Probing enzyme motions during catalysis

探测催化过程中酶的运动

• 批准号: 1151658
• 负责人: Michael Crowder
• 金额: $ 33万
• 依托单位: Miami University
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-09-01 至 2015-08-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=1151658&HistoricalAwards=false
• 关键词: Probing; enzyme; motions; during; catalysis

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.

有了这个奖项，生命过程的化学计划是支持教授迈克尔W。克劳德和大卫L.迈阿密大学的蒂尔尼。 教授克劳德和蒂尔尼将探测分子运动在酶催化过程中使用快速冷冻淬火双电子-电子共振（RFQ-DEER）光谱。 酶不是静止的生物分子;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的高度协调运动，以进行生化反应。虽然已经有许多技术可用于探测酶运动，但这些技术中的许多技术需要大量的酶和大量的假设来解释数据。在这项工作中要优化的技术是适合于低酶浓度的条件。在这些拟议的研究中开发的方法将解决影响该技术如何适用于其他系统的重要问题，例如（1）确定可以用该技术可靠地询问的反应时间范围，（2）探索不同的自旋标记以确定哪种最适合该技术，（3）优化Q-波段DEER样品收集的重复性过程，和（4）开发处理多亚基蛋白的方法。该项目的更广泛的影响包括在酶学，现代EPR光谱学和分子生物学的本科生和研究生的培训。 如果成功的话，所提出的方法可能会发现更广泛的科学影响，在催化过程中的酶运动的评估在一个显着的酶系统。他们将继续参与社区外展活动，例如为小学生举办年度科学周，并向当地非科学观众介绍抗生素耐药性。