mori silk fibroin was examined by (13)C cross-polarization magic angle spinning (CP/MAS), static (2)H and (19)F NMR and molecular mechanics calculations. Model peptides of the type (AG)(15) were synthesized with Tyr in a number of different positions, precipitated under conditions favoring either of the two characteristic protein conformations, and the resulting structures were assigned from their (13)C chemical shifts. Dialysis of native fibroin or the simple (AG)(15) peptide from a 9 M LiBr solution against water produces silk I (the structure of silk before spinning), whereas drying from formic acid yields silk II (fibrous structure after spinning). We found that the introduction one or more Tyr into (AG)(15) can have a dramatic effect not only on the local backbone conformation but also on the long-range intermolecular chain packing in the samples. The antiparallel beta-sheet conformation of silk II is able readily to accommodate a single Tyr residue. Interestingly, the beta-turn conformation of silk I only remains stable when Tyr is positioned near the chain terminus in (AG)(12)YG(AG)(2), but the conformation is driven towards silk II when Tyr is located in the central region of (AG)(7)YG(AG)(7). The role of H-bonding was tested by replacing Tyr with Phe or 4F-Phe, which are no longer compatible with silk I and fully induced a silk II conformation. In the presence of several Tyr residues a mixture of distorted beta-sheet and beta-turn conformations was obtained, regardless of the precipitation conditions. Static (2)H NMR of ring-deuterated [3',5'-(2)H(2)]Tyr located in the central region of (AG)(7)YG(AG)(7) showed that the side-chain is immobilized in both silk I and II, which was also observed by static (19)F NMR of the 4F-Phe analogue. To visualize the local packing around the Tyr side-chain, molecular mechanics calculations were performed on a mixture of (AG)(4) and AGAGYGAG, starting from either the beta-turn type II or the antiparallel beta-sheet structure. The resulting structures show that the intermolecular chain arrangement is significantly affected by Tyr, thus explaining the long-range packing effects in the semi-crystalline regions of silk fibers compared with the crystalline regions that are devoid of Tyr.

Amino Acid Sequence↗

PubMed2002

Fibroin-like substance is a major component of the outer layer of fertilization envelope via which carp egg adheres to the substratum.

Seven cDNA encoding silkworm fibroin homologues were cloned from a carp ovarian cDNA library. The encoded proteins are denoted as carp ovarian fibroin-like substances (FLS). FLS contain a repetitive domain consisting of tandem repeats of dipeptide of Gly-X, where X may be any amino acid. Each FLS has its own unique repeating sequence, such as GQGAGQGS, GQGMGQGM, GRGQGEGHGS, and GFGFGQGS, indicating a family of FLS genes exists in carp. FLS is exclusively expressed in oocytes and is stored in cortical granules. During cortical reaction, FLS is exocytosed to perivitelline space and then gradually added to the outer layer of the fertilization envelope (FEo). The FLS of fertilization envelope is conjugated with cystatin and cathepsin-like substance (CLS) and appears in multiple bands of molecular weights ranging from 40 to 205 kDa. After fertilization or artificial activation, carp eggs adhere firmly to the substratum via FEo. FLS is a major component of FEo. The presence of transglutaminase inhibitor, cadaverine or ethylene diaminetetraacetic acid, in the cortical reaction medium can impair or block the recruitment of FLS and other substances to FEo. As a consequence, FEo is not formed or is greatly reduced, resulting in a great reduction of egg adhesion.

Amino Acid Sequence↗

PubMed2001

Silk fibroin: structural implications of a remarkable amino acid sequence.

The amino acid sequence of the heavy chain of Bombyx mori silk fibroin was derived from the gene sequence. The 5,263-residue (391-kDa) polypeptide chain comprises 12 low-complexity "crystalline" domains made up of Gly-X repeats and covering 94% of the sequence; X is Ala in 65%, Ser in 23%, and Tyr in 9% of the repeats. The remainder includes a nonrepetitive 151-residue header sequence, 11 nearly identical copies of a 43-residue spacer sequence, and a 58-residue C-terminal sequence. The header sequence is homologous to the N-terminal sequence of other fibroins with a completely different crystalline region. In Bombyx mori, each crystalline domain is made up of subdomains of approximately 70 residues, which in most cases begin with repeats of the GAGAGS hexapeptide and terminate with the GAAS tetrapeptide. Within the subdomains, the Gly-X alternance is strict, which strongly supports the classic Pauling-Corey model, in which beta-sheets pack on each other in alternating layers of Gly/Gly and X/X contacts. When fitting the actual sequence to that model, we propose that each subdomain forms a beta-strand and each crystalline domain a two-layered beta-sandwich, and we suggest that the beta-sheets may be parallel, rather than antiparallel, as has been assumed up to now.

