Fig. 5.
Sequential assignment of A36–L38 shown in 13C–13C slices of 3D NCACX (green), 3D S3 i→i + 1 (purple), and i→i − 1 (orange) at the indicated 15N chemical shifts. Notably, the two S3 spectra show very different patterns. (A) In S3 i→i − 1, G37-A36 cross-peaks appeared at the glycine frequency in the indirect dimension (y axis) and were “read out” at alanine frequencies in the direct dimension (x axis), whereas in S3 i→i + 1, these frequencies were reversed. No S3 i→i − 1 intensity was detected at the glycine frequency, and no S3 i→i + 1 intensity was observed on the alanine slice, except for a diagonal peak representing G37Cα–N–Cα. (B) A similar pattern was observed for G37–L38 interresidue cross-peaks. (C) With the exception of some diagonal intensity, the observed cross-peak patterns were exactly as expected from the transfer diagram, demonstrating the excellent selectivity of S3 experiments. See SI Appendix for additional experimental details.