Sequential resonance assignment from two-dimensional inter- and intra-residue ¹⁵N-¹H correlation spectra

Sequential assignment of amide resonances in proteins and peptides can conveniently be derived from twodimensional inter-residue ¹⁵N_i –¹H^N_(i−1) and intra-residue ¹⁵N_i –¹H^N_i correlation spectra. The inter-residue  ¹⁵N_i –¹H^N_(i−1)correlation spectrum is generated by recording the ¹⁵N_i frequency evolution indirectly and subsequently transferring the magnetization to ¹H^N_(i−1) of the preceding residue for direct detection. The flow of coherence is established by the (H)N(COCAHA)-TOCSY pulse sequence. Following the path from intra-residue correlation via inter-residue correlation to the next intra-residue correlation results in sequential assignment in two dimensions. This kind of assignment protocol is most amenable to re-establishing sequential assignments of target proteins perturbed by binding of ligands or alternatively signals of peptide ligands can be traced in studies of structure–function relationships by NMR.