title: 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-(∼94 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 α-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 μL, concentration sensitivities of 2-20 μM 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.
authors.affiliation: National High Magnetic Field Laboratory, Florida State University, Tallahassee, FL 32310, USA.
authors.forename: Likai
authors.initials: L
authors.lastname: Song
authors.affiliation: Department of Chemistry, University of Florida, PO Box 117200, Gainesville, FL 32611, USA.
authors.forename: Zhanglong
authors.initials: Z
authors.lastname: Liu
authors.affiliation: National High Magnetic Field Laboratory, Florida State University, Tallahassee, FL 32310, USA; Department of Physics, Florida State University, Tallahassee, FL 32306, USA.
authors.forename: Pavanjeet
authors.initials: P
authors.lastname: Kaur
authors.affiliation: Department of Chemistry, University of Florida, PO Box 117200, Gainesville, FL 32611, USA.
authors.forename: Jackie M
authors.initials: JM
authors.lastname: Esquiaqui
authors.affiliation: School of Physics and Astronomy, University of St. Andrews, North Haugh, St. Andrews KY16 9SS, United Kingdom.
authors.forename: Robert I
authors.initials: RI
authors.lastname: Hunter
authors.affiliation: National High Magnetic Field Laboratory, Florida State University, Tallahassee, FL 32310, USA; Department of Physics, Florida State University, Tallahassee, FL 32306, USA.
authors.forename: Stephen
authors.initials: S
authors.lastname: Hill
authors.affiliation: School of Physics and Astronomy, University of St. Andrews, North Haugh, St. Andrews KY16 9SS, United Kingdom.
authors.forename: Graham M
authors.initials: GM
authors.lastname: Smith
authors.affiliation: Department of Chemistry, University of Florida, PO Box 117200, Gainesville, FL 32611, USA. Electronic address: fanucci@chem.ufl.edu.
authors.forename: Gail E
authors.initials: GE
authors.lastname: Fanucci
mesh_terms: D000465:Algorithms; D004574:Electromagnetic Fields; D004578:Electron Spin Resonance Spectroscopy / Q000295:instrumentation / Q000379:methods*; D005998:Glycine / Q000737:chemistry; D046911:Macromolecular Substances / Q000737:chemistry*; D011506:Proteins / Q000737:chemistry; D012329:RNA, Bacterial / Q000737:chemistry; D015203:Reproducibility of Results; D013113:Spin Labels; D014734:Vibrio cholerae / Q000737:chemistry
chemical_list: D046911:Macromolecular Substances; D011506:Proteins; D012329:RNA, Bacterial; D013113:Spin Labels; D005998:Glycine
keywords: EPR; High field; IDP; RNA; Spin label