Deciphering atomic-level enzymatic activity by time-resolved crystallography and computational enzymology

通过时间分辨晶体学和计算酶学破译原子级酶活性

• 批准号: 10507610
• 负责人: Wei Wang
• 金额: $ 10万
• 依托单位: COLUMBIA UNIVERSITY HEALTH SCIENCES
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-08-15 至 2024-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10507610
• 关键词: ATP Hydrolysis; ATP phosphohydrolase; ATPase Domain; Acceleration; Achievement; Actins; Automobile Driving; Award; Benchmarking; Binding; Binding Sites; Biochemical Reaction; Biological; Biological Models; Caring; Characteristics; Chemistry; Collaborations; Collection; Complex; Crystallization; Crystallography; Development; Disease; Electron Microscopy; Engineering; Ensure; Enzymatic Biochemistry; Enzymes; Event; Exhibits; Free Energy; Freezing; Geometry; Hand; Heat shock proteins; Hybrids; Hydrolysis; Impairment; In Vitro; Individual; Ions; Joints; Knowledge; Lead; Length; Life; Ligand Binding; Malignant Neoplasms; Mechanics; Mentors; Mentorship; Metabolism; Methods; Modeling; Modernization; Nucleotides; Organism; Pathway interactions; Persons; Phase; Physiological; Preparation; Process; Protons; Quantum Mechanics; Reaction; Reactive Oxygen Species; Research; Roentgen Rays; Series; Side; Solid; Structure; Sum; System; Temperature; Testing; Time; Time Study; Transportation; Universities; Water; Work; activation-induced cytidine deaminase; base; chemical reaction; electron diffraction; enzyme activity; enzyme substrate; experimental study; in vivo; innovation; insight; instrument; molecular mechanics; mutant; novel; operation; programs; quantum; receptor; restraint; skills; time interval; tool

Project Summary Enzymes are proteins that aid in the acceleration of metabolism or the chemical reactions in all living organisms. By synthesizing certain molecules and degrading others, enzymes can catalyze a range of biochemical reactions both in vivo and in vitro. When in collaboration with transporters and receptors, enzymes regulate almost all physiological functions in the body. Therefore, it is important to thoroughly understand ex- and in vivo enzyme activities. Among the many methods of studying enzyme activities, time-resolved macromolecular crystallography (TRX) has the advantage of investigating enzymatic reaction details on the fly. However, TRX theoretically can only capture the states where the enzymes are at local energetic saddle points. On the other hand, modern hybrid quantum mechanics/molecular mechanics (QM/MM) enables studying enzymatic reactions where new molecules are formed or destroyed. However, without the support from solid biological structures or if the transformation between reactant and product states is distinctively different, QM/MM cannot reach the authentic answer. This proposal aims to establish a new TRX- and QM/MM-based strategy to investigate enzymatic activities by joining the strength of those two territories. Notably, a recently elucidated allosteric controlling mechanism in 70- kDa heat shock proteins (Hsp70s) leads to an unparalleled opportunity of building a model system as a benchmark for developing the proposed TRX-QM/MM strategy. Specifically, in Aim 1, a groundbreaking discovery of oxygen radicals driving ATP hydrolysis will be examined by TRX experiments on the ATPase domain of bacterial Hsp70 DnaK. In Aim 2, I will use QM/MM to identify and verify the oxygen radical species, and depict the free energy landscape of the hydrolysis event in full scale. In Aim 3, I will use DnaK and actin to benchmark the development of three components that will significantly enhance the scope of the TRX-QM/MM method, including an automated freezing-and-quenching instrument, a new electron diffraction method for chasing proton transportation, and a new crystallographic refinement program that can handle open-shell systems. During the K99 phase (Aim 1 and 2), I will be mentored by Dr. Wayne Hendrickson, a leader in macromolecular crystallography, and Dr. Arieh Warshel, a leader in computational enzymology. This work will reveal a novel mechanism of ATP hydrolysis by Hsp70 in the short term. Still, most importantly, it will establish an unparalleled TRX-QM/MM method for broad enzymatic studies in the longer term.

项目摘要 酶是一种蛋白质，有助于加速所有生物体内的新陈代谢或化学反应。 通过合成某些分子并降解其他分子，酶可以催化一系列的生化反应。 无论是在体内还是体外。当与转运蛋白和受体协同工作时，酶调节几乎所有 身体的生理功能。因此，深入了解体内外酶是非常重要的。 活动。 在研究酶活性的众多方法中，时间分辨大分子结晶学(TRX) 具有在飞行中研究酶反应细节的优势。然而，TRX理论上只能 捕捉酶在当地能量鞍点的状态。另一方面，现代混合动力车 量子力学/分子力学(QM/MM)使研究酶反应成为可能 分子要么形成，要么被摧毁。然而，如果没有固体生物结构的支持，或者如果 反应物和产物状态之间的转变明显不同，QM/MM不能达到正品 回答。 这项建议旨在建立一种新的基于TRX和QM/MM的策略，通过以下方式研究酶活性 联合这两个领地的力量。值得注意的是，最近阐明的变构控制机制在70- KDA热休克蛋白(Hsp70)为构建模型系统提供了一个无与伦比的机会 制定拟议的TRX-QM/MM战略的基准。具体地说，在目标1中，一个开创性的 将通过ATPase结构域上的TRX实验来检验驱动ATP水解的氧自由基的发现 细菌Hsp70 DNAK.在目标2中，我将使用QM/MM来鉴定和验证氧自由基物种，以及 全面描绘了水解事件的自由能景观。在目标3中，我将使用DNAK和肌动蛋白来 对将显著扩大TRX-QM/MM范围的三个组成部分的开发进行基准 方法，包括自动冷冻和淬冷仪，一种新的电子衍射法 追逐质子传输，以及一种可以处理开放壳层的新晶体精炼程序 系统。 在K99阶段(目标1和目标2)，我将由大分子领域的领导者韦恩·亨德里克森博士指导 结晶学博士，以及计算酶学的领导者Arieh Warshel博士。这部作品将揭示一部小说 Hsp70在短期内对ATP的降解机制。尽管如此，最重要的是，它将建立一个无与伦比的 TRX-QM/MM方法用于长期的广泛的酶研究。