Conformational dynamics, binding and aggregation of intrinsically disordered proteins

摘要 内源性无序蛋白(IDPs)占真核蛋白质组的30%以上。他们执行的是至关重要的 细胞中的功能，如信号、转录和翻译，它们调节和控制细胞 周而复始。它们的故障导致了一些最具挑战性的疾病，越来越受到美国卫生部门的关注 护理系统，如癌症和神经变性。这些疾病的相关成本是一些 在美国是最高和增长最快的。国内流离失所者也在病毒的复制和传播中发挥关键作用。 病原体。为了正常运作，国内流离失所者必须(1)有效地与特定的有约束力的伙伴捆绑；和(2)避免 病理性聚集。在分子水平上了解IDP序列如何对这两种基因进行编码 过程，以及细胞中的突变和压力条件如何影响它们，将显著促进我们的 对治疗和预防此类疾病的理解和能力。 目前主要的试验性努力集中在：(1)解决国内流离失所者和 绑定机制(IdP结构/功能)；或(Ii)将IdP聚合机制分解为IdP 聚集成特定的淀粉样蛋白聚集体(IDP故障)。在这里，我们提出了一种完全不同的、物理的 化学方法，研究IDP构象动力学对结合机制的影响； 以及IdP相分离与聚集的定量关系。我们将通过使用HIGH 我们实验室开发的分辨率实验技术和方法，以及多尺度模拟。 由于大多数IDPs通过耦合折叠和结合结合，它们的构象动力学预计会 极大地影响了约束机制。结合IDP构象动力学的方法 研究将为理解IdP功能结合提供一个关键缺失的环节。 我们建议的研究建立在最近对一组境内流离失所者的结合机制的表征的基础上，以及 重点研究1)构象动力学对结合的影响；2)生理过程 液体相分离及其与病理性聚集的联系。我们将结合不同的高分辨率 实验技术-包括纳秒激光泵浦光谱-使用分子模拟来 描述Idp的结构和动态。新的方法将被用来定量液体相分离 国内流离失所者。目标I的结果：通过比较不同绑定的IdP动力学来验证我们的假设 情景；目标二：通过扰动序列和溶剂来调节结合机制，以及目标 III：量化疾病突变对构象动力学、相分离和 FUS_LC；的聚集将直接影响对IDP与卵巢相关的分子理解 和乳腺癌，副氧合病毒的复制和肌萎缩侧索硬化症 额颞部痴呆。这些结果有可能改变我们看待耦合折叠的方式 和结合，以及对IdP功能至关重要的液相分离。

Summary Intrinsically disordered proteins (IDPs) make up more than 30% of eukaryotic proteomes. They carry out vital functions in the cell, such as signaling, transcription and translation, and they regulate and control the cell cycle. Their malfunction leads to some of the most challenging diseases, of growing concern to the US health care system, such as cancer and neurodegeneration. The associated cost of these diseases are some of the highest and fastest growing in the US. IDPs also play a key role in the replication and spreading of viral pathogens. In order to function properly IDPs must (i) bind efficiently to specific binding partners; and (ii) avoid pathological aggregation. A molecular level understanding of how IDP sequences encode for these two processes, and how mutations and stress conditions in cells can affect them, will significantly advance our understanding and ability to treat and prevent such diseases. Major experimental efforts are currently focused on: (i) resolving the structural ensembles of IDPs and the binding mechanism (IDP structure/function); or (ii) resolving the mechanisms of IDP aggregation into IDP aggregation into specific amyloid aggregates (IDP malfunction). Here we propose a radically different, physical chemistry approach in which we study the effect of IDP conformational dynamics on the binding mechanism; and the quantitative relation between IDP phase separation and aggregation. We will do so by using high resolution experimental techniques and methods developed in our lab, along with the multiscale simulations. Because most IDPs bind through coupled folding and binding, their conformational dynamics is expected to greatly affect the binding mechanism. Our approach of incorporating IDP conformational dynamics in binding studies will provide a key missing link to understand IDP functional binding. Our proposed study builds on the recent characterization of the binding mechanism of a group of IDPs, and focuses on studying 1) the effect of conformational dynamics on binding; and 2) the physiological process of liquid phase-separation and its link to pathological aggregation. We will combine different high resolution experimental techniques -including nanosecond laser pump spectroscopy- with molecular simulations to characterize IDP structure and dynamics. Novel methods will be used to quantify liquid phase-separation of IDPs. Results from Aim I: Test our hypothesis by comparing IDP dynamics for different binding scenarios; Aim II: Modulating the binding mechanism by perturbing the sequence and solvent, and Aim III: Quantify the effect of a disease mutation on the conformational dynamics, phase separation and aggregation of FUS_LC; will have direct impact on the molecular understanding IDPs implicated in ovarian and breast cancer, in the replication of paramoxyviruses and in amyotrophic lateral sclerosis and frontotemporal dementia. These results have the potential of transforming our way of viewing coupled folding and binding, and liquid phase separation which are crucial for IDP function.