Using a set of six 1H-detected triple-resonance NMR experiments, we establish a method for sequence-specific backbone resonance assignment of magic angle spinning (MAS) nuclear magnetic resonance (NMR) spectra of 5-30 kDa proteins. The approach relies on perdeuteration, amide 2H/ 1H exchange, high magnetic fields, and high-spinning frequencies (ωr/2π ≥ 60 kHz) and yields high-quality NMR data, enabling the use of automated analysis. The method is validated with five examples of proteins in different condensed states, including two microcrystalline proteins, a sedimented virus capsid, and two membrane-embedded systems. In comparison to contemporary 13C/15N-based methods, this approach facilitates and accelerates the MAS NMR assignment process, shortening the spectral acquisition times and enabling the use of unsupervised state-of-the-art computational data analysis protocols originally developed for solution NMR.
Rapid proton-detected NMR assignment for proteins with fast magic angle spinning / E. Barbet-Massin, A.J. Pell, J.S. Retel, L.B. Andreas, K. Jaudzems, W..T. Franks, A.J. Nieuwkoop, M. Hiller, V. Higman, P. Guerry, A. Bertarello, M.J. Knight, M. Felletti, T. Le Marchand, S. Kotelovica, I. Akopjana, K. Tars, M. Stoppini, V. Bellotti, M. Bolognesi, S. Ricagno, J.J. Chou, R.G. Griffin, H. Oschkinat, A. Lesage, L. Emsley, T. Herrmann, G. Pintacuda. - In: JOURNAL OF THE AMERICAN CHEMICAL SOCIETY. - ISSN 0002-7863. - 136:35(2014 Sep 03), pp. 12489-12497. [10.1021/ja507382j]
Rapid proton-detected NMR assignment for proteins with fast magic angle spinning
M. Bolognesi;S. Ricagno;
2014
Abstract
Using a set of six 1H-detected triple-resonance NMR experiments, we establish a method for sequence-specific backbone resonance assignment of magic angle spinning (MAS) nuclear magnetic resonance (NMR) spectra of 5-30 kDa proteins. The approach relies on perdeuteration, amide 2H/ 1H exchange, high magnetic fields, and high-spinning frequencies (ωr/2π ≥ 60 kHz) and yields high-quality NMR data, enabling the use of automated analysis. The method is validated with five examples of proteins in different condensed states, including two microcrystalline proteins, a sedimented virus capsid, and two membrane-embedded systems. In comparison to contemporary 13C/15N-based methods, this approach facilitates and accelerates the MAS NMR assignment process, shortening the spectral acquisition times and enabling the use of unsupervised state-of-the-art computational data analysis protocols originally developed for solution NMR.File | Dimensione | Formato | |
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