ArticleJournal of biomolecular NMR2013
Differential isotope-labeling for Leu and Val residues in a protein by E. coli cellular expression using stereo-specifically methyl labeled amino acids.
Article in Journal of biomolecular NMR, 2013. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 18 papers.
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18 citing papers in PubMed, 41 citations in OpenAlex.
- Extended local deuteration of leucine enhances methyl NMR of large proteins expressed in eukaryotic cells.Chemical communications (Cambridge, England) · 2026Article
- Human cells for human proteins: Isotope labeling in mammalian cells in suspension for functional NMR studies.Protein science : a publication of the Protein Society · 2026Article
- Subunit-specific isotope labelling of heteromeric complexes using cell-free protein expression: application to the 760 kDa ClpXP molecular machine.RSC chemical biology · 2026Article
- Side-chain-selective deuterium labeling by a simple bio-expression method enhances resolution and simplifies spectra inJournal of biomolecular NMR · 2025Article
- Site-Specific Isotope Labeling of FliG for Studying Structural Dynamics Using Nuclear Magnetic Resonance Spectroscopy.Methods in molecular biology (Clifton, N.J.) · 2023Article
- Spectral editing of intra- and inter-chain methyl-methyl NOEs in protein complexes.Journal of biomolecular NMR · 2020Article
- Characterization of Internal Protein Dynamics and Conformational Entropy by NMR Relaxation.Methods in enzymology · 2019Article
- Deuteration and selective labeling of alanine methyl groups of βJournal of biomolecular NMR · 2018Article
- Current NMR Techniques for Structure-Based Drug Discovery.Molecules (Basel, Switzerland) · 2018Review
- Pressure dependence of side chainJournal of biomolecular NMR · 2017Article
- Methyl-Specific Isotope Labeling Strategies for NMR Studies of Membrane Proteins.Methods in molecular biology (Clifton, N.J.) · 2017Article
- Modern Technologies of Solution Nuclear Magnetic Resonance Spectroscopy for Three-dimensional Structure Determination of Proteins Open Avenues for Life Scientists.Computational and structural biotechnology journal · 2017Review
- Highly efficient residue-selective labeling with isotope-labeled Ile, Leu, and Val using a new auxotrophic E. coli strain.Journal of biomolecular NMR · 2016Article
- Exploiting E. coli auxotrophs for leucine, valine, and threonine specific methyl labeling of large proteins for NMR applications.Journal of biomolecular NMR · 2016Article
- Article
- An optimized NIntrinsically disordered proteins · 2015Article
- Scrambling free combinatorial labeling of alanine-β, isoleucine-δ1, leucine-proS and valine-proS methyl groups for the detection of long range NOEs.Journal of biomolecular NMR · 2015Article
- Specific labeling and assignment strategies of valine methyl groups for NMR studies of high molecular weight proteins.Journal of biomolecular NMR · 2013Article
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6 authors at 2 institutions in 1 country.
Funding
No grant is acknowledged in the PubMed record.
Abstract
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.
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