An efficient protocol for the complete incorporation of methyl-protonated alanine in perdeuterated protein

J Biomol NMR. 2009 Feb;43(2):111-9. doi: 10.1007/s10858-008-9294-7. Epub 2008 Dec 30.

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Alanine / chemistry
  • Alanine / metabolism
  • Carbon Isotopes / chemistry
  • Carbon Isotopes / metabolism
  • Culture Media
  • Deuterium / chemistry
  • Deuterium / metabolism
  • Escherichia coli / genetics
  • Hemiterpenes
  • Humans
  • Isoleucine / chemistry
  • Isoleucine / metabolism
  • Isotope Labeling / methods*
  • Keto Acids / chemistry
  • Keto Acids / metabolism
  • Malate Synthase / chemistry
  • Malate Synthase / metabolism
  • Metabolic Networks and Pathways
  • Nuclear Magnetic Resonance, Biomolecular*
  • Proteins / chemistry*
  • Proteins / genetics
  • Proteins / metabolism*
  • Succinic Acid / chemistry
  • Succinic Acid / metabolism
  • Ubiquitin / chemistry
  • Ubiquitin / genetics
  • Ubiquitin / metabolism

Substances

  • Carbon Isotopes
  • Culture Media
  • Hemiterpenes
  • Keto Acids
  • Proteins
  • Ubiquitin
  • Isoleucine
  • alpha-ketoisovalerate
  • Succinic Acid
  • Deuterium
  • Malate Synthase
  • Alanine