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

Using a set of six [superscript 1]H-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 [superscript 2]H/[superscript 1]H exchange, high magnetic fields, and high-spinning frequencies (ω[subscript r]/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 [superscript 13]C/[superscript 15]N-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.National Institutes of Health (U.S.) (NIH Grant EB-001960)National Institutes of Health (U.S.) (NIH Grant EB-002026