Tools4Cells:EAGER: CRYO-EM ANALYSIS OF THE CHLOROPLAST CLP PROTEASE SYSTEM THROUGH AFFINITY PURIFICATION OF ENDOGENOUS COMPLEXES

蛋白质之间的相互作用和蛋白质复合体对细胞的生存至关重要。单粒子低温电子显微镜(Cryo-EM)加快了这些纳米蛋白质复合体的高分辨结构测定的步伐。Cryo-EM主要用于细菌表达系统中产生的蛋白质复合体，以获得大量的这些蛋白质用于Cryo-EM分析。然而，对从其自然环境中获得的络合物进行结构分析将是有益的，因为它们代表了络合物最自然的状态。事实上，内源性复合体(即从其生理环境中获得的复合体)的冷冻EM的少数例子仅限于相对丰度较高的蛋白质。成功地开发和应用Cryo-EM工具并应用于从其自然细胞环境中纯化的低丰度内源复合体亲和力将极大地影响生物发现，包括对拟南芥等植物的发现。本项目致力于拟南芥叶绿体中CLP蛋白-伴侣系统的结构分析。这种CLP系统是叶绿体蛋白质动态平衡的中心；CLP活性不足对植物有害。这项拟议的研究将揭开这一独特的叶绿体CLP伴侣-蛋白酶系统的结构细节，并有助于了解其对叶绿体环境的进化适应。这项工作的更广泛影响包括蛋白酶系统的生物学重要性所产生的内在价值，以及所开发的方法学可能应用于其他需要植物低丰度内源复合体的结构数据的项目。此外，该项目还将为本科生和研究生提供培训机会。拟议的项目旨在通过冷冻-EM技术确定内源拟南芥叶绿体~1000 kDa CLP蛋白酶-伴侣复合体(ClpPRTC)的结构组织，该复合体由一个与六聚体伴侣环(C和D亚基)动态结合的十四聚体桶形蛋白酶(P、R、T亚基)组成。完整的复合体由WalkerB突变稳定，并将从转基因拟南芥植株中进行亲和纯化，随后进行Cryo-EM。这些(亚)复合体不能通过异源(如大肠杆菌)的过度表达获得，因为需要以正确的顺序和化学计量组装十几个亚基，还涉及翻译后修饰和蛋白质激活剂。该项目建立在生物材料(表达亲和力标记的ClpP3、ClpP5、ClpR4、ClpT1、2和ClpC1-TRAP的转基因拟南芥植株系)以及叶绿体蛋白生物化学、质谱学和低温EM方面的大量先前投资的基础上。由于CLP复合体的丰度非常中等(约为Rubisco全息复合体的100倍)，证明可以用Cryo-EM确定其3D结构将对植物研究产生重大影响，因为它表明在异源系统中不需要过度表达蛋白质复合体。此外，叶绿体蛋白分解和蛋白质动态平衡在植物胁迫反应、农业和分子农业以及合成生物学中至关重要。这一奖项反映了NSF的法定使命，并通过使用基金会的智力优势和更广泛的影响审查标准进行评估，被认为值得支持。

Protein-protein interactions and protein complexes are critical for cellular viability. Single particle cryogenic electron microscopy (Cryo-EM) has accelerated the pace of high-resolution structure determination of these nano-size protein complexes. Cryo-EM has mostly been used on protein complexes generated in bacterial expression systems to obtain large amount of these proteins for Cryo-EM analysis. However, structural analysis of complexes obtained from their natural environment would be beneficial since these represent the most natural state of the complex. Indeed, the few examples of Cryo-EM of endogenous complexes (i.e. complexes obtained from their physiological environment) is limited to proteins of high relative abundance. Successful tool development and application of Cryo-EM to low abundant endogenous complexes affinity purified from its natural cellular environment will greatly impact biological discoveries, including for plants such as Arabidopsis thaliana. This project is focused on structural analysis of the Clp protease-chaperone system in chloroplasts of Arabidopsis thaliana. This Clp system is central in chloroplast protein homeostasis; insufficient Clp activity is detrimental to the plant. The proposed research will unravel the structural details of this unique plastid Clp chaperone-protease system and help understand its evolutionary adaptation to the chloroplast environment. The Broader Impacts of the work include the intrinsic merit resulting from the biological importance of the protease system, as well as the potential application of the developed methodology for other projects which require structural data from low abundant endogenous complexes in plants. Additionally, the project will provide training opportunities for undergraduate and graduate students.The proposed project aims to determine the structural organization by Cryo-EM of the endogenous Arabidopsis chloroplast ~1000 kDa Clp protease-chaperone complex (ClpPRTC) consisting of a tetradecameric barrel-shaped protease (P,R,T subunits) that dynamically associates with hexameric chaperone rings (C and D subunits). The full complex is stabilized by WalkerB mutations and will be affinity purified from transgenic Arabidopsis plants followed by Cryo-EM. These (sub)complexes cannot be obtained by heterologous (e.g. E. coli) overexpression because more than a dozen subunits need to be assembled in the right order and stoichiometries, involving also post-translational modifications and protein activators. This project is built on extensive prior investments in biological materials (transgenic Arabidopsis plant lines expressing affinity-tagged ClpP3, ClpP5, ClpR4, ClpT1,2 and ClpC1-TRAP) and expertise in chloroplast protein biochemistry, mass spectrometry and Cryo-EM. Since the Clp complex is of very moderate abundance (~100x lower than the Rubisco holocomplex), demonstration that one can determine its 3D structure by Cryo-EM will have a big impact on plant research because it demonstrates that one does not need to use overexpression of protein complexes in heterologous systems. Furthermore, chloroplast proteolysis and protein homeostasis are critical in plant stress response, agriculture and molecular farming and synthetic biology.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.