SUPPORT OF H-1-NMR ASSIGNMENTS IN PROTEINS BY BIOSYNTHETICALLY DIRECTED FRACTIONAL C-13-LABELING

Biosynthetically directed fractional incorporation of C-13 into proteins results in nonrandom C-13-labeling patterns that can be investigated by analysis of the C-13-C-13 scalar coupling fine structures in heteronuclear C-13-H-1 or homonuclear C-13-C-13 correlation experiments. Previously this approach was used for obtaining stereospecific H-1 and C-13 assignments of the diastereotopic methyl groups of valine and leucine. In the present paper we investigate to what extent the labeling patterns are characteristic for other individual amino acids or groups of amino acids, and can thus be used to support the H-1 spin-system identifications. Studies of the hydrolysates of fractionally C-13-labeled proteins showed that the 59 aliphatic carbon positions in the 20 proteinogenic amino acids exhibit 16 different types of C-13-C-13 coupling fine structures. These provide support for the assignment of the resonances of all methyl groups in a protein, which arc otherwise often poorly resolved in homonuclear H-1 NMR spectra. In particular, besides the individual methyl assignments in Val and Leu, unambiguous distinctions are obtained between the methyl groups of Ala and Thr, and between the gamma- and delta-methyl groups of Ile. In addition to the methyl resonances, the gamma-CH2 groups of Glu and Gln can be uniquely assigned because of the large coupling constant with the delta-carbon, and the identification of most of the other spin systems can be supported on the basis of coupling patterns that are common to small groups of amino acid residues.