(H)N(COCA)NH and (HN)under-bar(COCA)NH experiments for H-1-N-15 backbone assignments in C-13/N-15-labeled proteins
Triple resonance HN(COCA)NH pulse sequences for correlating H-1((i)), H-15((i)), H-1((i-1)), and N-15((i-1)) spins that utilize overlapping coherence transfer periods provide increased sensitivity relative to pulse sequences that utilize sequential coherence transfer periods. Although the overlapping sequence elements reduce the overall duration of the pulse sequences, the principal benefit derives from a reduction in the number of 180 degrees pulses. Two versions of the technique are presented: a 3D (H)N(COCA)NH experiment that correlates N-15((i)), H-1((i-1)), and N-15((i-1)) spins, and a 3D experiment that correlates H-1((i)), N-15((i)), H-1((i-1)), and N-15((i-1)) spins by simultaneously encoding the H-1((i)) and N-15((i)) chemical shifts during the t(1) evolution period. The methods are demonstrated on a C-13/N-15-enriched sample of the protein ubiquitin and are easily adapted for application to H-2/C-13/N-15-enriched proteins.