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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

冷冻电镜是在生理条件下观察蛋白质的最有前途的方法。由于冷冻水合蛋白质产生的振幅对比度较低，因此相位对比度应随着大的散焦而增加。这就要求电子束的空间相干性好。我们利用我们开发的全息图像重建技术（HIRT）来补偿欠焦引起的图像模糊，并利用这种方法来显示细丝的三维结构，显示钙离子引起的肌钙蛋白的变化。我们用冷冻电镜和反投影图像分析技术重建了兔骨骼肌肌动蛋白-原肌球蛋白-肌钙蛋白复合物的三维结构。我们发现，在有Ca^<2+>存在的情况下，肌钙蛋白头部的质量位于肌动蛋白的内部结构域之上。另一方面，在缺乏Ca^<2+>的情况下，肌钙蛋白通过肌钙蛋白臂覆盖肌动蛋白的前表面，包括C-末端区域。 ...更多信息发现原肌球蛋白根据Ca离子的不同而不同地移位。根据这些新的数据，我们提出了一个新的钙调节肌肉收缩的模型，我们使用蛋白质工程产生突变肌动蛋白，在原肌球蛋白-肌钙蛋白和钙比野生型肌动蛋白高得多的情况下激活肌球蛋白ATP酶。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效果。我们解决了野生型肌动蛋白和突变体的原子结构，发现亮氨酸236的侧链更多地暴露于溶剂。Ca^2+ ATP结合位点的水结构表明肌动蛋白识别ATP腺嘌呤环的水合形式。我们发展了一种移动方法，通过结合FRET数据和其他可用的结构信息来确定荧光团的三维位置。利用这种方法，我们可以确定ATP诱导的截短的网囊藻肌球蛋白在溶液中的三维结构的变化。此外，该方法阐明了肌动蛋白Cys 374搬迁诱导的荧光染料标记。少