ArticleJournal of biomolecular NMR2009
An efficient protocol for the complete incorporation of methyl-protonated alanine in perdeuterated protein.
Article in Journal of biomolecular NMR, 2009. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 60 papers.
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60 citing papers in PubMed, 150 citations in OpenAlex.
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- Specific isotopic labelling and reverse labelling for protein NMR spectroscopy: using metabolic precursors in sample preparation.Biochemical Society transactions · 2022Review
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- Optimized precursor to simplify assignment transfer between backbone resonances and stereospecifically labelled valine and leucine methyl groups: application to human Hsp90 N-terminal domain.Journal of biomolecular NMR · 2021Article
- Sparse isotope labeling for nuclear magnetic resonance (NMR) of glycoproteins using 13C-glucose.Glycobiology · 2021Article
- Spectral editing of intra- and inter-chain methyl-methyl NOEs in protein complexes.Journal of biomolecular NMR · 2020Article
- Structural mechanism underlying G protein family-specific regulation of G protein-gated inwardly rectifying potassium channel.Nature communications · 2019Article
- Measurement of residual dipolar couplings in methyl groups via carbon detection.Journal of biomolecular NMR · 2019Article
- An Evaluation of the Potential of NMR Spectroscopy and Computational Modelling Methods to Inform Biopharmaceutical Formulations.Pharmaceutics · 2018Review
- Assigning methyl resonances for protein solution-state NMR studies.Methods (San Diego, Calif.) · 2018Article
- Methyl-selective isotope labeling using α-ketoisovalerate for the yeast Pichia pastoris recombinant protein expression system.Journal of biomolecular NMR · 2018Article
- Selective labeling and unlabeling strategies in protein solid-state NMR spectroscopy.Journal of biomolecular NMR · 2018Review
- Deuteration and selective labeling of alanine methyl groups of βJournal of biomolecular NMR · 2018Article
- Late metabolic precursors for selective aromatic residue labeling.Journal of biomolecular NMR · 2018Review
- Structural basis for the ethanol action on G-protein-activated inwardly rectifying potassium channel 1 revealed by NMR spectroscopy.Proceedings of the National Academy of Sciences of the United States of America · 2018Article
- Complete assignment of Ala, Ile, Leu, Met and Val methyl groups of human blood group A and B glycosyltransferases using lanthanide-induced pseudocontact shifts and methyl-methyl NOESY.Journal of biomolecular NMR · 2018Article
- Current NMR Techniques for Structure-Based Drug Discovery.Molecules (Basel, Switzerland) · 2018Review
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Authors and funding
5 authors at 4 institutions in 1 country.
Funding
No grant is acknowledged in the PubMed record.
Abstract
A strategy for the introduction of ((1)H,(13)C-methyl)-alanine into perdeuterated proteins is described. Specific protonation of alanine methyl groups to a level of 95% can be achieved by overexpressing proteins in M9/D(2)O based bacterial growth medium supplemented with 800 mg/l of 2-[(2)H], 3-[(13)C] L: -alanine. However, though simple, this approach results in undesired, non-specific background labeling due to isotope scrambling via different amino acid metabolic pathways. Following a careful analysis of known metabolic pathways we found that co-addition of perdeuterated forms of alpha-ketoisovalerate-d(7), succinate-d(4) and L: -isoleucine-d(10) with labeled L: -alanine, reduces undesired background labeling to <1%. When combined with recently developed methyl TROSY experiments, this methyl-specific labeling protocol permits the acquisition of excellent quality correlation spectra of alanine methyl groups in high molecular weight proteins. Our cost effective strategy offers a significant enhancement in the level of incorporation of methyl-labeled alanine in overexpressed proteins over previously reported methods.
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Read under generation 80e0d062 · epoch 390. Bibliography from PubMed, PubMed Central and OpenAlex; grants from NIH RePORTER; trial links from ClinicalTrials.gov; estimates, votes and beliefs from the OpenQuestion graph.