Native architecture of the chloroplast protein-import channel

叶绿体蛋白质输入通道的天然结构

• 批准号: 2887609
• 金额: --
• 依托单位: University of Oxford
• 依托单位国家: 英国
• 项目类别: Studentship
• 财政年份: 2023
• 资助国家: 英国
• 起止时间: 2023 至 无数据
• 项目状态: 未结题
• 来源: https://gtr.ukri.org/projects?ref=studentship-2887609
• 关键词: Native; architecture; chloroplast; protein; import

This Project aims to decipher the mechanisms of protein transport by the TOC-TIC system at a molecular level directly from native membranes. This Project is very timely with the recent developments made in native mass spectrometry8-9 and cryo-tomography10-12 techniques where samples are analysed directly from native lipid environment without chemical disruptions. These techniques are relatively new and come with significant challenges. From both Parties' perspective, the overall goal of the Project is to develop methodologies to solve one of the major challenges of cell biology that has fundamental importance and broad applications. The methods developed in this Project would be highly beneficial to other researchers working on organelle membrane complexes and help cement the relationships with the Diamond in the future.Molecular biology techniques including cloning, PCR, DNA and RNA extraction, protein purification, SDS-PAGE, Western Blotting (JRB); Plant genetics and generation of tagged transgenic lines (JRB, PJ); enriching complexes by sonication (JRB); mass spectrometry including bottom-up and top-down (JRB, CVR); cryo-ET and FIB-SEM (PZ and CM); Data and statistical analysis; mass spectrometry (evaluating complex spectra and performing quantitative analysis) (JRB, CVR) cryo-ET and FIB-SEM (subtomogram averaging, 3D image reconstructions and quantitative image analysis) (PZ and CM).

本项目旨在直接从天然膜上解读TOC-TIC系统在分子水平上的蛋白质运输机制。该项目非常及时，因为最近在天然质谱（8-9）和低温层析（10-12）技术方面取得了进展，这些技术可以直接从天然脂质环境中分析样品，而不需要化学干扰。这些技术相对较新，并面临重大挑战。从双方的角度来看，该项目的总体目标是开发方法来解决细胞生物学的主要挑战之一，具有根本性的重要性和广泛的应用。本项目开发的方法将对其他研究细胞器膜复合物的研究人员非常有益，并有助于在未来巩固与钻石的关系。分子生物学技术包括克隆、PCR、DNA和RNA提取、蛋白质纯化、SDS-PAGE、Western Blotting (JRB)；植物遗传与标记转基因株系的产生（JRB， PJ）超声富集配合物（JRB）；质谱法包括自下而上和自上而下（JRB， CVR）；cryo-ET和FIB-SEM (PZ和CM)；数据和统计分析；质谱（评估复杂光谱并进行定量分析）（JRB， CVR） cryo-ET和FIB-SEM（亚层析图平均，3D图像重建和定量图像分析）（PZ和CM）。