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.

内源性无序蛋白(IDPs)在真核生物中含量非常丰富，并在关键蛋白中发挥核心作用。 细胞调控途径和细胞的空间组织。大约一半的蛋白质 在人类蛋白质组中，蛋白质组要么完全无序，要么含有长无序区域(IDR)。蜂窝手机 无序蛋白的丰度受到严格调控，IDPs和IDRs的失调或突变 与癌症、糖尿病、心血管疾病等破坏性疾病有关 神经退行性疾病。无序的蛋白质具有高度的灵活性，并经历短暂的和动态的 分子内和分子间的相互作用是其调节功能的中心。分子 多种人类调节蛋白的水平特征，这些蛋白既包含结构化的，也包含 无序结构域对传统的结构生物学方法提出了巨大的挑战。 到目前为止，大多数研究都依赖于简化论、分而治之的方法，在这种方法中， 而无序区域被独立表达和孤立地研究。然而，在细胞内， 特定蛋白质的折叠和无序结构域协同作用，使其能够执行其生物学功能 功能和对潜在分子机制的充分理解只能通过 一种整体的方法，而不是简化主义的方法。我们研究的首要目标是利用非 简化论方法，辅以基于内含素的节段性同位素标记，以表征结构 同时含有折叠和无序的真核蛋白质的系综、动力学和相互作用 域名。这一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 相对简单地识别或设计无序区域内的最佳内含子剪接位点。 重要的是，无痕迹连接，在剪接处没有引入半胱氨酸或其他非天然残基 可使用Nrdj1内含子完成，从而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的探针位置的偶联。最初的努力将集中在 在全长180 kDa的抑癌基因P53上。目前关于P53的结构信息很有限。 无法解释无序和折叠区域如何协同作用于 控制P53活性。有大量不断增长的证据表明，本质上无序的区域 通过分子内和分子间的动态相互作用来调节其活性 受构造性和应力诱导的翻译后修饰的调制。这项研究将提供 在分子水平上对这种重要的肿瘤抑制因子的调控机制有了新的见解， 以及为大的真核调控分子的结构和动态表征提供新的工具 含有无序区域的蛋白质。