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.
Structural Biology Research Center, Graduate School of Science, Nagoya University, Furo-cho, Chikusa-ku, Nagoya, 464-8602, Japan.
Yohei
Y
Miyanoiri
Mitsuhiro
M
Takeda
Kosuke
K
Okuma
Akira M
AM
Ono
Tsutomu
T
Terauchi
Masatsune
M
Kainosho
D000596:Amino Acids / Q000737:chemistry; D002247:Carbon Isotopes; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D045782:Hemiterpenes; D007553:Isotope Labeling; D007651:Keto Acids / Q000737:chemistry; D007930:Leucine / Q000737:chemistry* / Q000378:metabolism; D019906:Nuclear Magnetic Resonance, Biomolecular / Q000379:methods; D014633:Valine / Q000737:chemistry* / Q000378:metabolism
D000596:Amino Acids; D002247:Carbon Isotopes; D029968:Escherichia coli Proteins; D045782:Hemiterpenes; D007651:Keto Acids; C001505:alpha-ketoisovalerate; D007930:Leucine; D014633:Valine