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

项目概要/摘要 表征生物分子及其复合物的结构和相互作用是生物分子研究的基础 在人类生理学、疾病和治疗学中的重要性。上个世纪的许多进步 生物分析技术的改进和对生物分析过程的控制， 他们例如，X射线晶体学、核磁共振光谱学和低温电子学 显微镜已经实现了原子级分辨率的结构，成千上万的蛋白质和蛋白质 复合物，这些方法往往是补充分子动力学研究，以进一步了解 生物分子结构和反应性。然而，这些方法对于非常小或非常高的应用来说可能是具有挑战性的。 异质样品或需要膜环境的样品。自然离子迁移率-质量 光谱法（IM-MS）是一种补充技术，其电离和转移完整的生物分子， - 将复合物直接从缓冲的水溶液转化为气相，用于质量和形状/尺寸分析，以及 现代样品制备和数据分析方法使其非常适合于脂质中的膜蛋白 环境以及异质和多分散样品。在常用的IM-MS中 碰撞诱导解离和解折叠用于通过以下方式激活天然生物分子离子： 不断地与中性缓冲气体碰撞，直到它们分解或展开， 诱导解离和展开通过与内部硬表面的单一受控碰撞激活离子。 质谱仪这些天然IM-MS方法对于分析样品的组成、大小 和形状的生物分子及其复合物与精致的化学特异性，灵敏度和速度。 然而，使用这些方法对生物分子进行准确定量解释的两个主要障碍是： 域、表面和界面结构缺乏灵活、鲁棒的计算和解释方法 引起观察到的结构变化所需的能量和缺乏可靠的基准值。在这里， 我们通过结合计算和实验的方法来应对这些挑战，旨在产生一种 “通用的”经验证的离子活化模型，可随时用于许多常用的天然MS 和IM-MS平台，并为原型的本地和大规模交互生成基准库 控制蛋白质解折叠、解离和表面标记。预期成果包括开放源码， 公开可用的软件，供世界各地的研究人员为自己的解折叠/解离能量学建模 样品，设计有效的气相表面标记试剂的化学方法，以及定量 理解白内障相关的人眼透镜蛋白异源寡聚化作为一个案例研究。很长的- 该项目的长期目标是促进决定性的结构和动力学的获取和解释 与人类健康相关的各种生物分子和复合物的信息。

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.