An essential role for the C-terminal domain of a dragline spider silk protein in directing fiber formation
An essential role for the C-terminal domain of a dragline spider silk protein in directing fiber formation
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DOI：
10.1021/bm060120k
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发表时间：
2006-06-01
影响因子：
6.2
通讯作者：
Gat, Uri
中科院分区：
文献类型：
--
作者：
Ittah, Shmulik;Cohen, Shulamit;Gat, Uri
我们采用杆状病毒介导的重组A. diadematus spider拖丝丝素蛋白rADF-4，以探索进化保守的C-末端结构域在蛋白质自组装成纤维中的作用。在这个独特的系统中，单体聚合发生在活细胞的细胞质中，产生超纤维，其类似于蜘蛛使用机械纺丝合成的天然拖丝纤维的一些特性。虽然含有rADF-4的C-末端自组装以在宿主昆虫细胞中产生复杂的纤维，但蛋白质的C-末端缺失形式（rADF-4-Delta C）自组装以产生聚集体，其保留了拖丝纤维的化学稳定性，但缺乏其形状。有趣的是，超微结构分析显示，rADF-4-Δ C单体确实形成了基本的纳米纤维，但与rADF-4的纳米纤维相比，这些纳米纤维短而粗糙，因此不支持rADF-4形式典型的高度紧凑和定向的“超级纤维”的形成。此外，使用热分析，我们显示的证据表明，rADF-4纤维，但不是rADF-4-Δ C聚集体包含结晶域，进一步建立了前者作为一个名副其实的模型，真正的拖丝纤维。因此，我们得出结论，拖丝的保守的C-末端结构域是重要的基本纳米纤维的正确结构，组装在一个定向的方式，形成最终的复杂的天然样拖丝纤维。
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-Delta C) 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-Delta C 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-Delta C 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.