, Volume 3, Issue 2, pp 185-204

Amino acid type determination in the sequential assignment procedure of uniformly 13C/15N-enriched proteins

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Summary

Experiments and procedures are described that greatly alleviate the sequential assignment process of uniformly 13C/15N-enriched proteins by determining the type of amino acid from experiments that correlate side chain with backbone amide resonances. A recently proposed 3D NMR experiment, CBCA(CO)NH, correlates Cα and Cβ resonances to the backbone amide 1H and 15N resonances of the next residue (Grzesiek, S. and Bax, A. (1992) J. Am. Chem. Soc., 114, 6291–6293). An extension of this experiment is described which correlates the proton Hβ and Hα resonances to the amide 1H and 15N resonances of the next amino acid, and a detailed product operator description is given. A simple 2D-edited constant-time HSQC experiment is described which rapidly identifies Hβ and Cβ resonances of aromatic or Asn/Asp residues. The extent to which combined knowledge of the Cα and Cβ chemical shift values determines the amino acid type is investigated, and it is demonstrated that the combined Cα and Cβ chemical shifts of three or four adjacent residues usually are sufficient for defining a unique position in the protein sequence.