A robust and cost-effective method for the production of Val, Leu, Ile (δ1) methyl-protonated 15N-, 13C-, 2H-labeled proteins

Natalie K. Goto, Kevin H. Gardner, Geoffrey A. Mueller, Randall C. Willis, Lewis E. Kay

Research output: Contribution to journalArticle

404 Citations (Scopus)

Abstract

A selective protonation strategy is described that uses [3-2H] 13C α-ketoisovalerate to introduce (1H-δ methyl)-leucine and (1H-γ methyl)- valine into 15N-, 13C-, 2H-labeled proteins. A minimum level of 90% incorporation of label into both leucine and valine methyl groups is obtained by inclusion of ≃100 mg/L α-ketoisovalerate in the bacterial growth medium. Addition of [3,3-2H2] α-ketobutyrate to the expression media (D2O solvent) results in the production of proteins with (1H-δ1 methyl)- isoleucine (>90% incorporation), 1H-13C HSQC correlation spectroscopy establishes that CH2D and CHD2 isotopomers are not produced with this method. This approach offers enhanced labeling of Leu methyl groups over previous methods that utilize Val as the labeling agent and is more cost effective.

Original languageEnglish (US)
Pages (from-to)369-374
Number of pages6
JournalJournal of Biomolecular NMR
Volume13
Issue number4
DOIs
StatePublished - 1999

Fingerprint

Valine
Labeling
Costs and Cost Analysis
Isoleucine
Protonation
Leucine
Labels
Costs
Spectrum Analysis
Proteins
Spectroscopy
Growth
leucine methyl ester
acetoacetic acid

Keywords

  • α-ketobutyrate
  • α-ketoisovalerate
  • Deuteration
  • Methyl protonation

ASJC Scopus subject areas

  • Spectroscopy
  • Biochemistry, Genetics and Molecular Biology(all)
  • Biochemistry

Cite this

A robust and cost-effective method for the production of Val, Leu, Ile (δ1) methyl-protonated 15N-, 13C-, 2H-labeled proteins. / Goto, Natalie K.; Gardner, Kevin H.; Mueller, Geoffrey A.; Willis, Randall C.; Kay, Lewis E.

In: Journal of Biomolecular NMR, Vol. 13, No. 4, 1999, p. 369-374.

Research output: Contribution to journalArticle

Goto, Natalie K. ; Gardner, Kevin H. ; Mueller, Geoffrey A. ; Willis, Randall C. ; Kay, Lewis E. / A robust and cost-effective method for the production of Val, Leu, Ile (δ1) methyl-protonated 15N-, 13C-, 2H-labeled proteins. In: Journal of Biomolecular NMR. 1999 ; Vol. 13, No. 4. pp. 369-374.
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AU - Willis, Randall C.

AU - Kay, Lewis E.

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