Alternative SAIL-Trp for robust aromatic signal assignment and determination of the [chi]^sub 2^ conformation by intra-residue NOEs

• Published in: Journal of biomolecular NMR 2011-12, Vol.51 (4), p.425
• Main Authors: Miyanoiri, Yohei, Takeda, Mitsuhiro, Jee, Jungoo, Ono, Akira M, Okuma, Kosuke, Terauchi, Tsutomu, Kainosho, Masatsune
• Format: Article
• Language: English
• Subjects: Amino acids; Residues
• ISSN: 0925-2738; 1573-5001
• DOI: 10.1007/s10858-011-9568-3

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-^sup 13^C,^sup 15^N]-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 ^sup 13^C-^sup 13^C and ^sup 13^C-^sup 1^H 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-^sup 2^H2; δ1,ε3,η2-^sup 13^C^sub 3^; ε1-^sup 15^N]-indole ring ([^sup 12^C^sub γ,^^sup 12^C^sub ε2^] SAIL-Trp), which provides a more robust way to correlate the ^sup 1^H^sub β^, ^sup 1^H^sub α^, and ^sup 1^H^sub N^ to the ^sup 1^H^sub δ1^ and ^sup 1^H^sub ε3^ through the intra-residue NOEs. The assignment of the ^sup 1^H^sub δ1^/^sup 13^C^sub δ1^ and ^sup 1^H^sub ε3^/^sup 13^C^sub ε3^ signals can thus be transferred to the ^sup 1^H^sub ε1^/^sup 15^N^sub ε1^ and ^sup 1^H^sub η2^/^sup 13^C^sub η2^ signals, as with the previous type of SAIL-Trp, which has an extra ^sup 13^C at the C^sub γ^ of the ring. By taking advantage of the stereospecific deuteration of one of the prochiral β-methylene protons, which was ^sup 1^H^sub β2^ in this experiment, one can determine the side-chain conformation of the Trp residue including the χ^sub 2^ angle, which is especially important for Trp residues, as they can adopt three preferred conformations. We demonstrated the usefulness of [^sup 12^C^sub γ^,^sup 12^C^sub ε2^] SAIL-Trp for the 12 kDa DNA binding domain of mouse c-Myb protein (Myb-R2R3), which contains six Trp residues.[PUBLICATION ABSTRACT]