设计通用蛋白骨架用于小蛋白的冷冻电镜 结构解析

  结构生物学通过揭示生物大分子的形状和结构，革新了我们对生命过程的认知,也为现代药物研发提供了数据基础。生物大分子的结构解析主要通过三种技术，X射线晶体学、核磁共振和冷冻电镜。这三种技术各有各自的优点和局限性：核磁共振技术探测溶液状态中的分子，可获得接近自然状态的分子构象，但是局限于解析分子量小于40 kDa的大分子；X射线晶体学在解析生物大分子原子水平分辨率的进程中发挥了主导作用，但是晶体学技术适用于部分构象稳定的蛋白质，对于构象多变且稳定性欠佳而难于结晶的蛋白适用困难；单颗粒冷冻电镜技术的革新使结构生物学进入了一个新的纪元，使得稳定性欠佳的大型动态复合物和膜蛋白的结构解析在无法结晶的情况下变得可能。

  但是，大部分利用此技术解析的近原子分辨率结构都是分子量大于100 kDa的蛋白质，而原核生物和真核生物中蛋白质分子量的中位数分别是29.9 kDa和45.6 kDa，远小于目前常规单颗粒冷冻电镜可解析蛋白的分子量下限。为了进一步拓展单颗粒冷冻电镜技术的应用范围，使得那些不易结晶的和存在多种构象状态的蛋白的结构也能够被解析出来，我们拟搭建一个通用的和模块化的骨架蛋白体系，包括底座蛋白和接头蛋白，通过解析骨架蛋白复合体与靶标蛋白的整体电镜结构从而获得靶标蛋白的结构。

  通过多轮设计和优化，目前我们已经获得：以Ferritin为底座，DARPin为接头蛋白的骨架蛋白与GFP结合，冷冻电镜下得到3.5 Å-4 Å分辨率的GFP的三维结构；以Dps为底座蛋白，Affitin为接头蛋白的骨架蛋白与DNA结合，冷冻电镜下得到3.1 Å的整体分辨率。这些结果为进一步优化骨架蛋白，进一步提高靶标蛋白的分辨率，奠定了坚实的基础。

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