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Amino-Acid Type Determination in the Sequential Assignment Procedure of Uniformly C-13/N-15-Enriched Proteins
Journal
Journal of Biomolecular NMR
Volume
3
Number
2
Pages / Article-Number
185-204
Abstract
Experiments and procedures are described that greatly alleviate the sequential assignment process of uniformly C-13/N-15-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(alpha) and C(beta) resonances to the backbone amide H-1 and N-15 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(beta) and H(alpha) resonances to the amide H-1 and N-15 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(beta) and C(beta) resonances of aromatic or Asn/Asp residues. The extent to which combined knowledge of the C(alpha) and C(beta) chemical shift values determines the amino acid type is investigated, and it is demonstrated that the combined C(alpha) and C(beta) chemical shifts of three or four adjacent residues usually are sufficient for defining a unique position in the protein sequence.
