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)光谱学的快速冷冻-淬火(RFQ)样品制备的工作。虽然RFQ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。RFQ在毫秒的时间尺度上阻止了酶的反应，以便于研究短暂的催化中间体物种。这项技术包括快速混合两种反应物(例如，酶及其底物)，并在已知的时间内(从5-1000毫秒)将得到的混合物通过将其喷洒到低温浴中进行冷冻。然而，高频电子顺磁共振通常使用的小波长和相应的小样品管和共振腔给RFQ技术的应用带来了困难，从而阻碍了这种光谱技术对RFQ样品的研究。这一目标将通过两条主要路线来实现。一种是对现有的RFQ进行改进，使其适用于小型HF-EPR毛细管样品管。另一方面，将致力于高频EPR仪器的改造，使其与现有的RFQ技术兼容。这将包括开发可接受标准的大型RFQ样品管的过模谐振式EPR腔。目前，大多数使用低频电子顺磁共振的实验室都在制备这些试管。这两种方法--使RFQ技术适应高频毛细管，以及开发接受标准RFQ管的过模谐振腔--都将使研究正常酶反应中的短暂中间体成为可能。在开发RFQ技术之前，此类研究的标准策略包括使用在中间阶段阻止反应的抑制剂或底物类似物。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用处较小，而电子性质是理解酶在反应中作用的关键。

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.