Alternative SAIL-Trp for robust aromatic signal assignment and determination of the Ï?2 conformation by intra-residue NOEs

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Alternative SAIL-Trp for robust aromatic signal assignment and determination of the Ï?2 conformation by intra-residue NOEs


Abstract 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 [Uâ??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 13Câ??13C and 13Câ??1H spin coupling networks (Kainosho et al. in Nature 440:52â??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 ([12Cγ, 12Cε2] 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 Ï?2 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.

• Content Type Journal Article
• Category Article
• Pages 1-11
• DOI 10.1007/s10858-011-9568-3
• Authors
  • Yohei Miyanoiri, Graduate School of Science, Structural Biology Research Center, Nagoya University, Furo-cho, Chikusa-ku, Nagoya, 464-8602 Japan
  • Mitsuhiro Takeda, Graduate School of Science, Structural Biology Research Center, Nagoya University, Furo-cho, Chikusa-ku, Nagoya, 464-8602 Japan
  • JunGoo Jee, Center for Priority Areas, Tokyo Metropolitan University, 1-1 Minami-ohsawa, Hachioji, 192-0397 Japan
  • Akira M. Ono, Center for Priority Areas, Tokyo Metropolitan University, 1-1 Minami-ohsawa, Hachioji, 192-0397 Japan
  • Kosuke Okuma, Center for Priority Areas, Tokyo Metropolitan University, 1-1 Minami-ohsawa, Hachioji, 192-0397 Japan
  • Tsutomu Terauchi, Center for Priority Areas, Tokyo Metropolitan University, 1-1 Minami-ohsawa, Hachioji, 192-0397 Japan
  • Masatsune Kainosho, Graduate School of Science, Structural Biology Research Center, Nagoya University, Furo-cho, Chikusa-ku, Nagoya, 464-8602 Japan

• • Journal Journal of Biomolecular NMR
  • Online ISSN 1573-5001
  • Print ISSN 0925-2738

Source: Journal of Biomolecular NMR