Protein resonance assignment by solid-state NMR based on 1H-detected 13C double-quantum spectroscopy at fast MAS

Abstract

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 (¹H) detection at fast magic-angle spinning (MAS) has considerably increased the analytical capabilities of the technique, enabling the acquisition of ¹H-detected fingerprint experiments in few hours. Here an approach based on double-quantum (DQ) ¹³C spectroscopy, detected on ¹H, 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 ¹H detected experiments correlating a ¹³C DQ dimension respectively to its intra-residue and sequential ¹⁵ N-¹H 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.