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 各种各样的亚细胞复合物由一个或多种本质上无序的蛋白质（IDP）组成 多价，灵活的，以动态，可逆的伴侣蛋白的结合为特征。一个 多价IDP组装的常见但研究不足的类型表现出独特的双工拓扑结构，其特征是 由无处不在的LC8轮毂蛋白可逆地交联的两个IDP链的平行排列 变构增强了对其他二价配体的亲和力。这些双链体可以用作类似梁的元素 在大型复合体中，充当传感器，并促进或“模板”形成大型上分子组件 （例如动力蛋白运动和核孔复合物）。这些系统的主要特征是在MPI中确定的 Barbar的实验室，但是对这种广泛功能的结构和生化基础的研究是 受到形成的复合物的多样性，内部流动性和异质性的挑战。 该建议将大大提高我们对多价LC8分子基础的理解 复杂的组件，通过整合一系列新颖和现有的计算建模方法 - 例如 加权安装分子动力学模拟 - 带有包括等温滴定量热法在内的实验 （ITC）和表面等离子体共振（SPR）以及结构表征，例如核磁性 共振（NMR），电子显微镜（EM）和天然质谱法（天然MS）。这些技术 被选中以解决该领域的关键未解决问题：多少构象和构图 异质性是这些可逆组装的双链体的固有的，它们如何避免无序状态？ LC8浓度如何由细胞紧密控制，调节异质性？什么是 变构效应和相关的机械途径表明对双工的调节？什么 在针对建筑与复杂界面与传感的建筑与复合体相关的双链系统中观察到差异 角色？为了解决这些问题，三个主要独立的目标将探究集成热力学 通过ITC和理论解剖物种种群，通过EM的构象合奏和整体理论 复杂到原子尺度，最后是模拟动力学和合作行为的原子基础 和动力学测量。 这些努力将由经验丰富的生物物理学团队指导，并具有广泛的补充专业知识 已经合作了几年 - 理论生物物理学专家（Zuckerman，MPI）；在LC8结构中 生物学，ITC和NMR（Barbar，MPI）；在电子显微镜中（Reichow，Co-I）；和本地MS（Prell，Co-I）。我们的 LC8结构功能关系的开创性工作的记录，在产生有用的蛋白质方面的成功 结构和处理这些复杂且部分无序的蛋白质以及团队在电池中的专业知识 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 非常适合在IDP多相关性研究中显着推进前沿的。