Structural studies of disorder-based protein-protein interactions

内含子无序蛋白（IDP）是一类独特的蛋白质，它们不具有良好的折叠结构，但仍保留功能。因此，它们挑战了经典的蛋白质结构-功能范式。重要的是，国内流离失所者在所有生物体中大量存在，并在许多重要的生物过程中发挥关键作用。 我的研究计划的长期目标是了解国内流离失所者的复杂结构-功能关系。我特别感兴趣的是参与细胞信号通路的IDP，在这些通路中，它们通常充当蛋白质-蛋白质相互作用网络的枢纽。我的团队旨在揭示IDPs的结构和动力学特征，以及它们在分子水平上与不同结合伙伴相互作用的方式。这些知识对于理解IDP如何发挥作用以及它们调节的细胞过程非常重要。在接下来的5年资助周期中，我的研究将集中在1）IDP在招募结合伙伴中作为蛋白质枢纽的机制，以及2）翻译后修饰，特别是磷酸化对IDP的结构和目标识别特性的影响。我们的研究将特别关注NRF 2。nrf 2是协调细胞对氧化应激反应的关键转录因子。它的活性通过蛋白质-蛋白质相互作用的级联来严格调节。许多这些相互作用是由磷酸化调节，增加了额外的复杂性水平的Nrf 2调控。 我们将结合联合收割机的实验和计算的方法在解剖Nrf 2与几个目标，发挥关键作用的Nrf 2调节相互作用的机制。将使用包括核磁共振（NMR）光谱、等温滴定量热法、遗传密码扩展和分子动力学（MD）模拟在内的技术。NMR光谱是在分子水平上研究IDP的最强大的生物物理技术。随着计算能力和模拟方法的进步，分子动力学模拟正迅速成为一种优秀的补充方法，用于获得IDP的分子见解。 我们还将研究磷酸化对Nrf 2结构和靶点识别的影响。尽管结构生物学不断取得进展，但由于无法合成具有高同质性和大量的位点特异性磷酸化蛋白，因此对磷酸化对蛋白质构象和靶结合的影响的分子理解受到阻碍。为了克服这一技术障碍，我们将使用酶促磷酸化和一种创新的遗传密码扩增方法，在Nrf 2的所需位置特异性地安装磷酸丝氨酸。这些研究将为Nrf 2磷酸化作为功能开关奠定结构基础。 总之，所提出的研究结果将为激动人心的IDP领域做出重大贡献。

Intrinsically disordered proteins (IDPs) are a unique class of proteins that do not adopt a well-folded structure but yet retain function. Therefore, they challenge the classical protein structure-function paradigm. Importantly, IDPs are highly abundant in all organisms and play crucial roles in many important biological processes. The long-term goal of my research program is to understand the complex structure--function relationship of IDPs. I am particularly interested in IDPs that are involved in cell signalling pathways, in which they often act as hubs in protein-protein interaction networks. My group aims to unravel the structural and dynamical characteristics of IDPs, and the ways they interact with different binding partners at the molecular level. The knowledge is important for understanding how IDPs function, and the cellular processes they regulate. In the next 5-year grant cycle, my research will be focused on 1) the mechanisms by which IDPs function as protein hubs in recruiting binding partners, and 2) the effects of post-translational modifications, especially phosphorylation, on the structural and target recognition properties of IDPs. Our studies will particularly focus on Nrf2. Nrf2 is the key transcription factor that orchestrates the cellular responses to oxidative stress. Its activity is tightly regulated via a cascade of protein-protein interactions. Many of these interactions are modulated by phosphorylation, adding an additional level of complexity to the Nrf2 regulation. We will combine experimental and computational approaches in dissecting the mechanisms by which Nrf2 interacts with several targets that play critical roles in Nrf2 regulation. Techniques including nuclear magnetic resonance (NMR) spectroscopy, isothermal titration calorimetry, genetic code expansion, and molecular dynamic (MD) simulation will be used. NMR spectroscopy is the most powerful biophysical technique for studying IDPs at the molecular level. With the advancements in computational power and simulation methodologies, MD simulation is quickly emerging as an excellent complementary approach for obtaining molecular insights of IDP. We will also investigate the effects of phosphorylation on the structure and target recognition of Nrf2. Despite the continued advances in structural biology, molecular understanding about the impact of phosphorylation on protein conformation and target binding is impeded by the inability to synthesize site-specifically phosphorylated proteins with high homogeneity and in large quantity. To overcome this technological barrier, we will use enzymatic phosphorylation and an innovative genetic code expansion method to site-specifically install phosphoserine at the desired locations in Nrf2. The studies will establish structural basis of phosphorylation as functional switches of Nrf2.  Together, results of the proposed research will make a significant contribution to the exciting IDP field.