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: 10198490
• 负责人: ELISAR J BARBAR
• 金额: $ 44.24万
• 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/10198490
• 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.

摘要 各种各样的亚细胞复合体由一个或多个内在无序蛋白(Idp)组成。 这是多价的，灵活的，并以动态的，可逆的多种伙伴蛋白结合为特征。一个 常见但未被研究的多价IDP组件呈现独特的双链拓扑结构，其特征为 由无处不在的LC8枢纽蛋白可逆地交联的两条IDP链的平行比对，其中IdP 变构增强对额外二价配体的亲和力。这些双层结构可以作为类似大梁的单元 在大的络合物中，充当传感器，促进或‘模板化’形成大的超分子组件 (如动力蛋白马达和核孔复合体)。在MPI中确定了这些系统的关键特性 但对这种广泛功能的结构和生化基础的研究是 受到形成的复合体的多样性、内部流动性和异质性的挑战。 这一提议将极大地促进我们对多价LC8分子基础的理解 通过集成一系列新的和现有的计算建模方法，例如 加权系综分子动力学模拟--包括等温滴定量热法实验 (ITC)和表面等离子激元共振(SPR)，以及核磁共振等结构表征 核磁共振(核磁共振)、电子显微镜(EM)和自然质谱(自然MS)。这些技术 被选中来解决该领域中尚未回答的关键问题：有多少构象和组成 异质性是这些可逆组装的双链的固有特性，它们如何避免无序状态？ 受细胞严格控制的Lc8浓度是如何调节异质性的？这是怎么回事 变构效应和相关的机械途径表明了双链体的调节？什么 观察到针对建筑和复杂脚手架与传感进行优化的双工系统之间的差异 角色？为了解决这些问题，三个基本独立的目标将探索整体热力学 通过ITC和理论对物种种群进行解剖，通过EM和理论从整体上分析了构象集成 从复杂到原子尺度，最后是通过模拟的动力学和合作行为的原子基础 和动力学测量。 这些工作将由一支经验丰富的生物物理学团队指导，该团队具有广泛的补充专业知识 已经合作多年-理论生物物理学(Zuckerman，MPI)专家；LC8结构 生物学，ITC和核磁共振(Barbar，MPI)；电子显微镜(Reicow，Co-I)；以及天然MS(Prell，Co-I)。我们的 Lc8结构-功能关系开创性工作的记录，成功地生产了两种有用的蛋白质 构建和处理这些复杂和部分无序的蛋白质，以及该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 特别适合于显著推进国内流离失所者多价性研究的前沿。