Conformational sub-states in enzyme catalysis: Applications to ribonuclease

酶催化中的构象亚状态：在核糖核酸酶中的应用

• 批准号: 9040996
• 负责人: Pratul K Agarwal
• 金额: $ 50.99万
• 依托单位: UNIVERSITY OF PITTSBURGH AT PITTSBURGH
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-04-01 至 2019-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9040996
• 关键词: Active Sites; Address; Architecture; Biophysics; Catalysis; Communities; Computer Simulation; Data; Data Set; Disease; Environment; Enzyme Kinetics; Enzymes; Eukaryota; Exhibits; Family; Family member; Foundations; Health; Human; Human Genome; Hydrolysis; Individual; Investigation; Joints; Kidney; Knowledge; Life; Medicine; Methodology; Methods; Minor; Modeling; Molecular; Molecular Conformation; Motion; Multienzyme Complexes; Nuclear Magnetic Resonance; Pancreas; Pathogenicity; Physiological; Play; Population; Proteins; RNA; Relaxation; Research; Research Personnel; Resolution; Ribonucleases; Role; Sampling; Shapes; Signal Transduction; Software Framework; Software Tools; Staging; Structure; System; Techniques; Theoretical model; Time; Titrations; Triad Acrylic Resin; United States National Institutes of Health; Validation; Virus; antiangiogenesis therapy; base; chemical reaction; conformer; design; enzyme mechanism; enzyme structure; improved; inhibitor/antagonist; insight; member; millisecond; molecular dynamics; molecular recognition; mutant; neurotoxicity; novel; protein function; protein structure; quantum; research study; scaffold; simulation; software development; theories; tool

DESCRIPTION (provided by applicant): Enzymes perform the designated function of catalyzing chemical reactions by serving more than a scaffold for bringing together the reactants. The role of structure in enzyme function has been known for more than a century now; however, more recent evidence suggests that a functioning enzyme exists in an ensemble of conformations under ambient physiological conditions. The ensemble view of enzyme structure suggests that it can sample conformational sub-states that exhibit function promoting structural and dynamical features. Further, evidence from experiments and computational modeling suggest that transitions between these conformational sub-states enable substrate recognition and catalysis. Quantitative insights into these functionally relevant sub-states remains challenging, particularly due to the wide range of time-scales involved, limited window of resolution for individual techniques and the fact that some of the sub-states can be potentially short-lived. We address these issues by developing a joint computational-experimental framework to identify and characterize such functionally relevant sub-states in the context of enzyme function. In addition to identifying structural intermediates, our framework will quantify the relative population of the conformations in various sub-states as well as enable their linkage to kinetics of enzyme function through the catalytic cycle. This integrated approach will be used to investigate the bio-medically relevant ribonuclease (RNase) family of proteins and enzymes. In particular, we will: (1) Develop a theoretical framework to identify and characterize the multi-scale hierarchy in the conformational landscape of proteins; (2) Utilize the developed framework to investigate the RNase fold members and their ability to access distinct conformational sub-states, including functionally relevant sub-states along the catalytic cycle; (3) Validate the developed model and predicted sub- states by integrating nuclear magnetic resonance (NMR) relaxation dispersion experiments. The developed methodology and models will be improved by iterative interaction between the 3 PIs with different expertise spanning theoretical biophysics, computational simulations and experimental techniques. Overall, our studies will have implications in the design of novel inhibitors of RNase function in the context of neurotoxicity, angiogenesis and anti-pathogenicity.

描述（由申请人提供）：酶不仅充当将反应物聚集在一起的支架，还执行催化化学反应的指定功能。结构在酶功能中的作用早已为人所知一个多世纪了。然而，最近的证据表明，在环境生理条件下，功能酶以构象集合的形式存在。酶结构的整体观点表明，它可以对表现出促进结构和动力学特征的功能的构象亚状态进行采样。此外，来自实验和计算模型的证据表明，这些构象亚状态之间的转变能够实现底物识别和催化。对这些功能相关的子状态的定量洞察仍然具有挑战性，特别是由于涉及的时间尺度范围广泛、单个技术的分辨率窗口有限以及某些子状态可能是潜在的 短暂的。我们通过开发联合计算实验框架来解决这些问题，以识别和表征酶功能背景下的此类功能相关的子状态。除了识别结构中间体之外，我们的框架还将量化各种亚状态中构象的相对数量，并通过催化循环将它们与酶功能的动力学联系起来。这种综合方法将用于研究生物医学相关的核糖核酸酶 (RNase) 家族的蛋白质和酶。具体来说，我们将：（1）开发一个理论框架来识别和表征蛋白质构象景观中的多尺度层次结构； (2) 利用开发的框架来研究 RNase 折叠成员及其访问不同构象亚状态的能力，包括催化循环中功能相关的亚状态； （3）通过整合核磁共振（NMR）弛豫色散实验来验证所开发的模型和预测的子状态。所开发的方法和模型将通过 3 位具有不同专业知识（涵盖理论生物物理学、计算模拟和实验技术）的 PI​​ 之间的迭代交互来改进。总体而言，我们的研究将对神经毒性、血管生成和抗致病性方面的新型 RNase 功能抑制剂的设计产生影响。