Journal of Biomolecular NMR

, Volume 2, Issue 4, pp 323–334

Support of1H NMR assignments in proteins by biosynthetically directed fractional13C-labeling

  • Thomas Szyperski
  • Dario Neri
  • Barbara Leiting
  • Gottfried Otting
  • Kurt Wüthrich
Research Papers


Biosynthetically directed fractional incorporation of13C into proteins results in nonrandom13C-labeling patterns that can be investigated by analysis of the13C−13C scalar coupling fine structures in heteronuclear13C−1H or homonuclear13C−13C correlation experiments. Previously this approach was used for obtaining stereospecific1H and13C 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 the1H spin-system identifications. Studies of the hydrolysates of fractionally13C-labeled proteins showed that the 59 aliphatic carbon positions in the 20 proteinogenic amino acids exhibit 16 different types of13C−13C coupling fine structures. These provide support for the assignment of the resonances of all methyl groups in a protein, which are otherwise often poorly resolved in homonuclear1H 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 γ- and δ-methyl groups of Ile. In addition to the methyl resonances, the γCH2 groups of Glu and Gln can be uniquely assigned because of the large coupling constant with the δ-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.


Protein structure NMR assignment Isotope labeling Biosynthetically directed fractional13C-labeling Stereospecific NMR assignment 



nuclear Overhauser effect

fractional13C labeling

biosynthetically directed fractional13C-labeling


total correlation spectroscopy


rotating frame Overhauser enhancement spectroscopy


two-dimensional13C−1H correlation spectroscopy


isotope isomer

P22 c2 repressor

c2 repressor of the salmonella phage P22 consisting of a polypeptide chain with 216 residues

P22 c2(1-76)

N-terminal domain of the P22 c2 repressor with residues 1–76


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Copyright information

© ESCOM Science Publishers B.V 1992

Authors and Affiliations

  • Thomas Szyperski
    • 1
  • Dario Neri
    • 1
  • Barbara Leiting
    • 1
  • Gottfried Otting
    • 1
  • Kurt Wüthrich
    • 1
  1. 1.Institut für Molekularbiologie und BiophysikEidgenössische Technische Hochschule-HönggerbergZürichSwitzerland

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