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

电子冷冻显微镜是显示生理条件下蛋白质的最有前景的方法。由于冷冻水合蛋白产生的幅度对比度较低，应在散焦较大的情况下增加相位对比度。这就要求电子束具有良好的空间相干性。利用我们开发的全息图像重建技术(HIRT)可以补偿由于欠聚焦造成的模糊。我们应用这种方法来可视化细丝的三维结构，并显示了钙诱导的肌钙蛋白的变化。我们用电子冷冻显微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-肌钙蛋白复合体的三维结构。我们发现在钙离子存在的情况下，肌钙蛋白的内部结构域上有大量肌钙蛋白头部。另一方面，肌钙蛋白以肌钙蛋白臂的形式覆盖在肌动蛋白的前表面，包括C末端。也叫t…更多的肌球蛋白根据钙离子的不同发生不同的移位。我们从这些新的数据中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来产生突变的肌动蛋白，在原肌球蛋白-肌钙蛋白和远高于野生型肌动蛋白的钙的存在下激活肌球蛋白ATPase。我们发现，将单一氨基酸丙氨酸230替换为酪氨酸就足以产生这种效果。我们解析了野生型和突变型肌动蛋白的原子结构，发现亮氨酸236的侧链更容易暴露在溶剂中。Ca^&lt；2+&gt；ATP结合部位的水结构表明，肌动蛋白识别ATP腺嘌呤环的水合形式。我们开发了一种结合FRET数据和其他现有结构信息来确定荧光团三维位置的移动方法。利用这种方法，我们可以确定ATP诱导的截短网柄网柄菌肌球蛋白在溶液中的三维结构的变化。此外，该方法还阐明了荧光染料标记引起的肌动蛋白Cys374的重新定位。较少

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