Innovative Native Ion Mobility Approaches for Transformational Measurements in Structural Biology

用于结构生物学转化测量的创新天然离子淌度方法

• 批准号: 10477459
• 负责人: Brian Clowers
• 金额: $ 28.85万
• 依托单位: TEXAS A&M UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-09-05 至 2024-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10477459
• 关键词: ATP Hydrolysis; ATP-Binding Cassette Transporters; Address; All-Trans-Retinol; Amyloidosis; Area; Binding; Biological; Biological Assay; Biological Process; Biophysics; Breathing; Catalysis; Cerebrospinal Fluid; Charge; Chemistry; Complement; Complex; Coupling; Cryoelectron Microscopy; Data; Development; Dissociation; Escherichia coli; Event; Formulation; Fourier Transform; Germ-Line Mutation; Goals; Homo; Individual; Investigation; Ions; Kinetics; Life; Ligand Binding; Ligands; Lipid Binding; Lipids; Mass Spectrum Analysis; Measurement; Membrane Proteins; Membrane Transport Proteins; Modernization; Modification; Molecular Conformation; Monitor; Motion; Noise; Nucleotides; Outcome; Peptide Conformation; Periodicity; Pharmaceutical Preparations; Physics; Physiological; Plasma; Positioning Attribute; Post-Translational Protein Processing; Prealbumin; Protein Dynamics; Proteins; Proteomics; Recombinants; Research; Research Personnel; Resolution; Roentgen Rays; Seminal; Shapes; Signal Transduction; Site; Structure; Surface; Techniques; Technology; Thermodynamics; Thyroxine; Time; Tube; X-Ray Crystallography; base; biophysical techniques; comparative; experience; frontier; improved; innovation; insight; instrument; instrumentation; ion mobility; mass analyzer; mass spectrometer; member; monomer; non-Native; novel; preservation; protein complex; protein expression; protein folding; protein purification; protein structure; protein structure function; prototype; rapid growth; self assembly; small molecule; stoichiometry; structural biology; success; technique development; time use

Project Summary Advancements in biophysical techniques, such as X-ray and cryoEM, have undoubtedly accelerated determination of protein structure. However, it still remains challenging to capture snapshots of protein folding intermediates, including non-native states, and breathing motions that protein assemblies undergo to perform their biological function. Moreover, understanding how molecules, such as lipids, modulate protein structure and function is of paramount biological importance. Over the past two decades, mass spectrometry (MS) of intact protein complexes, often referred to as native MS, has emerged as an indispensable biophysical technique whereby non-covalent interactions and protein structure are preserved within the mass spectrometer. Native MS is a rapid and sensitive technique that has already provided invaluable information on subunit stoichiometry and topology, allostery and cooperativity for individual ligand binding events, including their binding thermodynamics. The coupling with ion mobility (IM), a separation technique based on molecule charge and shape, further enhances the capabilities of native MS where it has enabled collision cross section (CCS) measurements for large protein complexes, identification of different conformations for peptides and stabilizing ligands using collision induced unfolding, and insight in folded and denatured structure(s) of proteins. However, low- resolution commercial IM-MS instrumentation has not changed since its introduction 12 years ago. Herein, this proposal seeks to develop transformative native IM-MS technologies with high-resolution IM and MS capabilities that can address modern questions in structural biology, such as conformational dynamics, including those that may have remained “hidden”, within membrane transporters under turnover conditions. In order to achieve these transformative goals, an interdisciplinary team of researchers whose expertise spans the fields of protein biophysics, expression and purification of proteins inclusive of membrane proteins, as well as traditional protein structure characterization, such as X-ray crystallography, has been assembled. Team members also possess decades of experience in the field of mass spectrometry inclusive of fundamental ion chemistry/physics, seminal contributions that have spawned MS proteomics, and related areas of analytical mass spectrometry and ion mobility- mass spectrometry. Collectively, the background and expertise of this research team is uniquely positioned to transform the field of IM-MS in the area of structural biology. In short, the proposed transformative research will lead to forefront IM-MS instrumentation that is poised to provide unprecedented insights into the structure and assembly of protein complexes and push the field into new frontiers of research.

项目概要 X 射线和冷冻电镜等生物物理技术的进步无疑加速了 然而，捕获蛋白质快照仍然具有挑战性。 折叠中间体，包括非天然状态，以及蛋白质组装的呼吸运动 此外，了解分子（例如脂质）如何发挥其生物学功能。 调节蛋白质结构和功能在过去两年中具有至关重要的生物学意义。 几十年来，完整蛋白质复合物的质谱 (MS)（通常称为天然 MS）已 成为一种不可或缺的生物物理技术，通过非共价相互作用和蛋白质 结构保留在质谱仪内 Native MS 是一种快速且灵敏的技术。 这已经提供了有关亚基化学计量和拓扑、变构和 单个配体结合事件的合作，包括它们的结合热力学。 离子淌度 (IM) 是一种基于分子电荷和形状的分离技术，进一步增强了 原生 MS 的功能，可实现大型碰撞截面 (CCS) 测量 蛋白质复合物，鉴定肽的不同构象并使用稳定配体 碰撞诱导展开，以及了解蛋白质的折叠和变性结构。 商用 IM-MS 仪器的分辨率自 12 年前推出以来一直没有改变。 在此，该提案旨在开发具有高分辨率的变革性原生 IM-MS 技术 IM 和 MS 功能可以解决结构生物学中的现代问题，例如构象 动态，包括那些可能仍然“隐藏”在膜转运蛋白内的动态 为了实现这些变革目标，需要一个跨学科团队。 研究人员的专业知识涵盖蛋白质生物物理学、表达和纯化领域 蛋白质，包括膜蛋白，以及传统的蛋白质结构表征，例如 作为X射线晶体学，已经聚集的团队成员也拥有数十年的经验。 在质谱领域，包括基础离子化学/物理学，具有开创性贡献 催生了 MS 蛋白质组学以及分析质谱和离子淌度的相关领域 - 总的来说，该研究团队的背景和专业知识是独一无二的。 简而言之，该提议旨在改变结构生物学领域的 IM-MS 领域。 变革性研究将带来最前沿的 IM-MS 仪器，有望提供 对蛋白质复合物的结构和组装的前所未有的见解，并将该领域推向 研究新领域。