Segmented nanofibers of spider dragline silk: Atomic force microscopy and single-molecule force spectroscopy

Despite its remarkable materials properties, the structure of spider dragline silk has remained unsolved. Results from two probe microscopy techniques provide new insights into the structure of spider dragline silk. A soluble synthetic protein from dragline silk spontaneously forms nanofibers, as observed by atomic force microscopy. These nanofibers have a segmented substructure. The segment length and amino acid sequence are consistent with a slab-like shape for individual silk protein molecules. The height and width of nanofiber segments suggest a stacking pattern of slab-like molecules in each nanofiber segment. This stacking pattern produces nano-crystals in an amorphous matrix, as observed previously by NMR and x-ray diffraction of spider dragline silk. The possible importance of nanofiber formation to native silk production is discussed. Force spectra for single molecules of the silk protein demonstrate that this protein unfolds through a number of rupture events, indicating a modular substructure within single silk protein molecules. A minimal unfolding module size is estimated to be around 14 nm, which corresponds to the extended length of a single repeated module, 38 amino acids long. The structure of this spider silk protein is distinctly different from the structures of other proteins that have been analyzed by single-molecule force spectroscopy, and the force spectra show correspondingly novel features.

尽管蜘蛛牵引丝（spider dragline silk）具备优异的材料性能，但其分子结构至今尚未被完全解析。两项探针显微技术的研究结果为该类丝蛋白的结构解析提供了全新视角。研究人员通过原子力显微镜（atomic force microscopy）观察发现，一种源自牵引丝的可溶性合成蛋白可自发形成纳米纤维（nanofibers），这类纳米纤维呈现分段式亚结构。纳米纤维的分段长度与氨基酸序列特征，与单个丝蛋白分子的板状结构相符；纳米纤维分段的高度与宽度数据表明，每个分段内的板状蛋白分子存在堆叠排布模式。该堆叠模式会在非晶基质中形成纳米晶体，这与此前通过蜘蛛牵引丝的核磁共振（NMR）与X射线衍射（X-ray diffraction）观测到的结果一致。本文还探讨了纳米纤维形成过程对天然丝蛋白合成的潜在重要意义。对单个丝蛋白分子的力谱测试结果显示，该蛋白的解折叠过程伴随多个断裂事件，这表明单个丝蛋白分子内部存在模块化亚结构；研究估算得到最小解折叠模块的尺寸约为14纳米，这一尺寸与单个由38个氨基酸残基构成的重复模块的伸展长度相符。该蜘蛛丝蛋白的结构与此前通过单分子力谱（single-molecule force spectroscopy）分析过的其他蛋白结构存在显著差异，其力谱数据也呈现出独特的全新特征。