Methyl-detected 'out-and-back' NMR experiments for simultaneous assignments of Alabeta and Ilegamma2 methyl groups in large proteins

J Biomol NMR. 2009 Apr;43(4):229-38. doi: 10.1007/s10858-009-9305-3. Epub 2009 Mar 10.

Abstract

A set of sensitive methyl-detected 'out-and-back' NMR experiments for simultaneous assignments of Alabeta and Ilegamma2 methyl positions in large proteins is described. The developed methodology is applied to an 82-kDa enzyme Malate Synthase G. Complete alanine beta and isoleucine gamma2 1H-13C methyl chemical shift assignments could be obtained from the set of new methyl-detected 'out-and-back' 3D experiments. The described methodology for methyl assignments should be applicable to protein molecules within approximately 100-kDa molecular weight range irrespective of the labeling strategy chosen to produce selectively protonated Alabeta and Ilegamma2 13CH3 sites on a deuterated background.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Alanine / chemistry*
  • Alanine / metabolism
  • Algorithms
  • Carbon Isotopes / chemistry*
  • Carbon Isotopes / metabolism
  • Computer Simulation
  • Escherichia coli / genetics
  • Isoleucine / chemistry*
  • Isoleucine / metabolism
  • Malate Synthase / chemistry
  • Methylation
  • Nuclear Magnetic Resonance, Biomolecular / methods*
  • Proteins / chemistry*
  • Pyruvic Acid / metabolism
  • Valine / chemistry
  • Valine / metabolism

Substances

  • Carbon Isotopes
  • Proteins
  • Isoleucine
  • Pyruvic Acid
  • Malate Synthase
  • Valine
  • Alanine