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: 10338723
• 负责人: Andrew L Lee
• 金额: $ 46.22万
• 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/10338723
• 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.

摘要 生物学是通过蛋白质的作用来驱动的。我们知道，结构通常为蛋白质的功能提供基础。 功能，但近年来已经清楚的是，蛋白质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， 我们对蛋白质的基本理解，以及在药物发现和蛋白质设计中的应用。虽然 有许多类型的功能，动力学-或构象灵活性-促进，两个功能原型 是酶催化和变构。这两种现象的机械基础， 尽管如此，它们在很大程度上仍然是神秘的，并且由于动力学可能的作用而引起了人们的广泛关注。李 一个实验室专注于研究动态和变构蛋白质使用核磁共振和其他生物物理方法 将近20年了该建议中概述的方法是将天然变构的研究与联合收割机的研究结合起来， 酶（领域1和2），致力于利用光遗传学将变构调节工程化为信号蛋白 (Area 3）。在过去的五年里，该实验室开发了两个互补的系统，用于核磁共振和生物物理 动力学和变构的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 分支酸合成酶（CM）和胸苷酸合成酶（TS），两者都是对称的同源二聚体， 功能性别构的CM（来自酵母）是一种经典的变构蛋白，表现出传统的变构蛋白的所有特征。 变构：S形活性曲线;对称四级结构;紧张（“T”）和松弛（“R”）构象; 以及上调或下调活性的小分子变构效应配体。与CM的积极 协同性，TS是负协同的，因为它是一半的网站反应。工作将在E。大肠杆菌（ecTS） 和人类（HTS）的形式，尽管它们的相似之处显示出非常不同的行为。人类的TS是 抗癌药物5-氟尿嘧啶（5-FU）靶向。CM、ecTS和hTS都具有突出的特征，可通过 溶液NMR，因为它们是高度可溶的、稳定的并且产生优异的光谱。未来五年的目标 分为三个主要领域：（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，这些酶的结构和动力学性质将 与关键感兴趣的功能行为有关，例如：变构通讯;载脂蛋白状态构象如何 比较T和R构象;单配位态中的原异构体不对称性;以及过渡的性质 状态(2)我们将通过引入化学分析技术， 使用点击化学进行原异构体的缀合。以这种方式产生的混合标记二聚体将有助于NMR研究 的前体间的相互作用，如变构，并改善NMR结构测定的同源二聚体。（三） 对于已经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。