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

项目摘要/摘要 结构生物学巧妙地为我们提供了完整的、恰当的- 组装的蛋白质；然而，我们关于该结构的结构信息极其有限。 这些美丽的建筑背后的过程。因为新生蛋白质是时间依赖的， 异质的，与核糖体和其他共同作用的细胞机制密切相关的 从翻译上讲，新生蛋白质超出了现有的结构和生物物理技术的掌握，这些技术 需要纯样本，并在体外进行。我们结构框架中的这个盲点令人担忧， 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 间隔区-负责囊性纤维化、神经变性、 衰老和癌症。事实上，因为大多数折叠研究都是通过对纯化的蛋白质进行重折叠来进行的 变性之后-与现实生活中蛋白质折叠的方式如此脱节，他们做出了贡献 相对较少的药物开发来治疗由蛋白质错误折叠和 装配错误。为了弥补这一差距，我的研究小组正在开发新的工具和方法来捕获 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。 具体地说，我们利用了交联质谱(XL-MS)--一种新的结构分析技术 生物学--通过交联剂将空间信息转化为共价键。距离 通过用质谱仪对产生的交联肽片段进行测序来推断限制条件。 然后，这些约束被用来对3-D蛋白质结构进行计算建模，或者将一个蛋白质与 尊重他人。本计划书中描述的创新展示了我们如何扩展XL-MS工具包 能够捕捉蛋白质折叠中间体在生物合成过程中的瞬时结构 细胞。 我们产生的技术将立即被其他研究蛋白质折叠的科学家所用， 翻译和质量控制；更广泛地说，它们将作为如何执行生物物理的范例 在其自然细胞环境中进行生物分子的实验。这一范式转变将使我们能够解决 一些紧迫的生物医学重要问题，具体地说：(1)基因突变是如何 疾病会导致蛋白质错误折叠？这些错综复杂的国家是什么结构，毒品可能是 被设计为在这个阶段进行干预？(2)当细胞老化时，蛋白质组到底发生了什么？(3)至于淀粉样蛋白- 形成蛋白质的细胞中可溶低聚物的结构和相互作用是什么？ 环境(到目前为止，现有技术还没有对其进行表征)，以及是什么使它们 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?