An essential role for the C-terminal domain of a dragline spider silk protein in directing fiber formation.
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.
Ittah
Shmulik
S
Department of Cell and Animal Biology, Silberman Life Sciences Institute, Edmond Safra Campus at Givat-Ram, The Hebrew University, Jerusalem 91904, Israel.
Cohen
Shulamit
S
Garty
Shai
S
Cohn
Daniel
D
Gat
Uri
U
D000818:Animals; D002152:Calorimetry, Differential Scanning; D002478:Cells, Cultured; D005349:Fibroins / Q000096:biosynthesis* / Q000737:chemistry / Q000302:isolation & purification; D011487:Protein Conformation; D017510:Protein Folding; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000737:chemistry* / Q000302:isolation & purification; D013112:Spiders / Q000737:chemistry* / Q000166:cytology / Q000235:genetics
D011994:Recombinant Proteins; D005349:Fibroins