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Optimal degree of protonation for 1H detection of aliphatic sites in randomly deuterated proteins as a function of the MAS frequency.
J. Biomol. NMR 54, 155-168 (2012)
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The H-1 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 H-1 sensitivity and linewidth of a microcrystalline protein, the SH3 domain of chicken alpha-spectrin, for samples, prepared with 5-25 % H2O 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 H-1 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 H-1 detected 1D H-1,C-13 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 similar to 20x less material is employed. The improved sensitivity is attributed to H-1 line narrowing due to fast MAS and to the increased efficiency of the 1.3 mm coil.
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Publikationstyp Artikel: Journalartikel
Dokumenttyp Wissenschaftlicher Artikel
Schlagwörter Magic Angle Spinning (mas) Solid-state Nmr ; Perdeuteration ; H-2-labeling ; Aliphaticresonances ; Linewidth ; Sensitivity ; 1.3 Mm Rotors ; Reduced Adjoining Protonation (rap); Solid-State Nmr; Angle-Spinning Nmr; Resolution Enhancement; Perdeuterated Proteins; Assignment Strategies; Pulse Sequences; Spectroscopy
ISSN (print) / ISBN 0925-2738
Zeitschrift Journal of Biomolecular NMR
Quellenangaben Band: 54, Heft: 2, Seiten: 155-168
Begutachtungsstatus Peer reviewed
Institut(e) Institute of Structural Biology (STB)