Characterization of the Protein Components ofNephila clavipesDragline Silk

Abstract

Spider silk is predominantly composed of structural proteins called spider fibroins or spidroins. The major ampullate silk that forms the dragline and the cobweb's frame threads of Nephila clavipes is believed to be a composite of two spidroins, designated as Masp 1 and 2. Specific antibodies indeed revealed the presence of Masp 1 and 2 specific epitopes in the spinning dope and solubilized threads. In contrast, sequencing of specific peptides obtained from solubilized threads or gland urea extracts were exclusively homologous to segments of Masp 1, suggesting that this protein is more abundantly expressed in silk than Masp 2. The strength of immunoreactivities corroborated this finding. Polypeptides reactive against both Masp 1 and 2 specific antibodies were found to be expressed in the epithelia of the tail and different gland zones and accumulated in the gland secreted material. Both extracts of gland secretion and solubilized threads showed a ladder of polypeptides in the size range of 260-320 kDa in gel electrophoresis under reducing conditions, whereas gel filtration chromatography yielded molecular masses of the proteins of approximately 300-350 kDa. In the absence of a reducing agent, dimeric forms of the spidroins were observed with estimated molecular masses of 420-480 kDa according to gel electrophoresis and 550-650 kDa as determined by gel filtration chromatography. Depending on the preparation, some silk material readily underwent degradation, and polypeptides down to 20 kDa in size and less were detectable.

Spidroin N-terminal Domain Promotes a pH-dependent Association of Silk Proteins during Self-assembly

“…Even though Nc.MaSp2-NTD has a histidine, it seems likely that the pH-dependent conformational change occurs through a mechanism similar to that of Nc.MaSp1A-NTD and Lm.MaSp1-NTD so its histidine may play little or no role in that regard. Additionally, all three homologs lack cysteine residues, and therefore MaSp-NTD dimers are not stabilized by disulfide bonds as is believed to be the case for MaSp-CTDs (11,20).…”

“…The fiber produced from the major ampullate gland is called dragline silk, and it is used in the main structural elements of an orb web and also as a hanging lifeline. Its primary protein constituents are major ampullate spidroins 1 and 2 (MaSp1 and MaSp2) (11)(12)(13). These proteins are highly repetitive molecules having a 30 -40-amino acid consensus sequence repeated ϳ100 times in tandem (14).…”

Recombinant Spider Silks—Biopolymers with Potential for Future Applications

“…Additionally, these spidroins have a high molecular weight, 200 to 350 kDa or even larger [9][10][11]. The spidroins are covalently linked via cysteine bridges in their termini and undergo further oligomerization due to their repetitive regions [12]. The repetitive regions have an unusual amino acid composition with more than 50% alanine (A) and glycine (G) residues.…”

“…The largest MaSp1 construct tested in such E. coli consisted of 96 copies of its repeating module giving a protein with a molecular weight of 285 kDa [34]. Thus it is now possible to bacterially produce spidroins with a size similar to that of natural spider silk proteins, and sufficient yields [12].…”

Production and processing of spider silk proteins

“…2). [17][18][19][20] The core contains two major proteins (Major Ampullate Spidroins 1 and 2) that are composed predominantly of glycine, alanine, and proline (although the quantity of the latter varies significantly between species). Major ampullate spidroins of Araneus diadematus and Nephila clavipes spiders are reminiscent of block copolymers containing blocks of polyalanine and either (GGX) n (where X is typically tyrosine, leucine or glutamine) or GPGXX.…”