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守门人，调节关键化学试剂的运输，并作为高选择性细胞外受体。因此，膜蛋白是许多疾病（包括许多癌症）的最有希望的潜在治疗靶点之一。许多膜蛋白质对广泛使用的技术如NMR和X射线晶体学的难治性已经导致关于这些关键分子机器的结构信息显著落后于我们对可溶性蛋白质的认识。膜蛋白结构通常高度依赖于局部的膜环境，这通常会对大多数结构探针产生难以克服的化学噪音。化学交联质谱法（CXL-MS）和离子迁移率质谱法（IM-MS）已经成为能够填补我们目前在膜蛋白复合物结构和拓扑结构方面的知识空白的技术，当与建模技术相结合时。最近的报告已经证明了如何从CXL-MS和IM-MS测量的距离和接触的限制，可以用来推断拓扑结构的高度复杂的大分子膜蛋白机器，挫败了所有其他的尝试详细的结构表征。将这些方法的使用扩展到几个例子之外，并利用MS技术最近展示的用于膜蛋白结构测定的前景，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物的新结构质谱技术。我们将使用模型线粒体蛋白TspO来构建我们的方法，并研究膜和洗涤剂条件对其结构的影响。从IM和CXL-MS获得的结构约束将被用来建立更准确的模型TspO及其多聚体，以及定义的蛋白质和脂质的化学计量的系统。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.