HIGH-RESOLUTION DETECTION OF 5 FREQUENCIES IN A SINGLE 3D SPECTRUM - HNHCACO - A BIDIRECTIONAL COHERENCE TRANSFER EXPERIMENT

A new triple-resonance pulse sequence, 3D HNHCACO, is introduced and d iscussed, which identifies sequential correlations of the backbone nuc lei (H-alpha(i-1), C-alpha(i-1), C'(i-1), NH(i), N(i)) of doubly label ed proteins in H2O. The three-dimensional (3D) method utilizes a recor ding of N-15 and C-13 resonances in a single indirect time domain, the C-13' resonance 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, resultin g in a single high-resolution 3D spectrum that contains the frequencie s of all five backbone nuclei. The experiment was applied to the 12.3 kDa ribonuclease from Bacillus intermedius (Binase).