Tkach Igor, Bejenke Isabel, Hecker Fabian, Kehl Annemarie, Kasanmascheff Müge, Gromov Igor, Prisecaru Ion, Höfer Peter, Hiller Markus, Bennati Marina
Research Group EPR Spectroscopy, Max Planck Institute for Biophysical Chemistry, Am Fassberg 11, 37077 Göttingen, Germany.
Research Group EPR Spectroscopy, Max Planck Institute for Biophysical Chemistry, Am Fassberg 11, 37077 Göttingen, Germany.
J Magn Reson. 2019 Jun;303:17-27. doi: 10.1016/j.jmr.2019.04.001. Epub 2019 Apr 3.
We present and discuss the performance of H electron-nuclear double resonance (ENDOR) at 263 GHz/9.4 T by employing a prototype, commercial quasi optical spectrometer. Basic instrumental features of the setup are described alongside a comprehensive characterization of the new ENDOR probe head design. The performance of three different ENDOR pulse sequences (Davies, Mims and CP-ENDOR) is evaluated using the H BDPA radical. A key feature of 263 GHz spectroscopy - the increase in orientation selectivity in comparison with 94 GHz experiments - is discussed in detail. For this purpose, the resolution of H ENDOR spectra at 263 GHz is verified using a representative protein sample containing approximately 15 picomoles of a tyrosyl radical. Davies ENDOR spectra recorded at 5 K reveal previously obscured spectral features, which are interpreted by spectral simulations aided by DFT calculations. Our analysis shows that seven internal proton couplings are detectable for this specific radical if sufficient orientation selectivity is achieved. The results prove the fidelity of 263 GHz experiments in reporting orientation-selected H ENDOR spectra and demonstrate that new significant information can be uncovered in complex molecular systems, owing to the enhanced resolution combined with high absolute sensitivity and no compromise in acquisition time.
我们展示并讨论了使用原型商用准光学光谱仪在263 GHz/9.4 T下进行的氢电子 - 核双共振（ENDOR）实验的性能。介绍了该装置的基本仪器特性，并对新型ENDOR探头设计进行了全面表征。使用氢BDPA自由基评估了三种不同ENDOR脉冲序列（戴维斯、米姆斯和CP - ENDOR）的性能。详细讨论了263 GHz光谱的一个关键特性——与94 GHz实验相比，取向选择性增加。为此，使用含有约15皮摩尔酪氨酸自由基的代表性蛋白质样品验证了263 GHz下氢ENDOR光谱的分辨率。在5 K下记录的戴维斯ENDOR光谱揭示了以前模糊不清的光谱特征，通过DFT计算辅助的光谱模拟对其进行了解释。我们的分析表明，如果实现足够的取向选择性，对于这种特定自由基可检测到七个内部质子耦合。结果证明了263 GHz实验在报告取向选择的氢ENDOR光谱方面的准确性，并表明由于分辨率提高、绝对灵敏度高且采集时间无妥协，在复杂分子系统中可以发现新的重要信息。
