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

项目摘要 酶是帮助加速新陈代谢或所有生物体中的化学反应的蛋白质。 通过合成某些分子和降解其他分子，酶可以催化一系列生化反应 无论是在体内还是体外。当与转运蛋白和受体合作时，酶调节几乎所有 身体的生理功能。因此，深入了解体外和体内酶是非常重要的。 活动 在研究酶活性的许多方法中，时间分辨大分子晶体学（TRX） 具有在飞行中研究酶促反应细节的优点。然而，TRX理论上只能 捕获酶在局部能量鞍点的状态。另一方面，现代混合动力汽车 量子力学/分子力学（QM/MM）使研究酶反应，其中新的 分子形成或被破坏。然而，如果没有固体生物结构的支持， 反应物和产物状态之间的转化是明显不同的，QM/MM不能达到真实的 答案 该提案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： 将这两个地区的力量结合起来。值得注意的是，最近阐明的变构控制机制，在70- kDa的热休克蛋白（Hsp 70）导致了一个无与伦比的机会，建立一个模型系统， 制定拟议TRX-QM/MM战略的基准。具体而言，在目标1中， 氧自由基驱动ATP水解的发现将通过ATP酶域上的TRX实验来检验 细菌Hsp 70 DnaK。在目标2中，我将使用QM/MM来识别和验证氧自由基物种， 描绘了水解事件的自由能全景。在目标3中，我将使用DnaK和肌动蛋白来 对三个组件的开发进行基准测试，这三个组件将显著增强TRX-QM/MM的范围 方法，包括一个自动冷冻和淬火仪器，一个新的电子衍射方法， 追逐质子运输，以及一个新的晶体学精炼程序，可以处理开壳层 系统. 在K99阶段（目标1和2），我将接受大分子领域的领导者韦恩亨德里克森博士的指导。 晶体学和Arieh Warshel博士，计算酶学的领导者。这部作品将揭示一部小说 Hsp 70在短期内水解ATP的机制。最重要的是，它将建立一个无与伦比的 TRX-QM/MM方法用于长期广泛的酶研究。

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.