The goal of the proposed research is to develop, validate, and apply electron paramagnetic resonance (EPR) methodology for measuring distances in non-crystalline biological systems. The unique power of EPR among the many techniques for measuring distances is that the paramagnetic centers may be native or added via site-directed mutagenesis, the electron spin dipole moment is large enough to yield long-range interactions, and EPR is sensitive only to the paramagnetic centers, even deeply buried in vast numbers of nuclear spins or light-absorbing species. The distance information is in the dipolar interaction between electron spins, which can be measured by continuous wave (CW) or pulsed EPR techniques. This proposal emphasizes X-band and Q-band pulse techniques because they measure the impact of spin-spin interaction on spin packets directly, rather than requiring interpretation of broadened CW spectra. Q-band offers enhanced sensitivity, spectral dispersion, and orientation selection. In the current funding period we developed saturation recovery and spin echo methods for measuring distances between rapidly-relaxing heme Fe(III) and a nitroxyl radical. In the proposed funding period we will address unanswered questions related to these techniques and extend them to other iron centers - the iron-sulfur cluster in electron transfer flavoprotein ubiquinone oxidoreductase (ETF-QO) and the non-heme iron in iron enterobactin (FeEnt) bound to iron protein A (FepA). These systems provide the opportunity to examine the effect of electron spin delocalization (ETF-QO) on the distance measurements and the effect of iron energy level splittings comparable to the EPR quantum (FepA). Pulsed double electron-electron resonance (DEER) is a complementary method to measure distances between two slowly relaxing paramagnetic centers, and we propose to enhance its utility for biological samples. Two inter-related themes extend across the three specific aims - orientation selection and conformational flexibility. The methods that we develop will permit us to address important biological questions raised by our collaborators. We will determine the conformational change in the tonB box of iron protein A, characterize structure changes in the redox active sites of electron transfer flavoprotein ubiquinone reductase, and elucidate the mechanism of DNA melting by large tumor antigen.

Agency
National Institute of Health (NIH)
Institute
National Institute of Biomedical Imaging and Bioengineering (NIBIB)
Type
Research Project (R01)
Project #
5R01EB002807-29
Application #
7149135
Study Section
Biophysical Chemistry Study Section (BBCB)
Program Officer
Mclaughlin, Alan Charles
Project Start
1977-07-01
Project End
2008-12-31
Budget Start
2007-01-01
Budget End
2007-12-31
Support Year
29
Fiscal Year
2007
Total Cost
$319,374
Indirect Cost
Name
University of Denver
Department
Chemistry
Type
Schools of Arts and Sciences
DUNS #
007431760
City
Denver
State
CO
Country
United States
Zip Code
80208
Biller, Joshua R; Mitchell, Deborah G; Tseytlin, Mark et al. (2016) Rapid Scan Electron Paramagnetic Resonance Opens New Avenues for Imaging Physiologically Important Parameters In Vivo. J Vis Exp :
Eaton, Sandra S; Eaton, Gareth R (2015) Multifrequency Pulsed EPR and the Characterization of Molecular Dynamics. Methods Enzymol 563:37-58
Mitchell, Deborah G; Rosen, Gerald M; Tseitlin, Mark et al. (2013) Use of rapid-scan EPR to improve detection sensitivity for spin-trapped radicals. Biophys J 105:338-42
Meyer, Virginia; Eaton, Sandra S; Eaton, Gareth R (2013) Temperature Dependence of Electron Spin Relaxation of 2,2-diphenyl-1-picrylhydrazyl in Polystyrene. Appl Magn Reson 44:509-517
Rajca, Andrzej; Wang, Ying; Boska, Michael et al. (2012) Organic radical contrast agents for magnetic resonance imaging. J Am Chem Soc 134:15724-7
Biller, Joshua R; Meyer, Virginia; Elajaili, Hanan et al. (2011) Relaxation times and line widths of isotopically-substituted nitroxides in aqueous solution at X-band. J Magn Reson 212:370-7
Olankitwanit, Arnon; Kathirvelu, Velavan; Rajca, Suchada et al. (2011) Calix[4]arene nitroxide tetraradical and octaradical. Chem Commun (Camb) 47:6443-5
Swanson, Michael A; Kathirvelu, Velavan; Majtan, Tomas et al. (2011) Electron transfer flavoprotein domain II orientation monitored using double electron-electron resonance between an enzymatically reduced, native FAD cofactor, and spin labels. Protein Sci 20:610-20
Rajca, Andrzej; Kathirvelu, Velavan; Roy, Sandip K et al. (2010) A spirocyclohexyl nitroxide amino acid spin label for pulsed EPR spectroscopy distance measurements. Chemistry 16:5778-82
Eaton, Gareth R; Eaton, Sandra S; Quine, Richard W et al. (2010) A signal-to-noise standard for pulsed EPR. J Magn Reson 205:109-13

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