The physical underpinnings of intrinsically disordered proteins: conformations, dynamics and interactions

本质无序蛋白质的物理基础：构象、动力学和相互作用

• 批准号: RGPIN-2017-06030
• 负责人: Gradinaru, Claudiu
• 金额: $ 3.28万
• 依托单位: University of Toronto
• 依托单位国家: 加拿大
• 项目类别: Discovery Grants Program - Individual
• 财政年份: 2020
• 资助国家: 加拿大
• 起止时间: 2020-01-01 至 2021-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=712012
• 关键词: physical; underpinnings; intrinsically; disordered; proteins

Complex dynamic behavior dictates many key aspects of protein function. Intrinsically disordered proteins (IDPs), which are involved in cancer and neurodegenerative diseases, exhibit conformational changes on nanosecond to millisecond timescales which are essential for their regulatory mechanisms. Despite an intense multidisciplinary focus on IDPs in recent years, a reasonable understanding of the physical mechanisms underlying their structural disorder and the dynamic nature of their interactions is still lacking. Sic1 is a cyclin-dependent kinase inhibitor involved in the regulation of the yeast cell cycle, which upon multi-site phosphorylation binds to a single site on its acceptor protein, Cdc4. NMR studies indicated the presence of a dynamic complex of Sic1:Cdc4, with Sic1 remaining predominantly disordered upon phosphorylation and binding. The physical basis for this dynamic, multivalent binding of Sic1 to Cdc4 is unknown, although electrostatic interactions are thought to play an important role. In the nervous system, the cap-dependent translation is regulated by the interaction of eukaryotic initiation factor 4E (eIF4E) with disordered eIF4E binding proteins (4E-BPs) in a phosphorylation-dependent manner. One of those proteins, 4E-BP2, acts like a regulatory switch: in the non-phosphorylated state is largely disordered and bound to eIF4E, while in the multi-phosphorylated state it folds and detaches, allowing another protein (eIF4G) to dock onto eIF4E and initiate translation. The main question for this system concerns the role of phosphate groups, whether they stabilize the folding of 4E-BP2, or destabilize the interaction with eIF4E, or both. We propose a research program focused on studying the conformational states and the dynamics of fluorescently labelled Sic1 and 4E-BP2 using single-molecule fluorescence techniques, such as single-molecule Förster Resonance Energy Transfer (smFRET) and Fluorescence Correlation Spectroscopy (FCS). Our previous smFRET studies point to a rather shallow, but rugged energy landscape of IDPs and to electrostatics modulating their compaction and conformational dynamics. Building on our recent progress, polymer physics models and computational modelling will be used to infer global dimensions of IDPs from smFRET data. Our main goals are to quantify the distribution of conformations, local and global dynamics, binding kinetics and energetics for two paradigmatic IDPs, at first in vitro and then in live cells. The results will help change the classic structure-function paradigm in biology to more dynamic alternatives, will provide insight into the variety of creative regulatory mechanisms provided by IDPs, and will be an important step towards designing synthetic or biologic drugs against their aberrant activities.

复杂的动态行为决定了蛋白质功能的许多关键方面。固有无序蛋白(IDPs)与癌症和神经退行性疾病有关，在纳秒到毫秒的时间尺度上表现出构象变化，这是其调控机制所必需的。尽管近年来国内流离失所者受到多学科的高度关注，但对其结构紊乱的物理机制及其相互作用的动态性质仍然缺乏合理的了解。 SIC1是一种细胞周期蛋白依赖性的激酶抑制因子，参与酵母细胞周期的调控，通过多位点的磷酸化结合到其受体蛋白CDC4上的单一位点。核磁共振研究表明，SIC1：CdC4存在一个动态的络合物，SIC1在磷酸化和结合时主要保持无序。尽管静电相互作用被认为起着重要的作用，但这种动态的、多价的SIC1与CdC4结合的物理基础尚不清楚。在神经系统中，帽依赖的翻译受真核细胞起始因子4E(EIF4E)与无序的eIF4E结合蛋白(4E-BPS)以磷酸化依赖的方式相互作用的调节。其中一种蛋白质4E-BP2起着调控开关的作用：在非磷酸化状态下，它在很大程度上是无序的，并与eIF4E结合，而在多磷酸化状态下，它折叠并分离，允许另一种蛋白质(EIF4G)对接到eIF4E上并启动翻译。这个系统的主要问题涉及磷酸基团的作用，它们是稳定4E-BP2的折叠，还是破坏与eIF4E的相互作用，或者两者兼而有之。 我们提出了一个研究计划，重点是利用单分子荧光技术，如单分子Förster共振能量转移(SmFRET)和荧光相关光谱(FCS)，研究荧光标记的SIC1和4E-BP2的构象和动力学。我们之前的smFRET研究指出了国内流离失所者相当浅但崎岖的能量格局，以及静电学调节他们的压实和构象动力学。在我们最新进展的基础上，将使用聚合物物理模型和计算模型从smFRET数据推断国内流离失所者的全球维度。我们的主要目标是量化两种典型的内毒素的构象分布、局部和全球动力学、结合动力学和能量学，首先是在体外，然后是在活细胞中。这些结果将有助于改变生物学中经典的结构-功能范式，转向更动态的替代方案，将提供对国内流离失所者提供的各种创造性调节机制的洞察，并将是设计针对其异常活性的合成或生物药物的重要一步。