Development of Rapid Freeze-Quench Sample Preparation Techniques for Use in High Frequency Electron Paramagnetic Resonance

该奖项将支持其目标是允许使用高频电子顺磁共振和电子-核双共振（HF-EPR/ENDOR）光谱的快速冷冻淬灭（EHF）样品制备的工作。虽然EPR/ENDOR技术被广泛地与传统的低频EPR/ENDOR光谱仪结合使用，但技术上的困难阻碍了其在高频下的最佳使用。 它能在毫秒级的时间尺度上阻止酶的反应，以促进对短寿命催化中间体的研究。该技术涉及快速混合两种反应物（e。G.酶及其底物），并通过将所得混合物喷入低温浴中在已知时间段（5 - 1000毫秒）内冷冻所得混合物。然而，通常用于高频EPR的小波长和相应的小样品管和谐振腔使得拉曼光谱技术的应用变得困难，并且因此阻止了使用这种光谱技术对拉曼光谱样品的研究。将通过两条总的路线实现这一目标。一个是修改现有的电子探针，使其适应小HF-EPR毛细管样品管。另一个更大的努力将致力于修改高频EPR仪器，使其与现有的EPR技术兼容。这将涉及到多模谐振EPR腔的发展，接受标准，大的样品管。 这些试管目前在大多数使用低频EPR的实验室中制备。这两种方法-适应高频毛细管的微波技术和发展的过模谐振腔，接受标准微波管-将允许在正常的酶促反应中的短寿命的中间体的研究。 在开发生物技术之前，此类研究的标准策略包括使用抑制剂或底物类似物，在中间阶段阻止反应。 虽然这些中间体可以提供有关酶的重要结构信息，但它们在提供有关电子性质的信息方面不太有用，而这些电子性质是理解酶在反应中的作用的关键。

This award will support work whose objective is to permit the use of rapid freeze-quench (RFQ) sample preparation with high frequency electron paramagnetic resonance and electron-nuclear double resonance (HF-EPR/ENDOR) spectroscopy. While the RFQ technique is extensively used in conjunction with conventional low frequency EPR/ENDOR spectrometers, technical difficulties have prevented its optimal use at high frequencies. RFQ arrests the reaction of enzymes on the millisecond time scale to facilitate the investigation of short-lived catalytic intermediate species. The technique involves rapidly mixing two reactants (e. g. an enzyme and its substrate) and freezing the resulting mixture within a known time period (from 5 - 1000 milliseconds) by spraying it into a cryogenic bath. However, the small wavelengths and correspondingly small sample tubes and resonant cavities commonly used for high-frequency EPR make the application of RFQ techniques difficult, and have thus prevented the study of RFQ samples with this spectroscopic technique. The objective will be pursued via two general routes. One is the modification of existing RFQ by adapting them to the small HF-EPR capillary sample tubes. The other, larger effort will be devoted to modification of high frequency EPR instrumentation so that it is compatible with existing RFQ technology. This will involve the development of overmoded resonant EPR cavities that accept the standard, large RFQ sample tubes. These tubes are currently prepared in most laboratories that use low frequency EPR. Both approaches - the adaptation of RFQ techniques to high-frequency capillary tubes and the development of overmoded resonant cavities that accept standard RFQ tubes - will permit the study of short-lived intermediates in normal enzymatic reactions. Prior to the development of RFQ techniques, standard strategies for such studies involved the use of inhibitors or substrate analogues that arrested reactions at an intermediate stage. While such intermediates can provide important structural information about enzymes, they are less useful in providing information about electronic properties that are key to understanding the enzyme's role in the reaction.