[en] The extensive collection of NOE constraint data involving the aromatic ring signals is essential for accurate protein structure determination, although it is often hampered in practice by the pervasive signal overlapping and tight spin couplings for aromatic rings. We have prepared various types of stereo-array isotope labeled phenylalanines (ε- and ζ-SAIL Phe) and tyrosine (ε-SAIL Tyr) to overcome these problems (Torizawa et al. 2005), and proven that these SAIL amino acids provide dramatic spectral simplification and sensitivity enhancement for the aromatic ring NMR signals. In addition to these SAIL aromatic amino acids, we recently synthesized δ-SAIL Phe and δ-SAIL Tyr, which allow us to observe and assign δ-¹³C/¹H signals very efficiently. Each of the various types of SAIL Phe and SAIL Tyr yields well-resolved resonances for the δ-, ε- or ζ-¹³C/¹H signals, respectively, which can readily be assigned by simple and robust pulse sequences. Since the δ-, ε-, and ζ-proton signals of Phe/Tyr residues give rise to complementary NOE constraints, the concomitant use of various types of SAIL-Phe and SAIL-Tyr would generate more accurate protein structures, as compared to those obtained by using conventional uniformly ¹³C, ¹⁵N-double labeled proteins. We illustrated this with the case of an 18.2 kDa protein, Escherichia coli peptidyl-prolyl cis-trans isomerase b (EPPIb), and concluded that the combined use of ζ-SAIL Phe and ε-SAIL Tyr would be practically the best choice for protein structural determinations.

[en] The ¹H–¹³C HMQC signals of the ¹³CH₃ moieties of Ile, Leu, and Val residues, in an otherwise deuterated background, exhibit narrow line-widths, and thus are useful for investigating the structures and dynamics of larger proteins. This approach, named methyl TROSY, is economical as compared to laborious methods using chemically synthesized site- and stereo-specifically isotope-labeled amino acids, such as stereo-array isotope labeling amino acids, since moderately priced, commercially available isotope-labeled α-keto acid precursors can be used to prepare the necessary protein samples. The Ile δ₁-methyls can be selectively labeled, using isotope-labeled α-ketobutyrates as precursors. However, it is still difficult to prepare a residue-selectively Leu and Val labeled protein, since these residues share a common biosynthetic intermediate, α-ketoisovalerate. Another hindering drawback in using the α-ketoisovalerate precursor is the lack of stereo-selectivity for Leu and Val methyls. Here we present a differential labeling method for Leu and Val residues, using four kinds of stereo-specifically ¹³CH₃-labeled [U–²H;¹⁵N]-leucine and -valine, which can be efficiently incorporated into a protein using Escherichia coli cellular expression. The method allows the differential labeling of Leu and Val residues with any combination of stereo-specifically isotope-labeled prochiral methyls. Since relatively small amounts of labeled leucine and valine are required to prepare the NMR samples; i.e., 2 and 10 mg/100 mL of culture for leucine and valine, respectively, with sufficient isotope incorporation efficiency, this approach will be a good alternative to the precursor methods. The feasibility of the method is demonstrated for 82 kDa malate synthase G

[en] 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,¹²C_ε2] SAIL-Trp), which provides a more robust way to correlate the ¹H_β, ¹H_α, and ¹H_N to the ¹H_δ1 and ¹H_ε3 through the intra-residue NOEs. The assignment of the ¹H_δ1/¹³C_δ1 and ¹H_ε3/¹³C_ε3 signals can thus be transferred to the ¹H_ε1/¹⁵N_ε1 and ¹H_η2/¹³C_η2 signals, as with the previous type of SAIL-Trp, which has an extra ¹³C at the C_γ of the ring. By taking advantage of the stereospecific deuteration of one of the prochiral β-methylene protons, which was ¹H_β2 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 [¹²C_γ,¹²C_ε2] SAIL-Trp for the 12 kDa DNA binding domain of mouse c-Myb protein (Myb-R2R3), which contains six Trp residues.