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.

项目总结/摘要 大分子功能的核心是它们为了实现其功能而经历的构象动力学。 生物功能。天然质谱（MS），其中非共价相互作用保留在质谱中。 质谱仪，正在成为研究蛋白质化学计量，拓扑结构，动力学， 动力学和蛋白质-配体相互作用。原生MS加上离子迁移率（IM-MS），报告 大分子形状，是革命性的蛋白质和蛋白质复合物的大构象是 分析和理解。然而，现有的商业IM-MS仪器受限于相对低的成本。 分辨能力，使它们能够根据形状的差异描绘不同的结构。 本文介绍了一种小型多程选择泛频迁移率谱仪的研制方案 （M-SOMS）装置，其可插入通常用于结构研究的商业平台中。的 目标是在第一年将分辨率提高2至5倍;最终， M-SOMS装置将是可调谐的，使得高分辨率光谱（具有大于100 nm的分辨能力）可以被测量。 一个数量级大于目前可用）将访问任何研究人员使用 商业平台。M-SOMS装置将使用单体进行开发、优化和验证。 和低聚泛素系统（以及金属硫蛋白-金属络合物），并应用于解决更大， 更复杂的蛋白质和蛋白质-配体系统。具体而言，高分辨率将使其能够 辨别与胍核苷酸复合的癌蛋白RAS的小构象差异， 类似物以及其他效应蛋白。最后，膜蛋白水通道蛋白， 通道，与脂质复合物将与M-SOMS仪器进行研究。后两项研究将 这是对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-SOMS将 在结构生物学和相关领域中具有显著的影响， 被捕获并为分子建模提供高分辨率的迁移率限制。