Fig. 1.
High resolution DNP spectra of bR. (a) RFDR20 correlation spectrum of 14 mg of [14–13C]retinal, [ε-13C]lysine-labeled bR in the dark-adapted state with 1D direct 13C dimension slices overlaid on the 2D contours. 12.5 days worth of acquisition time from mixing times between 8 and 28 ms were averaged together. The DNP enhancement is ~40, relative to the signal recorded in the absence of microwaves. ωr/2π = 8000 Hz and the temperature is 93 K. The resulting signal-to-noise negated the need for any line-broadening. The bR568 correlation in the upper left has a linewidth of 1.2 ppm (115 Hz). (b) Expansion of the lower right portion of the spectrum in (a) demonstrating the narrow linewidths achievable with MAS DNP. Whereas the bR555 correlation is slightly broader due to the presence of two conformations of bR555, the bR568 resonance is extremely narrow with a linewidth of 1.0 ppm (95 Hz). (c) Carbon–nitrogen 2D spectrum of uniformly 13C, 15N-labeled bR568,16 ωr/2π = 6000 Hz. After an indirect chemical shift evolution on the resolved Schiff base (165 ppm in the 15N dimension), the magnetization is transferred along the retinal chain all the way to C11, and also in the other direction directly to Cε. The carbon resonances show relatively narrow linewidths (1.2–2.2 ppm).