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

复杂的动态行为决定了蛋白质功能的许多关键方面。内在无序蛋白（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，这对其调节机制至关重要。尽管近年来对国内流离失所者进行了多学科的关注，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理解。***Sic1是一种周期蛋白依赖性激酶抑制剂，参与酵母细胞周期的调控，其在多位点磷酸化后与其受体蛋白Cdc4上的单个位点结合。核磁共振研究表明存在Sic1:Cdc4的动态复合物，Sic1在磷酸化和结合时主要保持无序。Sic1与Cdc4的这种动态多价结合的物理基础尚不清楚，尽管静电相互作用被认为起着重要作用。在神经系统中，帽依赖翻译是由真核起始因子4E （eIF4E）与无序eIF4E结合蛋白（4E- bp）以磷酸化依赖的方式相互作用调节的。其中一种蛋白质，4E-BP2，就像一个调节开关：在非磷酸化状态下，它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）停靠在eIF4E上并启动翻译。该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项目，重点研究荧光标记Sic1和4E-BP2的构象状态和动力学使用单分子荧光技术，如单分子Förster共振能量转移（smFRET）和荧光相关光谱（FCS）。我们之前的smFRET研究指出，IDPs的能量景观相当浅，但崎岖不平，并且静电调节了它们的压实和构象动力学。基于我们最近的进展，聚合物物理模型和计算模型将用于从smFRET数据推断IDPs的全球维度。我们的主要目标是量化两种典型IDPs的构象分布，局部和全局动力学，结合动力学和能量学，首先在体外，然后在活细胞中。这些结果将有助于将生物学中的经典结构-功能范式转变为更动态的替代方案，将提供对IDPs提供的各种创造性调节机制的见解，并将是设计针对其异常活动的合成或生物药物的重要一步。

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.