Quantitative Determination of High-Order Protein Structure with Native Ion Mobility-Mass Spectrometry and Computational Chemistry

项目摘要/摘要 表征生物分子及其络合物的结构和相互作用具有基础性意义 在人类生理学、疾病和治疗学中的重要性。上个世纪在这些方面的许多进步 这些领域归因于生物分析技术和控制基础过程的改进。 他们。例如，x射线结晶学、核磁共振波谱和低温电子 显微镜已经实现了对数千种蛋白质和蛋白质结构的原子级分辨率 这些方法通常得到分子动力学研究的补充，以进一步了解 生物分子结构和反应性。然而，对于非常小或非常高的情况，这些方法的使用可能会具有挑战性 异质样品或需要膜环境的样品。天然离子迁移率-质量 光谱分析(IM-MS)是一种补充技术，它电离和转移完整的生物分子和 从缓冲的水溶液直接进入气相进行质量和形状/尺寸分析的络合物，以及 现代的样品制备和数据分析方法使其非常适合于脂类中膜蛋白的分析 环境以及非均质和多分散的样品。在常见的IM-MS中 仪器、碰撞诱导解离和去折叠被用来激活天然生物分子离子 将它们与中性缓冲气体反复碰撞，直到它们解离或解开，以及最近引入的表面 通过与内部硬表面的一次受控碰撞，诱导解离和展开激活离子 质谱仪。这些本地IM-MS方法对于分析成分、尺寸 以及生物分子及其络合物的形状，具有精致的化学专一性、敏感性和速度。 然而，使用这些方法准确、定量地解释生物分子有两个主要障碍 域、表面和界面结构缺乏灵活、健壮的计算和解释方法 导致观察到的结构变化所需的能量，以及缺乏可靠的基准值。这里, 我们通过计算和实验相结合的方法来应对这些挑战，旨在产生一个 经过验证的“通用”离子激活模型，可在许多常用的本机MS中轻松使用 和IM-MS平台，并通过为典型的本地和大规模交互生成基准库 它们控制蛋白质的展开、解离和表面标记。预期结果包括开源、 世界各地的研究人员可以公开获得的软件来为他们自己的展开/解离能量建模 样品，设计有效的气相表面标记试剂的启发式方法，以及一种定量 理解与白内障相关的人眼晶状体蛋白异寡聚化作为个案研究。长的- 该项目的长期目标是促进获得和解释决定性的结构和动力 一系列与人类健康相关的生物分子和复合体的信息。

PROJECT SUMMARY/ABSTRACT Characterizing the structures and interactions of biomolecules and their complexes is of fundamental importance in human physiology, disease, and therapeutics. Many of the advances of the last century in these areas are attributed to improvements in bioanalytical techniques and controlling the processes that underlie them. For example, x-ray crystallography, nuclear magnetic resonance spectroscopy, and cryoelectron microscopy have achieved atomic-level resolution of the structure of many thousands of proteins and protein complexes, and these methods are often complemented by Molecular Dynamics studies to further understand biomolecule structure and reactivity. However, these methods can be challenging to use for very small or highly heterogeneous samples or samples that require a membrane environment. Native Ion Mobility-Mass Spectrometry (IM-MS) is a complementary technique that ionizes and transfers intact biomolecules and complexes directly from buffered, aqueous solution into the gas-phase for mass and shape/size analysis, and modern sample preparation and data analysis methods make it highly suitable for membrane proteins in lipid environments as well as heterogeneous and polydisperse samples. In commonly available IM-MS instrumentation, Collision Induced Dissociation and Unfolding are used to activate native biomolecular ions by colliding them repeatedly with neutral buffer gas until they dissociate or unfold, and recently-introduced Surface Induced Dissociation and Unfolding activate ions via a single, controlled collision with a hard surface inside the mass spectrometer. These native IM-MS methods can be extremely useful for profiling the composition, size, and shape of biomolecules and their complexes with exquisite chemical specificity, sensitivity, and speed. However, two major hurdles to the use of these methods for accurate, quantitative interpretation of biomolecule domain, surface, and interface structure are the lack of a flexible, robust method for computing and interpreting the energy required to induce the observed structural changes and a dearth of reliable benchmark values. Here, we tackle these challenges with a combined computational and experimental approach aimed at producing a “universal,” validated ion activation model that can be readily used for across many commonly used native MS and IM-MS platforms and by producing a benchmark library for prototypical local and large-scale interactions that govern protein unfolding, dissociation, and surface labeling. Expected outcomes include open-source, publicly available software for researchers world-wide to model unfolding/dissociation energetics for their own samples, heuristics for the design of effective gas-phase surface-labeling reagents, and a quantitative understanding of cataract-associated human eye lens protein heterooligomerization as a case study. The long- term goal of the project is to facilitate the acquisition and interpretation of decisive structural and dynamical information for a wide range of biomolecules and complexes relevant to human health.