Development of high resolution mobility measurements for structural biology

结构生物学高分辨率迁移率测量的开发

• 批准号: 9383630
• 负责人: DAVID E. CLEMMER
• 金额: $ 47.77万
• 依托单位: TRUSTEES OF INDIANA UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-07-15 至 2021-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9383630
• 关键词: Back; Benchmarking; Binding; Biological Process; Complex; Complex Analysis; Coupled; Cysteine; Development; Devices; Disease; Guanidines; Ions; KRAS2 gene; Kinetics; Laboratories; Lead; Ligand Binding; Ligands; Lipids; Malignant Neoplasms; Mass Spectrum Analysis; Measurement; Membrane Proteins; Metallothionein; Molecular Conformation; Molecular Models; Monomeric GTP-Binding Proteins; Mutation; Nucleotides; Oncoproteins; Performance; Program Development; Protein Conformation; Protein Dynamics; Proteins; Reporting; Research Personnel; Resolution; Sampling; Shapes; Spectrometry; Structure; System; Techniques; Technology; Time; Ubiquitin; Ubiquitination; Validation; Work; base; design; experimental study; improved; instrument; instrumentation; ion mobility; macromolecule; mass spectrometer; metal complex; molecular modeling; monomer; novel; nucleotide analog; programs; protein complex; protein function; restraint; stoichiometry; structural biology; water channel

Project Summary/Abstract Central to the function of macromolecules are the conformational dynamics they undergo in order to carry out biological function. Native mass spectrometry (MS), whereby non-covalent interactions are preserved in the mass spectrometer, is emerging as a powerful technique to study protein stoichiometry, topology, dynamics, and kinetics and protein-ligand interactions. Native MS coupled with ion mobility (IM-MS), which reports on macromolecule shape, is revolutionizing how large conformations of proteins and protein complexes are analyzed and understood. However, existing commercial IM-MS instrumentation is limited by relatively low resolving powers that render their ability to delineate different structures based on differences in their shapes. This proposal describes a program for developing a small, multipass selected overtone mobility spectrometry (M-SOMS) device that can be inserted into commercial platforms commonly used for structural studies. The aim is to improve resolving power in the first year by a factor of 2 to 5 fold; ultimately the resolving power of the M-SOMS device will be tunable, such that high-resolution spectra (having resolving powers that are more than an order of magnitude greater than currently available) will be accessible to any researchers using the commercial platforms. The M-SOMS device will be developed, optimized, and validated using the monomeric and oligomeric ubiquitin system (as well as metallothionein–metal complexes) and applied to tackle larger, more complex protein and protein-ligand systems. Specifically, the high-resolving power will make it possible to discern small conformational differences for the oncoprotein RAS in complex with guanidine nucleotides and analogs, as well as other effector proteins. Lastly, the membrane protein aquaporin, a tetrameric water channel, in complex with lipids will be investigated with the M-SOMS instrument. The latter two studies will represent the first high-resolution mobility study of an intact protein complex. We envisage that M-SOMS will have a significant impact in structural biology and related fields by enabling a number for conformational states to be captured and providing high-resolution mobility restraints for molecular modeling.

项目摘要/摘要 大分子功能的核心是他们进行的构象动力学 生物功能。天然质谱法（MS），从而保留了非共价相互作用 质谱仪正在成为研究蛋白质化学计量，拓扑，动力学， 以及动力学和蛋白质 - 配体相互作用。本机MS与离子迁移率（IM-MS）相结合，报告 大分子形状正在彻底改变蛋白质和蛋白质复合物的大构象 分析和理解。但是，现有的商业IM-MS仪器受到相对较低的限制 解决能力，从而使他们根据其形状差异来描述不同结构的能力。 该提案描述了一个用于开发小型多量选定的泛音移动光谱法的程序 （M-SOM）可以插入常用于结构研究的商业平台。 目的是在第一年提高解决能力，提高2至5倍。最终的解决力 M-SOMS设备将是可调节的，因此高分辨率光谱（具有超过超过的解决功率 任何研究人员都可以使用比目前可用的数量级。 商业平台。 M-SOMS设备将使用单体开发，优化和验证 和寡聚泛素系统（以及金属氨酸金属络合物），并应用于更大的问题 更复杂的蛋白质和蛋白质 - 配体系统。具体而言，高分辨率的力量将使 辨别与鸟嘌呤核苷酸复合物中的癌蛋白RA的小小的协商差异， 类似物以及其他效应蛋白。最后，膜蛋白水通道蛋白，四聚体水 通道将使用M-SOMS仪器进行研究。后两个研究将 代表完整蛋白质复合物的首次高分辨率迁移率研究。我们设想M-SOM会 通过启用会议状态的数字，对结构生物学和相关领域产生重大影响 捕获并为分子建模提供高分辨率的迁移率限制。