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（通过应用程序提供）：酶机制的确定是生物分子系统研究中的一个核心主题，因为它们参与了生物组织中的大多数过程。新的实验和计算生物物理学方法的开发，这些方法允许这些过程的新观点和更详细的观点不仅从基础科学的角度来看，而且由于方法的广泛应用以及从中得出的知识而言，这也具有至关重要的重要性。该提案的目的是创建一部“分子电影”，在该电影中，系统中每个原子的位置，运动和能量随着反应的过程遵循。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LAUE晶体学和（ii）HMGR反应途径的模拟。该方法的应用将允许访问有关酶化学的一系列详细知识，而拟议方法依赖于可以通过时间分辨的晶体学和计算方法访问的时间表的新兴收敛性。我们将使用我们最近开发过的复印的辅助因子通过LAUE晶体学产生结构“快照”，其时间分辨率为1-100 µs。这些快照产生的中间状态的集合将使用奇异值分解（SVD）进行反驳，并使用长时间的分子动力学（MD）模拟连接，以提供结构，动态和能量的洞察力，可在完整的反应途径中。量子引导的分子机制（Q2mm）将产生可极化和非极化过渡状态力场（TSFF）。 TSFF的使用比广泛使用的QM/mm方法快102-104倍，从而允许大量采样，并以一致的水平处理整个系统，从而阻止了QM/MM接口区域引起的众所周知的问题。晶体结构的迭代循环 - > MD模拟 - > Markov状态集合生成 - > laue数据的SVD分析 - >新的时间分析结构将用于研究一个复杂的反应途径，可以将其分解为较小的步骤，以促进实验和计算方法。这种组合的方法将应用于假单胞菌Mevalonii HMGR的情况，HMGR具有广泛的生物医学兴趣，因为它是广泛使用的统计数据的靶标，也是通过新的抗生素类别的抗菌治疗的潜在靶标，但是在该建议中开发的方法主要适用于系统的广泛范围。为了促进引入的实验和计算创新的使用，将为更广泛的科学界提供所有工具化合物和要开发的计算代码。