A 'just-in-time' HN(CA)CO experiment for the backbone assignment of large proteins with high sensitivity.

Among the suite of commonly used backbone experiments, HNCACO presents an unresolved sensitivity limitation due to fast 13CO transverse relaxation and passive 13Calpha-13Cbeta coupling. Here, we present a high-sensitivity 'just-in-time' (JIT) HN(CA)CO pulse sequence that uniformly refocuses 13Calpha-13Cbeta coupling while collecting 13CO shifts in real time. Sensitivity comparisons of the 3-D JIT HN(CA)CO, a CT-HMQC-based control, and a HSQC-based control with selective 13Calpha inversion pulses were performed using a 2H/13C/15N labeled sample of the 29 kDa HCA II protein at 15 degrees C. The JIT experiment shows a 42% signal enhancement over the CT-HMQC-based experiment. Compared to the HSQC-based experiment, the JIT experiment is 16% less sensitive for residues experiencing proper 13Calpha refocusing and13Calpha-13Cbeta decoupling. However, for the remaining residues, the JIT spectrum shows a 106% average sensitivity gain over the HSQC-based experiment. The high-sensitivity JIT HNCACO experiment should be particularly beneficial for studies of large proteins to provide 13CO resonance information regardless of residue type.