Dielectric Resonator-Based Flow and Stopped-Flow EPR with Rapid Field Scanning: A Methodology for Increasing Kinetic Information

Structural Information from CW-EPR Spectra of Dipolar Coupled Nitroxide Spin Labels

“…This fact, together with the high sensitivity of electron paramagnetic resonance (EPR) spectroscopy, the ability to determine structural properties of proteins in their native environments (i.e. in solution, in membranes, etc), and the ability to observe the time course of structural transitions on msec and longer timescales (Shin et al, 1993;Qu et al, 1997;Sienkiewicz et al, 1999) have all contributed to the emergence of double site directed spin labeling (DSDSL) as the method of choice for many different structural problems which are not amenable to characterization using classical techniques such as nuclear magnetic resonance or X-ray crystallography. The applications of DSDSL reported to date have demonstrated the utility and flexibility of the approach and have opened the door for a vast array of future applications as discussed at length in recent reviews (Millhauser, 1992;Hubbell and Altenbach, 1994b;Hubbell et al, 1998;Feix and KIug, 1999;Hustedt and Beth, 1999) and by other authors in this volume.…”

An advanced EPR stopped-flow apparatus based on a dielectric ring resonator

“…Tests of the function of the EPR-SF accessory under flow conditions have been performed using the chemical reduction of the stable nitroxide radical 4-hydroxy-2,2,6,6-tetra-methyl-piperidine-N-oxyl (TEMPOLE) in aqueous solution by (i) sodium ascorbate (for second kinetics) [1][2][3] and (ii) sodium dithionite (for millisecond kinetics) [4].…”

“…Examples are the decay of stable nitroxide radicals during reduction by ascorbate [1][2][3] or dithionite [4], and formation and decay of semi-dehydro ascorbate radicals during the oxidation of ascorbate by Ce 4+ [5,1]. Radicals that are not EPR-detectable in the liquid state due to short relaxation times T 1 (linewidth broadening), as e.g., hydroxyl (OH Å ), superoxide ðO ÅÀ 2 Þ, or alkyl thiyl (R-S Å ), cannot be studied directly by EPR-SF techniques.…”

“…The resonator employed in [2,3] contained two stacked dielectric rings with a spacer between them, so that the frequency could be varied by the thickness of the spacer by about 1 GHz. The smaller size of the EPR-active volume in comparison with the standard waveguide H 102 cavity together with a higher H 1 field and an almost absent E field at the sample position in the dielectric resonator, allowing a higher filling factor, led to a considerable enhancement of the sensitivity by a factor of about 30, particularly for small samples of aqueous solution, e.g., of nitroxide radicals or Mn 2+ [2,20,21].…”

“…In R2 mutants of E. coli RNR the life-times of transient tryptophan radicals were determined by EPR-SF [10]. Using nitroxide radicals, kinetics of conformational changes (folding and unfolding of proteins) in spin-labeled proteins (via line shape changes of the EPR spectra) [3,4,11,12], and the kinetics of diffusion of lipid-like spin-probes in biomembranes have been reported [13,14]. In cases where paramagnetic transition metal ions or their complexes are EPR-active in the liquid state, due to sufficiently long spin-lattice relaxation times, the kinetics of a valence change or a spin-state change upon redox reactions, or the kinetics of a ligand exchange, can also be studied by EPR-SF technique, see, e.g.…”