A 4D TROSY-based pulse scheme for correlating 1HNi,15Ni,13Cαi,13C′i-1 chemical shifts in high molecular weight, 15N,13C, 2H labeled proteins

A 4D TROSY-based triple resonance experiment, 4D-HNCO(i-1)CA(i), is presented which correlates intra-residue (HN)-H-1, N-15, C-13(alpha) chemical shifts with the carbonyl (C-13') shift of the preceding residue. The experiment is best used in concert with recently described 4D TROSY-HNCOCA and -HNCACO experiments [Yang, D. and Kay, L.E. (1999) J. Am. Chem. Soc., 121, 2571-2575]. In cases where degeneracy of ((HN)-H-1,N-15) spin pairs precludes assignment using the HNCOCA and HNCACO, the HNCO(i-1)CA(i) often allows resolution of the ambiguity by linking the C-13(alpha) and C-13' spins surrounding the ((HN)-H-1,N-15) pair. The experiment is demonstrated on a sample of N-15, C-13, H-2 labeled maltose binding protein in complex with beta-cyclodextrin that tumbles with a correlation time of 46 ns.