Department of Genetics and Biochemistry, Clemson University, Clemson, South Carolina 29634, USA.
J Biol Chem. 2010 Dec 24;285(52):40745-53. doi: 10.1074/jbc.M110.163121. Epub 2010 Oct 19.
Spider silks are spun from concentrated solutions of spidroin proteins. The appropriate timing of spidroin assembly into organized fibers must be highly regulated to avoid premature fiber formation. Chemical and physical signals presented to the silk proteins as they pass from the ampulle and through the tapered duct include changes in ionic environment and pH as well as the introduction of shear forces. Here, we show that the N-terminal domain of spidroins from the major ampullate gland (MaSp-NTDs) for both Nephila and Latrodectus spiders associate noncovalently as homodimers. The MaSp-NTDs are highly pH-responsive and undergo a structural transition in the physiological pH range of the spider duct. Tryptophan fluorescence of the MaSp-NTDs reveals a change in conformation when pH is decreased, and the pH at which the transition occurs is determined by the amount and type of salt present. Size exclusion chromatography and pulldown assays both indicate that the lower pH conformation is associated with a significantly increased MaSp-NTD homodimer stability. By transducing the duct pH signal into specific protein-protein interactions, this conserved spidroin domain likely contributes significantly to the silk-spinning process. Based on these results, we propose a model of spider silk assembly dynamics as mediated through the MaSp-NTD.
蜘蛛丝是由浓缩的蜘蛛丝蛋白溶液纺制而成的。为了避免过早形成纤维，蜘蛛丝蛋白必须高度有序地组装成有组织的纤维。当丝蛋白从壶腹通过锥形管道时，它们会接收到化学和物理信号，包括离子环境和 pH 值的变化以及剪切力的引入。在这里，我们表明，来自主要壶腹腺（MaSp-NTDs）的两种蜘蛛的丝蛋白的 N 端结构域（MaSp-NTDs）以非共价的方式形成同源二聚体。MaSp-NTDs 对 pH 高度敏感，并在蜘蛛管道的生理 pH 范围内发生结构转变。MaSp-NTDs 的色氨酸荧光在 pH 降低时会发生构象变化，而发生转变的 pH 值取决于存在的盐的量和类型。凝胶过滤色谱和下拉实验均表明，较低 pH 构象与 MaSp-NTD 同源二聚体稳定性显著增加有关。通过将管道 pH 信号转导为特定的蛋白质-蛋白质相互作用，这个保守的丝蛋白结构域可能对丝纺过程有重要贡献。基于这些结果，我们提出了一个通过 MaSp-NTD 介导的蜘蛛丝组装动力学模型。
