Optimal degree of protonation for 1H detection of aliphatic sites in randomly deuterated proteins as a function of the MAS frequency

The ¹H dipolar network, which is the major obstacle for applying proton detection in the solid-state, can be reduced by deuteration, employing the RAP (Reduced Adjoining Protonation) labeling scheme, which yields random protonation at non-exchangeable sites. We present here a systematic study on the optimal degree of random sidechain protonation in RAP samples as a function of the MAS (magic angle spinning) frequency. In particular, we compare ¹H sensitivity and linewidth of a microcrystalline protein, the SH3 domain of chicken ??-spectrin, for samples, prepared with 5?C25?% H₂O in the E. coli growth medium, in the MAS frequency range of 20?C60?kHz. At an external field of 19.96?T (850?MHz), we find that using a proton concentration between 15 and 25?% in the M9 medium yields the best compromise in terms of sensitivity and resolution, with an achievable average ¹H linewidth on the order of 40?C50?Hz. Comparing sensitivities at a MAS frequency of 60 versus 20?kHz, a gain in sensitivity by a factor of 4?C4.5 is observed in INEPT-based ¹H detected 1D ¹H,¹³C correlation experiments. In total, we find that spectra recorded with a 1.3?mm rotor at 60?kHz have almost the same sensitivity as spectra recorded with a fully packed 3.2?mm rotor at 20?kHz, even though ~20×?less material is employed. The improved sensitivity is attributed to ¹H line narrowing due to fast MAS and to the increased efficiency of the 1.3?mm coil.