We describe a quasioptical 94 GHz kW pulsed electron paramagnetic resonance spectrometer featuring pi/2 pulses as short as 5 ns and an instantaneous bandwidth of 1 GHz in nonresonant sample holders operating in induction mode and at low temperatures. Low power pulses can be as short as 200 ps and kilowatt pulses as short as 1.5 ns with timing resolution of a few hundred picoseconds. Phase and frequency can be changed on nanosecond time scales and complex high power pulse sequences can be run at repetition rates up to 80 kHz with low dead time. We demonstrate that the combination of high power pulses at high frequencies and nonresonant cavities can offer excellent concentration sensitivity for orientation selective pulsed electron double resonance (double electron-electron resonance), where we demonstrate measurements at 1 microM concentration levels.

The design, synthesis and application of a nine-coordinate gadolinium(III)-containing spin label, [Gd.sTPATCN]-SL, for use in nanometer-distance measurement experiments by EPR spectroscopy is presented. The spin label links to cysteines via a short thioether tether and has a narrow central transition indicative of small zero-field splitting (ZFS). A protein homodimer, TRIM25cc, was selectively labeled with [Gd.sTPATCN]-SL (70%) and a nitroxide (30%) under mild conditions and measured using the double electron electron resonance (DEER) technique with both commercial Qband and home-built W-band spectrometers. The label shows great promise for increasing the sensitivity of DEER measurements through both its favorable relaxation parameters, and the large DEER modulation depth at both Q-and W-band for the inter-Gd(III) DEER measurement which, at 9%, is the largest recorded under these conditions.

Using low temperature dynamic nuclear polarisation (DNP) in conjunction with dissolution makes it possible to generate highly polarised nuclear spin systems for liquid state applications of nuclear magnetic resonance spectroscopy. However, in its current implementation, which requires the transfer of the solute between two different magnets, the hyperpolarisation strategy is limited to spin systems with relatively long longitudinal relaxation time constants. Here we describe the design and construction of a dedicated spectrometer for DNP applications that is based on a magnet with two isocentres. DNP enhancement is carried out in the upper compartment of this magnet in a low temperature environment at 3.35 T, while a 9.4 T isocentre in the lower compartment is used for high resolution NMR spectroscopy. The close proximity (85 cm) of the two isocentres makes it possible to transfer the sample in the solid state with very little loss of spin polarisation. In first performance tests this novel experimental set-up proved to be superior to the strategy involving two separate magnets.

No abstract

Pulsed electron-electron double resonance (PELDOR or DEER) spectroscopy is frequently used to determine distances between spin centres in biomacromolecular systems. Experiments where mutual orientations of the spin pair are selectively excited provide the so-called orientation-selective PELDOR data. This data is characterised by the orientation dependence of the modulation depth parameter and of the dipolar frequencies. This dependence has to be taken into account in the data analysis in order to extract distance distributions accurately from the experimental time traces. In this work, a fitting algorithm for such data analysis is discussed. The approach is tested on PELDOR data-sets from the literature and is compared with the previous results.

High-field, high-frequency electron paramagnetic resonance (EPR) spectroscopy at W-(~95 GHz) and D-band (~140 GHz) is important for investigating the conformational dynamics of flexible biological macromolecules because this frequency range has increased spectral sensitivity to nitroxide motion over the 100 ps to 2 ns regime. However, low concentration sensitivity remains a roadblock for studying aqueous samples at high magnetic fields. Here, we examine the sensitivity of a non-resonant thin-layer cylindrical sample holder, coupled to a quasi-optical induction-mode W-band EPR spectrometer (HiPER), for continuous wave (CW) EPR analyses of: (i) the aqueous nitroxide standard, TEMPO; (ii) the unstructured to -helical transition of a model IDP protein; and (iii) the base-stacking transition in a kink-turn motif of a large 232 nt RNA. For sample volumes of ~50 L, concentration sensitivities of 2-20 µM were achieved, representing a ~10-fold enhancement compared to a cylindrical TE 011 resonator on a commercial Bruker W-band spectrometer. These results therefore highlight the sensitivity of the thin-layer sample holders employed in HiPER for spin-labeling studies of biological macromolecules at high fields, where applications can extend to other systems that are facilitated by the modest sample volumes and ease of sample loading and geometry.2

This work demonstrates the feasibility of making sensitive nanometer distance measurements between Fe(III) heme centers and nitroxide spin labels in proteins using the double electron–electron resonance (DEER) pulsed EPR technique at 94 GHz. Techniques to measure accurately long distances in many classes of heme proteins using DEER are currently strongly limited by sensitivity. In this paper we demonstrate sensitivity gains of more than 30 times compared with previous lower frequency (X-band) DEER measurements on both human neuroglobin and sperm whale myoglobin. This is achieved by taking advantage of recent instrumental advances, employing wideband excitation techniques based on composite pulses and exploiting more favorable relaxation properties of low-spin Fe(III) in high magnetic fields. This gain in sensitivity potentially allows the DEER technique to be routinely used as a sensitive probe of structure and conformation in the large number of heme and many other metalloproteins.

In this paper, a smoothly profiled horn was designed and manufactured to replace a corrugated output horn for a W-band gyro-TWA with improved ultra-high vacuum compatibility. It was optimized for high vacuum integrity, low reflection, high fundamental Gaussian mode content, low side lobe levels and high directivity over the frequency bandwidth of 90 -100 GHz. Over this operating frequency band the reflectivity was better than -37 dB and the coupling to a fundamental Gaussian mode was above 97%. The far field pattern showed a directivity of approximately 27 dB in the measurement with side lobes lower than -30 dB.