The determination of protein structures using NMR achieved using the WATERGATE technique ( 17) . Water saturation is minimized by keeping some of the water spectroscopy relies heavily on the evaluation of interpromagnetization along the z axis during acquisition with the ton distances that are derived from nuclear-Overhauseruse of water-selective 90Њ pulses ( 4 ) . If the NOESY seceffect data. The quality of the resulting structure depends tion is omitted from the pulse sequence in Fig. 1, a 2D critically on the number of unambiguously assigned H ( N ) CO spectrum can be obtained to correlate the chemi-NOEs. Since assignment of NOEs is difficult and time cal shifts of 1 H NH with the 13 C resonances in the preceding consuming, many pulse sequences have been developed residue. for simplifying this process ( 1 -14 ) . The 3D 15 N-edited The resolution of degenerate 1 H NH and 15 N NH resonances ( 1 -4 ) and 4D 13 C / 15 N-edited ( 5 ) NOESY experiments using the NOESY-H ( N ) CO experiment was accomare efficient for obtaining NOE constraints involving 1 H NH plished for the SH2 domain of Hck in 90% H 2 O / 10% D 2 O resonances, while the 3D 13 C-edited ( 6 -9 ) and 4D 13 C / at pH 6.4. A sample uniformly labeled with 15 N ( Ç3 mM) 13 C-edited ( 10 -12 ) NOESY experiments are the methods was used for the 2D 1 H -15 N HSQC and the 3D 15 N-edited of choice for determining NOEs involving protons NOESY-HSQC experiments ( 3 ) while a 13 C / 15 N dually attached to 13 C nuclei. Two different heteronucleus-edited labeled sample ( Ç3 mM) was used for the 2D 1 H -13 C NOESY experiments can be recorded simultaneously us-H ( N ) CO and 3D 13 C -edited NOESY-H ( N ) CO experiing the pulse sequences developed by Pascal et al. ( 13) ments ( Fig. 1 ) . Data were acquired using a Varian UNITY and Farmer et al. ( 14 ) . In large proteins, however, some 500 NMR spectrometer equipped with a 5 mm 1 H { 13 C, residues have both overlapping 1 H NH and 15 N NH reso-15 N } PFG probe. The 2D 1 H -15 N HSQC spectrum is nances. In this situation, it is difficult to determine the shown in Fig. 2, while the spectrum obtained using the origin of some NOE cross peaks using only 15 N-edited H ( N ) CO section of the 13 C -edited NOESY-H ( N ) CO se-NOESY experiments. Editing of the backbone 1 H NH using quence is shown in Fig. 3. Although the dispersion in both the chemical shift of a different heteronucleus, e.g., carthe 1 H and 15 N dimensions of the HSQC spectrum is good, bonyl ( 13 C ) in the preceding residue, may be advantasome overlap still occurs. Deriving unambiguous NOE geous for those cases where the 13 C frequencies do not constraints from the 15 N-edited NOESY spectrum alone overlap.
is difficult for cross peaks involving degenerate 1 H NH and In this Communication, we present a 3D 13 C-edited 15 N NH . For example, the cross peaks for L20 and H59 of NOESY-H(N)CO experiment for resolving NOEs from dethe Hck SH2 overlap in the 2D 1 H -15 N HSQC spectrum, generate 1 H NH that are attached to degenerate 15 N NH resorendering 15 N-edited NOESY experiments ineffective for nances. The utility of this method is demonstrated for the evaluating distance constraints for these residues. How-107 residue SH2 domain of a Src-family tyrosine kinase, ever, the chemical shifts of 13 C in the preceding residues Hck (15). of L20 and H59 are distinct, giving rise to completely The pulse sequence for the 3D 13 C -edited NOESYseparated peaks in the 2D 1 H -13 C H ( N ) CO spectrum H ( N ) CO experiment is depicted in Fig. 1. The detection ( Fig. 3 ) . Similar results are observed for several other period of a NOESY sequence is replaced with an HNCO residues with degenerate 1 H NH and 15 N NH resonances in sequence in which the 15 N evolution is omitted ( 16 ) .
the 2D 1 H -15 N HSQC spectrum. The resolution of NOEs Pulsed-field-gradient pulses are incorporated to reduce involving L20 and H59 using the 13 C -edited NOESYphase cycling and remove artifacts originating from im-H ( N ) CO experiment is shown in Fig. 4. A plane taken perfect 180Њ pulses ( 8, 12 ) , while water suppression is from a 3D 15 N-edited NOESY-HSQC spectrum at the ( degenerate ) 15 N chemical shifts of L20 and H59 ( 117.6 ppm) is shown in Fig. 4A. Figures 4B and4C show slices in