Enhancing solution structural analysis of large molecular proteins through optimal stereo array isotope labeling of aromatic amino acids.

The observation of side-chain peaks of aromatic amino acids is the prerequisite for a high-resolution three-dimensional structure determination of proteins by NMR. However, it becomes difficult with increasing molecular size due to an increased transverse relaxation and the control of the relaxation pathway is needed to achieve the observation. We demonstrated that even for the large molecular size of 82 kDa Malate synthase G (MSG), the aromatic 13C-1H (CH) peaks of Tryptophan (Trp) and Phenylalanine (Phe) residues can be observed with high quality using a systematic stable isotope labeling scheme, Stereo-Array Isotope Labeling (SAIL) method. However, the sequence specific assignments of these peaks relied on the use of amino acid substitutions, employing an inefficient method that required many isotopes labeled samples. In this study, we developed novel SAIL amino acids that allow for the observation of the aromatic ring δ,ζ and the aliphatic ß position peak of Phe residues. The application of TROSY-based experiment to the isolated CH moieties resulted in the successful observation of discernible and resolved CH peaks in Phe residues in MSG. In MSG, the sequence-specific assignments of the backbone and Cß positions have already been confirmed. Therefore, using this labeling method, the δ and ß position peaks of Phe residues can be clearly assigned in a sequence-specific and stereospecific manner through experiments based on intra-residue NOE. Furthermore, the NOESY experiment also allows for the acquisition of information pertaining to the conformation of Phe residues, such as the χ1 dihedral angle, providing valuable insights for the determination of accurate protein structures and in dynamic analysis. This new SAIL amino acids open an avenue to achieve a variety of NMR analysis of large molecular proteins, including a high-resolution structure determination and dynamics and interaction analysis.

Differential isotope-labeling for Leu and Val residues in a protein by E. coli cellular expression using stereo-specifically methyl labeled amino acids.

The (1)H-(13)C HMQC signals of the (13)CH3 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 (13)CH3-labeled [U-(2)H;(15)N]-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.

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(ß), (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 ß-methylene protons, which was (1)H(ß2) 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.

Stereoselective synthesis of triply isotope-labeled Ser, Cys, and Ala: amino acids for stereoarray isotope labeling technology.

Efficient access to highly enantioselective isotope-labeled serine, cysteine, and alanine for stereoarray isotope labeling (SAIL) is described.