Radiolytic Footprinting Methods for Structural Mass Spectrometry

结构质谱的放射分解足迹方法

• 批准号: 8070036
• 负责人: MARK R CHANCE
• 金额: $ 27.16万
• 依托单位: CASE WESTERN RESERVE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-05-10 至 2014-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8070036
• 关键词: Actins; Algorithms; Amino Acids; Binding; Biological; Biology; Cell membrane; Cells; Chemistry; Collaborations; Complex; Computing Methodologies; Coupled; Data; Detection; Development; Disease; Dissociation; Docking; Dose; Electron Transport; Environment; Exhibits; Filament; Future; G Protein-Coupled Receptor Genes; G-Protein-Coupled Receptors; GTP-Binding Proteins; Goals; Health; Hydroxyl Radical; Ions; Label; Laboratories; Lead; Location; Mass Spectrum Analysis; Mediating; Medicine; Membrane Proteins; Methods; Modeling; Modification; Monitor; Myosin ATPase; Noise; Pharmaceutical Preparations; Post-Translational Protein Processing; Process; Property; Protein Footprinting; Proteins; Reaction; Resolution; Rhodopsin; Side; Signal Transduction; Signaling Protein; Site; Solutions; Solvents; State Interests; Structure; Techniques; Technology; Time; Transducin; Water; Work; alanylglutamine; aspartylglutamate; base; cofilin; density; dimer; innovation; instrumentation; lysylglutamic acid; macromolecular assembly; millisecond; monomer; multiple reaction monitoring; novel; novel strategies; protein complex; public health relevance; research study; response; technology development

DESCRIPTION (provided by applicant): We have developed innovative methods of structural mass spectrometry based on hydroxyl radical modification of proteins. These structural mass spectrometry methods have recently gained widespread acceptance and are ripe for further development. In this proposal we will: increase the sensitivity of protein footprinting methods ~1000 fold; integrate docking approaches with protein footprinting data to probe the structure of protein complexes and develop methods to examine the dynamics of water in proteins using footprinting. Our preliminary data has shown feasibility for the examination of the G- protein coupled receptor rhodopsin in its ground and photo-activated states using increased x-ray flux density and shorter exposure times. Within Aim 1 we will use increased x-ray flux density to further explore the structural and solvent dynamics accompanying GPCR activation and the structural mechanism of signaling that mediates information to downstream signaling proteins. Our guiding hypothesis is that highly conserved structural motifs that include bound waters are reorganized to provide a highly controlled signaling channel. In Aim 2 we will further develop a novel O18 water labeling- radiolysis technique to examine the locations and dynamics of structural waters and the exchange properties of bulk water in multiple biological states of interest for rhodopsin and actin. In Aim 3 we will perfect targeted MS approaches to detect low abundance modifications in protein footprinting experiments to enhance the number of amino acids side chains routinely detected by these experiments. In Aim 4 we will develop computational methods of docking that incorporate footprinting data in structure determination. Our hypothesis is that use of footprinting data will drive correct selection of the correct structure among competing docking solutions that are of comparable energies. Optimized algorithmic approaches will be used to model complexes of myosin or cofilin with actin and complexes of rhodopsin and transducin. PUBLIC HEALTH RELEVANCE: G-protein coupled receptors are the targets of close to half of all drugs; a better understanding of their activation mechanisms would have a major impact on health and disease. Advanced technologies to examine the structure and dynamics of all proteins are needed in biology and medicine to better correlate structure and function.

描述（由申请人提供）：我们开发了基于蛋白质羟基自由基修饰的结构质谱的创新方法。这些结构质谱方法最近获得了广泛的接受，并且已经为进一步发展做好了准备。在本提案中，我们将： 将蛋白质足迹方法的灵敏度提高约 1000 倍；将对接方法与蛋白质足迹数据相结合，以探测蛋白质复合物的结构，并开发使用足迹来检查蛋白质中水动态的方法。我们的初步数据显示了使用增加的 X 射线通量密度和更短的曝光时间检查基态和光激活状态的 G 蛋白偶联受体视紫红质的可行性。在目标 1 中，我们将利用增加的 X 射线通量密度来进一步探索伴随 GPCR 激活的结构和溶剂动力学，以及将信息介导到下游信号蛋白的信号传导结构机制。我们的指导性假设是，包括结合水在内的高度保守的结构基序被重组，以提供高度受控的信号通道。在目标 2 中，我们将进一步开发一种新型 O18 水标记辐射分解技术，以检查结构水的位置和动态以及视紫红质和肌动蛋白在多种感兴趣的生物状态下散装水的交换特性。在目标 3 中，我们将完善靶向 MS 方法来检测蛋白质足迹实验中的低丰度修饰，以增加这些实验常规检测到的氨基酸侧链的数量。在目标 4 中，我们将开发对接计算方法，将足迹数据纳入结构确定中。我们的假设是，足迹数据的使用将推动在具有可比能量的竞争对接解决方案中正确选择正确的结构。优化的算法方法将用于模拟肌球蛋白或丝切蛋白与肌动蛋白的复合物以及视紫红质和转导蛋白的复合物。 公共健康相关性：G 蛋白偶联受体是近一半药物的靶标；更好地了解它们的激活机制将对健康和疾病产生重大影响。生物学和医学需要先进的技术来检查所有蛋白质的结构和动力学，以更好地关联结构和功能。