Determination of structure, dynamics and energetics of enzyme reactions

酶反应的结构、动力学和能量学测定

• 批准号: 9278009
• 负责人: OLAF G WIEST
• 金额: $ 29.62万
• 依托单位: UNIVERSITY OF NOTRE DAME
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-07-01 至 2019-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9278009
• 关键词: Anti-Bacterial Agents; Antibiotics; Basic Science; Biochemistry; Biological; Biophysics; Chemicals; Chemistry; Cholesterol; Code; Combined Modality Therapy; Communities; Complex; Computing Methodologies; Crystallization; Crystallography; Data; Developed Countries; Development; Disease; Drug Targeting; Enzymes; Evolution; Free Energy; Generations; Goals; Health; Human; Hydroxymethylglutaryl-CoA reductase; Knowledge; Life; Maps; Methodology; Methods; Modeling; Molecular; Molecular Conformation; Molecular Structure; Movement; Nature; Noise; Organism; Pathway interactions; Pharmaceutical Preparations; Positioning Attribute; Process; Proteins; Pseudomonas; Reaction; Resolution; Roentgen Rays; Role; Running; Sampling; Science; Signal Transduction; Structure; System; Systems Analysis; Time; Validation; Weight; Work; base; biophysical chemistry; biophysical techniques; cofactor; computer studies; design; electron density; electronic structure; enzyme mechanism; improved; innovation; insight; interest; irradiation; millisecond; molecular dynamics; molecular mechanics; molecular scale; movie; new therapeutic target; prevent; public health relevance; quantum; simulation; tool

 DESCRIPTION (provided by applicant): The determination of enzyme mechanisms is a central topic in the study of biomolecular systems because they are involved in most processes in living organisms. The development of new experimental and computational biophysics methods that allow new and ever more detailed views of these processes is of fundamental importance not just from the basic science point of view, but also due to the wide range of applications of the methods and the knowledge derived from them. The goal of the proposal is to create a "molecular movie" where the position, movement and energy of every atom in the system followed over the course of the reaction. This will be achieved by pursuing two Specific Aims: (i) Time-Resolved Laue Crystallography of HMG-CoA Reductase (HMGR) and (ii) Simulation of the Reaction Pathway of HMGR. The application of the methodology will allow access to a level of detailed knowledge about enzyme chemistry that was not attainable up to now. The proposed approach relies on the emerging convergence of the timescales accessible by time- resolved crystallography and computational methods. We will use our recently developed photocaged cofactors to generate structural "snapshots" with a time resolution of 1-100 µs by Laue crystallography. The ensembles of intermediate states generated by these snapshots will be deconvoluted using singular value decomposition (SVD) and connected using long timescale molecular dynamics (MD) simulations to provide structural, dynamic, and energetic insights into the complete reaction pathway. Polarizable and non- polarizable transition state force fields (TSFF) will be generated by the quantum-guided molecular mechanics (Q2MM). The use of TSFFs is 102-104 times faster than the widely used QM/MM methods, thus allowing extensive sampling, and treats the entire system at a consistent level, thus preventing the well- known problems resulting from the QM/MM interface region. Iterative cycles of crystal structure -> MD simulation -> Markov State ensemble generation -> SVD analysis of Laue data -> new time resolved structures will be used to study a complex reaction pathway, which can be broken down into smaller steps to facilitate both experimental and computational approaches. This combined methodology will be applied to the case of Pseudomonas mevalonii HMGR, which has a complex reaction mechanism involving three chemical steps, six large-scale conformational changes and two cofactor exchange steps. HMGR is of broad biomedical interest because it is the target of the widely used statins and a potential target for antibacterial treatments by new classes of antibiotics, but the methodology developed in this proposal is in principle applicable to a wide range of systems. To promote the use of the experimental and computational innovations introduced, all tool compounds and computational codes to be developed will be made available to the broader scientific community.

 描述(由申请人提供)：酶机制的确定是生物分子系统研究的中心课题，因为它们涉及生物有机体中的大多数过程。发展新的实验和计算生物物理学方法，使人们能够对这些过程有新的和更详细的看法，不仅从基础科学的角度来看，而且由于这些方法及其衍生的知识的广泛应用，具有根本的重要性。该提议的目标是创建一部“分子电影”，在这部电影中，系统中每个原子的位置、运动和能量都会在整个反应过程中得到跟踪。这将通过追求两个具体目标来实现：(I)HMG-CoA还原酶(HMGR)的时间分辨劳厄结晶学(HMGR)和(Ii)HMGR反应路径的模拟。该方法的应用将使人们能够获得迄今为止无法获得的关于酶化学的详细知识。所提出的方法依赖于时间分辨结晶学和计算方法可获得的时间尺度的新兴收敛。我们将使用我们最近开发的光笼辅助因子来生成时间分辨率为1-100微米S的结构快照。由这些快照生成的中间态的系综将使用奇异值分解(SVD)进行去卷积，并使用长时间尺度分子动力学(MD)模拟进行连接，以提供对完整反应路径的结构、动力学和能量方面的见解。量子引导分子力学(Q2 MM)将产生可极化和不可极化的过渡态力场(TSF)。TSFF的使用速度比广泛使用的QM/MM方法快102-104倍，从而允许广泛的采样，并以一致的水平对待整个系统，从而防止了QM/MM界面区域导致的众所周知的问题。晶体结构的迭代循环--&gt；MD模拟-gt；马尔可夫状态系综生成-gt；劳厄数据的奇异值分解分析--&gt；新的时间分辨结构将被用来研究复杂的反应路径，它可以被分解成更小的步骤，以便于实验和计算方法。这种组合方法将应用于梅瓦隆假单胞菌HMGR的案例，其反应机理复杂，涉及三个化学步骤、六个大规模构象变化和两个辅因子交换步骤。HMGR引起了广泛的生物医学兴趣，因为它是广泛使用的他汀类药物的靶点，也是新类别抗生素抗菌治疗的潜在靶点，但本提案中开发的方法学原则上适用于广泛的系统。为了促进采用所采用的实验和计算创新方法，将向更广泛的科学界提供将要开发的所有工具化合物和计算代码。