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We demonstrate that adiabatic and fast passage ultra-wideband (UWB) pulses can achieve inversion over several hundreds of MHz and thus enhance the measurement sensitivity, as shown by two selected experiments. Technically, frequency-swept pulses are generated by a 12 GS/s arbitrary waveform generator and upconverted to X-band frequencies. This pulsed UWB source is utilized as an incoherent channel in an ordinary pulsed EPR spectrometer. We discuss experimental methodologies and modeling techniques to account for the response of the resonator, which can strongly limit the excitation bandwidth of the entire non-linear excitation chain. Aided by these procedures, pulses compensated for bandwidth or variations in group delay reveal enhanced inversion efficiency. The degree of bandwidth compensation is shown to depend critically on the time available for excitation. As a result, we demonstrate optimized inversion recovery and double electron electron resonance (DEER) experiments. First, virtually complete inversion of the nitroxide spectrum with an adiabatic pulse of 128ns length is achieved. Consequently, spectral diffusion between inverted and non-inverted spins is largely suppressed and the observation bandwidth can be increased to increase measurement sensitivity. Second, DEER is performed on a terpyridine-based copper (II) complex with a nitroxide-copper distance of 2.5nm. As previously demonstrated on this complex, when pumping copper spins and observing nitroxide spins, the modulation depth is severely limited by the excitation bandwidth of the pump pulse. By using fast passage UWB pulses with a maximum length of 64ns, we achieve up to threefold enhancement of the modulation depth. Associated artifacts in distance distributions when increasing the bandwidth of the pump pulse are shown to be small., (Copyright © 2013 Elsevier Inc. All rights reserved.)

We demonstrate the application of continuous wave dynamic nuclear polarization (DNP) at 0.35 T for site-specific water accessibility studies on spin-labeled membrane proteins at concentrations in the 10-100 μM range. The DNP effects at such low concentrations are weak and the experimentally achievable dynamic nuclear polarizations can be below the equilibrium polarization. This sensitivity problem is solved with an optimized home-built DNP probe head consisting of a dielectric microwave resonator and a saddle coil as close as possible to the sample. The performance of the probe head is demonstrated with both a modified pulsed EPR spectrometer and a dedicated CW EPR spectrometer equipped with a commercial NMR console. In comparison to a commercial pulsed ENDOR resonator, the home-built resonator has an FID detection sensitivity improvement of 2.15 and an electron spin excitation field improvement of 1.2. The reproducibility of the DNP results is tested on the water soluble maltose binding protein MalE of the ABC maltose importer, where we determine a net standard deviation of 9% in the primary DNP data in the concentration range between 10 and 100 μM. DNP parameters are measured in a spin-labeled membrane protein, namely the vitamin B(12) importer BtuCD in both detergent-solubilized and reconstituted states. The data obtained in different nucleotide states in the presence and absence of binding protein BtuF reveal the applicability of this technique to qualitatively extract water accessibility changes between different conformations by the ratio of primary DNP parameters ϵ. The ϵ-ratio unveils the physiologically relevant transmembrane communication in the transporter in terms of changes in water accessibility at the cytoplasmic gate of the protein induced by both BtuF binding at the periplasmic region of the transporter and ATP binding at the cytoplasmic nucleotide binding domains., (Copyright © 2012 Elsevier Inc. All rights reserved.)

The construction and performance of a cryogenic 35GHz pulse electron nuclear double resonance (ENDOR) probehead for large samples is presented. The resonator is based on a rectangular TE(102) cavity in which the radio frequency (rf) B(2)-field is generated by a two turn saddle ENDOR coil crossing the resonator along the sample axis with minimal distance to the sample tube. An rf power efficiency factor is used to define the B(2)-field strength per square-root of the transmitted rf power over the frequency range 2-180MHz. The distributions of the microwave B(1)- and E(1)-field, and the rf B(2)-field are investigated by electromagnetic field calculations. All dielectrics, the sample tube, and coupling elements are included in the calculations. The application range of the probehead and the advantages of using large sample sizes are demonstrated and discussed on a number of paramagnetic samples containing transition metal ions.