Optimal degree of protonation for ¹H detection of aliphatic sites in randomly deuterated proteins as a function of the MAS frequency.
The (1)H 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 (1)H sensitivity and linewidth of a microcrystalline protein, the SH3 domain of chicken α-spectrin, for samples, prepared with 5-25 % H(2)O in the E. coli growth medium, in the MAS frequency range of 20-60 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 (1)H linewidth on the order of 40-50 Hz. Comparing sensitivities at a MAS frequency of 60 versus 20 kHz, a gain in sensitivity by a factor of 4-4.5 is observed in INEPT-based (1)H detected 1D (1)H,(13)C 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 (1)H line narrowing due to fast MAS and to the increased efficiency of the 1.3 mm coil.
Helmholtz-Zentrum München (HMGU), Deutsches Forschungszentrum für Gesundheit und Umwelt (HMGU), Ingolstädter Landstr. 1, 85764 Neuherberg, Germany.
Sam
S
Asami
Kathrin
K
Szekely
Paul
P
Schanda
Beat H
BH
Meier
Bernd
B
Reif
D002247:Carbon Isotopes; D003903:Deuterium; D009587:Nitrogen Isotopes; D019906:Nuclear Magnetic Resonance, Biomolecular / Q000379:methods*; D011506:Proteins / Q000737:chemistry*; D011522:Protons; D013049:Spectrin / Q000737:chemistry*; D018909:src Homology Domains
D002247:Carbon Isotopes; D009587:Nitrogen Isotopes; D011506:Proteins; D011522:Protons; D013049:Spectrin; D003903:Deuterium