title: Continuous wave W- and D-band EPR spectroscopy offer "sweet-spots" for characterizing conformational changes and dynamics in intrinsically disordered proteins.
abstract: Site-directed spin labeling (SDSL) electron paramagnetic resonance (EPR) spectroscopy is a powerful tool for characterizing conformational sampling and dynamics in biological macromolecules. Here we demonstrate that nitroxide spectra collected at frequencies higher than X-band (∼9.5 GHz) have sensitivity to the timescale of motion sampled by highly dynamic intrinsically disordered proteins (IDPs). The 68 amino acid protein IA3, was spin-labeled at two distinct sites and a comparison of X-band, Q-band (35 GHz) and W-band (95 GHz) spectra are shown for this protein as it undergoes the helical transition chemically induced by tri-fluoroethanol. Experimental spectra at W-band showed pronounced line shape dispersion corresponding to a change in correlation time from ∼0.3 ns (unstructured) to ∼0.6 ns (α-helical) as indicated by comparison with simulations. Experimental and simulated spectra at X- and Q-bands showed minimal dispersion over this range, illustrating the utility of SDSL EPR at higher frequencies for characterizing structural transitions and dynamics in IDPs.
authors.affiliation: Department of Chemistry, University of Florida, PO Box 117200, Gainesville, FL 32611, USA.
authors.forename: Thomas M
authors.initials: TM
authors.lastname: Casey
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: 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: Department of Chemistry, University of Florida, PO Box 117200, Gainesville, FL 32611, USA.
authors.forename: Natasha L
authors.initials: NL
authors.lastname: Pirman
authors.affiliation: Department of Chemistry, University of Florida, PO Box 117200, Gainesville, FL 32611, USA.
authors.forename: Eugene
authors.initials: E
authors.lastname: Milshteyn
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; D004578:Electron Spin Resonance Spectroscopy / Q000379:methods*; D011487:Protein Conformation; D011506:Proteins / Q000737:chemistry* / Q000648:ultrastructure*; D013194:Staining and Labeling / Q000379:methods
chemical_list: D011506:Proteins
keywords: EPR spectroscopy; High-field EPR; Intrinsically disordered proteins; Multi-frequency EPR; Site-directed spin-labeling