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于细胞活力至关重要。单个颗粒低温电子显微镜（Cryo-EM）加速了这些纳米大小蛋白质复合物的高分辨率结构测定的速度。冷冻EM主要用于细菌表达系统中产生的蛋白质复合物，以获取大量这些蛋白质进行冷冻EM分析。但是，从其自然环境中获得的复合物的结构分析将是有益的，因为它们代表了综合体的最自然状态。实际上，内源性复合物的低温EM（即从其生理环境获得的复合物）的几个例子仅限于相对丰度高的蛋白质。从其自然细胞环境中纯化纯净的工具开发和应用冷冻EM在低丰富的内源性复合物中的亲和力将极大地影响生物学发现，包括拟南芥等植物。该项目的重点是拟南芥叶绿体中CLP蛋白酶 - 链酮系统的结构分析。该CLP系统在叶绿体蛋白稳态中是中心的。 CLP活性不足对植物有害。拟议的研究将揭示此独特的质体CLP伴侣 - 蛋白酶系统的结构细节，并帮助了解其对叶绿体环境的进化适应。这项工作的更广泛的影响包括蛋白酶系统的生物学重要性产生的内在优点，以及开发方法对其他项目的潜在应用，这些项目需要从植物中低丰富的内源性复合物中进行结构性数据。此外，该项目还将为本科和研究生提供培训机会。拟议的项目旨在确定内源性拟南芥叶绿体的冷冻结构组织〜1000 kDa Clp蛋白酶 - 蛋白酶 - 蛋白酶 - 蛋白酶 - CLPPRTC（CLPPRTC），由四型桶形桶形形成蛋白酶（P，R，T亚基）与六聚体伴侣环（C和D亚基）动态关联。完整的复合物通过步行者突变稳定，并将是从转基因拟南芥植物中纯化的，然后是冷冻EM。这些（子）复合物不能通过异源（例如大肠杆菌）过表达获得，因为需要以正确的顺序和石化核法规组装十几个亚基，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（表达亲和力标记的CLPP3，CLPP5，CLPR4，CLPR4，CLPT1,2和CLPC1-TRAP）和叶绿体蛋白质生物化学，质谱和冰冻质量的植物生物学植物系和叶绿体蛋白质生物化学，质谱和冷冻 - 叶绿体蛋白质生物化学方面的专业知识。由于CLP复合物的丰度非常中等（比Rubisco全胶质复合物低约100倍），因此证明可以通过Cryo-EM确定其3D结构对植物研究的影响很大，因为它表明不需要使用过度表达异源系统中的蛋白质复合物。此外，叶绿体蛋白水解和蛋白稳态对于植物压力反应，农业和分子种植和合成生物学至关重要。该奖项反映了NSF的法定任务，并被认为是通过基金会的知识分子优点和更广泛的影响审查标准来通过评估来通过评估来支持的。