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

Tools4Cells:EAGER：通过内源复合物的亲和纯化对叶绿体 CLP 蛋白酶系统进行冷冻电镜分析

• 批准号: 2222495
• 负责人: Klaas van Wijk
• 金额: $ 30万
• 依托单位: Cornell University
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-06-15 至 2025-05-31
• 项目状态: 未结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=2222495&HistoricalAwards=false
• 关键词: Tools4Cells; EAGER; CRYO; EM; ANALYSIS

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.

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）加快了这些纳米尺寸蛋白质复合物的高分辨率结构测定的步伐。Cryo-EM主要用于细菌表达系统中产生的蛋白质复合物，以获得大量的这些蛋白质用于Cryo-EM分析。然而，从其自然环境中获得的复合物的结构分析将是有益的，因为这些代表了复合物的最自然状态。事实上，内源性复合物（即从其生理环境中获得的复合物）的Cryo-EM的少数实例仅限于相对丰度高的蛋白质。成功的工具开发和Cryo-EM对从其天然细胞环境中纯化的低丰度内源性复合物的亲和力的应用将极大地影响生物学发现，包括对植物如拟南芥的发现。本项目主要研究拟南芥叶绿体Clp蛋白酶-分子伴侣系统的结构分析。该Clp系统在叶绿体蛋白质稳态中是中心的;不足的Clp活性对植物是有害的。这项研究将揭示这种独特的质体Clp分子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重要性所带来的内在价值，以及所开发的方法在其他项目中的潜在应用，这些项目需要植物中低丰度内源性复合物的结构数据。该项目旨在通过Cryo-EM确定拟南芥叶绿体~1000 kDa Clp蛋白酶-伴侣蛋白复合物（ClpPRTC）的结构组织，该复合物由一个十四聚体的桶形蛋白酶（P，R，T亚基）和六聚体的伴侣蛋白环（C和D亚基）组成。完整的复合物通过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Cryo-EM。这些（亚）复合物不能通过异源（例如E.大肠杆菌）过表达，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。该项目建立在对生物材料（表达亲和标记的ClpP 3，ClpP 5，ClpR 4，ClpT 1，2和ClpC 1-TRAP的转基因拟南芥植物系）的广泛投资以及叶绿体蛋白生物化学，质谱和Cryo-EM的专业知识基础上。由于Clp复合物的丰度非常适中（比Rubisco全复合物低约100倍），因此可以通过Cryo-EM确定其3D结构的证明将对植物研究产生重大影响，因为它表明不需要在异源系统中使用蛋白质复合物的过表达。此外，叶绿体蛋白水解和蛋白质稳态在植物胁迫反应、农业、分子农业和合成生物学中至关重要。该奖项反映了NSF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。