Solution structure of eggcase silk protein and its implications for silk fiber formation
Solution structure of eggcase silk protein and its implications for silk fiber formation
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DOI：
10.1073/pnas.0813255106
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发表时间：
2009-06-02
影响因子：
11.1
通讯作者：
Yang, Daiwen
中科院分区：
文献类型：
--
作者：
Lin, Zhi;Huang, Weidong;Yang, Daiwen
蜘蛛丝以其优异的机械性能、仿生和工业潜力而闻名。它们是由丝腺中具有α -螺旋和随机线圈结构的水溶性丝素自然再折叠成主要具有β -结构的不溶性纤维形成的。丝素蛋白的原子分辨率结构和成丝机制在很大程度上仍然是未知的。在这里，我们报告了一个约366 kda的卵状丝蛋白的单个结构域的三维结构，该蛋白由20个相同的1型重复结构域，1个2型重复结构域以及保守的非重复N端和c端结构域组成。用核磁共振技术测定了溶液中各结构域的结构。利用核磁共振和动态光散射技术研究了畴间相互作用。用电子显微镜观察了胶束和宏观纤维的形成。我们发现，与至少一个重复结构域共价连接的末端结构域中的任何一个都可以自发形成胶束状结构，并且可以在>= 37℃和蛋白质浓度为> 0.1 wt%时进一步转化为纤维。我们的生物物理和生化实验表明，亲水性较差的末端结构域启动蛋白质的组装并形成胶束的外层，而亲水性较强的重复结构域则嵌入其中，以确保胶束样结构的形成，这是丝绸形成过程中必不可少的中间体。我们的研究结果确定了单个丝蛋白结构域在纤维形成中的作用，并为设计用于生产人造丝的微型丝素提供了依据。
Spider silks are renowned for their excellent mechanical properties and biomimetic and industrial potentials. They are formed from the natural refolding of water-soluble fibroins with alpha-helical and random coil structures in silk glands into insoluble fibers with mainly beta-structures. The structures of the fibroins at atomic resolution and silk formation mechanism remain largely unknown. Here, we report the 3D structures of individual domains of a approximate to 366-kDa eggcase silk protein that consists of 20 identical type 1 repetitive domains, one type 2 repetitive domain, and conserved nonrepetitive N- and C-terminal domains. The structures of the individual domains in solution were determined by using NMR techniques. The domain interactions were investigated by NMR and dynamic light-scattering techniques. The formation of micelles and macroscopic fibers from the domains was examined by electron microscopy. We find that either of the terminal domains covalently linked with at least one repetitive domain spontaneously forms micelle-like structures and can be further transformed into fibers at >= 37 degrees C and a protein concentration of > 0.1 wt%. Our biophysical and biochemical experiments indicate that the less hydrophilic terminal domains initiate the assembly of the proteins and form the outer layer of the micelles whereas the more hydrophilic repetitive domains are embedded inside to ensure the formation of the micelle-like structures that are the essential intermediates in silk formation. Our results establish the roles of individual silk protein domains in fiber formation and provide the basis for designing miniature fibroins for producing artificial silks.