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: 10680611
• 负责人: Wei Wang
• 金额: $ 11.46万
• 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/10680611
• 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; Classical Mechanics; Collaborations; Collection; Complex; 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; Length; Life; Ligand Binding; Malignant Neoplasms; 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; chemical reaction; electron diffraction; enzyme activity; enzyme substrate; experimental study; in vivo; innovation; insight; instrument; molecular mechanics; mutant; novel; operation; programs; 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 酶是蛋白质，可帮助所有生物体中的代谢加速或化学反应的加速。 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 体内和体外。与转运蛋白和受体合作时，酶几乎调节 体内的生理功能。因此，重要的是要彻底了解外体和体内酶 活动。 在研究酶活性的众多方法中，时间分辨的大分子晶体学（TRX） 具有研究酶促反应细节的优势。但是，TRX理论上只能 捕获酶处于局部能量鞍点处的状态。另一方面，现代混合动力车 量子力学/分子力学（QM/mm）可以研究新的酶促反应 分子形成或破坏。但是，没有固体生物结构的支持或 反应物和产品状态之间的转换截然不同，QM/mm无法达到真实 回答。 该建议旨在建立一种新的TRX和QM/MM策略，以通过 加入这两个领土的力量。值得注意的是，最近阐明了70-中的变构控制机制 KDA热休克蛋白（HSP70S）导致无与伦比的机会将模型系统构建为 制定拟议的TRX-QM/MM策略的基准。具体来说，在AIM 1中，是开创性的 驱动ATP水解的氧自由基的发现将通过ATPase结构域的TRX实验检查 细菌HSP70 DNAK。在AIM 2中，我将使用QM/mm来识别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AIM 3中，我将使用DNAK和ATCIN进行 基准制定三个组件的开发将显着提高TRX-QM/mm的范围 方法，包括一种自动冻结和淬火仪器，一种新的电子衍射方法 追逐质子运输和一个新的晶体学改进程序，可以处理开放式壳 系统。 在K99阶段（AIM 1和2），我将由大分子的领导者Wayne Hendrickson博士指导 晶体学和计算酶学领导者Arieh Warshel博士。这项工作将揭示一本小说 HSP70在短期内通过ATP水解的机理。不过，最重要的是，它将建立无与伦比的 从长远来看，用于广泛酶促研究的TRX-QM/MM方法。