The 3D HN(CO)C experiment allows the simultaneous measurement of J(CO-CB) and J(CO-CG) coupling constants in ¹³C,¹⁵N-labeled proteins using the quantitative J-correlation approach.

Implementation on AVANCE spectrometers equipped with a third channel and pulsed field gradients (PFGs).

The experiment has been described in 97JACS6360 . The magnetization transfer is as follows:

Initial transfer from NH protons to ¹⁵N via ¹J_NH using an INEPT scheme

Chemical shift evolution of ¹⁵N during a variable t1 period.

Magnetization transfer to ¹³CO via ¹J_NCO.

Magnetization transfer from ¹³CO to other ¹³C nuclei via ²J_CH and ³J_CH.

Chemical Shift evolution of aliphatic carbons

Transfer back to the NH protons and detection as usual.

The 3D HN(CO)C experiment affords a 3D map with (F1,F2,F3=N,C,NH) in which coupling constants are extracted from intensity ratio of the corresponding CO-C cross-peak and a reference external peak. For each NH(i) amide proton, up to five correlations can be observed:

Weak cross peak to CA(i-1) due to ¹J_COCA.

Cross-peak with CA(i) and CB(i-1) resulting from ²J_COCA and ³J_COCB.

Cross-peaks with CG(i-1) and CB(i) due to ³J_COCG and ³J_COCB.