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来驱动的。我们知道结构通常为蛋白质提供基础 功能，但近年来，很明显蛋白质功能也关键取决于动态， 或结构运动。动力学启用如何启用功能现在是限制蛋白质生物学的核心问题 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。那里 动态或构象灵活性的许多类型的功能都促进了两种功能原型 对于动力学，是酶催化和变构。这两个现象的机械基础，普遍 就像他们一样，仍然在很大程度上神秘，并引起了人们对动态的可能作用的极大关注。李 实验室专注于使用NMR和其他生物物理方法研究蛋白质的动力学和变构。 将近20年。该提案中概述的方法是结合对天然变构的研究 酶（区域1和2），努力使用光遗传学来对信号蛋白进行变构调节 （区域3）。在过去的五年中，该实验室为NMR和生物物理开发了两个完整的系统 对于解决这些机械性问题的高度适合的动力学和变构的研究， 重要的是，正在开发研究基间变构沟通的方法。这两个系统是 酶酸化突变酶（CM）和胸苷酸合酶（TS），这是两个对称同型二聚体 功能变构。 CM（来自酵母）是一种经典的变构蛋白，表现出传统的所有标志 变构：乙状结肠活性曲线；对称的第四纪结构；时态（“ t”）和放松（“ r”）构象； 以及上调或下调活性的小分子变构效应配体。与CM的积极相反 合作性，TS是负合作的，因为它具有半个位置的反应性。工作将在大肠杆菌（ECTS）上 和人类（HTS）形式，它们的相似性表现出截然不同的行为。人类TS是 抗癌药物5-氟鲁西尔（5-FU）的靶标。 CM，ECTS和HTS都具有出色的研究功能 溶液NMR由于它们具有高度固体，稳定并产生出色的光谱。未来五年的目标 属于三个主要区域：（1）通过使用NMR光谱，分子动力学模拟， 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有关，例如：变构交流； Apo状态构象如何 与T和R构象相比；单一配体状态下的生产者不对称；和过渡的本质 状态。 （2）我们将通过引入化学技术来推进NMR研究蛋白质同二聚体的研究 使用点击化学结合剂的​​结合。产生的混合标记的二聚体将有助于NMR研究 跨局体相互作用（例如变构和改进的NMR结构的测定）的同二聚体的确定。 （3） 对于已人为地置于光遗传控制的工程GTPase，变构机制 将使用NMR方法确定。