Mechanisms and dynamics of allosteric function in proteins

摘要 生物是通过蛋白质的作用来驱动的。我们知道，结构往往为蛋白质的 功能，但近年来变得明显的是，蛋白质的功能也严重依赖于动力学， 或结构的运动。动力学如何实现功能现在是蛋白质生物学的一个中心问题，它限制了 我们对蛋白质的基本了解，以及在药物发现和蛋白质设计中的应用。在那里的时候 动态-或构象灵活性-促进的许多类型的功能，两个功能原型 动力学方面有酶催化和变构作用。这两种现象的机制基础是普遍存在的 事实上，它们在很大程度上仍然是神秘的，并因动力学可能扮演的角色而吸引了许多关注。李氏家族 实验室一直致力于利用核磁共振和其他生物物理方法研究蛋白质的动力学和变构。 已经将近20年了。这项建议中概述的方法是将天然变构研究结合起来 酶(区域1和区域2)，利用光遗传学将变构调节设计为信号蛋白 (第三区)。在过去的五年里，该实验室开发了两个互补的核磁共振和生物物理系统 对动力学和变构的研究，这些研究非常适合解决这些机械问题， 重要的是，开发研究亚基间变构通讯的方法。这两个系统是 分支酸变位酶(CM)和胸苷酸合酶(TS)，这两种对称的同源二聚体 功能变构的Cm(来自酵母)是一种经典的变构蛋白，具有传统 变构：S型活性曲线；对称的四元结构；紧张(T)和松弛(R)构象； 以及上调或下调活性的小分子变构效应配体。与CM的正面对比 协作性，TS是负协作性，因为它是半个位置的反应性。工作将在大肠杆菌(ECTS)上进行 和人类(HTS)形式，尽管它们相似，但表现出非常不同的行为。人类的TS是 抗癌药物5-氟尿嘧啶(5-FU)的靶点。CM、ECTS和HTS都有值得研究的突出特性 溶液核磁共振，因为它们是高度可溶的，稳定的，并产生出色的光谱。今后五年的奋斗目标 主要分为三个方面：(1)通过使用核磁共振波谱、分子动力学模拟、 量热学、x射线结晶学和生物化学，这些酶的结构和动态性质将 与关键兴趣的功能行为有关，例如：变构通讯；载脂蛋白状态如何构象 与T和R构象的比较；单连接状态下的原构象不对称性；以及转变的性质 州政府。(2)我们将通过引入一种化学技术来推进蛋白质均二聚体的核磁共振研究 利用点击化学进行原核糖体的偶联。以这种方式产生的混合标记二聚体将有助于核磁共振研究 对原核糖体间相互作用的研究，如变构，以及改进同质二聚体的核磁共振结构测定。(3) 对于人工放置在光遗传控制下的工程GTP酶，变构机制 将使用核磁共振方法来确定。

Abstract Biology is driven through the action of proteins. We know that structure often provides the foundation for proteins’ function, but in recent years it has become clear that protein function is also critically dependent on dynamics, or movements of structure. How dynamics enables function is now a central question in protein biology that limits our basic understanding of proteins, as well as applications in drug discovery and protein design. While there are many types of functions that dynamics – or conformational flexibility – promotes, two functional archetypes for dynamics are enzyme catalysis and allostery. The mechanistic bases for these two phenomena, pervasive as they are, remain largely mysterious and have attracted much attention for the likely role of dynamics. The Lee laboratory has focused on studying dynamics and allostery in proteins using NMR and other biophysical methods for nearly 20 years. The approach outlined in this proposal is to combine investigation of natural allosteric enzymes (Areas 1 and 2) with efforts to engineer allosteric regulation into signaling proteins using optogenetics (Area 3). In the last five years, the lab has developed two complementary systems for NMR and biophysical studies of dynamics and allostery that are highly amenable for addressing these mechanistic questions and, importantly, developing approaches to study intersubunit allosteric communication. The two systems are the enzymes chorismate mutase (CM) and thymidylate synthase (TS), both symmetric homodimers that are functionally allosteric. CM (from yeast) is a classically allosteric protein, exhibiting all the hallmarks of traditional allostery: sigmoidal activity curve; symmetric quaternary structure; tense (“T”) and relaxed (“R”) conformations; and small molecule allosteric effector ligands that either up- or down-regulate activity. In contrast to CM’s positive cooperativity, TS is negatively cooperative because it is half-the-sites reactive. Work will be on the E. coli (ecTS) and human (hTS) forms, which, despite their similarities show very different behaviors. The human TS is the target of anticancer drug 5-fluoro-uracil (5-FU). CM, ecTS, and hTS all have outstanding features for study by solution NMR since they are highly soluble, stable, and yield excellent spectra. The goals for the next five years fall into three main areas: (1) Through the use of NMR spectroscopy, molecular dynamics simulations, calorimetry, x-ray crystallography, and biochemistry, the structural and dynamic properties of these enzymes will be related to functional behaviors of key interest, such as: allosteric communication; how apo state conformations compare to T and R conformations; protomer asymmetry in singly liganded states; and the nature of the transition state. (2) We will advance the study of protein homodimers by NMR by introducing a technology for chemical conjugation of protomers using click chemistry. Mixed labeled dimers produced this way will facilitate NMR study of interprotomer interactions, such as allostery, and improve NMR structure determination of homodimers. (3) For engineered GTPases that have been artificially placed under optogenetic control, the allosteric mechanisms will be determined using an NMR approach.