Innovative Native Ion Mobility Approaches for Transformational Measurements in Structural Biology

项目摘要 生物物理技术的进步，如X射线和低温电子显微镜，无疑加速了 蛋白质结构的测定。然而，捕捉蛋白质的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 来执行它们的生物功能。此外，了解分子，如脂类， 调节蛋白质的结构和功能具有极其重要的生物学意义。在过去两年中 几十年来，完整蛋白质复合体的质谱学(MS)，通常被称为天然MS，已经 作为一种不可或缺的生物物理技术，非共价相互作用和蛋白质 结构被保存在质谱计内。自然多发性硬化症是一种快速而敏感的技术 它已经提供了关于亚基化学计量和拓扑、变构和 单个配体结合事件的协作性，包括它们的结合热力学。联结 离子迁移率(IM)是一种基于分子电荷和形状的分离技术，它进一步增强了 本机MS的功能，其中它启用了对大型 蛋白质复合体、多肽和稳定配体不同构象的鉴定 碰撞诱导蛋白质展开，洞察蛋白质折叠和变性结构(S)。然而，低- 分辨率商业IM-MS仪器自12年前推出以来一直没有改变。 在此，该提案寻求开发具有变革性的高分辨率原生IM-MS技术 IM和MS能力，可以解决结构生物学中的现代问题，如构象 动力学，包括那些可能仍然“隐藏”的，在膜转运体内 周转条件。为了实现这些变革性的目标，一个由 研究人员的专业知识横跨蛋白质生物物理、表达和纯化 包括膜蛋白在内的蛋白质，以及传统的蛋白质结构特征，如 作为X射线结晶学，已经组装完毕。团队成员还拥有数十年的经验 在包括基础离子化学/物理在内的质谱学领域，开创性的贡献 它们催生了MS蛋白质组学，以及分析质谱学和离子迁移率的相关领域- 质谱学。总体而言，这个研究团队的背景和专业知识是独一无二的 定位于改变结构生物学领域的IM-MS领域。简而言之，建议的 变革性研究将带来最前沿的IM-MS仪器，有望提供 对蛋白质复合体的结构和组装有前所未有的洞察力，并将该领域推向 研究的新前沿。

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.