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Comprehensive and cost-effective NMR spectroscopy of methyl groups in large proteins

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  • Renee Otten, University of Groningen, Groningen Biomolecular Sciences and Biotechnology Institute, Netherlands
  • Byron Chu, University of Calgary, Canada
  • Karla D. Krewulak, University of Calgary, Canada
  • Hans J. Vogel, University of Calgary, Canada
  • F. A A Mulder

An NMR approach is described which yields the methyl resonance assignments of alanine, threonine, valine, leucine, and isoleucine residues in proteins with high sensitivity and excellent resolution. The method relies on protein samples produced by bacterial expression using [1H,13C]-D-glucose and ∼100% D2O, which is cost-effective and ensures the isotopic enrichment of all possible methyl groups. Magnetization transfer throughout the methyl-containing side chains is possible with this labeling scheme due to the high level of deuteration along the amino acid side chain, coupled with the selection of the favorable CHD2 methyl isotopomer for detection. In an application to the 34 kDa periplasmic binding protein FepB 164 out of 195 methyl groups (85%) were assigned sequence-specifically and stereospecifically. This percentage increases to 91 % when taking into account that not all backbone assignments are available for this system. The remaining unasslgned methyl groups belong to six leucine residues, caused by low cross-peak intensities, and four alanine residues due to degeneracy of the 13C α/13Oβ frequencies. Our results demonstrate that NMR spectroscopic investigations of protein structure, dynamics, and interactions can be extended to include all methyl-containing amino acids also for larger proteins.

Original languageEnglish
JournalJournal of the American Chemical Society
Volume132
Issue9
Pages (from-to)2952-2960
Number of pages9
ISSN0002-7863
DOIs
Publication statusPublished - 10 Mar 2010
Externally publishedYes

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