Two-dimensional electron spin resonance and slow motions

The slow rotational dynamics of a polyproline peptide with a nitroxide labeled at one end in a glassy medium is probed using two-dimensional (2D) electron spin resonance (ESR). The contributions to the homogeneous relaxation time, T-2, from the overall and/or the internal rotations of the nitroxide is elucidated from the COSY spectra. The use of pure absorption spectra allows the variation of T-2 across the spectrum to be monitored. It is shown from simulations that the model of anisotropic Brownian diffusion provides semiquantitative agreement with such a variation. In the 2D ELDOR experiment several mechanisms can lead to spectral diffusion, which yields a broadening of the hyperfine (hf) auto-peaks with mixing time. We call these spectral diffusion (SD) cross-peaks. It is shown that at higher temperatures the principal mechanism for the formation of SD cross-peaks is the slow reorientation of the molecule, which modulates the N-14 hf and g tensor interactions. A procedure is shown for extracting a correlation time, tau(c), by monitoring this growth of SD cross-peaks, which is in good agreement with theory. An anomalous temperature dependence of the experimental tau(c), at very low temperatures, is tentatively attributed to the fast internal rotations of the methyl groups on the nitroxide, which leads to spin-flips of the protons on these methyl groups. The use of pure absorption spectra in 2D ELDOR enhances the sensitivity to these cross-peaks.