As aromatic residues very often are part of the hydrophobic essential for an accurate and precise structure determination. core of proteins, the unambiguous assignment of the aromatic Therefore, methods for the unambiguous assignment of aroproton resonances is essential for an accurate and precise structure matic ring protons are highly desirable. The reverse labeling determination. Instead of transferring 1 H b coherence to the arotechnique introduced by Vuister et al. (5) facilitates the matic protons via 13 C g like in a number of published methods, in assignment process, but requires an (expensive) additional our new experiments the 13 C g resonances are first correlated with sample. The conventional HCCH-COSY (6, 7) and the the 1 H b chemical shifts in one experiment and then with the arothree-dimensional 1 H-TOCSY-relayed ct-[ 13 C, 1 H]-HMQC matic proton resonances in four other experiments. Their short (8) experiments correlate carbons and protons within the coherence transfer pathways make the experiments applicable aromatic ring, but they do not establish the connection with to proteins with a molecular weight larger than 20 kDa, as is the aliphatic carbons and protons. An HCCH-TOCSY demonstrated for Fusarium solani pisi cutinase (214 residues). The dispersion of the C g chemical shifts between different aro-(9, 10) experiment optimized for the aromatic resonances is matic residue types is obvious, but even the dispersion within one not suited for transferring coherence between the band type is sufficient to combine the experiments using only the C g g-carbon spins of aromatic residues, because of the large chemical shift and to assign nearly all aromatic proton resonances chemical shift differences between these nuclei. Several exof cutinase. α§ 1998 Academic Press periments have been proposed to achieve transfer between Key Words: heteronuclear NMR; resonance assignment; aro-13 C b and 13 C g atoms, among which are the polarizationmatic side chain; protein; cutinase.