Watching Proteins Fold (or Misfold) in vivo with Mass Spectrometry

项目总结/摘要 结构生物学非常擅长为我们提供高分辨率的完整图像，正确的- 组装的蛋白质;然而，我们对"结构“的结构信息非常有限。 这些美丽的建筑是如何形成的。因为新生蛋白质是时间依赖性的， 异质性，并密切相关的核糖体和其他细胞机制，共同作用， 从理论上讲，新生蛋白质超出了现有结构和生物物理技术的掌握， 需要纯样品，并在体外进行。我们结构框架中的这个盲点令人不安， 因为新生阶段是蛋白质最有可能错误折叠或被错误定位为不正确的蛋白质的时候。 隔室-负责多种病理的过程，如囊性纤维化，神经变性， 衰老和癌症事实上，因为大多数关于折叠的研究--通过重新折叠纯化的蛋白质来进行 它们与蛋白质在真实的生命中如何折叠是如此的脱节， 相对较少的药物开发，以治疗由蛋白质错误折叠引起的许多疾病， 错误装配。为了解决这一差距，我的研究小组正在开发新的工具和方法， 蛋白质折叠中间体在活细胞中合成时的结构和动力学信息。 具体而言，我们利用交联质谱法（XL-MS）-一种新兴的结构分析技术， 生物学-其中空间信息通过交联剂转化为共价键。距离 通过用质谱法对所得交联肽片段进行测序来推断限制。 这些约束然后被用于计算模型的3-D蛋白质结构或对接一个蛋白质与 尊重另一个。本提案中描述的创新显示了我们如何扩展XL-MS工具包 能够捕捉蛋白质折叠中间体的瞬时结构，因为它们在生物合成中， 细胞 我们所产生的技术将立即用于其他研究蛋白质折叠的科学家， 翻译和质量控制;更广泛地说，它们将作为如何进行生物物理 生物分子在其自然细胞环境中的实验。这种模式的转变将使我们能够解决一个 一些紧迫的问题，生物医学的重要性，具体而言：（1）如何突变的遗传 疾病导致蛋白质错误折叠？这些错误折叠状态的结构是什么，药物是否可能是 在这个阶段进行干预吗(2)随着细胞衰老，蛋白质组到底发生了什么？(3)至于淀粉样蛋白- 形成蛋白质，它们的细胞中可溶性寡聚体形式的结构和相互作用是什么？ 环境（到目前为止，这些环境还没有被现有的技术所描述），是什么使它们 细胞毒性？

Project Summary/Abstract Structural biology is exquisitely adept at providing us with high-resolution images of completed, properly- assembled proteins; however, we have extremely limited structural information about the `construction process' behind how these beautiful structures come to be. Because nascent proteins are time-dependent, heterogeneous, and intimately associated with the ribosome and other cellular machineries that act co- translationally, nascent proteins are beyond the grasp of existing structural and biophysical techniques, which require pure samples and are carried out in vitro. This blind-spot in our structural framework is troubling, because the nascent stage is when proteins are most likely to misfold or become mistargeted to an incorrect compartment – processes responsible for pathologies as diverse as cystic fibrosis, neurodegeneration, ageing, and cancer. Indeed, because most studies of folding – carried out by refolding a purified protein following denaturation – are so disconnected from how proteins fold in real life, they have contributed relatively little to the development of medicines to treat the many diseases caused by protein misfolding and misassembly. To address this gap, my research group is developing new tools and approaches to capture structural and kinetic information of protein folding intermediates as they are being synthesized in living cells. Specifically, we utilize crosslinking mass spectrometry (XL-MS) – an emerging technique in structural biology – in which spatial information is converted into covalent bonds via crosslinking agents. Distance restraints are inferred by sequencing the resulting crosslinked peptide fragments with mass spectrometry. These restraints are then used to computationally model a 3-D protein structure or dock one protein with respect to another. The innovations described in this proposal show how we are expanding the XL-MS toolkit to be able to capture transient structures of protein folding intermediates as they are being biosynthesized in cells. The technologies that we generate will be of immediate use to other scientists studying protein folding, translation, and quality control; more broadly, they will serve as a paradigm of how to perform biophysical experiments on biomolecules in their native cellular milieu. This paradigm shift will enable us to address a number of pressing questions of biomedical importance, specifically: (1) How do mutations in genetic diseases cause proteins to misfold? What is the structure of these misfolded states, and could drugs be designed to intervene at this stage? (2) What actually happens to proteomes as cells age? (3) As for amyloid- forming proteins, what are the structures and interactions of the soluble oligomeric forms in their cellular environment (which have so far eluded characterization by existing techniques), and what makes them cytotoxic?