Three-dimensional (1)H-TOCSY-relayed ct-[(13)C, (1)H]-HMQC for aromatic spin system identification in uniformly (13)C-labeled proteins.

Three-dimensional (1)H-TOCSY-relayed ct-[(13)C,(1)H]-HMQC is a novel experiment for aromatic spin system identification in uniformly (13)C-labeled proteins, which is implemented so that it correlates the chemical shift of a given aromatic proton with those of the directly attached carbon and all vicinal protons. The ct-HMQC scheme is used both for overlay of the indirect (1)H and (13)C chemical shift evolution periods and for the generation of (1)H-(1)H antiphase magnetization to accelerate the (1)H-TOCSY magnetization transfer at short mixing times. As an illustration, data recorded for the 18 kDa protein cyclophilin A are presented. Since transverse relaxation of (13)C-(1)H zero-quantum and double-quantum coherences is to first order insensitive to (13)C-(1)H heteronuclear dipolar relaxation, the new experiment should work also for proteins with molecular weights above 20 kDa.