CO_H(N)CACB experiments for assigningbackbone resonances in 13C/15N-labeledproteins

A triple resonance NMR experiment, denoted CO_H(N)CACB, correlates¹H^N and ¹³CO spins with the¹³C and¹³C spins of adjacent amino acids. Thepulse sequence is an out-and-back design that starts with¹H^N magnetization and transfers coherence viathe ¹⁵N spin simultaneously to the ¹³CO and¹³C spins, followed by transfer to the¹³C spin. Two versions of the sequence arepresented: one in which the ¹³CO spins are frequency labeledduring an incremented t₁ evolution period prior to transfer ofmagnetization from the ¹³C to the¹³C resonances, and one in which the¹³CO spins are frequency labeled in a constant-time mannerduring the coherence transfer to and from the¹³C resonances. Because ¹³COand ¹⁵N chemical shifts are largely uncorrelated, thetechnique will be especially useful when degeneracy in the¹H^N-¹⁵N chemical shifts hindersresonance assignment. The CO_H(N)CACB experiment is demonstrated usinguniformly ¹³C/¹⁵N-labeled ubiquitin.