Amino Acid Sequence↗

PubMed1995

Attachment and growth of fibroblast cells on silk fibroin.

The attachment and growth of fibroblast cells (L-929) on matrices of silk fibroin from Bombyx mori domestic silkworm (DSF) and Antheraea pernyi wild silkworm (WSF) were studied by a cell culture method. The performance of the two kinds of silk fibroin was compared to that of collagen. DSF exhibited as high a cell attachment and growth as collagen did. The cells attached to DSF were extensively spread out and their filopodia were visible in the SEM pictures. WSF, which contains the tripeptide sequence Arg-Gly-Asp (believed to be a specific interaction site for cell-attachment), displayed much higher cell attachment and growth compared to DSF. The cells attached on WSF became virtually flat and their filopodia could be seen, indicating that the cells were very strongly held on the surface.

Amino Acid Sequence↗

PubMed2001

A repeated beta-turn structure in poly(Ala-Gly) as a model for silk I of Bombyx mori silk fibroin studied with two-dimensional spin-diffusion NMR under off magic angle spinning and rotational echo double resonance.

The structure of a crystalline form of Bombyx mori silk fibroin, commonly found before the spinning process (known as silk I), was proposed by combining data obtained from two-dimensional spin-diffusion nuclear magnetic resonance under off magic angle spinning, rotational-echo double-resonance (REDOR), previously reported X-ray diffraction analyses and 13C NMR chemical shifts. Instead of B. mori silk fibroin with silk I structure, we used the sequential model peptide (Ala-Gly)15. The structure of the sequential model peptide is characterized as silk I after dissolving the peptide in 9 M LiBr and then dialyzing against water. Moreover, 13C or 15N-labeled sites may be introduced easily at any position in (Ala-Gly)(15) by the solid phase synthesis method for these NMR experiments. The torsional angles of (Ala-Gly)15 with silk I structure were determined as (-60(+/-5) degrees, 130(+/-5) degrees ) and (70(+/-5) degrees, 30(+/-5) degrees ) for Ala and Gly residues, respectively. The formation of the intra-molecular hydrogen bonding along the chain was confirmed from REDOR NMR by determination of the inter-atomic distance between the nitrogen and carbon atoms comprising the intra-molecular hydrogen bonding. The structure is named a repeated beta-turn type II-like structure.

Alanine↗

PubMed1986

Low temperature causes accumulation of unspliced fibroin mRNA precursor molecules in silkworm larvae.

Silk fibroin premessenger mRNA is a large (17 kb) molecule containing a single intron. Previous S1 mapping studies yielded evidence of processing cleavages at sites within the intron, in addition to the major cleavages at the intron/exon boundaries. We have performed S1 mapping experiments using RNA from animals which had been incubated at low temperature. These experiments show a marked accumulation of full-length fibroin mRNA precursor relative to controls, and a reduction in the abundance of molecules lacking portions of the intron. These results demonstrate a partial uncoupling of transcription and splicing in vivo.

Animals↗

PubMed1994

Investigation of uranium recovery from dilute aqueous solutions using silk fibroin.

The uranium uptake ability of silk fibroin was investigated. High ability to uptake uranium from nonsaline water containing 2.500 mg of uranium was observed with the silk fibroin tested. The uranium uptake was very rapid and was dependent on pH, uranium concentration, temperature, and retention time. Almost all uranium taken up is easily eluted with 1 mol/L CH3COONH4. This biomatrix, therefore, appears to have potential for use in a commercial process for uranium recovery from uranium-containing waste water.

Fibroins↗

PubMed2003

Chitin-silk fibroin interactions: relevance to calcium carbonate formation in invertebrates.

In mineralized tissues chitin is almost always associated with proteins, many of which are known to have chitin recognition consensus sequences. It has been observed in some mollusk shells that there is a well-defined spatial relation between the crystallographic axes of the crystals and the chitin fibrils. This implies that the chitin functions directly or indirectly as a template for nucleation of the mineral phase. It is thus of much interest to understand the exact nature of the interface between the chitin and the proteins at the molecular level in mineralized tissues. Chitin/silk fibroin interactions were studied in vitro at the molecular level using homogenous films composed of the two macromolecules. The results show that the silk fibroin intercalates between the molecular planes of the chitin, and that the interactions are mainly through the chitin acetyl groups. Published X-ray diffraction patterns and infrared spectra of mineralized tissue organic matrices, as well as infrared spectra reported here of the squid pen and lobster cuticle, all show that in vivo the chitin and protein are not intimately mixed, but exist as two phases. We deduce that there is an interfacial plane between them in which the interactions are through the amide groups.

Animals↗

PubMed1989

Primary structure of the silk fibroin light chain determined by cDNA sequencing and peptide analysis.

