Molecular architecture and mechanism of the chloroplast's beta-barrel assembly machinery (ChlAM)

细胞生物学和生物化学中的一个基本问题是β-桶膜蛋白如何组装成革兰氏阴性细菌和内共生细胞器（线粒体和叶绿体）的外膜。这是非常重要的，因为许多这些细胞器的功能取决于参与运输离子，代谢物和蛋白质的必需β-桶蛋白的正确组装。因此，对这一过程的完整机制理解可能使我们能够修改或控制β桶生物发生，以帮助开发针对耐药细菌或线粒体疾病的特定疗法。此外，这一认识将有助于提高作物产量（因为叶绿体负责光合作用），从而解决人口需求和气候变化的压力。我们目前对细菌和线粒体β桶组装机制的了解使我们难以辨别三个模型系统之间的共同基本原则、共性和差异。此外，许多关键的机械步骤仍然不清楚，包括组装机器如何识别，折叠和释放基板。这主要是由于我们无法捕捉这个过程中涉及的瞬态相互作用。基于我最近的发现，本机质谱（MS）可以有效地捕捉这些瞬态相互作用和所获得的信息，可以指导结构分析，我建议破译themolecular架构和机制的叶绿体β-桶装配机械，从而有助于更好地理解一个统一的机械模型。这个雄心勃勃的项目整合了结构生物学中的几个最新突破性发现，如原生MS，自上而下MS，质量光度学和cryo-EM。在过去一年，我已奠定了基础，并制定了一个管道，以促进这些研究，并证明了该项目的整体可行性。

A fundamental question in cell biology and biochemistry is how beta-barrel membrane proteins assemble into the outer membrane of Gram-negative bacteria and endosymbiotic organelles (mitochondria and chloroplasts). This is of profound importance because the function of many of these organelles depends upon the proper assembly of essential beta-barrel proteins involved in transporting ions, metabolites and proteins. Thus, a complete mechanistic understanding of this process may enable us to modify or control beta-barrel biogenesis to aid the development of specific therapies targeting resistant bacteria or mitochondrial diseases. Moreover, thisunderstanding will contribute to the development of crops with enhanced yields (since chloroplasts are responsible for photosynthesis), thereby addressing population needs and pressure from climate change.Our current knowledge of bacterial and mitochondrial beta-barrel assembly machineries makes it difficult to discern common underlying principles, generalities, and disparities among three model systems. Furthermore, many critical mechanistic steps remain unclear, including how the assembly machineries recognise, fold, and release substrates. This is primarily due to our inability to capture the transient interactions involved within this process. Based on my recent discovery that native mass spectrometry (MS) can efficiently capture these transient interactions and the information obtained that can guide structural analysis, I propose to decipher themolecular architecture and mechanism of the chloroplast beta-barrel assembly machinery and thereby contribute to a greater understanding of a unified mechanistic model. This ambitious project integrates several recent breakthrough discoveries in structural biology, such as native MS, top-down MS, mass photometry, and cryo-EM. Over the last year, I have laid the foundation and developed a pipeline to facilitate these studies and have demonstrated the project's overall feasibility.