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

Abstract

A 4D TROSY-based triple resonance experiment, 4D-HNCO_i−1CA_i, is presented which correlates intra-residue ¹HN, ¹⁵N, ¹³ C^α chemical shifts with the carbonyl (¹³C′) 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 (¹HN,¹⁵N) spin pairs precludes assignment using the HNCOCA and HNCACO, the HNCO_i−1CA_i often allows resolution of the ambiguity by linking the ¹³C^α and ¹³C′ spins surrounding the (¹HN,¹⁵N) pair. The experiment is demonstrated on a sample of ¹⁵N, ¹³C, ² H labeled maltose binding protein in complex with β-cyclodextrin that tumbles with a correlation time of 46 ns.