title: A 'just-in-time' HN(CA)CO experiment for the backbone assignment of large proteins with high sensitivity.
abstract: 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.
authors.affiliation: Department of Biochemistry, Duke University Medical Center, Durham, NC 27710, USA.
authors.forename: Jon W
authors.initials: JW
authors.lastname: Werner-Allen
authors.forename: Ling
authors.initials: L
authors.lastname: Jiang
authors.forename: Pei
authors.initials: P
authors.lastname: Zhou
mesh_terms: D002247:Carbon Isotopes; D019906:Nuclear Magnetic Resonance, Biomolecular / Q000379:methods*; D011506:Proteins / Q000737:chemistry*; D012680:Sensitivity and Specificity
chemical_list: D002247:Carbon Isotopes; D011506:Proteins