Alternative SAIL-Trp for robust aromatic signal assignment and determination of the χ(2) conformation by intra-residue NOEs.

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