New Structural Mass Spectrometry Tools Applied to the Mitochondrial Membrane Prot

描述（由申请人提供）：膜蛋白及其复合物作为细胞看门人，调节关键化学制剂的运输，并作为高选择性的细胞外受体。因此，膜蛋白是包括许多癌症在内的许多疾病最有希望的潜在治疗靶点之一。许多膜蛋白对广泛使用的核磁共振和x射线晶体学等技术的难降解性导致这些关键分子机器的结构信息远远落后于我们对可溶性蛋白的了解。膜蛋白结构通常高度依赖于局部膜环境，这通常会对大多数结构探针产生不可克服的化学噪声。化学交联质谱法（CXL-MS）和离子迁移质谱法（IM-MS）结合建模技术，填补了我们目前在膜蛋白复合物结构和拓扑知识方面的空白。最近的报告表明，从CXL-MS和IM-MS测量中得出的距离和接触约束如何用于推断高度复杂的大分子膜蛋白机器的拓扑结构，这些机器在详细结构表征方面的所有其他尝试都失败了。扩大这些方法的应用范围，并利用质谱技术最近在膜蛋白结构测定方面所展示的前景，将需要克服几个关键挑战。我们建议优化新的线粒体膜蛋白复合物结构质谱技术。我们将使用线粒体蛋白TspO模型来构建我们的方法，并将研究膜和洗涤剂条件对其结构的影响。从IM和CXL-MS中获得的结构约束将用于建立更精确的TspO及其多聚体模型，并定义该系统的蛋白质和脂质化学计量学。然后，我们将扩展我们的技术，以发现几个线粒体膜蛋白复合物的结构，在癌症病因学中很重要。

DESCRIPTION (provided by applicant): Membrane proteins and their complexes act as cellular gatekeepers, regulating the traffic of critical chemical agents and acting as highly-selective extracellular receptors. As such, membrane proteins are amongst the most promising potential therapeutic targets for a host of diseases, including many cancers. The refractory nature of many membrane proteins to widely- used technologies such as NMR and X-ray crystallography has resulted in structural information on these critical molecular machines lagging significantly behind our knowledge of soluble proteins. Membrane protein structure is often highly dependent on the local membranous environment, which often generates an insurmountable level of chemical noise for most structural probes. Chemical cross-linking mass spectrometry (CXL-MS) and Ion Mobility-Mass Spectrometry (IM-MS) have emerged as technologies capable of filling our current gap in knowledge of membrane protein complex structure and topology when combined with modeling techniques. Recent reports have demonstrated how distance and contact constraints derived from CXL-MS and IM-MS measurements can be used to deduce topologies for highly-complex macromolecular membrane protein machines that have frustrated all other attempts at detailed structural characterization. Expanding the use of these approaches beyond a few examples and capitalizing on the recent promise demonstrated by MS technologies for membrane protein structure determination will require overcoming several key challenges. We propose to optimize new structural mass spectrometry technologies for mitochondrial membrane protein complexes. We will use the model mitochondrial protein TspO to build our approach, and will also study the influence of membrane and detergent conditions on its structure. The structural constraints obtained from IM and CXL-MS will be used to build more accurate models of TspO and its multimers, as well as define the protein and lipid stoichiometries for the system. We will then extend our technologies to discover the structure of several mitochondrial membrane protein complexes, important in cancer etiology.