Rapid Proton-Detected NMR Assignment for Proteins with Fast Magic Angle Spinning

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• 作者：Emeline Barbet-Massin ; Andrew J. Pell ; Joren S. Retel ; Loren B. Andreas ; Kristaps Jaudzems ; W. Trent Franks ; Andrew J. Nieuwkoop ; Matthias Hiller ; Victoria Higman ; Paul Guerry ; Andrea Bertarello ; Michael J. Knight ; Michele Felletti ; Tanguy Le Marchand ; Svetlana Kotelovica ; Inara Akopjana ; Kaspars Tars ; Monica Stoppini ; Vittorio Bellotti ; Martino Bolognesi ; Stefano Ricagno ; James J. Chou ; Robert G. Griffin ; Hartmut Oschkinat ; Anne Lesage ; Lyndon Emsley ; Torsten Herrmann ; Guido Pintacuda
• 刊名：Journal of the American Chemical Society
• 出版年：2014
• 出版时间：September 3, 2014
• 年：2014
• 卷：136
• 期：35
• 页码：12489-12497
• 全文大小：554K
• ISSN：1520-5126

文摘

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鈥?0 kDa proteins. The approach relies on perdeuteration, amide ²H/¹H exchange, high magnetic fields, and high-spinning frequencies (蠅_r/2蟺 鈮?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.