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

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 C^α and C^β 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 H^β and H^α 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 H^β and C^β resonances of aromatic or Asn/Asp residues. The extent to which combined knowledge of the C^α and C^β chemical shift values determines the amino acid type is investigated, and it is demonstrated that the combined C^α and C^β chemical shifts of three or four adjacent residues usually are sufficient for defining a unique position in the protein sequence.