Web spiders use specialized glands to produce silk proteins, so-called spidroins, which assemble into extraordinarily tough silk fibers through tightly regulated phase and structural transitions. A crucial step in the polymerization of spidroins is the pH-triggered assembly of their N-terminal domains (NTDs) into tight dimers. Major ampullate spidroin NTDs contain an unusually high content of the amino acid methionine. We previously showed that the simultaneous mutation of the six hydrophobic core methionine residues to leucine in the NTD of the major ampullate spidroin 1 from Euprosthenops australis, a nursery web spider, yields a protein (L6-NTD) retaining a three-dimensional fold identical to the wildtype (WT) domain, yet with a significantly increased stability. Further, the dynamics of the L6-NTD are significantly reduced and the ability to dimerize is severely impaired compared to the WT domain. These properties lead to significant changes in the NMR spectra between WT and L6-NTD so that the previously available WT-NTD assignments cannot be transferred to the mutant protein. Here, we thus report the de novo NMR backbone and side chain assignments of the major ampullate spidroin 1 L6-NTD variant from E. australis as a prerequisite for obtaining further insights into protein structure and dynamics.

中文翻译：

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动态干扰和二聚化的NMR分配抑制了来自澳大利亚大肠埃希氏菌的蜘蛛丝蛋白的N末端结构域变体。

蜘蛛网利用专门的腺体生产丝蛋白，即所谓的蜘蛛蛋白，它们通过严格调控的相和结构转变而组装成非常坚韧的丝纤维。spidroins聚合过程中的关键步骤是将其N末端域（NTD）的pH触发组装成紧密的二聚体。主要的壶腹spidroin NTDs含有异常高含量的氨基酸蛋氨酸。我们以前表明，在主要的壶腹spidroin 1来自澳大利亚真细菌的NTD的六个疏水核心蛋氨酸残基同时突变为亮氨酸。育苗网蜘蛛，产生的蛋白质（L6-NTD）保留与野生型（WT）域相同的三维折叠，但稳定性显着提高。此外，与WT域相比，L6-NTD的动力学显着降低，并且二聚化能力严重受损。这些性质导致WT和L6-NTD之间的NMR光谱发生显着变化，因此先前可用的WT-NTD指定不能转移到突变蛋白上。因此，在这里，我们报道了来自澳大利亚大肠埃希菌的主要壶腹spidroin 1 L6-NTD变异体的从头NMR骨架和侧链分配，这是获得深入了解蛋白质结构和动力学的先决条件。