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

作者：Miyanoiri Yohei; Takeda Mitsuhiro; Okuma Kosuke; Ono Akira M; Terauchi Tsutomu; Kainosho Masatsune^*

来源：Journal of Biomolecular NMR, 2013, 57(3): 237-249.

DOI：10.1007/s10858-013-9784-0

• Leu/Val differential labeling
• Stereo-specifically methyl labeled Leu/Val
• Methyl-methyl NOEs
• Large protein

The H-1-C-13 HMQC signals of the (CH3)-C-13 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 alpha-keto acid precursors can be used to prepare the necessary protein samples. The Ile delta(1)-methyls can be selectively labeled, using isotope-labeled alpha-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, alpha-ketoisovalerate. Another hindering drawback in using the alpha-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 (CH3)-C-13- labeled [U-H-2; N-15]-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.

• 出版日期2013-11