Funds are requested to complete the purchase of a Bruker EleXsys 580 FT-EPR system at Vanderbilt University. This equipment, which is not currently available on campus or at any nearby institution, will be utilized by six different established research groups to make time-domain EPR measurements on a wide range of nitroxide spin-labeled proteins that are already being studied using conventional cw-EPR approaches and site-directed spin-labeling. Recent advances in instrumentation and methodology have positioned timedomain EPR as an important and in many cases an essential complement to cw-EPR for characterizing the structures and dynamics of spin-labeled proteins. In the eight NIH funded projects that constitute the core user group of this application, the requested equipment will be utilized to: 1) measure """"""""long"""""""" (> 20 - 25 angstroms) inter-probe distances in proteins by DEER and/or double quantum coherence; 2) measure T1e's of spin-labeled side chains in the presence and absence of paramagnetic relaxation agents as a direct index of side-chain accessibility; and 3) measure T1e's for spin-labeled proteins that are being studied by non-linear methods including saturation transfer EPR. In each of the eight ongoing projects, the requested instrumentation will have an immediate and substantial impact by providing unique new measurement capabilities for full characterization of the structures and dynamics of wide range of soluble and membrane bound proteins that are of clear biomedical importance, The requested instrumentation will also be made available to other investigators at Vanderbilt and in the surrounding area as they develop new projects that involve spinlabeling. Equitable utilization of the equipment will be assured by an internal oversight committee and day to- day operation will be handled by an experienced EPR spectroscopist.
| Stein, Richard A; Beth, Albert H; Hustedt, Eric J (2015) A Straightforward Approach to the Analysis of Double Electron-Electron Resonance Data. Methods Enzymol 563:531-67 |
| Dixit, Mrinalini; Kim, Sunghoon; Matthews, Gage F et al. (2013) Structural arrangement of the intracellular Ca2+ binding domains of the cardiac Na+/Ca2+ exchanger (NCX1.1): effects of Ca2+ binding. J Biol Chem 288:4194-207 |
| Borbat, Peter P; Surendhran, Kavitha; Bortolus, Marco et al. (2007) Conformational motion of the ABC transporter MsbA induced by ATP hydrolysis. PLoS Biol 5:e271 |
| Zhou, Zheng; DeSensi, Susan C; Stein, Richard A et al. (2007) Structure of the cytoplasmic domain of erythrocyte band 3 hereditary spherocytosis variant P327R: band 3 Tuscaloosa. Biochemistry 46:10248-57 |
