Structural and dynamics studies of intrinsically disordered proteins

蛋白质的功能与其结构、灵活性以及与细胞中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、动力学和目标识别对于理解其功能具有根本的重要性。蛋白质内部无序蛋白（IDP）是一类独特的蛋白质，其不采用明确的结构，而是在生理条件下作为快速相互转换构象体的集合体存在。重要的是，这些蛋白质在我们的身体中非常丰富，并参与重要的生物功能。 我的研究计划的长期目标是寻求一个完整的理解的复杂的结构-功能关系的国内流离失所者。我们对参与信号通路的IDP特别感兴趣。为了实现这一目标，我们将广泛的结构倾向，动力学特征，和目标结合机制的不同IDP与不同的结构特性和不同的条件下。通过使用一系列的生物物理技术，包括核磁共振光谱，等温滴定量热法，圆二色光谱偏振，以及分子动力学模拟，我们的目标是确定的结构和动力学特性的IDP在分子水平上，并将它们与它们的生物功能。 基于我们以前工作中获得的知识，在本资助期间，研究将集中在1）IDP与蛋白质中心相互作用的机制，以及2）在拥挤环境中IDP的结构和动力学行为。最近的研究表明，许多折叠良好的蛋白质通过特异性靶向IDP而在蛋白质-蛋白质相互作用网络中起关键枢纽作用。阐明这些枢纽和IDP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功能是必不可少的。 通过广泛的研究与14-3-3枢纽蛋白在这里提出的内在无序Cby之间的相互作用，我们试图获得一个更好的理解的因素，支配的亲和力和特异性的结合之间的IDP和IDP-枢纽。 这项建议的另一个目标是从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在稀释的缓冲溶液中用低浓度的纯化蛋白质进行。然而，这些条件与蛋白质执行其功能的细胞环境显著不同。高浓度的大分子，包括蛋白质和DNA的存在，在细胞中创造了拥挤的条件。这种现象，通常被称为分子拥挤，对蛋白质的行为有显着的影响。为了研究分子拥挤效应对IDP的结构、动力学和分子识别的影响，将使用高浓度的惰性试剂来模拟拥挤的细胞环境，并且将通过NMR和其他生物物理技术来研究在这些条件下具有不同结构特征的不同IDP的行为。这项工作将揭示国内流离失所者如何在拥挤的蜂窝环境中发挥作用。 最近关于无序蛋白质的令人兴奋的实验发现引起了世界范围内对其功能和性质的研究兴趣。这项研究的结果将为这一令人兴奋的领域做出重大贡献。

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.