A cDNA clone, pFL18, carrying a putative full-length fibroin light chain (L-chain) sequence was isolated and its nucleotide sequence was determined. This revealed the presence of an open reading frame corresponding to a polypeptide with 262 amino acid residues. The sequence was concluded to be that of the L-chain with its signal peptide because corresponding amino acid sequences for the seven tryptic and the four chymotryptic peptides from the purified L-chain were all included and an N-terminal region having typical properties of a signal peptide was present. The N terminus of the mature form of L-chain was identified as N-acetyl serine by analyzing the acyl-dansylhydrazide derived from the N-acyl-amino acid which had been released from the N-terminal blocked chymotryptic peptide by the acylamino acid-releasing enzyme. It was suggested that a signal peptide had cleaved between Pro18 and Ser19, yielding a mature L-chain polypeptide consisting of 244 amino acid residues. The molecular weight of the L-chain was calculated to be 25,800 including the N-acetyl group. The L-chain contained three Cys residues, two of which were suggested to form an intramolecular disulfide linkage, leaving the third one at the most C-terminal position and in a relatively hydrophilic region as the most probable site of disulfide linkage with the fibroin heavy chain.

Amidohydrolases↗

PubMed1990

Physico-chemical properties of silk fibroin membrane as a biomaterial.

A water-insoluble silk fibroin membrane was prepared by immersing a silk fibroin membrane as cast in 50 vol% aqueous methanol solution for different periods of time at 25 degrees C. To use the membrane as a biomaterial, oxygen and water vapour permeability, transparency, mechanical property and enzymatic degradation behaviour in vitro of the membrane in the wet state were investigated. These physico-chemical properties changed according to the condition of the methanol treatment. The membrane had oxygen permeability, water vapour permeability, transparency and biodegradability.

Biocompatible Materials↗

PubMed1992

Development of an antibody against a 170-kDa fragment of fibroin isolated from cocoon fibres of Bombyx mori.

A simple method for the isolation of denatured fragments of the fibroin protein from cocoon fibres by alkali solubilization is discussed. This 170-kDa antigen has been purified and used to raise the polyclonal antibody in rabbit. The specificity of this antibody to the purified cocoon protein has suggested strong immunoreactivity up to a titre of 1:5000 dilution of the antibody. Further, dot-blot analysis with the tissue extracts from silk glands of different larval stages (3rd to 5th) reveals that this antibody reacts showing a stage-specific increase in the intensity of the colour, correlating well with the in-vivo expression of the silk protein. This study suggests the availability of a specific polyclonal antibody that detects the native fibroin with no crossreactivity with other tissue proteins.

Animals↗

PubMed1984

Sequence polymorphisms in the 5'-upstream region of the fibroin H-chain gene in the silkworm, Bombyx mori.

DNA fragments containing the fibroin H-chain gene from two different strains of Bombyx mori, J-139 and Nd (2) were cloned into phage lambda Charon 4A. Comparison of the restriction sites in these cloned DNAs revealed that in addition to the known polymorphism in the region coding for the repetitive amino acid sequence of the fibroin H-chain [Manning and Gage, J. Biol. Chem. 255 (1980) 9451-9457], at least two other types of polymorphism were present, one around the 5' end of the structural gene, and the other in the far upstream region of the gene. Restriction sites around the 5' end of the gene were well conserved between these strains, but some heterogeneity, suggesting the presence of small insertions, deletions or base changes, was noted. In contrast, DNA sequences of the region 2-4 kb upstream from the 5' end of the gene were markedly different between these two strains, indicating that either a deletion or an insertion of a DNA sequence longer than 2 kb had occurred in this region. Comparison with several other strains suggested that the observed changes in the far-upstream region were unique to the Nd(2) strain.

Animals↗

PubMed1989

Enhancement of transcription from the Ad2 major late promoter by upstream elements of the fibroin- and sericin-1-encoding genes in silk gland extracts.

The activity of the upstream promoter element (UPE) of the Bombyx mori fibroin-encoding gene was analyzed by using the major late promoter (MLP) of adenovirus 2 (Ad2) as the basal promoter. The UPE reveals remarkable stimulatory activity on the transcription of Ad2 MLP in tissue extracts prepared from the posterior silk gland where the fibroin-encoding gene is specifically expressed. This stimulation of transcription is due to an increase of initiation complex formation as shown by a one-round transcription assay. The UPE of the sericin-1-encoding gene, which is specifically expressed in the middle silk gland, also stimulates Ad2 MLP transcription in a similar manner. The results of the competition experiments indicate that the stimulatory effect is mediated by trans-acting factors which bind to these UPE. These two UPE are also active in tissue extracts of the middle silk gland, but are nearly inactive in extracts from the ovarian tissue and an embryonic cell line, Bm-e21. These observations further support the hypothesis that a silk-gland-specific factor(s) is responsible for this tissue-specific transcription in vitro. The possible molecular basis of this silk-gland specificity is discussed in light of the various trans-acting factors known to bind these UPE.

