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 (∼9.5GHz) 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 (35GHz) and W-band (95GHz) 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 ∼0.3ns (unstructured) to ∼0.6ns (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.
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