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

项目总结 固有无序蛋白(IDPs)，指的是在 溶液，约占整个人类蛋白质组的三分之一，它们的错误折叠是导致 几种严重的疾病。尽管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，准确衡量境内流离失所者结构的需要与现有的差距仍然很大 工具。目前的工具要么提供的信息内容不够多，就不能对许多国内流离失所者有重大的了解 结构存在于整个系综中，或者由于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，此应用程序的目标是应用原生离子 韦伯实验室开发的用于分析折叠良好的蛋白质的迁移率/质谱仪(IM/MS)工具 与分子动力学相结合来表征国内流离失所者的结构集合，并内在地 具有构象和蛋白形式特异性的无序区(IDR)，并确定其对 翻译后修饰的结构集合(PTM)。 本申请中的拟议工作预计将回答以下生物学问题：1)什么 IDP构象是否在整个整体中？将识别交叉链接和标记的国内流离失所者/国内流离失所者 在类似天然的条件下确定作为溶液构象函数的结构系综(通过IM和 Ms)。将使用交叉链接/标签标识来确定国内流离失所者/国内流离失所者的结构组合 分子动力学。2.)翻译后修饰和配体结合如何影响IDP结构？ 这些方法将被用来测量PTM和小分子结合如何影响构象 国内流离失所者群体，将结构变化与其职能和聚合联系起来。3.)哪种构象 组蛋白的状态和蛋白形式将表观遗传伙伴联系在一起？组蛋白尾巴是有许多装饰的IDR 控制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，在完整的组蛋白上 核小体，并在核小体上结合修饰物和阅读器蛋白来测量结构的变化 合唱团。 这项研究表明，通过使用综合解决方案，实质上偏离了现状 (交联和标记)和气相MS结构工具包，以提供国内流离失所者/国内流离失所者的结构细节 通过蛋白质形式和构象独立测量。这项研究意义重大，因为预计它将 弥合可用于国内流离失所者构象研究的工具与基础和 生物医学需要描述它们的结构。最终，IDP的构象特征很可能导致 为了更广泛地了解细胞生物学以及国内流离失所者/国内流离失所者作为相互作用中心和 并更好地了解它们在疾病和治疗中的作用。

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.