Nitroxide side-chain dynamics in a spin-labeled helix-forming peptide revealed by high-frequency (139.5-GHz) EPR spectroscopy

High-frequency electron paramagnetic resonance (EPR) spectroscopy has been performed on a nitroxide spin-labeled peptide in fluid aqueous solution. The peptide, which follows the single letter sequence Ac-(AAAAK)(2)CAAAKA-NH2 3K-11, was reacted with the methanethiosulfonate spin label at the cysteine sulfur, The spin sensitivity of high-frequency EPR is excellent with less than 20 pmol of sample required to obtain spectra with good signal-to-noise ratios, Simulation of the temperature-dependent spectral lineshapes reveals the existence of local anisotropic motion about the nitroxide N-O bond with a motional anisotropy tau(perpendicular to)/tau(parallel to) (equivalent to N) approaching 2.6 at 306 K, Comparison with previous work on rigidly labeled peptides suggests that the spin label is reorienting about its side-chain tether, This study demonstrates the feasibility of performing 140-GHz EPR on biological samples in fluid aqueous solution. (C) 1999 Academic Press.