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射线和冷冻电镜，无疑加速了 蛋白质结构的测定然而，捕获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 以履行其生物功能。此外，了解分子，如脂质， 调节蛋白质结构和功能具有极其重要的生物学意义。过去两 几十年来，完整蛋白质复合物的质谱（MS），通常被称为天然MS， 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 结构被保留在质谱仪内。天然MS是一种快速、灵敏的技术 它已经提供了关于亚基化学计量和拓扑结构、变构和 单个配体结合事件的协同性，包括它们的结合热力学。耦合 离子迁移率（IM）是一种基于分子电荷和形状的分离技术， 本地MS的能力，其中它使碰撞截面（CCS）测量大 蛋白质复合物、肽和稳定配体的不同构象的鉴定 碰撞诱导的解折叠，以及对蛋白质折叠和变性结构的了解。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，该提议寻求开发具有高分辨率的变革性本地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， 周转条件。为了实现这些变革性目标，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， 包括膜蛋白在内的蛋白质，以及传统的蛋白质结构表征，如 像X射线晶体学一样，已经组装好了。团队成员还拥有数十年的经验 在质谱领域，包括基本离子化学/物理学，开创性的贡献 产生了MS蛋白质组学，以及分析质谱和离子迁移率的相关领域- 质谱分析法来总的来说，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 变革性的研究将导致前沿的IM-MS仪器，准备提供 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 研究的新领域。

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.