Structural characterization of large eukaryotic proteins containing both folded and disordered domains

含有折叠和无序结构域的大型真核蛋白质的结构表征

• 批准号: 10552345
• 负责人: PETER Edwin WRIGHT
• 金额: $ 45.25万
• 依托单位: SCRIPPS RESEARCH INSTITUTE, THE
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-09-21 至 2028-07-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10552345
• 关键词: Amino Acid Sequence; Biological Process; Cardiovascular Diseases; Cells; Coupling; Cysteine; DNA Damage; Diabetes Mellitus; Disease; Engineering; Eukaryota; Goals; Human; Isotope Labeling; Length; Ligation; Malignant Neoplasms; Mediating; Methods; Molecular; Mutation; Neurodegenerative Disorders; Play; Post-Translational Protein Processing; Protein Dynamics; Proteins; Proteome; RNA Splicing; Regulatory Pathway; Research; Role; Site; Spin Labels; Stress; Structure; TP53 gene; Tumor Suppressor Proteins; cancer prevention; flexibility; fluorophore; genetic regulatory protein; human disease; insight; intein; intermolecular interaction; non-Native; novel strategies; response; structural biology; tool

Intrinsically disordered proteins (IDPs) are highly abundant in eukaryotes and play a central role in key cellular regulatory pathways and in the spatial organization of the cell. Approximately half of the proteins in the human proteome are either fully disordered or contain long disordered regions (IDRs). The cellular abundance of disordered proteins is tightly regulated and dysregulation or mutation of IDPs and IDRs is associated with devastating diseases such as cancer, diabetes, cardiovascular disease, and neurodegenerative disease. Disordered proteins are highly flexible and undergo transient and dynamic intramolecular and intermolecular interactions that are central to their regulatory functions. Molecular level characterization of the numerous human regulatory proteins that contain both structured and disordered domains represents an enormous challenge to the traditional methods of structural biology. Most studies to date have relied upon a reductionist, divide-and-conquer approach, in which the ordered and disordered regions are expressed independently and studied in isolation. However, within the cell, the folded and disordered domains of a given protein act synergistically to allow it to perform its biological function and a full understanding of the underlying molecular mechanism can only be achieved through a holistic, rather than reductionist, approach. An overarching goal of our research is to utilize a non- reductionist approach, aided by intein-based segmental isotope labeling, to characterize the structural ensemble, dynamics, and interactions of eukaryotic proteins containing both folded and disordered domains. This strategy is broadly applicable to large, dynamic proteins with disordered domains since it is relatively straightforward to identify or engineer optimal intein splice sites within disordered regions. Importantly, traceless ligation, where no cysteine or other non-native residues are introduced at the splice site, can be accomplished using the Nrdj1 intein, allowing retention of the native protein sequence and cysteine-mediated coupling of spin labels or fluorophores at desired probe sites. Initial efforts will focus on the full-length, 180 kDa tumor suppressor p53. Current structural information on p53 is largely limited to isolated domains and fails to explain how the disordered and folded regions function synergistically to control p53 activity. There is a large and growing body of evidence that the intrinsically disordered regions of p53 regulate its activity through dynamic intramolecular and intermolecular interactions that are modulated by constitutive and stress-induced post-translational modifications. This research will provide new molecular-level insights into the mechanisms by which this important tumor suppressor is regulated, as well as providing new tools for structural and dynamic characterization of large eukaryotic regulatory proteins that contain disordered regions.

内在无序蛋白 (IDP) 在真核生物中含量丰富，在关键生物体中发挥着核心作用。 细胞调节途径和细胞的空间组织。大约一半的蛋白质 人类蛋白质组要么完全无序，要么包含长无序区域（IDR）。蜂窝式 无序蛋白质的丰度受到严格调控，IDP 和 IDR 的失调或突变 与癌症、糖尿病、心血管疾病等毁灭性疾病有关 神经退行性疾病。无序蛋白质具有高度的灵活性并经历瞬态和动态 分子内和分子间相互作用是其调节功能的核心。分子 大量人类调节蛋白的水平表征，其中包含结构化和 无序结构域对结构生物学的传统方法提出了巨大的挑战。 迄今为止，大多数研究都依赖于还原论、分而治之的方法，其中有序 和无序区域是独立表达和孤立研究的。然而，在细胞内， 给定蛋白质的折叠和无序结构域协同作用，使其能够发挥其生物学功能 功能和对潜在分子机制的充分理解只能通过 整体而非简化的方法。我们研究的首要目标是利用非 还原论方法，借助基于内含肽的片段同位素标记，来表征结构 包含折叠和无序的真核蛋白质的整体、动力学和相互作用 域。该策略广泛适用于具有无序结构域的大型动态蛋白质，因为它 在无序区域内识别或设计最佳内含肽剪接位点相对简单。 重要的是，无痕连接，即在剪接处不引入半胱氨酸或其他非天然残基 位点，可以使用 Nrdj1 内含肽完成，允许保留天然蛋白质序列和 半胱氨酸介导的自旋标记或荧光团在所需探针位点的偶联。初步努力将集中 位于全长 180 kDa 肿瘤抑制因子 p53 上。目前关于 p53 的结构信息在很大程度上是有限的 到孤立的域，并且无法解释无序和折叠区域如何协同作用 控制p53活性。有大量且不断增长的证据表明，本质上无序的区域 p53 通过动态分子内和分子间相互作用调节其活性 由组成型和应激诱导的翻译后修饰调节。这项研究将提供 对这种重要的肿瘤抑制因子的调节机制的新的分子水平见解， 以及为大型真核生物调控的结构和动态表征提供新工具 含有无序区域的蛋白质。