New Structural Mass Spectrometry Tools Applied to the Mitochondrial Membrane Prot

应用于线粒体膜保护的新型结构质谱工具

基本信息

批准号：
8690922
负责人：
Philip C Andrews
金额：
$ 34.96万
依托单位：
UNIVERSITY OF MICHIGAN AT ANN ARBOR
依托单位国家：
美国
项目类别：
财政年份：
2013
资助国家：
美国
起止时间：
2013-07-01 至 2017-05-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8690922
关键词：
Binding Binding Proteins Biological Models Cancer Etiology Cell Communication Cell Membrane Permeability Cells Chemical Agents Chemicals Chemistry Collaborations Complex Data Data Analyses Detergents Development Disease Environment Frequencies Gases Gatekeeping Goals Heating Knowledge Lead Link Lipids Macromolecular Complexes Malignant Neoplasms Maps Mass Spectrum Analysis Measurement Membrane Membrane Proteins Methodology Methods Michigan Mitochondria Mitochondrial Membrane Protein Mitochondrial Proteins Modeling Molecular Machines Nature Noise Phase Physiological Preparation Procedures Protein Dynamics Proteins Proteomics Protocols documentation Reagent Refractory Reporting Solutions Structural Models Structure System Techniques Technology Toxic effect Transmembrane Domain Tyrosine Universities Validation Work X-Ray Crystallography Yeasts base crosslink design extracellular improved instrument ion mobility method development mitochondrial membrane new technology protein complex protein folding protein structure public health relevance receptor small molecule stem stoichiometry structural biology surfactant therapeutic target tool trafficking

项目摘要

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.

描述（由申请人提供）：膜蛋白及其复合物充当细胞看门人，调节关键化学试剂的运输并充当高度选择性的细胞外受体。因此，膜蛋白是许多疾病（包括许多癌症）最有希望的潜在治疗靶点之一。许多膜蛋白对核磁共振和 X 射线晶体学等广泛使用的技术的难处理特性导致这些关键分子机器的结构信息明显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，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 (CXL-MS) 和离子淌度质谱 (IM-MS) 与建模技术相结合，能够填补我们目前在膜蛋白复合物结构和拓扑方面的知识空白。最近的报告展示了如何使用从 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机器的拓扑结构，这些拓扑结构挫败了所有其他详细结构表征的尝试。将这些方法的使用扩展到几个例子之外，并利用 MS 技术最近在膜蛋白结构测定方面所展现的前景，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，还将研究膜和去垢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构建更准确的 TspO 及其多聚体模型，并定义系统的蛋白质和脂质化学计量。然后，我们将扩展我们的技术来发现几种线粒体膜蛋白复合物的结构，这在癌症病因学中很重要。