Roughly 95% of the proteins that make up the chloroplast must be imported from the cytoplasm. The machinery responsible for the translocation of these cargo proteins is called the translocon at the outer membrane of chloroplast (TOC). The TOC core consists of three proteins, Toc34, Toc75, and Toc159; no high-resolution structure has been solved of fully assembled TOC from plants. Efforts toward determining the structure of the TOC have been hindered almost entirely by difficulties in producing sufficient yields for structural studies. In this study, we introduce an innovative method that utilizes synthetic antigen binding fragments (sABs) to isolate TOC directly from wild-type plant biomass including A. thaliana and P. sativum. Binding between the sABs and the POTRA domains was characterized by size-exclusion chromatography coupled with small-angle X-ray scattering (SEC-SAXS), X-ray crystallography, and isothermal titration calorimetry. We also demonstrate the isolation of the TOC from P. sativum, laying the framework for large-scale isolation and purification of TOC for functional and structural studies.

构成叶绿体的大约 95% 的蛋白质必须从细胞质中输入。负责这些货物蛋白易位的机制称为叶绿体外膜易位子 (TOC)。TOC核心由三种蛋白质组成：Toc34、Toc75和Toc159；尚未解析植物完全组装的 TOC 的高分辨率结构。确定 TOC 结构的努力几乎完全由于难以获得足够的结构研究产量而受到阻碍。在这项研究中，我们介绍了一种创新方法，利用合成抗原结合片段 (sAB) 直接从野生型植物生物质（包括拟南芥和苜蓿）。sAB 和 POTRA 结构域之间的结合通过尺寸排阻色谱结合小角 X 射线散射 (SEC-SAXS)、X 射线晶体学和等温滴定量热法进行表征。我们还展示了从P. sativum中分离 TOC，为大规模分离和纯化 TOC 用于功能和结构研究奠定了框架。