ArticleJournal of biomolecular NMR2016
Exploiting E. coli auxotrophs for leucine, valine, and threonine specific methyl labeling of large proteins for NMR applications.
Article in Journal of biomolecular NMR, 2016. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 16 papers.
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Who cites it
16 citing papers in PubMed, 30 citations in OpenAlex.
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- Imatinib can act as an Allosteric Activator of Abl Kinase.Journal of molecular biology · 2022Article
- Progress toward automated methyl assignments for methyl-TROSY applications.Structure (London, England : 1993) · 2022Article
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- Large Multidomain Protein NMR: HIV-1 Reverse Transcriptase Precursor in Solution.International journal of molecular sciences · 2020Review
- Conformational states dynamically populated by a kinase determine its function.Science (New York, N.Y.) · 2020Article
- Bioinformatic Mapping of Radical S-Adenosylmethionine-Dependent Ribosomally Synthesized and Post-Translationally Modified Peptides Identifies New Cα, Cβ, and Cγ-Linked Thioether-Containing Peptides.Journal of the American Chemical Society · 2019Article
- Characterization of Internal Protein Dynamics and Conformational Entropy by NMR Relaxation.Methods in enzymology · 2019Article
- Measuring Entropy in Molecular Recognition by Proteins.Annual review of biophysics · 2018Review
- Structures of chaperone-substrate complexes docked onto the export gate in a type III secretion system.Nature communications · 2018Article
- Automatic methyl assignment in large proteins by the MAGIC algorithm.Journal of biomolecular NMR · 2017Article
- Recognition and targeting mechanisms by chaperones in flagellum assembly and operation.Proceedings of the National Academy of Sciences of the United States of America · 2016Article
Corrections and comments
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Authors and funding
7 authors at 3 institutions in 2 countries.
Funding
Abstract
A simple and cost effective method to independently and stereo-specifically incorporate [(1)H,(13)C]-methyls in Leu and Val in proteins is presented. Recombinant proteins for NMR studies are produced using a tailored set of auxotrophic E. coli strains. NMR active isotopes are routed to either Leu or Val methyl groups from the commercially available and scrambling-free precursors α-ketoisovalerate and acetolactate. The engineered strains produce deuterated proteins with stereospecific [(1)H,(13)C]-methyl labeling separately at Leu or Val amino acids. This is the first method that achieves Leu-specific stereospecific [(1)H,(13)C]-methyl labeling of proteins and scramble-free Val-specific labeling. Use of auxotrophs drastically decreases the amount of labeled precursor required for expression without impacting the yield. The concept is extended to Thr methyl labeling by means of a Thr-specific auxotroph that provides enhanced efficiency for use with the costly L-[4-(13)C,2,3-(2)H2,(15)N]-Thr reagent. The Thr-specific strain allows for the production of Thr-[(13)CH3](γ2) labeled protein with an optimal isotope incorporation using up to 50 % less labeled Thr than the traditional E. coli strain without the need for (2)H-glycine to prevent scrambling.
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Registered trials
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