Mechanical chaperonin : Mechanical folding of proteins at the single molecular level

机械伴侣蛋白：蛋白质在单分子水平上的机械折叠

• 批准号: 10490016
• 负责人: IKAI Atsushi
• 金额: $ 8.06万
• 依托单位: Tokyo Institute of Technology
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (B)
• 财政年份: 1998
• 资助国家: 日本
• 起止时间: 1998 至 1999
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-10490016/
• 关键词: protein structure; folding; unfolding; chaperonin; nano-mechanics; single molecule measurement; atomic force micro-scope; mechanical chaperonin; フォールディング反応; ナノカ学

The purpose of the study is to use the atomic force microscope (AFM) to extend a single protein molecule after sandwiching it between the crystalline substrate and the tip of the AFM. We chose carbonic dehydratase as a model enzyme and derivatized it so that it had cysteine residues at its N- and C-termini. Extending the protein using the AFM revealed that the protein was pulled to about 20 nm and further application of the extending force abruptly broke the 3D structure of the protein. After the rupture of the 3D structure, the protein behaved as a randomly coiled chain. The process may be regarded as a mechanical denaturation of a protein molecule. Retraction of the distance between the tip and substrate relaxed the protein to resume certain structural elements which may or may not be parts of the native 3D structure. The time allowance in this experiment was in the order of 100 ms and we are extending the time to 10 s so that more structure formation should be observed. A mutant protein that would not form the "knot" structure when pulled from the two ends revealed that the knot formation was indeed a factor to characterize the mechanical behavior of the wild type enzyme. We also conducted experiments on a synthetic polypeptide that forms alpha-helix at a lower pH.

该研究的目的是使用原子力显微镜（AFM）将单个蛋白质分子夹在晶体基质和 AFM 尖端之间后进行延伸。我们选择碳酸脱水酶作为模型酶并将其衍生化，使其在 N 端和 C 端具有半胱氨酸残基。使用 AFM 延伸蛋白质表明蛋白质被拉至约 20 nm，进一步施加延伸力会突然破坏蛋白质的 3D 结构。 3D 结构断裂后，蛋白质表现为随机卷曲的链。该过程可被视为蛋白质分子的机械变性。尖端和基底之间距离的收缩使蛋白质松弛，以恢复某些结构元件，这些元件可能是也可能不是天然 3D 结构的一部分。本实验允许的时间约为 100 毫秒，我们将时间延长至 10 秒，以便观察到更多的结构形成。当从两端拉动时不会形成“结”结构的突变蛋白表明，结的形成确实是表征野生型酶的机械行为的一个因素。我们还对在较低 pH 下形成 α 螺旋的合成多肽进行了实验。

项目成果

Xu, Xue-ming and Ikai, A.: "Recovery and amplification of plasmid DNA with atomic force microscopy and the polymerase chain reaction"Anal. Chim. Acta. 361. 1-7 (1998)

Xu、Xue-ming 和 Ikai, A.：“用原子力显微镜和聚合酶链式反应回收和扩增质粒 DNA”。

Wang, T. and Ikai, A.: "Protein Stretching III. Force expention curves of tethered bovine carbonic anhydrase B to the silicon substrate under native, ntermediate and denaturing conditions"Jap. J. App. Phys.. 38. 3912-3917 (1999)

Wang, T. 和 Ikai, A.：“蛋白质拉伸 III。在天然、中间和变性条件下束缚牛碳酸酐酶 B 对硅基质的力扩展曲线”

Toshiya Osada: "Atomic force microscopy of histological sections using a new electron beam etching method"Journal of Microscopy. 184. 43-49 (1998)

Toshiya Osada：“使用新的电子束蚀刻方法进行组织切片的原子力显微镜”《显微镜杂志》。

Atsushi Ikai: "A recombinant bait region mutant of human alpha-2-macroglobulin exhibiting an altered proteinase-inhibiting spectrum"Cytotechnology. 31. 53-60 (1999)

Atsushi Ikai：“人类 α-2-巨球蛋白的重组诱饵区域突变体表现出改变的蛋白酶抑制谱”细胞技术。

Osada, T., Takezawa, S., Itoh, A., Arakawa, H., Ichikawa, M. and Ikai, A.: "The distribution of sugar chains on the vomeronasal epithelium observed with the atomic force microscope"Chemical Senses. 24. 1-6 (1999)

Osada, T.、Takezawa, S.、Itoh, A.、Arakawa, H.、Ichikawa, M. 和 Ikai, A.：“用原子力显微镜观察到的犁鼻上皮上糖链的分布”化学感官。