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.
|Titolo:||Rapid proton-detected NMR assignment for proteins with fast magic angle spinning|
|Parole Chiave:||Chemistry (all); Catalysis; Biochemistry; Colloid and Surface Chemistry|
|Settore Scientifico Disciplinare:||Settore BIO/10 - Biochimica|
|Data di pubblicazione:||3-set-2014|
|Digital Object Identifier (DOI):||10.1021/ja507382j|
|Appare nelle tipologie:||01 - Articolo su periodico|