An Essential Role for the C-Terminal Domain of A Dragline Spider Silk Protein in Directing Fiber Formation

Abstract

We have employed baculovirus-mediated expression of the recombinant A. diadematus spider dragline silk fibroin rADF-4 to explore the role of the evolutionary conserved C-terminal domain in self-assembly of the protein into fiber. In this unique system, polymerization of monomers occurs in the cytoplasm of living cells, giving rise to superfibers, which resemble some properties of the native dragline fibers that are synthesized by the spider using mechanical spinning. While the C-terminal containing rADF-4 self-assembled to create intricate fibers in the host insect cells, a C-terminal deleted form of the protein (rADF-4-DeltaC) self-assembled to create aggregates, which preserved the chemical stability of dragline fibers, yet lacked their shape. Interestingly, ultrastructural analysis showed that the rADF-4-DeltaC monomers did form rudimentary nanofibers, but these were short and crude as compared to those of rADF-4, thus not supporting formation of the highly compact and oriented "superfiber" typical to the rADF-4 form. In addition, using thermal analysis, we show evidence that the rADF-4 fibers but not the rADF-4-DeltaC aggregates contain crystalline domains, further establishing the former as a veritable model of authentic dragline fibers. Thus, we conclude that the conserved C-terminal domain of dragline silk is important for the correct structure of the basic nanofibers, which assemble in an oriented fashion to form the final intricate natural-like dragline silk fiber.

The role of salt and shear on the storage and assembly of spider silk proteins

“…We ( Huemmerich et al, 2004b) and subsequently others ( Huang et al, 2006, Ittah et al, 2007, Ittah et al, 2006, Lin et al, 2009and Stark et al, 2007 have shown the C-terminal NR domains to adopt a predominantly α-helical conformation in vitro. These domains have been postulated to form the outer layer of droplet-like structures observed in the spinning dope in vivo ( Jin and Kaplan, 2003, Lin et al, 2009and Vollrath and Knight, 1999.…”

A Novel Model System for Design of Biomaterials Based on Recombinant Analogs of Spider Silk Proteins

“…The synthesis of the whole artificial gene appears to be a very complex problem, which will be solved during further experiments. Some authors (Jin and Kaplan 2003;Ittah et al 2006) suggested that hydrophilic N-and C-termini are necessary for nanofibril's and micelle's formation. Nevertheless, the proteins obtained from expression of the artificial genes that contain only the hydrophobic core regions possess all characteristic properties of the full-length spidroins.…”

Spider silk proteins: recent advances in recombinant production, structure–function relationships and biomedical applications

“…Transgenic production in mammary glands and secretion into milk has been tried in mice [71] and goats [72], but the fairly low yields [7] do not justify the long development times and high production cost. Insects cells have proven to be important tools for the study of assembly properties of spidroin fragments in the cytoplasm [73,74], but these appear to be less suitable for large-scale production. Motivated by structural similarities between spider and silkworm silk, the expression of spidroins in cell lines and larvae of B. mori have been tried, with solubility as the primary limitation [75].…”

“…Biochemical studies of recombinant ADF-3 and ADF-4 fragments that have been lyophilized, resolubilized in guanidinium thiocyanate and dialysed revealed that the presence of the non-repetitive C-terminal domain affects aggregation behaviour [78]. Upon expression in the cytosol of insect cells, ADF-4 fragments lacking the C-terminal domain do not self-assemble into nano fibers, as do their counterparts containing the C-terminal domain, but instead form aggregates [74]. The presence of amphipathic helices in the C-terminal domain has been proposed to be important for fibre formation [104].…”

“…The C-terminal homodimer is stabilized by a disulphide bridge. In miniature spidroins, the cysteine that forms the disulphide can be exchanged for serine without loss of ability to form fibres in the cytosol of insect cells [74] or in the test tube [93].…”