Sequence-specific assignment of histidine and tryptophan ring 1H, 13C and 15N resonances in 13C/15N- and 2H/13C/15N-labelled proteins.

Methods are described to correlate aromatic 1H(delta)2/13C(delta)2 or 1H(epsilon)1/15N(epsilon)1 with aliphatic 13C(beta) chemical shifts of histidine and tryptophan residues, respectively. The pulse sequences exclusively rely on magnetization transfers via one-bond scalar couplings and employ [15N, 1H]- and/or [13C, 1H]-TROSY schemes to enhance sensitivity. In the case of histidine imidazole rings exhibiting slow HN-exchange with the solvent, connectivities of these proton resonances with beta-carbons can be established as well. In addition, their correlations to ring carbons can be detected in a simple [15N, 1H]-TROSY-H(N)Car experiment, revealing the tautomeric state of the neutral ring system. The novel methods are demonstrated with the 23-kDa protein xylanase and the 35-kDa protein diisopropyl-fluorophosphatase, providing nearly complete sequence-specific resonance assignments of their histidine delta-CH and tryptophan epsilon-NH groups.