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.

项目概要 本质无序蛋白质 (IDP)，即不呈现首选折叠构象的蛋白质 溶液，约占整个人类蛋白质组的三分之一，它们的错误折叠是 几种严重的疾病。尽管一系列生物物理工具已应用于结构分析 对于国内流离失所者而言，准确衡量国内流离失所者结构的需要与现有的 工具。当前的工具提供的信息内容不够，无法深入了解许多 IDP 结构存在于整个系综中，或者由于相互转换只能提供系综平均值 实验期间的构象。因此，本应用的目标是应用原生离子 Webb 实验室开发的用于分析折叠良好的蛋白质的迁移率/质谱 (IM/MS) 工具 结合分子动力学来表征 IDP 的结构整体，并本质上 具有构象和蛋白质形式特异性的无序区域（IDR），并确定对这些区域的影响 翻译后修饰（PTM）的结构整体。 本申请中提出的工作预计将回答以下生物学问题：1.）什么 IDP 构象是整体整体中的吗？将识别交联和标记的 IDP/IDR 在类似本地的条件下确定结构系综作为解决方案构象的函数（通过 IM 和 多发性硬化症）。交联/标记鉴定将用于确定 IDP/IDR 的结构整体 分子动力学。 2.) 翻译后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 国内流离失所者的集合，将结构变化与其功能和聚集联系起来。 3.) 哪种构象 组蛋白的状态和蛋白质形式结合表观遗传伴侣？组蛋白尾部是 IDR，上面装饰有许多 调节 DNA 获取的 PTM 组合。我们将在单个组蛋白、完整组蛋白上使用我们的工具 核小体，以及结合修饰剂和阅读器蛋白的核小体，以测量结构变化 合奏团。 这项研究通过使用组合解决方案与现状有了实质性的不同 （交联和标记）和气相 MS 结构工具包，提供 IDP/IDR 的结构细节 通过蛋白质组和构象独立测量。这项研究意义重大，因为它预计 弥合可用于国内流离失所者构象研究的工具与基础和研究工具之间的差距 生物医学需要表征其结构。最终，IDP 构象表征可能会导致 对细胞生物学以及 IDP/IDR 作为互动中心的重要性有更广泛的了解 监管者，并更好地了解他们在疾病和治疗中的作用。