Time-resolved crystallography of enzyme-catalyzed reactions

我将专注于不可逆酶反应的时间分辨晶体学（TRX），通过追求3个特定领域，每个领域由蛋白质代表：1）提高氟乙酸脱卤酶（FAcD）TRX的时间分辨率，专注于键的形成和断裂，2）探索谷胱甘肽还原酶（GR）的新触发机制，3）研究H-ras P21的笼状底物的光解。所有3种蛋白质在晶体中具有活性，同时保持晶体完整性。我们将使用一个芯片，在TRX实验中提供许多数据集。项目1）和2）需要高端同步加速器光束线，项目3）需要X射线自由电子激光器（XFEL）。 18 TRX晶体结构，从30 ms到30 s，覆盖不可逆FAcD反应的4个完整催化循环，提供了半位点反应性的第一个视觉证据，并揭示了催化步骤和分子呼吸运动之间的相关性，水网络的结构和蛋白质模具的微小变化。研究结果还为键合形成和断裂的发生设定了相当窄的界限。我们将研究SN 2取代和酯水解的过程，大大提高了时间分辨率。 黄素蛋白的宽吸收光谱禁止通过笼状前体的光解引发反应。我提出了两种方法来启动这样的TRX反应：1）使用基于超声的混合技术和2）由温度（T）跳跃引起的快速pH值变化。仅基于可接近的T位移的反应速率的变化太小，然而，对于某些缓冲液，20 C位移使pH改变2个单位，使休眠系统催化。T的变化可以降低或增加pH值。虽然存在快速降低pH的化合物（笼状质子），但没有笼状碱在光照下反应足够快。GR催化两个连续的半反应，NADPH氧化和谷胱甘肽还原，这也可以停止在中间状态。应用pL混合和pH位移，我们将分析GR的催化步骤，并将它们与晶体内的维斯光谱和电子密度图的变化相关联。 3)第三个子项目是最雄心勃勃的一个，2-硝基苄基和1-（2-硝基苯基）乙基-笼状基团光解后的暗反应的结构研究。虽然前者已成功地用于相当多的TRX研究，例如附在？尽管H-ras P21对GTP的磷酸化研究，但文献中仍讨论了酸性硝基和双环中间体转化为亚硝基酮和游离化合物的确切机制。TR-光谱表明的时间范围？这些笼群的暗反应的各个步骤的s到ms，使它们可以用于TRX实验。早期的工作已经表明，H-ras P21蛋白与笼状GTP的复合物可以结晶，并且晶体将耐受脱笼和GTP水解过程而不解体。

I will focus exclusively on time-resolved crystallography (TRX) of irreversible enzyme reactions by pursuing 3 specific areas, each represented by a protein: 1) improving the time-resolution of TRX of fluoroacetate dehalogenase (FAcD), focusing on bond formation and breakage, 2) exploring new triggering mechanisms with glutathione reductase (GR), 3) investigating the photolysis of caged substrates with H-ras P21. All 3 proteins are active in crystals while preserving crystal integrity. We will use a chip that delivered many data sets in TRX experiments. Projects 1) and 2) need access to high-end synchrotron beamlines, project 3) requires an X-ray Free Electron Laser (XFEL). 18 TRX crystal structures, from 30 ms to 30 s, covering 4 full catalytic cycles of the irreversible FAcD reaction, provide the first visual proof of half-of-the-sites reactivity and reveal correlations between the catalytic steps and molecular breathing motions, the structure of a water network and small changes of the protein mold. The results also set rather narrow boundaries for when bond formation and breakage occur. We will investigate the processes of SN2 substitution and ester hydrolysis with much improved time resolution. The broad absorption spectra of flavoproteins forbid triggering reactions by photolysis of caged precursors. I propose 2 methods to start such TRX reactions: 1) use of an ultrasound-based mixing technique and 2) fast pH shifts caused by temperature (T) jumps. Changes in reaction rates based on accessible T shifts alone are too small, however, for some buffers, 20 C shifts change pH by 2 units, taking a dormant system to catalysis. T changes can both lower or increase the pH value. While compounds exist that lower pH quickly (caged protons') no caged bases' react sufficiently fast upon illumination. GR catalyzes two successive half-reactions, NADPH oxidation and glutathione reduction, which can also be stopped at intermediate states. Applying pL mixing and pH shifts, we will analyze GR's catalytic steps and correlate them to in-crystal VIS spectra and changes to the electron density maps. 3) The third sub-project is the most ambitious one, the structural investigation of the dark reaction following the photolysis of the 2-nitrobenzyl- and 1-(2-nitrophenyl)ethyl- caging groups. While the former has been used successfully in quite a number of TRX studies, e.g. attached to the ?-phosphate of GTP investigating GTP hydrolysis by H-ras P21, the exact mechanism of transformation of aci-nitro- and bicyclic intermediates to nitrosoketone and free compound are still discussed in the literature. TR-spectroscopy indicates time ranges of ?s to ms for the various steps for the dark reactions of these caging groups, making them accessible to TRX experiments. Earlier work has shown that a complex of H-ras P21 protein with caged GTP can be crystallized and the crystals will tolerate the decaging and GTP hydrolysis processes without disintegration.