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: 8888788
• 负责人: OLAF G WIEST
• 金额: $ 30.95万
• 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/8888788
• 关键词: Anti-Bacterial Agents; Antibiotics; Basic Science; Biochemistry; Biological; Biophysics; Chemicals; Chemistry; Cholesterol; Code; Combined Modality Therapy; Communities; Complex; Computing Methodologies; 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; 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; cofactor; computer studies; design; electron density; electronic structure; enzyme mechanism; improved; innovation; insight; interest; irradiation; millisecond; molecular dynamics; molecular mechanics; movie; 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，因为它们参与了生物体中的大多数过程。新的实验和计算生物物理学方法的发展，使新的和更详细的意见，这些过程是至关重要的，不仅从基础科学的角度来看，但也由于广泛的应用的方法和知识来自他们。 该提案的目标是创建一个“分子电影”，其中系统中每个原子的位置，运动和能量在反应过程中跟随。这将通过追求两个特定目标来实现：（i）HMG-CoA还原酶（HMGR）的时间分辨劳厄晶体学和（ii）HMGR反应途径的模拟。该方法的应用将允许获得酶化学的详细知识，这是迄今为止无法达到的水平。 所提出的方法依赖于时间分辨晶体学和计算方法可访问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因子通过劳厄晶体学产生时间分辨率为1-100 µs的结构“快照”。由这些快照生成的中间状态的集合将使用奇异值分解（SVD）进行解卷积，并使用长时间尺度分子动力学（MD）模拟进行连接，以提供对完整反应途径的结构，动态和能量见解。量子导引分子力学（Q2 MM）将产生可极化和不可极化的过渡态力场（TSFF）。TSFF的使用比广泛使用的QM/MM方法快102-104倍，因此允许广泛的采样，并以一致的水平对待整个系统，从而防止由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->马尔可夫状态系综生成->劳厄数据的SVD分析->新的时间分辨结构将用于研究复杂的反应途径，该反应途径可以分解为更小的步骤以促进实验和计算方法。 这种组合的方法将被应用到的情况下，假单胞菌mevalonii HMGR，它有一个复杂的反应机制，涉及三个化学步骤，六个大规模的构象变化和两个辅因子交换步骤。HMGR具有广泛的生物医学意义，因为它是广泛使用的他汀类药物的靶点，也是新型抗生素抗菌治疗的潜在靶点，但本提案中开发的方法原则上适用于广泛的系统。为了促进使用所介绍的实验和计算创新，将向更广泛的科学界提供所有有待开发的工具化合物和计算代码。