Toward increased concentration sensitivity for continuous wave EPR investigations of spin-labeled biological macromolecules at high fields

Abstract

High-field, high-frequency electron paramagnetic resonance (EPR) spectroscopy at W-(∼94GHz) and D-band (∼140GHz) is important for investigating the conformational dynamics of flexible biological macromolecules because this frequency range has increased spectral sensitivity to nitroxide motion over the 100ps to 2ns 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 α-helical transition of a model IDP protein; and (iii) the base-stacking transition in a kink-turn motif of a large 232nt RNA. For sample volumes of ∼50μL, concentration sensitivities of 2–20μM 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.