CO_H(N)CACB experiments for assigning backbone resonances in C-13/N-15-labeled proteins
A triple resonance NMR experiment, denoted CO_H(N)CACB, correlates H-1(N) and (CO)-C-13 spins with the C-13(alpha) and C-13(beta) spins of adjacent amino acids. The pulse sequence is an 'out-and-back' design that starts with H-1(N) magnetization and transfers coherence via the N-15 spin simultaneously to the (CO)-C-13 and C-13(alpha) spins, followed by transfer to the C-13(beta) spin. Two versions of the sequence are presented: one in which the (CO)-C-13 spins are frequency labeled during an incremented t(1) evolution period prior to transfer of magnetization from the C-13(alpha) to the C-13(beta) resonances, and one in which the (CO)-C-13 spins are frequency labeled in a constant-time manner during the coherence transfer to and from the C-13(beta) resonances. Because (CO)-C-13 and N-15 chemical shifts are largely uncorrelated, the technique will be especially useful when degeneracy in the H-1(N)-N-15 chemical shifts hinders resonance assignment. The CO_H(N)CACB experiment is demonstrated using uniformly C-13/N-15-labeled ubiquitin.