Structural studies of disorder-based protein-protein interactions

内在无序蛋白（IDPs）是一类独特的蛋白质，它们不具有良好的折叠结构，但仍保持功能。因此，它们挑战了经典的蛋白质结构-功能范式。重要的是，IDPs在所有生物中都非常丰富，在许多重要的生物过程中起着至关重要的作用。我的研究项目的长期目标是了解国内流离失所者复杂的结构-功能关系。我对参与细胞信号通路的IDPs特别感兴趣，它们通常在蛋白质-蛋白质相互作用网络中充当枢纽。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IDPs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)IDPs作为招募结合伙伴的蛋白质枢纽的机制，以及2)翻译后修饰，特别是磷酸化，对IDPs结构和靶标识别特性的影响。我们的研究将特别关注Nrf2。Nrf2是调控细胞对氧化应激反应的关键转录因子。它的活性通过一系列的蛋白质相互作用受到严格的调控。许多这些相互作用是由磷酸化调节的，这增加了Nrf2调控的额外复杂性。我们将结合实验和计算方法来剖析Nrf2与几个在Nrf2调控中起关键作用的靶标相互作用的机制。技术包括核磁共振（NMR）光谱，等温滴定量热法，遗传密码扩展和分子动力学（MD）模拟将被使用。核磁共振波谱是在分子水平上研究IDPs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，MD模拟正迅速成为获得IDP分子洞察力的一种极好的补充方法。我们还将研究磷酸化对Nrf2结构和靶标识别的影响。尽管结构生物学不断取得进展，磷酸化对蛋白质构象和靶标结合影响的分子理解仍受到无法大量合成高均匀性、特异位点磷酸化蛋白的阻碍。为了克服这一技术障碍，我们将使用酶磷酸化和一种创新的遗传密码扩展方法，在Nrf2的所需位置特异性地安装磷酸丝氨酸。这些研究将建立Nrf2作为功能开关的磷酸化的结构基础。总之，拟议的研究结果将对令人兴奋的国内生产总值领域作出重大贡献。

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.