Multiscale Characterization of a Unique Class of Duplex, Multivalent IDP systems

一类独特的双工、多价 IDP 系统的多尺度表征

• 批准号: 10461032
• 负责人: ELISAR J BARBAR
• 金额: $ 41.54万
• 依托单位: OREGON HEALTH & SCIENCE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-08-03 至 2025-07-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10461032
• 关键词: Address; Affinity; Amino Acid Sequence; Architecture; Binding; Binding Proteins; Biochemical; Biological; Biological Assay; Biological Process; Biophysics; Calorimetry; Cell Cycle; Cells; Collaborations; Comparative Study; Complex; Computer Models; Computing Methodologies; Cooperative Behavior; Custom; Data; Disease; Dynein ATPase; Ebola; Electron Microscopy; Elements; Event; Exhibits; Formulation; Gene Expression Regulation; Geometry; Heterogeneity; Kinetics; Length; Ligands; Malignant Neoplasms; Mass Spectrum Analysis; Measurement; Methods; Mitotic spindle; Modeling; Molecular; Molecular Conformation; Molecular Motors; Motor; Negative Staining; Nerve Degeneration; Neurodegenerative Disorders; Nuclear Magnetic Resonance; Nuclear Pore; Pathway interactions; Phase; Play; Population; Positioning Attribute; Prevalence; Process; Property; Proteins; Rabies; Regulation; Regulatory Element; Resolution; Role; Structure; Surface Plasmon Resonance; Syndrome; System; Techniques; Technology; Theoretical model; Thermodynamics; Titrations; Transcriptional Regulation; Ursidae Family; Validation; Variant; Viral Proteins; Virus Diseases; Work; base; cell growth regulation; combinatorial; crosslink; experience; experimental study; flexibility; frontier; innovation; link protein; molecular assembly/self assembly; molecular dynamics; molecular imaging; novel; protein complex; recruit; scaffold; sensor; simulation; single molecule; structural biology; success; theories

Summary A wide variety of subcellular complexes are composed of one or more intrinsically disordered proteins (IDPs) that are multivalent, flexible, and characterized by dynamic, reversible binding of diverse partner proteins. A common but understudied type of multivalent IDP assembly exhibits a unique duplex topology, characterized by parallel alignment of two IDP chains reversibly cross-linked by the ubiquitous LC8 hub protein, where the IDPs allosterically enhance affinity for additional bivalent ligands. These duplexes can serve as a girder-like element in large complexes, act as sensors, and facilitate or `template' the formation of large supra-molecular assemblies (such as the dynein motor and nucleopore complex). Key features of these systems were identified in MPI Barbar's lab, but studies of the structural and biochemical basis for this wide range of functionalities are challenged by the diversity, internal mobility, and heterogeneity of the complexes formed. This proposal will significantly advance our understanding of the molecular underpinnings of multivalent LC8 complex assemblies, by integrating an array of novel and existing methods of computational modeling - such as weighted-ensemble molecular dynamics simulation - with experiments including isothermal titration calorimetry (ITC) and surface plasmon resonance (SPR), and structural characterization such as nuclear magnetic resonance (NMR), electron microscopy (EM), and native mass spectrometry (native MS). These techniques were selected to address critical unanswered questions in the field: How much conformational and compositional heterogeneity is intrinsic to these reversibly assembled duplexes, and how do they avoid a disordered state? How does LC8 concentration, which is tightly controlled by the cell, modulate the heterogeneity? What do the allosteric effects and associated mechanistic pathways indicate about regulation of the duplexes? What differences are observed among duplex systems optimized for architectural vs. complex-scaffolding vs. sensing roles? To address these questions, three largely independent aims will probe the ensemble thermodynamics via ITC and theory dissecting species populations, the conformational ensemble via EM and theory from whole complex- to atomistic-scale, and finally the atomistic basis of kinetic and cooperative behavior via simulations and kinetics measurements. The efforts will be guided by an experienced biophysics team with a wide-range of complementary expertise who have been collaborating for several years - experts in theoretical biophysics (Zuckerman, MPI); in LC8 structural biology, ITC and NMR (Barbar, MPI); in electron microscopy (Reichow, Co-I); and in native MS (Prell, Co-I). Our track record of pioneering work on structure-function relations of LC8, success in both producing useful protein constructs and handling these complex and partially disordered proteins, and the team's expertise in the battery of computational, structural, biophysical, and biochemical techniques required to probe these systems, make us uniquely suited to significantly advance the frontiers in the study of IDP multivalency.

概括 多种亚细胞复合物由一种或多种本质无序蛋白 (IDP) 组成 它们是多价的、灵活的，其特征是动态、可逆地结合不同的伴侣蛋白。一个 常见但尚未研究的多价 IDP 组装类型表现出独特的双工拓扑，其特征在于 两条 IDP 链通过普遍存在的 LC8 中心蛋白可逆交联的平行排列，其中 IDP 变构增强对其他二价配体的亲和力。这些双工可以作为梁状元件 在大型复合物中，充当传感器，并促进或“模板”大型超分子组装体的形成 （例如动力蛋白马达和核孔复合体）。 MPI 中确定了这些系统的主要特征 Barbar 的实验室，但对这种广泛功能的结构和生化基础的研究正在进行 所形成的复合物的多样性、内部流动性和异质性面临着挑战。 该提案将显着增进我们对多价 LC8 分子基础的理解 复杂的组件，通过集成一系列新颖的和现有的计算建模方法 - 例如 加权系综分子动力学模拟 - 实验包括等温滴定量热法 (ITC) 和表面等离子体共振 (SPR)，以及核磁等结构表征 共振 (NMR)、电子显微镜 (EM) 和天然质谱 (天然 MS)。这些技巧 被选中来解决该领域尚未解答的关键问题：构象和成分有多少 异质性是这些可逆组装的双链体所固有的，它们如何避免无序状态？ 由细胞严格控制的 LC8 浓度如何调节异质性？做什么的 变构效应和相关的机制途径表明双链体的调节？什么 在针对建筑与复杂脚手架与传感进行优化的双工系统之间观察到差异 角色？为了解决这些问题，三个基本上独立的目标将探讨系综热力学 通过 ITC 和理论剖析物种种群，通过 EM 和整体理论得到构象集合 复杂到原子尺度，最后通过模拟获得动力学和合作行为的原子基础 和动力学测量。 这些工作将由经验丰富的生物物理学团队指导，该团队拥有广泛的互补专业知识， 多年来一直合作 - 理论生物物理学专家（Zuckerman、MPI）； LC8结构 生物学、ITC 和 NMR（Barbar、MPI）；电子显微镜（Reichow，Co-I）；和天然 MS（Prell，Co-I）。我们的 在 LC8 结构-功能关系方面的开创性工作记录，在生产有用蛋白质方面取得了成功 构建和处理这些复杂且部分无序的蛋白质，以及团队在电池方面的专业知识 探测这些系统所需的计算、结构、生物物理和生化技术，使我们 非常适合显着推进 IDP 多价研究的前沿。