Development of structural analysis and electron microscopy to visualize single motor protein

开发结构分析和电子显微镜以可视化单运动蛋白

• 批准号: 06558098
• 负责人: WAKABAYASHI Takeyuki
• 金额: $ 5.38万
• 依托单位: University of Tokyo
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Developmental Scientific Research (B)
• 财政年份: 1994
• 资助国家: 日本
• 起止时间: 1994 至 1995
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-06558098/
• 关键词: Muscle; Myosin; Actin; Tubulin; Kinesin; Electron microscopy; X-ray scattering; Site-directed mutagenesis; クライオ電子顕微鏡法; 対物レンズ; 傾斜像シリーズ; 画像解析; ミオシン; 筋収縮; アンデカゴールド

Our goal is to understand the molecular mechanism of motor proteins on the basis of atomic structure of proteins.First, we inproved an electron microscope with a cold field emission gun. Especially, we developed a new objective lenz, by which we can observe high-tilted images, an anti-contaminator, a new minimum dose system.Second, we developed new image analysis techniques. The first one is electron tomography to reconstruct three-dimensional images of cross-bridge in contracting muscle.The second one is visualization of single motor protein using a holographic image reconstruction technique. The third one is three-dimensional reconstruction without using helical symmetry in order to reconstruct three-dimensional structure of thin filaments. The forth one is a new three-dimensional structure using helical symmetry.Third, by electron cryo-microscopy, we analyzed three-dimensional structure of kinesin-tubulin complexes, thin filaments, native thin filaments, actomyosin complexes, and actin filaments.Last, we identified the position of the myosin N-terminus in the actin-myosin rigor complexes using protein engineering and cryo-electron microscopy.

我们的目标是从蛋白质的原子结构出发来理解马达蛋白的分子机制。首先，我们改进了一台带有冷场发射枪的电子显微镜。特别是我们研制了一种新的物镜伦茨，通过它我们可以观察到高倾斜的图像，一个抗污染的，一个新的最小剂量系统。第一个是电子断层扫描重建收缩肌肉中横桥的三维图像，第二个是使用全息图像重建技术显示单个运动蛋白。第三种是不使用螺旋对称性的三维重建，以重建细丝的三维结构。第三，利用冷冻电镜技术分析了驱动蛋白-微管蛋白复合物、细丝、天然细丝、肌动球蛋白复合物和肌动蛋白细丝的三维结构，最后利用蛋白质工程技术和冷冻电镜技术确定了肌动蛋白-肌球蛋白刚性复合物中肌球蛋白N-末端的位置。

项目成果

Ishikawa, T.& Wakabayashi T.: "Calcium induced change in three-dimensional structure of thin filaments of rabbit skeletal muscle as revealed by cryo-electron microscopy" Biochem.Biophys.Res.Commun.203. 951-958 (1994)

石川，T.

Kikkawa, M., Ishikawa, T., Miyazawa, A., Fujiyoshi, Y., Wakabayashi, T.& Hirokawa, N.: "Direct visualization of the microtubule lattice seam both in vitro and in vivo." J Cell Biol.127. 1965-1971 (1994)

吉川，M.，石川，T.，宫泽，A.，藤吉，Y.，若林，T.

佐伯 他: "Troporaysin-bindina site(s) on the Dictyosteriun activl surface asidentified by site-dirocted mutageusis" Biochemistry. 35. 14465-14472 (1996)

Saeki 等人：“通过定点诱变鉴定的网基网藻活性表面上的原丝蛋白结合位点”生物化学 35. 14465-14472 (1996)。

Saeki, K., Sutoh, K.& Wakabayashi, T.: "T., Tropomyosin-binding site(s) on the Dictyosterium Actin surface as identify by site-directed mutagenesis" Biochemistry. 35. 14465-14472 (1996)

佐伯，K.，须藤，K.

Meng, Y. et al.: "Detemination of the Protein Components of Native Thin Filaments Isolated from Natural actomyosin: Nebulin and a-Actinin Are Associated with Actin Filaments" J. Biochem.118. 422-427 (1995)

孟，Y.等人：“从天然肌动球蛋白中分离出的天然细丝的蛋白质成分的测定：星云蛋白和α-肌动蛋白与肌动蛋白丝相关”J.Biochem.118。