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

电子冷冻显微镜是在生理条件下观察蛋白质最有前途的方法。由于冷冻水合蛋白产生的振幅对比度较低，因此在散焦较大的情况下应增加相衬。这就要求电子束的空间相干性好。利用我们开发的全息图像重建技术（HIRT）可以补偿由于聚焦不足而产生的模糊。我们应用这种方法可视化了细纤维的三维结构，并显示了钙诱导的肌钙蛋白的变化。利用电子冷冻显微镜和背投影成像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Ca^<2+>存在的情况下，肌钙蛋白的质量在肌动蛋白的内部结构域上方。另一方面，在没有Ca^<2+>的情况下，肌钙蛋白通过肌钙蛋白臂覆盖在肌动蛋白的正面，包括c端区域。更多的原肌凝蛋白被发现根据钙离子的不同而不同地转移。我们根据这些新数据提出了钙调节肌肉收缩的新模型。我们使用蛋白质工程技术生产突变型肌动蛋白，在原肌球蛋白-肌钙蛋白和钙的存在下激活肌凝蛋白atp酶，比野生型肌动蛋白高得多。我们发现单氨基酸丙氨酸230替换为酪氨酸足以产生这种效果。我们对野生型和突变型肌动蛋白的原子结构进行了解析，发现亮氨酸236侧链更容易暴露于溶剂。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。我们开发了一种移动方法，通过结合FRET数据和其他结构信息来确定荧光团的三维位置。利用这种方法，我们可以测定atp诱导的截断盘基骨菌肌球蛋白在溶液中的三维结构变化。该方法还阐明了荧光染料标记诱导肌动蛋白Cys374的定位。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