Intrinsically Disordered Protein Structural Dynamics from Combined Solution and Gas-Phase Approaches

结合溶液和气相方法的本质无序蛋白质结构动力学

• 批准号: 10714896
• 负责人: Ian Webb
• 金额: $ 39.26万
• 依托单位: INDIANA UNIV-PURDUE UNIV AT INDIANAPOLIS
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-07-15 至 2028-04-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10714896
• 关键词: Affect; Binding; Biological; Cell physiology; Cells; Cellular biology; DNA; Degenerative Disorder; Disease; Epigenetic Process; Gases; Goals; Histones; Human; Individual; Knowledge; Label; Laboratories; Ligand Binding; Link; Mass Spectrum Analysis; Measures; Methods; Mission; Molecular Biology; Molecular Conformation; Neurodegenerative Disorders; Nucleosomes; Patients; Phase; Post-Translational Protein Processing; Protein Conformation; Proteins; Proteome; Reader; Research; Role; Specificity; Structure; Tail; Time; United States National Institutes of Health; Work; biophysical tools; crosslink; experimental study; insight; ion mobility; molecular dynamics; protein folding; protein structure; small molecule; tool

PROJECT SUMMARY Intrinsically disordered proteins (IDPs), proteins that do not assume a preferred folded conformation in solution, make up around one-third of the entire human proteome, and their misfolding is a causative agent of several serious diseases. Although an array of biophysical tools has been applied to the structural analysis of IDPs, there is still a significant gap between the need to accurately measure IDP structures and the available tools. Current tools provide either not enough information content to gain significant insight on the many IDP structures present in the overall ensemble or can only provide an ensemble average due to interconversion of conformations over the time frame of the experiment. Therefore, the goal of this application is to apply native ion mobility/mass spectrometry (IM/MS) tools developed in the Webb laboratory for analyzing well-folded proteins in combination with molecular dynamics to characterize the structural ensembles of IDPs and intrinsically disordered regions (IDRs) with conformational and proteoform specificity and to determine the effect on those structural ensembles of post-translational modifications (PTMs). The proposed work in this application is expected to answer the following biological questions: 1.) What are the IDP conformations in the overall ensemble? Crosslinked and labeled IDPs/IDRs will be identified under native-like conditions to determine structural ensembles as a function of solution conformation (by IM and MS). Crosslinking/labeling identifications will be used to determine structural ensembles of IDPs/IDRs with molecular dynamics. 2.) How do post translational modifications and ligand binding affect IDP structure? These methods will be used to measure how PTMs and small molecule binding affects conformational ensembles of IDPs, linking structural changes to their functions and to aggregation. 3.) Which conformational states and proteoforms of histones bind epigenetic partners? Histone tails are IDRs decorated with many combinations of PTMs that regulate access to DNA. We will use our tools on individual histones, on the intact nucleosome, and on nucleosomes upon binding modifier and reader proteins to measure changes in structural ensembles. This research represents a substantive departure from the status quo by using a combined solution (crosslinking and labeling) and gas-phase MS structural toolkit to provide structural details of IDPs/IDRs measured independently by proteoform and conformation. The research is significant because it is expected to bridge the gap between the tools available for the conformational study of IDPs and the fundamental and biomedical need to characterize their structures. Ultimately, IDP conformational characterization is likely to lead to a much broader understanding of cellular biology and the importance of IDPs/IDRs as interaction hubs and regulators and a better understanding of their roles in disease and treatment.

项目摘要 本质上无序的蛋白质（IDP），不假定在 解决方案，构成整个人类蛋白质组的三分之一，它们的错误折叠是一种原因 几种严重的疾病。尽管一系列生物物理工具已应用于 IDPS，准确测量IDP结构的需求与可用的需要之间存在显着差距 工具。当前工具提供的信息内容不足以获得许多IDP的重要见解 整体合奏中存在的结构，或者仅能提供合奏的平均值 实验时间范围内的构象。因此，此应用的目的是应用本地离子 韦伯实验室开发的移动性/质谱（IM/MS）工具用于分析折叠式蛋白质 结合分子动力学结合表征IDP的结构集合和本质 具有构象和蛋白质成型特异性的无序区域（IDR），并确定对那些的影响 翻译后修饰（PTM）的结构集合。 预计本应用程序中的拟议工作将回答以下生物学问题：1。） 整体合奏中的IDP构象是否？将确定交联和标记的IDP/IDR 在类似天然的条件下，确定结构合奏是溶液构象的函数（通过IM和 多发性硬化症）。交联/标签标识将用于确定IDP/IDR的结构集合 分子动力学。 2.）后翻译修饰和配体结合如何影响IDP结构？ 这些方法将用于测量PTM和小分子结合如何影响构象 IDP的合奏，将结构变化与其功能联系起来和聚集。 3.）哪种构象 组蛋白的状态和蛋白质相结合表观遗传伴侣？组蛋白的尾巴是装饰许多的IDR 调节DNA访问的PTM的组合。我们将在单个组蛋白上使用我们的工具，完整 结合修饰剂和读取器蛋白质的核小体和核小体上的核小体，以测量结构的变化 合奏。 这项研究代表了通过使用组合解决方案的实质性偏离现状 （交联和标签）和气相MS结构工具包，以提供IDP/IDR的结构细节 通过蛋白质成型和构象独立测量。这项研究很重要，因为预计 弥合可用于IDP构象研究的工具与基本的构象研究之间的差距 生物医学需要表征其结构。最终，IDP构象表征可能会导致 更广泛地了解细胞生物学以及IDP/IDR作为相互作用中心的重要性 监管机构，更好地了解其在疾病和治疗中的作用。