New Structural Mass Spectrometry Tools Applied to the Mitochondrial Membrane Prot

描述(申请人提供)：膜蛋白及其复合体起细胞守门人的作用，调节关键化学物质的运输，并作为高度选择性的细胞外受体。因此，膜蛋白是许多疾病最有希望的潜在治疗靶点之一，包括许多癌症。许多膜蛋白对核磁共振和X射线结晶学等广泛使用的技术的耐受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质的了解。膜蛋白结构通常高度依赖于局部膜环境，对于大多数结构探针来说，这往往会产生难以克服的化学噪声。化学交联质谱(CXL-MS)和离子迁移率-质谱仪(IM-MS)作为一种能够填补目前膜蛋白复杂结构和拓扑知识的空白的技术，与建模技术相结合已经出现。最近的报告展示了如何使用CXL-MS和IM-MS测量得出的距离和接触约束来推断高度复杂的大分子膜蛋白质机器的拓扑结构，这些拓扑结构已经挫败了所有其他详细结构表征的尝试。除了几个例子之外，扩大这些方法的使用，并利用MS技术最近展示的膜蛋白质结构测定的前景，将需要克服几个关键挑战。我们建议优化线粒体膜蛋白复合体的新结构质谱学技术。我们将使用线粒体蛋白TSPO的模型来建立我们的方法，并研究膜和洗涤剂条件对其结构的影响。从IM和CXL-MS获得的结构约束将用于建立更准确的TSPO及其多聚体模型，以及定义系统的蛋白质和脂肪化学计量。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体的结构，这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.