Two-dimensional NMR experiments for the assignment of aromatic side chains in 13C labeled proteins

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 transferring1Hßcoherence to the aromatic protons via13C¿like in a number of published methods, in our new experiments the13C¿resonances are first correlated with the1Hßchemical 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 C¿chemical shifts between different aromatic residue types is obvious, but even the dispersion within one type is sufficient to combine the experiments using only the C¿chemical shift and to assign nearly all aromatic proton resonances of cutinase. Author Keywords: heteronuclear NMR; resonance assignment; aromatic side chain; protein; cutinase