Two-Dimensional NMR Experiments for the Assignment of Aromatic Side Chains in13C-labeled Proteins

Abstract

As aromatic residues very often are part of the hydrophobic core of proteins, the unambiguous assignment of the aromatic proton resonances is essential for an accurate and precise structure determination. Instead of transferring1Hβcoherence to the aromatic protons via13Cγlike in a number of published methods, in our new experiments the13Cγresonances are first correlated with the1Hβchemical shifts in one experiment and then with the aromatic proton resonances in four other experiments. Their short coherence transfer pathways make the experiments applicable to proteins with a molecular weight larger than 20 kDa, as is demonstrated forFusarium solani pisicutinase (214 residues). The dispersion of the Cγchemical shifts between different aromatic residue types is obvious, but even the dispersion within one type is sufficient to combine the experiments using only the Cγchemical shift and to assign nearly all aromatic proton resonances of cutinase.