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

• Published in: Journal of biomolecular NMR 2013-11, Vol.57 (3), p.237-249
• Main Authors: Miyanoiri, Yohei, Takeda, Mitsuhiro, Okuma, Kosuke, Ono, Akira M., Terauchi, Tsutomu, Kainosho, Masatsune
• Format: Article
• Language: English
• Subjects: Amino acids; Amino Acids - chemistry; Biochemistry; Biological and Medical Physics; Biophysics; Carbon Isotopes; E coli; Escherichia coli - genetics; Escherichia coli - metabolism; Escherichia coli Proteins - chemistry; Escherichia coli Proteins - genetics; Escherichia coli Proteins - metabolism; Gene Expression; Isotope Labeling; Keto Acids - chemistry; Leucine - chemistry; Leucine - metabolism; Nuclear Magnetic Resonance, Biomolecular - methods; Physics; Physics and Astronomy; Residues; Spectroscopy/Spectrometry; Valine - chemistry; Valine - metabolism
• ISSN: 0925-2738; 1573-5001
• DOI: 10.1007/s10858-013-9784-0

The 1 H– 13 C HMQC signals of the 13 CH 3 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 δ 1 -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 CH 3 -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.