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

Three-dimensional H-1-TOCSY-relayed ct-[C-13,H-1]-HMQC is a novel experiment for aromatic spin system identification in uniformly C-13-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 H-1 and C-13 chemical shift evolution periods and for the generation of H-1-H-1 antiphase magnetization to accelerate the H-1-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 C-13-H-1 zero-quantum and double-quantum coherences is to first order insensitive to C-13-H-1 heteronuclear dipolar relaxation, the new experiment should work also for proteins with molecular weights above 20 kDa.