Protein resonance assignment by solid-state NMR based on H-detected C double-quantum spectroscopy at fast MAS.

Solid-state NMR spectroscopy is a powerful technique to study insoluble and non-crystalline proteins and protein complexes at atomic resolution. The development of proton (H) detection at fast magic-angle spinning (MAS) has considerably increased the analytical capabilities of the technique, enabling the acquisition of H-detected fingerprint experiments in few hours. Here an approach based on double-quantum (DQ) C spectroscopy, detected on H, is proposed for fast MAS regime (> 60 kHz) to perform the sequential assignment of insoluble proteins of small size, without any specific deuteration requirement. By combining two three-dimensional H detected experiments correlating a C DQ dimension respectively to its intra-residue and sequential  N-H pairs, a sequential walk through DQ (Ca + CO) resonance is obtained. The approach takes advantage of fast MAS to achieve an efficient sensitivity and the addition of a DQ dimension provides spectral features useful for the resonance assignment process.