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

蛋白质-蛋白质相互作用和蛋白质复合物对细胞活力至关重要。单粒子低温电子显微镜（Cryo-EM）加快了这些纳米级蛋白质复合物的高分辨率结构测定的步伐。冷冻电镜主要用于细菌表达系统中产生的蛋白质复合物，以获得大量用于冷冻电镜分析的蛋白质。然而，从其自然环境中获得的配合物的结构分析将是有益的，因为它们代表了配合物的最自然状态。事实上，内源性复合物（即从其生理环境中获得的复合物）的低温电子显微镜的少数例子仅限于相对丰度高的蛋白质。低温电镜（Cryo-EM）工具的成功开发和应用将极大地影响生物学的发现，包括拟南芥等植物。本项目主要研究拟南芥叶绿体中Clp蛋白酶-伴侣系统的结构分析。该Clp系统在叶绿体蛋白稳态中起中心作用；Clp活性不足对植物有害。该研究将揭示这种独特的叶绿体伴侣-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。这项工作的广泛影响包括蛋白酶系统的生物学重要性所带来的内在价值，以及开发的方法在其他项目中的潜在应用，这些项目需要来自植物中低丰度内源性复合物的结构数据。此外，该项目将为本科生和研究生提供培训机会。本项目旨在通过低温电镜技术确定内源拟南芥叶绿体~1000 kDa Clp蛋白酶-伴侣复合物（ClpPRTC）的结构组织，该复合物由一个四聚体桶状蛋白酶（P，R，T亚基）组成，该蛋白酶与六聚体伴侣环（C和D亚基）动态结合。整个复合物通过WalkerB突变稳定，并将从转基因拟南芥植物中亲和纯化，然后进行冷冻电镜分析。这些（亚）复合物不能通过异源（如大肠杆菌）过表达获得，因为需要以正确的顺序和化学计量学组装十多个亚基，还涉及翻译后修饰和蛋白质激活剂。该项目建立在对生物材料（表达亲和标记的ClpP3、ClpP5、ClpR4、ClpT1、2和ClpC1-TRAP的转基因拟南芥植物品系）和叶绿体蛋白生物化学、质谱和低温电镜技术的广泛投资基础上。由于Clp复合物的丰度非常中等（比Rubisco整体复合物低约100倍），通过Cryo-EM确定其3D结构的证明将对植物研究产生重大影响，因为它表明人们不需要在异种系统中使用蛋白质复合物的过表达。此外，叶绿体蛋白质水解和蛋白质稳态在植物逆境响应、农业和分子农业以及合成生物学中起着至关重要的作用。该奖项反映了美国国家科学基金会的法定使命，并通过使用基金会的知识价值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.