Structural and dynamics studies of intrinsically disordered proteins

蛋白质的功能与其结构、柔韧性以及与细胞内其他分子相互作用的方式密切相关。因此，研究蛋白质的折叠、结构、动力学和靶标识别对于理解蛋白质的功能是至关重要的。内在无序蛋白（IDPs）是一类独特的蛋白质，它们不采用明确的结构，而是在生理条件下作为快速相互转换的构象集合存在。重要的是，这类蛋白质在我们体内非常丰富，并参与重要的生物功能。我的研究计划的长期目标是寻求对国内流离失所者复杂的结构-功能关系的完整理解。我们对参与信号通路的idp特别感兴趣。为了实现这一目标，我们将广泛表征不同结构性质和不同条件下不同IDPs的结构倾向、动态特征和靶结合机制。通过使用一系列生物物理技术，包括核磁共振波谱，等温滴定量热法，圆二色光谱偏振，以及分子动力学模拟，我们的目标是在分子水平上识别IDPs的结构和动力学特征，并将它们与生物功能联系起来。基于我们之前工作中获得的知识，在这个资助期，研究将集中在1)IDPs与蛋白质中心相互作用的机制，以及2)拥挤环境中IDPs的结构和动态行为。最近的研究表明，许多折叠良好的蛋白质通过特异性靶向IDPs在蛋白质-蛋白质相互作用网络中发挥关键枢纽作用。阐明这些枢纽和IDPs之间的结合机制对于破译通过这些蛋白质-蛋白质相互作用执行的生物学功能至关重要。通过本研究提出的内在无序Cby与14-3-3 hub蛋白之间相互作用的广泛研究，我们试图更好地了解控制IDPs与idp -hub之间结合的亲和力和特异性的因素。本提案的另一个目的是在分子水平上剖析大分子拥挤对国内流离失所者的影响。蛋白质的结构研究通常用低浓度的纯化蛋白质在稀释的缓冲溶液中进行。然而，这些条件与蛋白质执行其功能的细胞环境有很大不同。包括蛋白质和DNA在内的高浓度大分子的存在，在细胞中造成了拥挤的状况。这种现象，通常被称为分子拥挤，对蛋白质的行为有显著的影响。为了研究分子拥挤对IDPs结构、动力学和分子识别的影响，我们将使用高浓度的惰性剂模拟拥挤的细胞环境，并通过核磁共振和其他生物物理技术研究具有不同结构特征的不同IDPs在这些条件下的行为。这项工作将揭示IDPs如何在拥挤的蜂窝环境中发挥作用。最近关于无序蛋白的令人兴奋的实验发现引起了全世界对其功能和特性的研究兴趣。所提出的研究结果将对这一令人兴奋的领域作出重大贡献。

The function of a protein is intimately related to its structure, flexibility, and the ways it interacts with other molecules in cells. Studying of the folding, structure, dynamics, and target recognition of a protein is therefore with fundamental importance in understanding how it functions. Intrinsically disordered proteins (IDPs) are a unique class of proteins that do not adopt a well-defined structure but exist as an ensemble of fast inter-converting conformers under physiological conditions. Importantly, such proteins are highly abundant in our bodies and are involved in important biological functions. The long-term goal of my research program is to seek a complete understanding of the complex structure-function relationship of IDPs. We are particularly interested in IDPs that are involved in signaling pathways. To achieve this goal, we will extensively characterize the structural propensity, dynamic feature, and target-binding mechanism of different IDPs with varying structural properties and under different conditions. By using an array of biophysical techniques including nuclear magnetic resonance spectroscopy, isothermal titration calorimetry, circular dichroism spectropolarimety, as well as molecular dynamics simulations, we aim to identify the structural and dynamical characteristics of IDPs at the molecular level and correlate them to their biological functions. Building on the knowledge acquired from our previous work, in this grant period, the research will be focused on 1) the mechanisms by which IDPs interact with protein hubs, and 2) the structural and dynamical behaviors of IDPs in crowded environments. Recent studies revealed that a number of well-folded proteins function as critical hubs in the protein-protein interaction network by specifically targeting for IDPs. Elucidating the binding mechanisms between these hubs and IDPs are essential for deciphering the biological functions executed through these protein-protein interactions. Through the extensive studies of the interaction between intrinsically disordered Cby with the 14-3-3 hub protein proposed here, we seek to obtain a better understanding of the factors that govern the affinity and specificity of binding between IDPs and IDP-hubs. Another objective of this proposal is to dissect the effects of macromolecular crowding on IDPs at the molecular level. Structural studies of proteins are usually performed with low concentrations of purified protein in dilute buffer solutions. These conditions, however, are significantly different from that of the cellular environments where proteins carry out their functions. The presence of high concentrations of macromolecules, including protein and DNA, creates a crowded condition in cells. This phenomenon, commonly referred to as molecular crowding, has significant effects on the behaviors of proteins. To investigate the molecular crowding effects on the structure, dynamics, and molecular recognition of IDPs, high concentration of inert agents will be used to mimic the crowded cellular environments and the behaviors of different IDPs with distinct structural characteristics under these conditions will be studied by NMR and other biophysical techniques. The work will shed light on how IDPs functions in crowded cellular environments.Exciting recent experimental discoveries about disordered proteins have generated worldwide research interest in their functions and properties. The results of the proposed research will make a significant contribution to this exciting field.