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Default Towards Increased Concentration Sensitivity for Continuous Wave EPR Investigations of Spin-Labeled Biological Macromolecules at High Fields

Towards Increased Concentration Sensitivity for Continuous Wave EPR Investigations of Spin-Labeled Biological Macromolecules at High Fields


Publication date: Available online 15 February 2016
Source:Journal of Magnetic Resonance

Author(s): Likai Song, Zhanglong Liu, Pavanjeet Kaur, Jackie M. Esquiaqui, Robert I. Hunter, Stephen Hill, Graham M. Smith, Gail E. Fanucci

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 ?M were achieved, representing a ~10-fold enhancement compared to a cylindrical TE011 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.
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Source: Journal of Magnetic Resonance
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