Thursday, November 21, 2024
11/21/2024
Project Summary/Abstract We request funds to purchase a Bruker Biospin Corp. Q-Band Elexsys E580 Pulse EPR Spectrometer (Pulse EPR) to enable structural dynamics studies of proteins and their complexes and the training of students and researchers. To maximize sensitivity, this instrument is equipped with a 300 W TWT Q-band pulse amplifier, ER5106QT-II resonator, SpinJet AWG, and Stinger cryogen-free low-temperature system. The high sensitivity of the requested pulse EPR enables us to support projects from user communities that are not feasible otherwise. In addition, combined with our current pulse EPR instrument, having access to a second pulse EPR with enhanced sensitivity and capabilities is essential, not only to manage our throughput, but also to serve as a regional pulse EPR facility for the state of Missouri and nearby states. DEER spectroscopy as a powerful pulse EPR technique provides an effective nanometer distance ruler to measure conformational changes in biomacromolecules under relevant physiological conditions. With increasing technological advances in this ensemble-oriented method, atomic resolution structural information can be directly linked to conformational sampling in solution. Thus, mechanistic models for protein function can be obtained and DEER spectroscopy is imperative to establish such models. The instrument will be housed in the Department of Biochemistry and Molecular Biology at the Saint Louis University School of Medicine. The instrument will support basic research of NIH-funded investigators at both Saint Louis University and Washington University in St. Louis. We describe projects from 8 major and minor users with NIH grants that will benefit from the requested instrumentation. The user projects span a range of membrane transporters, unconventional protein secretion, blood clotting processes, biomolecular interactions in DNA repair and recombination, immunothrombosis, regulation of protein translation, antimicrobial peptide transport, ligand-gated ion channels, and membrane protein oligomerization. These projects focus on protein structure, functional dynamics, and interactions, with majority involving low spin concentration samples. Thus, the high- sensitivity Pulse EPR will be critical for these studies. Training for the Pulse EPR will be primarily handled by members of the Dastvan research group who have more than a decade of experience using the instrumentation, sample preparation, and data analysis. Users (graduate students, postdoctoral fellows, and research associates) will be trained to independently use the Pulse EPR, and instrument time will be determined using the existing scheduling system for departmental instrumentation. A local advisory committee will provide oversight of the Pulse EPR and its operations. The high-sensitivity Pulse EPR will be an integral component of the biophysical instrumentation portfolio at Saint Louis University and will have a significant impact on the NIH-funded research in the region.
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