Enabling ultra-high resolution hydrogen/deuterium exchange for challenging biomedical systems

为具有挑战性的生物医学系统实现超高分辨率氢/氘交换

• 批准号: EP/P012701/1
• 负责人: Anastasia Zhuravleva
• 金额: $ 12.84万
• 依托单位: University of Leeds
• 依托单位国家: 英国
• 项目类别: Research Grant
• 财政年份: 2017
• 资助国家: 英国
• 起止时间: 2017 至 无数据
• 项目状态: 已结题
• 来源: https://gtr.ukri.org/projects?ref=EP%2FP012701%2F1
• 关键词: Enabling; ultra; resolution; hydrogen; deuterium

Whilst protein structure and conformation have been traditionally accomplished by traditional X-ray crystallography, NMR, and Cryo-EM, these structural techniques are often extremely labour intensive and have significant technical limitations for many challenging protein systems. Hydrogen/deuterium exchange (HDX) has emerged as a complement, valuable and rapid technique for probing protein structure, functional flexibility, conformational variability, allosteric changes, as well as for monitoring and characterising protein interactions with other macromolecules, small metabolites and drugs. Moreover, HDX applications also have focused on monitoring protein stability in response of post-translational modifications and covalent ligand/drug binding as well as protein engineering. Despite the significant progress in HDX method developments, all current HDX techniques are limited by either the system size and complexity or resolution, with which HDX data can be obtained. As a result, the traditional high-resolution (HR) HDX techniques have only restricted (if any) applicability for many challenging biological systems, such as multidomain proteins, transient and multicomponent protein-protein complexes, large intrinsically disordered proteins/regions, and membrane proteins. We propose to develop a time- and cost-efficient HDX 3D NMR approach, applicable for the high-resolution characterisation of large challenging proteins. The proposed project will significantly enhance the current HDX capabilities, improving its effectiveness, efficiency and applicability to the biological and biomedical systems, currently inaccessible via the other HR HDX techniques. It will allow the collection and analysis of high resolution HDX data, obtained with the use of physiological protein concentrations (as low as sub-uM) and physiological buffers (without limitations on salt concentration and buffer composition). We will performed read-out 3D NMR experiments in aprotic organic solvent DMSO that preserves HDX patterns obtained under physiological conditions, but drastically improves the quality of NMR spectra by unfolding the protein of interest. We will exploit the most recent developments in ultra-resolution fast multidimensional NMR to allow time-optimised, semi-automatic data acquisitions, NMR assignments and analysis of HDX read-out 3D NMR spectra. We will vigorously test and optimise the proposed approach using several model systems, which represent the most common types of challenging protein systems, including multidomain proteins, intrinsically disordered proteins, transient protein complexes, and membrane proteins. Next, we will examine the accuracy and applicability of the HDX 3D NMR approach for drug developments, particularly, for monitoring conformational perturbations induced by small molecule binding. As model systems we will use molecular chaperones BIP and Hsp90 that undergo subtle, but functionally important conformational changes upon binding to small molecule inhibitors. This project will provide a long-awaited high-resolution method for structural and dynamic characterisation of challenging and complex biological systems in the near-native context. In a long term, it will provide new opportunities for elucidation of molecular mechanisms of action for large, multicomponent biological machines and facilitate future drug developments for such systems.

虽然蛋白质的结构和构象传统上是通过传统的X射线结晶学、核磁共振和低温电子显微镜来完成的，但这些结构技术往往是极其劳动密集型的，并且对于许多具有挑战性的蛋白质系统具有重大的技术限制。氢/氢交换(HDX)已成为一种补充、有价值的快速技术，用于探测蛋白质的结构、功能灵活性、构象变化、变构变化，以及监测和表征蛋白质与其他大分子、小代谢物和药物的相互作用。此外，HDX的应用还侧重于监测蛋白质在翻译后修饰、共价配体/药物结合以及蛋白质工程中的稳定性。尽管HDX方法的发展取得了重大进展，但目前所有的HDX技术都受到系统大小和复杂性或获得HDX数据的分辨率的限制。因此，传统的高分辨率(HR)HDX技术仅限制了(如果有的话)许多具有挑战性的生物系统的适用性，如多结构域蛋白质、瞬时和多组分蛋白质-蛋白质复合体、大的内在无序的蛋白质/区域和膜蛋白质。我们建议开发一种既省时又省钱的HDX 3D核磁共振方法，适用于具有挑战性的大型蛋白质的高分辨率表征。拟议的项目将显著增强目前的HDX能力，提高其效力、效率和对生物和生物医学系统的适用性，目前无法通过其他人力资源HDX技术获得这些系统。它将允许收集和分析通过使用生理蛋白质浓度(低至亚微米)和生理缓冲液(对盐浓度和缓冲液成分没有限制)而获得的高分辨率HDX数据。我们将在非质子有机溶剂DMSO中进行读出3D核磁共振实验，该溶剂保留了在生理条件下获得的HDX图案，但通过展开感兴趣的蛋白质来极大地提高核磁共振谱的质量。我们将利用超分辨率快速多维核磁共振的最新发展，实现时间优化的半自动数据采集、核磁共振分配和HDX读出的3D核磁共振谱分析。我们将使用几个模型系统来大力测试和优化所提出的方法，这些模型系统代表了最常见的具有挑战性的蛋白质系统类型，包括多结构域蛋白质、内在无序蛋白质、瞬时蛋白质复合体和膜蛋白质。接下来，我们将检查HDX 3D核磁共振方法在药物开发中的准确性和适用性，特别是在监测小分子结合引起的构象扰动方面。作为模型系统，我们将使用分子伴侣BIP和Hsp90，它们在与小分子抑制剂结合时会发生微小但功能上重要的构象变化。该项目将提供一种期待已久的高分辨率方法，用于在近原生环境中描述具有挑战性的复杂生物系统的结构和动态特征。从长远来看，它将为阐明大型、多组分生物机器的分子作用机制提供新的机会，并促进此类系统未来的药物开发。