Structural Biology Brussels, Vrije Universiteit Brussel, VUB, Brussels, Belgium.
VIB-VUB Center for Structural Biology, VIB, Brussels, Belgium.
Nat Methods. 2021 Jan;18(1):60-68. doi: 10.1038/s41592-020-01001-6. Epub 2021 Jan 6.
Nanobodies are popular and versatile tools for structural biology. They have a compact single immunoglobulin domain organization, bind target proteins with high affinities while reducing their conformational heterogeneity and stabilize multi-protein complexes. Here we demonstrate that engineered nanobodies can also help overcome two major obstacles that limit the resolution of single-particle cryo-electron microscopy reconstructions: particle size and preferential orientation at the water-air interfaces. We have developed and characterized constructs, termed megabodies, by grafting nanobodies onto selected protein scaffolds to increase their molecular weight while retaining the full antigen-binding specificity and affinity. We show that the megabody design principles are applicable to different scaffold proteins and recognition domains of compatible geometries and are amenable for efficient selection from yeast display libraries. Moreover, we demonstrate that megabodies can be used to obtain three-dimensional reconstructions for membrane proteins that suffer from severe preferential orientation or are otherwise too small to allow accurate particle alignment.
纳米抗体是结构生物学中一种流行且多功能的工具。它们具有紧凑的单免疫球蛋白结构域组织，能够以高亲和力结合靶蛋白，同时降低其构象异质性并稳定多蛋白复合物。在这里，我们证明了工程化纳米抗体还可以帮助克服限制单颗粒冷冻电子显微镜重构分辨率的两个主要障碍：颗粒大小和在水-气界面上的优先取向。我们通过将纳米抗体嫁接到选定的蛋白质支架上，开发并表征了称为巨抗体的构建体，从而增加了它们的分子量，同时保留了完整的抗原结合特异性和亲和力。我们表明，巨抗体设计原则适用于不同的支架蛋白和具有兼容几何形状的识别结构域，并且可以从酵母展示文库中进行高效选择。此外，我们证明了巨抗体可用于获得三维重构，这些三维重构针对的是那些严重优先取向或太小而无法进行准确粒子对准的膜蛋白。
