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

项目概要 酶是有助于加速所有生物体新陈代谢或化学反应的蛋白质。 通过合成某些分子并降解其他分子，酶可以催化一系列生化反应 体内和体外。当与转运蛋白和受体合作时，酶调节几乎所有 体内的生理功能。因此，彻底了解体外和体内酶非常重要 活动。 在研究酶活性的众多方法中，时间分辨大分子晶体学 (TRX) 具有即时研究酶反应细节的优点。然而，TRX理论上只能 捕获酶处于局部能量鞍点的状态。另一方面，现代混合动力 量子力学/分子力学 (QM/MM) 能够研究新的酶促反应 分子形成或破坏。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无法达到真实的 回答。 该提案旨在建立一种新的基于 TRX 和 QM/MM 的策略来研究酶活性 联合这两个领土的力量。值得注意的是，最近阐明了 70- kDa 热休克蛋白 (Hsp70s) 为构建模型系统提供了无与伦比的机会 制定拟议的 TRX-QM/MM 战略的基准。具体来说，在目标 1 中， 驱动 ATP 水解的氧自由基的发现将通过 ATPase 结构域的 TRX 实验进行检验 细菌 Hsp70 DnaK。在目标 2 中，我将使用 QM/MM 来识别和验证氧自由基种类，并且 全面描绘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 对三个组件的开发进行基准测试，这将显着扩大 TRX-QM/MM 的范围 方法，包括自动冷冻和淬灭仪器、一种新的电子衍射方法 追逐质子传输，以及可以处理开壳的新晶体细化程序 系统。 在K99阶段（目标1和2），我将得到大分子领域的领导者Wayne Hendrickson博士的指导。 晶体学，以及计算酶学领域的领导者 Arieh Warshel 博士。本作将揭露一部小说 Hsp70短期内水解ATP的机制。但最重要的是，它将建立一个无与伦比的 TRX-QM/MM 方法用于长期广泛的酶学研究。

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.