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 内含子无序蛋白（IDP），即在细胞内不呈现优选折叠构象的蛋白质。 蛋白质组约占整个人类蛋白质组的三分之一，它们的错误折叠是导致蛋白质组异常的原因之一。 几种严重的疾病。虽然一系列生物物理工具已被应用于结构分析， 关于国内流离失所者，在准确衡量国内流离失所者结构的需要与现有的 工具.目前的工具提供的信息内容要么不足以深入了解许多国内流离失所者 存在于整体系综中的结构，或者由于以下结构的相互转换而只能提供系综平均值： 在实验的时间范围内的构象。因此，本申请的目标是将原生离子 韦伯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的结构集合， 具有构象和蛋白质形式特异性的无序区（IDR），并确定对那些 翻译后修饰的结构集合（PTM）。 本申请中拟议的工作预计将回答以下生物学问题：1.）什么 IDP构象在整体中是什么样的将识别交联和标记的IDP/IDR 在类似天然的条件下，以确定作为溶液构象的函数的结构系综（通过IM和 MS）。交联/标记鉴定将用于确定IDP/IDR的结构集合， 分子动力学2.）的情况。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。 将结构变化与其职能和聚集联系起来。3.）第三章哪个构象 状态和组蛋白的蛋白质形式结合表观遗传的合作伙伴？组蛋白尾部是由许多 调节DNA访问的PTM的组合。我们将使用我们的工具对单个组蛋白，对完整的 核小体上，并在结合修饰剂和阅读器蛋白质的核小体上，以测量结构变化。 合奏。 这项研究代表了一个实质性的脱离现状，通过使用一个组合的解决方案 （交联和标记）和气相MS结构工具包，以提供IDP/IDR的结构细节 通过蛋白质形式和构象独立测量。这项研究意义重大，因为它预计将 弥合国内流离失所者构象研究可用的工具与基本和 生物医学需要表征其结构。最终，IDP构象表征可能导致 更广泛地理解细胞生物学以及国内流离失所者/国内流离失所者作为互动中心的重要性， 监管机构和更好地了解他们在疾病和治疗中的作用。