Selective methyl group protonation of perdeuterated proteins

Michael K. Rosen, Kevin H. Gardner, Randall C. Willis, Wendy E. Parris, Tony Pawson, Lewis E. Kay

Research output: Contribution to journalArticle

241 Citations (Scopus)

Abstract

Deuteration of aliphatic sites in proteins has shown great potential to increase the range of molecules amenable to study by NMR spectroscopy. One problem inherent in high-level deuterium incorporation is the loss of 1H-1H distance information obtainable from NOESY spectra of the labeled proteins. In the limit of perdeuteration, the available NH-NH NOEs are insufficient in many cases to define the three-dimensional structure of a folded protein. We describe here a method of producing proteins that retains all the advantages of perdeuteration, while enabling observation of many NOEs absent from spectra of fully deuterated samples. Overexpression of proteins in bacteria grown in 2H2O medium containing protonated pyruvate as the sole carbon source results in complete deuteration at C(α) and > 80% deuteration at C(β) positions of nearly all amino acids. In contrast, the methyl groups of Ala, Val, Leu and Ile (γ2 only) remain highly protonated. This labeling pattern can be readily understood from analysis of bacterial pathways for pyruvate utilization and amino acid biosynthesis. As Ala, Val, Leu and Ile are among the most highly represented residue types in protein hydrophobic cores and at protein-protein interfaces, selectively methyl-protonated samples will be useful in many areas of structural analysis of larger molecules and molecular complexes by NMR.

Original languageEnglish (US)
Pages (from-to)627-636
Number of pages10
JournalJournal of Molecular Biology
Volume263
Issue number5
DOIs
StatePublished - Nov 15 1996

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Proteins
Pyruvic Acid
Amino Acids
Deuterium
Magnetic Resonance Spectroscopy
Carbon
Observation
Bacteria

Keywords

  • Deuteration
  • NMR
  • Protein structure determination

ASJC Scopus subject areas

  • Virology

Cite this

Selective methyl group protonation of perdeuterated proteins. / Rosen, Michael K.; Gardner, Kevin H.; Willis, Randall C.; Parris, Wendy E.; Pawson, Tony; Kay, Lewis E.

In: Journal of Molecular Biology, Vol. 263, No. 5, 15.11.1996, p. 627-636.

Research output: Contribution to journalArticle

Rosen, Michael K. ; Gardner, Kevin H. ; Willis, Randall C. ; Parris, Wendy E. ; Pawson, Tony ; Kay, Lewis E. / Selective methyl group protonation of perdeuterated proteins. In: Journal of Molecular Biology. 1996 ; Vol. 263, No. 5. pp. 627-636.
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