Molecular Biophysics and Integrated Bioimaging Division, Lawrence Berkeley National Laboratory, Berkeley, CA, United States.
Molecular Biophysics and Integrated Bioimaging Division, Lawrence Berkeley National Laboratory, Berkeley, CA, United States.
Methods Enzymol. 2023;688:307-348. doi: 10.1016/bs.mie.2023.07.009. Epub 2023 Aug 16.
The ultrashort (10s of femtoseconds) X-ray pulses generated by X-ray free electron lasers enable the measurement of X-ray diffraction and spectroscopic data from radiation-sensitive metalloenzymes at room temperature while mostly avoiding the effects of radiation damage usually encountered when performing such experiments at synchrotron sources. Here we discuss an approach to measure both X-ray emission and X-ray crystallographic data at the same time from the same sample volume. The droplet-on-tape setup described allows for efficient sample use and the integration of different reaction triggering options in order to conduct time-resolved studies with limited sample amounts. The approach is illustrated by two examples, photosystem II that catalyzes the light-driven oxidation of water to oxygen, and isopenicillin N synthase, an enzyme that catalyzes the double ring cyclization of a tripeptide precursor into the β-lactam isopenicillin and can be activated by oxygen exposure. We describe the necessary steps to obtain microcrystals of both proteins as well as the operation procedure for the drop-on-tape setup and details of the data acquisition and processing involved in this experiment. At the end, we present how the combination of time-resolved X-ray emission spectra and diffraction data can be used to improve the knowledge about the enzyme reaction mechanism.
X 射线自由电子激光产生的超短（10 飞秒）X 射线脉冲使人们能够在室温下测量对辐射敏感的金属酶的 X 射线衍射和光谱数据，而在同步加速器源上进行此类实验时通常会遇到辐射损伤的影响。在这里，我们讨论了一种从同一样品体积同时测量 X 射线发射和 X 射线晶体学数据的方法。所描述的液滴在带上设置允许有效地使用样品，并集成了不同的反应触发选项，以便在有限的样品量下进行时间分辨研究。该方法通过两个示例来说明，即催化水光氧化为氧的光系统 II，以及催化三肽前体双环环化生成β-内酰胺异青霉素的异青霉素 N 合酶，并且可以通过暴露于氧气来激活。我们描述了获得两种蛋白质微晶体的必要步骤，以及液滴在带上设置的操作程序，以及涉及此实验的数据采集和处理的详细信息。最后，我们展示了如何将时间分辨的 X 射线发射光谱和衍射数据结合起来，以提高对酶反应机制的认识。
