[1H,13C]-NOESY-HSQC

In the 3D spectrum diagonal (at H and H diagonal) and cross peaks are expected. Only cross-peaks confer spatial information.

The 3D ¹³C NOESY-HSQC experiment is specifically designed to obtain carbon-edited NOESY spectra of carbon-labeled biomolecules and protein-ligand complexes from which homonuclear ¹H-¹H NOEs can be clearly assigned even in overcrowded regions (the mechanism involves ¹H-¹H NOE step followed by a heteronuclear transfer via ¹J_CH).

To start with, magnetization is exchanged between all hydrogens using the NOE. The NOESY experiment yields a 2D proton-proton correlation map with two different types of signals: a.) autocorrelation diagonal peaks, and b.) off-diagonal cross-peaks correlating spins close to each other. NOESY spectra are represented in phase-sensitive mode in order to distinguish exchange cross-peaks and unwanted anti-phase COSY contributions.

After NOESY the magnetization is transferred to neighboring ¹³C nuclei and back to ¹H for detection. This acquires the chemical shift of the carbon attached to the corresponding hydrogen, which shifts the NOESY cross-peak into the carbon plane. Peaks that were previously too close to be resolved in the NOESY spectrum become discrete. Transfer either occurs to/from the aliphatic ¹³C nuclei or to/from the aromatic ¹³C nuclei (but not both) depending on the ¹³C frequency used during the pulse sequence.

Each strip in the CH plane contains NOE’s from one CH group to all other hydrogens nearby. This experiment yields constraints that can be used to calculate the 3D protein structure after carbon assignation has been done using other NMR methods.

Figure 1: Original PDF paper pulse sequence with accompanying explanation

Figure 2: Bruker pulse sequence to record 3D ¹³C NOESY-HSQC experiments with with sensitivity-improved building block (PEP)

Muhandiram, D.R., Farrow, N. A., Xu, G. Y., Smallcombe, S. H., & Kay, L. E. (1993). Agradient 13 C NOESY-HSQC experiment for recording NOESY spectra of 13 C-labeledproteins dissolved in H 2 O. Journalof Magnetic Resonance, Series B, 102(3),317-321.