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

总结 各种各样的亚细胞复合体由一种或多种内在无序蛋白（IDP）组成 其是多价的、柔性的，并且特征在于动态的、可逆的结合不同的伴侣蛋白。一 一种常见但研究不足的多价IDP组装体表现出独特的双链体拓扑结构，其特征在于： 通过普遍存在的LC 8枢纽蛋白可逆交联的两条IDP链的平行比对，其中IDP 变构增强对另外的二价配体的亲和力。这些双链体可以作为一个梁状元件 在大的复合物中，作为传感器，并促进或“模板”形成大的超分子组装体 (such作为动力蛋白马达和核孔复合物）。这些系统的关键特征在MPI中被识别 Barbar的实验室，但对这种广泛功能的结构和生物化学基础的研究， 挑战的多样性，内部流动性，和异质性的复杂形成。 这一提议将大大推进我们对多价LC 8分子基础的理解。 复杂的装配，通过整合一系列新颖的和现有的计算建模方法，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），以及结构表征如核磁共振（NMR）。 在一些实施方案中，所述方法包括核磁共振（NMR）、电子显微镜（EM）和天然质谱法（天然MS）。这些技术 被选为解决关键的悬而未决的问题，在该领域：有多少构象和组成 异质性是这些可逆组装的双链体固有的，它们如何避免无序状态？ 受细胞严格控制的LC 8浓度如何调节异质性？是什么 变构效应和相关的机制途径表明关于双链体的调节？什么 在针对结构与复杂支架与传感优化的双重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合奏热力学 通过ITC和理论解剖物种种群，构象合奏通过EM和理论从整体 复杂到原子尺度，最后通过模拟动力学和合作行为的原子基础 和动力学测量。 这些工作将由一个经验丰富的生物物理学团队指导，该团队具有广泛的互补专业知识， 我已经合作了几年-理论生物物理学专家（Zuckerman，MPI）;在LC 8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 在LC 8的结构-功能关系方面的开创性工作记录，成功生产有用的蛋白质 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， 计算，结构，生物物理和生物化学技术需要探测这些系统，使我们 独特地适合于显著推进IDP多价性研究的前沿。

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.