Mechanisms and dynamics of allosteric function in proteins

蛋白质变构功能的机制和动力学

• 批准号: 10691713
• 负责人: Andrew L Lee
• 金额: $ 7.7万
• 依托单位: UNIV OF NORTH CAROLINA CHAPEL HILL
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-07-01 至 2027-06-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10691713
• 关键词: Address; Affect; Allosteric Regulation; Antineoplastic Agents; Area; Attention; Behavior; Binding Sites; Biochemistry; Biology; Calorimetry; Catalysis; Chemicals; Chemistry; Chorismate Mutase; Communication; Distal; Engineering; Enzymes; Escherichia coli; Exhibits; Fluorouracil; Foundations; Goals; Guanosine Triphosphate Phosphohydrolases; Human; Investigation; Label; Laboratories; Ligand Binding; Ligands; Metabolism; Molecular Conformation; Movement; NMR Spectroscopy; Nature; Pathway interactions; Property; Protein Engineering; Proteins; Protomer; Regulation; Role; Signal Transduction; Signaling Protein; Site; Structure; System; Technology; Thymidylate Synthase; Work; X-Ray Crystallography; base; biophysical analysis; biophysical techniques; dimer; drug development; drug discovery; falls; flexibility; improved; interest; molecular dynamics; optogenetics; protein function; small molecule

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.

抽象的 生物学是通过蛋白质的作用驱动的。我们知道，结构通常为蛋白质的功能提供基础。 但近年来人们已经清楚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限制蛋白质生物学的一个核心问题。 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。 动力学或构象灵活性促进了许多类型的功能，两种功能原型 对于动力学来说，酶催化和变构是这两种现象普遍存在的机制基础。 事实上，它们在很大程度上仍然是神秘的，并且由于动力的可能作用而引起了人们的广泛关注。 实验室专注于使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 该提案概述的方法是结合自然变构的研究。 酶（区域 1 和区域 2），努力利用光遗传学将变构调节工程化到信号蛋白中 （区域 3）在过去五年中，实验室开发了两个互补的 NMR 和生物物理系统。 动力学和变构研究非常适合解决这些机械问题，并且， 重要的是，开发研究亚基间变构通讯的方法是这两个系统的关键。 分支酸变位酶 (CM) 和胸苷酸合酶 (TS)，都是对称同源二聚体 CM（来自酵母）是一种经典的变构蛋白，具有传统的所有特征。 变构：S形活性曲线；对称四级结构；紧张（“T”）和松弛（“R”）构象； 与 CM 的阳性活性相反，小分子变构效应配体可以上调或下调活性。 合作性，TS 是负合作性的，因为它的工作将在大肠杆菌 (ecTS) 上进行。 和人类（hTS）形式，尽管它们有相似之处，但表现出非常不同的行为。 抗癌药物5-氟尿嘧啶（5-FU）的靶点、ecTS、hTS均具有值得研究的突出特点。 溶液 NMR，因为它们具有高度可溶性、稳定性，并且能产生出色的谱图 未来五年的目标。 分为三个主要领域：（1）通过使用核磁共振波谱、分子动力学模拟， 通过量热法、X 射线晶体学和生物化学，这些酶的结构和动态特性将 与关键兴趣的功能行为相关，例如：apo 状态构象的变构通讯； 比较单配体状态下的 T 和 R 构象不对称性以及转变的性质； (2) 我们将通过引入化学分析技术来推进通过NMR对蛋白质同源二聚体的研究 使用点击化学方法缀合原聚体将有助于 NMR 研究。 原体间相互作用，例如变构，并改进同二聚体的 NMR 结构测定 (3)。 对于人工置于光遗传学控制下的工程化 GTP 酶，变构机制 将使用NMR方法测定。