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: 9897100
• 负责人: OLAF G WIEST
• 金额: $ 33.57万
• 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 2024-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9897100
• 关键词: Active Sites; Anti-Bacterial Agents; Biochemistry; Biological; Biology; Biophysics; Catalysis; Chemicals; Cholesterol; Code; Collaborations; Combined Modality Therapy; Communities; Complex; Computational Biology; Computing Methodologies; Couples; Coupling; Crystallization; Crystallography; Data; Developed Countries; Development; Disease; Drug Targeting; Enzymatic Biochemistry; Enzymes; Equilibrium; Free Energy; Freezing; Goals; Grant; Health; Human; Hydroxymethylglutaryl-CoA reductase; Knowledge; Life; Link; Machine Learning; Maps; Methodology; Methods; Modeling; Molecular; Molecular Conformation; Movement; Mutagenesis; Nature; Oxidoreductase; Pathway interactions; Pharmaceutical Preparations; Positioning Attribute; Process; Protein Dynamics; Proteins; Pseudomonas; Public Health; Reaction; Research Personnel; Resolution; Roentgen Rays; Role; Running; Science; Structure; System; Techniques; Time; Validation; Work; base; biophysical chemistry; cofactor; computer studies; design; electron density; enzyme mechanism; experience; experimental study; improved; innovation; machine learning method; millisecond; molecular dynamics; molecular mechanics; molecular scale; movie; new therapeutic target; particle; protein structure; quantum; simulation; structural biology; synthetic biology; theories; tool

Project Summary Understanding enzyme mechanisms is of paramount importance from both the basic biophysics perspective of understanding life processes and the role of enzymes in diseases. To achieve a detailed understanding of enzyme catalysis, the effects of protein structure and dynamics on the reaction energetics need to be elucidated. We propose a combined computational and experimental approach that combines the synthetic, computational and structural biology expertise of a team of investigators that has been working together for >15 years to create a “molecular movie” where the position, movement and energy of every atom in the system followed over the entire reaction pathway. The proposal exploits the emerging convergence of timescales accessible by molecular simulation using GPUs and time resolved structural biology. Specific Aim 1 describes the simulation of the complete reaction pathway of Pseudomonas mevalonii (Pm) HMGCoA Reductase (HMGR) and will use transition state force fields (TSFFs) generated by the quantum guided molecular mechanics method to allow the µsec MD simulations of the chemical steps. TSFFs not only circumvent the well-known boundary problem of QM/MM, but are also 102-104 times faster. This allows a realistic modeling of the coupling of µsec dynamics and catalysis that was demonstrated in the last grant period to be essential for understanding the reaction. Together with accelerated MD simulations of the conformational changes involved in the reaction using standard force fields, these computational studies cover the fsec to µsec timescale. In Specific Aim 2, the computational results will be merged with the results of a three-tiered approach to obtain structural snapshots with progressively increasing time resolution: (i) “Frozen” intermediates that map out the overall pathway on long timescales, (ii) time resolved Laue crystallography using pH jump initiation on the msec timescale and (iii) use of photocaged substrates to allow time resolved Laue experiments on the µsec timescale. This approach will be applied to the study of HMGR, an enzyme of high biophysical and biomedical significance that has a complex reaction mechanism involving three chemical steps, six large-scale conformational changes and two cofactor exchange steps. The project is highly innovative because it (i) uses a combination of MD simulations using TSFFs and time resolved crystallography to span timescales of at least 12 orders of magnitude, (ii) iteratively couples the Markov State analysis of long timescale trajectories to the Singular Value Decomposition used to analyze time resolved crystallography data, thus providing new tools to generate and experimentally validate trial structures (iii) applies global optimization and machine learning techniques to allow the automated fitting of TSFFs for proteins, which will enhance the application of this powerful method to other proteins and (iv) provides new photocaged substrates for the study of enzyme mechanisms to the chemical biology community. All tool compounds, methods and codes developed in this project will be made available to the scientific community.

项目摘要 从两个基本的生物物理学角度来看，了解酶的机制是至关重要的 了解生命过程和酶在疾病中的作用。要详细了解 酶催化、蛋白质结构和动力学对反应能量学的影响还有待阐明。 我们提出了一种计算和实验相结合的方法，它结合了合成的、计算的 和结构生物学专业知识的研究团队，他们已经合作了15年，创造了 这是一部“分子电影”，其中系统中每个原子的位置、运动和能量都跟随着 整个反应路径。该提议利用了分子可访问的时间尺度的新兴趋同 使用GPU和时间分辨结构生物学进行模拟。具体目标1描述了对 梅瓦隆假单胞菌(Pm)HMGCoA还原酶(HMGR)的完整反应途径及其应用 由量子引导分子力学方法产生的过渡态力场(TSFF)允许 微秒MD模拟化学步骤。TSFF不仅绕过了众所周知的边界问题 QM/MM，但速度也是102-104倍。这允许对微秒动态和 在上一次授权期中证明的催化作用对于理解反应是必不可少的。同舟共济 用标准作用力加速MD模拟反应中的构象变化 这些计算研究涵盖了从秒到微秒的时间尺度。在具体目标2中，计算结果 将与三层方法的结果合并，以逐步获得结构快照 提高时间分辨率：(I)“冰冻”中间体，绘制出长时间尺度上的总体路径；(Ii) 利用毫秒级pH跃迁引发的时间分辨劳厄结晶学和(Iii)光笼的使用 基片，允许在微秒时间刻度上进行时间分辨的劳厄实验。此方法将应用于 具有复杂反应的高生物物理和生物医学意义的酶--HMGR的研究 三个化学步骤、六个大范围构象变化和两个辅因子交换的机制 台阶。该项目具有很高的创新性，因为它(I)结合了使用TSFF和TIME的MD模拟 分辨结晶学以跨越至少12个数量级的时间尺度，(Ii)迭代地耦合马尔科夫 长时间尺度轨迹的状态分析用奇异值分解来分析时间分辨 结晶学数据，从而提供了生成和实验验证试验结构的新工具(III)应用 全局优化和机器学习技术，以允许TSFF自动匹配蛋白质，这 将加强这一强大的方法在其他蛋白质上的应用，以及(Iv)提供新的光笼 用于研究酶机制的底物，以化学生物界。所有工具化合物， 将向科学界提供在该项目中开发的方法和代码。