The 3D NOESY-[ ¹ H, ¹⁵ N, ¹ H]-ZQ-TROSY NMR experiment with diagonal peak suppression

In our 3D NOESY-[ 1 H, 15 N, 1 H]-ZQ-TROSY experiment, the TROSY principle (transverse relaxation-optimized spectroscopy) is used in three-dimensional (3D) 15 N-resolved nuclear Overhauser enhancement spectroscopy (NOESY), which enables resonance assignments by sequential nuclear Overhauser effects and the collection of structural constraints in large 15 N- or 2 H, 15 N-labeled proteins. Our experiment affords optimization of the transverse relaxation in all three frequency dimensions, provides suppression of the strong diagonal autorelaxation peaks, which otherwise tend to interfere with the analysis of nearby informative crosspeaks, and yields improved resolution for the entire spectrum when compared with conventional 3D 15 N-resolved-[ 1 H, 1 H]-NOESY, because of the narrower lineshapes along both proton dimensions. The key element of this experiment is an approach for correlating the 15 N and 1 H chemical shifts with two-dimensional ZQ-[ 15 N, 1 H]-TROSY, where zero-quantum (ZQ) coherence is generated and the remote cross-correlation between the 1 H and 15 N chemical shift anisotropy interactions is used to reduce transverse relaxation during 15 N evolution. Practical applications are illustrated with spectra of a protein with a molecular mass of 110,000 Da.