Adenoviruses, Human↗

PubMed2005

Unusual protein behavior illustrated with silk fibroin.

We investigated the interaction between phospholipid membranes and silk fibroin recovered from the posterior silk gland of the silkworm. Observations of the planar lipid bilayer membrane and electron microscopic observations of liposomes showed that newly constructed silk fibroin, existing in the form of filaments, quickly penetrates phospholipid membranes without bursting them.

Fibroins↗

PubMed2005

The conserved C-termini contribute to the properties of spider silk fibroins.

Spider silk fibroins can adopt different structural states at high protein concentrations. They are soluble within the spinning dope of the glands, but readily converted into insoluble polymers upon extrusion. A contribution of the C-termini to the maintenance and conversion of these states is suggested by their predicted secondary structures and biochemical behavior in vitro. Special sequence parts endow the C-termini with the capability to promote both the solubility and aggregation of the fibroins depending on the environmental conditions.

Amino Acid Sequence↗

PubMed2004

Sequence variability in the fibroin-H intron of domesticated and wild silk moths.

The single intron of the heavy-chain fibroin gene in domesticated (Bombyx mori) and wild (B. mandarina) silk moths has a length of approximately 1000 nucleotides. It is located only 57 bp from the gene's core promoter and harbors multiple AT-rich regulatory elements that have been found to enhance the basal level of transcription in vitro. In this work, the intronic nucleotide variability among members of both Bombyx species is analyzed. The intron sequences of B. mori strains k120 and Nistari, as well as B. mandarina specimens from Japan and Korea, were obtained. This information was compared with the previously reported sequences of B. mori strains p50 and C-108, as well as an additional B. mandarina specimen collected in Japan. We found a total of 26 variant positions, including variants shared by members of both species and species-specific changes. The potential functional role of these variants was investigated by using the program MatInspector to search for putative binding sites of transcription factors within the intron. We detected a multitude of putative binding elements distributed along the entire intronic sequence. Among them, 22 correspond to protein binding domains that are known to regulate fibroin transcription. The mapping of multiple variant positions within these putative binding sequences as well as in known regulatory elements of the intron argue for functional significance on the regulation of transcription.

Animals↗

PubMed2004

X-ray structural study of noncrystalline regenerated Bombyx mori silk fibroin.

X-ray diffraction measurements of regenerated Bombyx mori silk fibroin were carried out to determine its structural characteristic from an analysis of differential radial distribution functions (DRDFs). The temperature dependence of X-ray diffraction patterns from noncrystalline and crystal structures of regenerated silk fibroin was investigated using a high temperature furnace. Time resolved X-ray diffraction profiles were also obtained to construct kinematical models of structural changes caused by the addition of water. DRDFs, calculated from the experimental data, were compared with the DRDFs simulated on the basis of the Monte Carlo method. In order to model the noncrystalline structures, structural units were assumed to be parts of the crystalline structure of silk and those with appropriate structural defects reported previously. From the comparison of experimental and simulated DRDFs, it was determined that noncrystalline regenerated silk consisted of locally ordered atomic sheets similar to the atomic arrangement in the silk I crystal (Type-I sheets), and the final state of the structural change was noncrystalline, consisting of small crystallites, the structure of which is similar to that of silk II (Type-II crystallites). Time resolved DRDFs were also qualitatively interpreted by both the ordering of Type-I sheets and structural changes from Type-I to Type-II. The formation of the small Type-II crystallites obtained in this study was consistent with the nucleation of silk II by birefringence measurements of silk glands and the spinneret of Bombyx mori silkworm reported previously. X-ray diffraction should be a useful technique to understand the structural characteristics of noncrystalline organic materials.

Animals↗

PubMed2005

A study on the flow stability of regenerated silk fibroin aqueous solution.

The flow stability of silk fibroin (SF) aqueous solutions with different concentrations under different temperatures was investigated. It was found that the flow stability decreased quickly with the increase of solution concentration and temperature. X-ray diffraction, Fourier transform infrared (FTIR) and Raman spectroscopy analysis showed that silk fibroin in aqueous solution was mainly in random coil and alpha-helix conformation. However, it turned into alpha-helix and beta-sheet conformation after gelation, and both silk I and silk II crystalline structures appeared accordingly. The investigation implies that the original dilute regenerated SF aqueous solution should be stored under low temperature and concentrated just before spinning.

Dose-Response Relationship, Drug↗