Probing enzyme motions during catalysis

有了这个奖项，生命过程化学项目将支持迈阿密大学教授迈克尔·w·克劳德和大卫·l·蒂尔尼的研究。克劳德教授和蒂尔尼教授将使用快速冷冻淬火双电子-电子共振（RFQ-DEER）光谱技术来探测酶在催化过程中的分子运动。酶不是静态的生物分子；相反，大多数酶的催化活性需要酶的环、螺旋和其他结构特征高度协调的运动才能进行生化反应。虽然已有许多技术可用于探测酶的运动，但其中许多技术需要大量的酶和大的假设来解释数据。本研究优化的工艺适用于低酶浓度条件。在这些提出的研究中开发的方法将解决影响该技术在其他系统中的适用性的重要问题，例如(1)确定可以用该技术可靠地询问的反应时间范围，(2)探索不同的自旋标签以确定哪种标签最适合该技术，(3)优化RFQ过程以收集q波段DEER的样品，以及(4)开发处理多亚基蛋白质的方法。该项目的广泛影响包括培养酶学、现代EPR光谱学和分子生物学方面的本科生和研究生。如果成功，所提出的方法可以在酶系统催化过程中酶运动的评估中找到更广泛的科学影响。研究人员将继续参与社区外展活动，例如为小学生举办一年一度的科学周，并向当地非科学观众介绍抗生素耐药性。

With this award, the Chemistry of Life Processes program is supporting the research of Professors Michael W. Crowder and David L. Tierney of Miami University. Professors Crowder and Tierney will probe molecular motions in enzymes during catalysis using rapid freeze quench double electron-electron resonance (RFQ-DEER) spectroscopy. Enzymes are not static biomolecules; rather, the catalytic activities of most enzymes require highly coordinated motions of loops, helices, and other structural features of the enzymes for the biochemical reactions to proceed. While there are a number of techniques already available to probe enzyme motions, many of these techniques require large quantities of enzyme and large assumptions to interpret the data. The technique to be optimized in this work is amenable to conditions of low enzyme concentration. The methodology developed in these proposed studies will address important issues that affect how applicable the technique will be to other systems, such as (1) determining the range of reaction times that can be reliably interrogated with this technique, (2) exploring varying spin labels to ascertain which work best for this technique, (3) optimizing the RFQ process for the collection of samples for Q-band DEER, and (4) developing methods to handle multi-subunit proteins. The Broader Impacts of this project include training of undergraduate and graduate students in enzymology, modern EPR spectroscopy, and molecular biology. If successful, the proposed methodology could find broader scientific impact in the evaluation of enzyme motions during catalysis across a significant range of enzyme systems. The investigtors will continue their involvement in community outreach, such as hosting an annual Science Week for elementary school children and making presentations on antibiotic resistance to local non-science audiences.