High-resolution detection of five frequencies in a single 3D spectrum: HNHCACO--a bidirectional coherence transfer...

A new triple-resonance pulse sequence, 3D HNHCACO, is introduced and discussed, which identifies sequential correlations of the backbone nuclei (H alpha (i-1), C alpha (i-1), C(i-1), NH(i), N(i)) of doubly labeled proteins in H2O. The three-dimensional (3D) method utilizes a recording of 15N and 13C resonances in a single indirect time domain, the 13C' resonance in another indirect time domain, and detects both NH and H alpha protons. A bidirectional coherence transfer (NH(i) <--> N(i) <--> C(i-1) <--> C alpha (i-1) <--> H alpha (i-1)) is effectuated, resulting in a single high-resolution 3D spectrum that contains the frequencies of all five backbone nuclei. The experiment was applied to the 12.3 kDa ribonuclease from Bacillus intermedius (Binase).