Journal of Biomolecular NMR

, Volume 51, Issue 4, pp 425–435 | Cite as

Alternative SAIL-Trp for robust aromatic signal assignment and determination of the χ2 conformation by intra-residue NOEs

  • Yohei Miyanoiri
  • Mitsuhiro Takeda
  • JunGoo Jee
  • Akira M. Ono
  • Kosuke Okuma
  • Tsutomu Terauchi
  • Masatsune KainoshoEmail author


Tryptophan (Trp) residues are frequently found in the hydrophobic cores of proteins, and therefore, their side-chain conformations, especially the precise locations of the bulky indole rings, are critical for determining structures by NMR. However, when analyzing [U–13C,15N]-proteins, the observation and assignment of the ring signals are often hampered by excessive overlaps and tight spin couplings. These difficulties have been greatly alleviated by using stereo-array isotope labeled (SAIL) proteins, which are composed of isotope-labeled amino acids optimized for unambiguous side-chain NMR assignment, exclusively through the 13C–13C and 13C–1H spin coupling networks (Kainosho et al. in Nature 440:52–57, 2006). In this paper, we propose an alternative type of SAIL-Trp with the [ζ2,ζ3-2H2; δ1,ε3,η2-13C3; ε1-15N]-indole ring ([12C γ, 12 Cε2] SAIL-Trp), which provides a more robust way to correlate the 1Hβ, 1Hα, and 1HN to the 1Hδ1 and 1Hε3 through the intra-residue NOEs. The assignment of the 1Hδ1/13Cδ1 and 1Hε3/13Cε3 signals can thus be transferred to the 1Hε1/15Nε1 and 1Hη2/13Cη2 signals, as with the previous type of SAIL-Trp, which has an extra 13C at the Cγ of the ring. By taking advantage of the stereospecific deuteration of one of the prochiral β-methylene protons, which was 1Hβ2 in this experiment, one can determine the side-chain conformation of the Trp residue including the χ2 angle, which is especially important for Trp residues, as they can adopt three preferred conformations. We demonstrated the usefulness of [12Cγ,12Cε2] SAIL-Trp for the 12 kDa DNA binding domain of mouse c-Myb protein (Myb-R2R3), which contains six Trp residues.


SAIL-Trp Aromatic ring CH assignment Intra-residue NOE χ2 conformation 



We thank Prof. Yoshifumi Nishimura and Dr. Aritaka Nagadoi, of Yokohama City University, for providing the Myb-R2R3 gene, and Dr. Frank Löhr, Institute of Biophysical Chemistry and Center of Biological Magnetic Resonance, Goethe-University, for his kind help in providing the NMR sequence of the 1H–13C TROSY experiments. This work was supported in part by the Targeted Protein Research Program (MEXT) to M.K., a Grant-in-Aid for Young Scientists (B) (23770109) to M.T. and a Grant-in-Aid for Young Scientists (B) (23770111) to Y.M.


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Copyright information

© Springer Science+Business Media B.V. 2011

Authors and Affiliations

  • Yohei Miyanoiri
    • 1
  • Mitsuhiro Takeda
    • 1
  • JunGoo Jee
    • 2
  • Akira M. Ono
    • 2
    • 3
  • Kosuke Okuma
    • 2
    • 3
  • Tsutomu Terauchi
    • 2
    • 3
  • Masatsune Kainosho
    • 1
    • 2
    Email author
  1. 1.Graduate School of Science, Structural Biology Research CenterNagoya UniversityNagoyaJapan
  2. 2.Center for Priority AreasTokyo Metropolitan UniversityHachiojiJapan
  3. 3.SAIL Technologies Co., Inc.YokohamaJapan

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