Study of functions of motor protein by protein engineering and holographic electron cryo-microscopy

蛋白质工程和全息电子冷冻显微镜研究运动蛋白的功能

基本信息

批准号：
09102006
负责人：
WAKABAYASHI Takeyuki
金额：
$ 164.48万
依托单位：
University of Tokyo
依托单位国家：
日本
项目类别：
Grant-in-Aid for Specially Promoted Research
财政年份：
1997
资助国家：
日本
起止时间：
1997 至 2000
项目状态：
已结题

项目摘要

The electron cryo-microscopy is the most promising method to visualize proteins under the physiological conditions. Because the amplitude contrast produced by the frozen hydrated proteins is low, phase-contrast should be increased with large defocus. This requires that the spatial coherence of electron beam is good. We could compensate the blurring due to underfocus using the holographic image reconstruction technique(HIRT) we developed.We applied this method to visualize the three-dimensional structure of thin filaments and showed the calcium-induced changes of troponin. We reconstructed three-dimensional structure of actin-tropomyosin-troponin complex from rabbit skeletal muscle by electron cryo microscopy and image analysis using back projection. We found the mass of troponin head over the inner domain of actin in the presence of Ca^<2+>. On the other hand, troponin covered the frontal surface of actin in the absence of Ca^<2+> including the C-terminal region by troponin-arm. Also t … More ropomyosin was found to be shifted differentially depending upon Ca ions. We proposed a new model of calcium regulation of muscle contraction from these new data.We use protein engineering to produce the mutant actins that activate myosin ATPase in a presence of tropomyosin-troponin and calcium much higher than the wild-type actin. We found that the replacement of single amino acid alanine230 to tyrosine is sufficient to produce this effect. We solved the atomic structure of the wild-type actin and mutant ones and found that the side chain of leucine236 is more exposed to solvent. The water structure in the Ca^<2+> ATP binding site suggests that actin recognizes the hydrated form of the adenine ring of ATP.We developed a move method to determine the three-dimensional positions of fluorophores by combining the FRET data and other structural information available. Using this method, we could determine the ATP-induced changes of three-dimensional structure of truncated Dictyostelium myosin in solution. Also the method elucidated actin Cys374 relocation induced by labelling of fluorescent dyes. Less

电子冷冻 - 微观镜检查是在生理条件下可视化蛋白质的最有前途的方法。由于冷冻水合蛋白产生的放大器对比度很低，因此应大量的散焦增加相对比。这要求电子束的空间相干性很好。我们可以使用全息图像重建技术（HIRT）来补偿由于焦点不焦点的模糊，我们开发了这种方法来可视化薄丝的三维结构，并显示了钙诱导的肌钙蛋白变化。我们通过电子冷冻显微镜从兔子骨骼肌肉中重建了肌动蛋白 - 肌球蛋白 - 丝氨酸蛋白复合物的三维结构，并使用背部投影进行了图像分析。在存在Ca^<2+>的情况下，我们发现肌动蛋白内部结构域的肌钙蛋白头部质量。另一方面，在没有Ca^<2+>的情况下，肌钙蛋白覆盖了肌动蛋白的额叶表面，包括肌钙蛋白臂的C末端区域。同样……发现更多的ropomyosin因CA离子而有所不同。我们提出了从这些新数据中对肌肉收缩的钙调节的新模型。我们使用蛋白质工程来产生突变肌动蛋白，该突变肌动蛋白在存在肌球蛋白 - 肌球蛋白和钙的情况下激活肌球蛋白ATPase，远高于野生型肌动蛋白。我们发现，将单个氨基酸丙氨酸230替换为酪氨酸足以产生这种作用。我们解决了野生型肌动蛋白和突变体的原子结构，发现Leucine236的侧链更暴露于求解中。 Ca^<2+> ATP结合位点中的水结构表明，肌动蛋白识别ATP的腺嘌呤环的水合形式。我们开发了一种移动方法来确定荧光团的三维位置，通过将FRET数据和其他可用的结构信息组合在一起。使用这种方法，我们可以确定ATP诱导的溶液中截短骨肌球蛋白的三维结构的变化。该方法还阐明了通过荧光染料的标记引起的肌动蛋白Cys374。较少的