Probing enzyme motions during catalysis

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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.