Distance and dynamics determination by W-band DEER and W-band ST-EPR.

To explore high-field EPR in biological applications we have compared measurements of dynamics with X-band (9 GHz) and W-band (94 GHz) saturation transfer EPR (ST-EPR) and distance determination by X and W-band DEER. A fourfold increase of sensitivity was observed for W-band ST-EPR compared with X-band. The distance measurements at both fields showed very good agreement in both the average distances and in the distance distributions. Multifrequency EPR thus provides an additional experimental dimension to facilitate extraction of distance populations. However, the expected orientational selectivity of W-band DEER to determine the relative orientation of spins has not been realized, most likely because of the large orientational disorder of spin labels on the protein surface.

Solute effects on spin labels at an aqueous-exposed site in the flap region of HIV-1 protease.

The effects of solutes on spin-label mobility and protein conformation have been investigated with X-band continuous-wave and pulsed electron paramagnetic resonance (EPR) spectroscopy for spin labels attached to an aqueous-exposed site in the beta-hairpin flap region of HIV-1 protease. Specifically, we examined the effects of glycerol, sucrose, PEG3000, and Ficoll400 for four commonly used nitroxide spin labels and found that the largest perturbations to the EPR line shapes occur for solutions containing PEG3000 and glycerol. From comparisons of the spectral line shapes and distance distribution profiles of spin-labeled HIV-1 protease with and without inhibitor, it was concluded that solutes such as glycerol and PEG3000 alter the line shapes of the spin label in the beta-hairpin flaps of HIV-1 PR by modulation of spin-label mobility through changes in preferential interactions with the solutes. It is noteworthy that the high osmolality of the 40% glycerol solution did not alter the conformation of the flaps as determined from pulsed EPR distance measurements.

Suppression of electron spin-echo envelope modulation peaks in double quantum coherence electron spin resonance.

We show the use of the observer blind spots effect for the elimination of electron spin-echo envelope modulation (ESEEM) peaks in double quantum coherence (DQC) electron spin resonance (ESR). The suppression of ESEEM facilitates the routine and unambiguous extraction of distances from DQC-ESR spectra. This is also the first demonstration of this challenging methodology on commercial instrumentation.

EPR dosimetric properties of 2-methylalanine: EPR, ENDOR and FT-EPR investigations.

To find an EPR dosimeter material that is sensitive enough for clinical use, the substance 2-methylalanine (2MA) with the chemical structure (CH(3))(2)C(NH(3)(+))COO(-) was tested for its sensitivity to ionizing radiation, dose response, and radical stability over time. At equal and moderate settings of microwave power and modulation amplitude, 2MA was found to be 70% more sensitive than L-alpha-alanine, which is the most common EPR dosimeter material today. The dose response is linear, at least in the dose range of interest (0.5-00 Gy), and the time-dependent variations in signal intensity are very small and may be corrected for easily. The energy dependence of the stopping power and energy absorption was calculated and was found to be similar to that of alanine. The dependence of the signal intensity on microwave power and modulation amplitude was investigated, and the optimal settings were found to be 25 mW (Bruker ER 4102ST) and 12 gauss, respectively. Single crystals of 2MA were analyzed using ENDOR and ENDOR-induced EPR to identify the radiation-induced radicals that formed. Only one radical, in which the amino group is detached from the original molecule, was identified. This radical is obviously dominating and is apparently the only one relevant for dosimetry purposes. The complete set of coupling parameters for three hyperfine couplings is reported. The power saturation properties and spectral line width are ruled by the relaxation times T(1) and T(2). To determine the relaxation times of 2MA, pulsed EPR experiments were performed on single crystals. Two different values of T(1) were obtained, one in the range 1-3 micros, shown to be of importance for the dosimetry properties, and another that is strongly anisotropic with a value between 10 and 35 micros that does not seem to affect the saturation behavior. T(2) was estimated to be of the order of 200-300 ns.