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Biomolecular NMR

Methyl and Amino Acid Type Labeling

Selective Isotope-Labeling Methods for Protein Structural Studies

  • Selective Isotope-Labeling Methods for Protein Structural Studies
  • Production of U-[2H], Thy-γ2[13CH3] Labeled Proteins for methyl-TROSY NMR (Application Note 39)
  • Isotope Labeling of Alanine Methyl Groups on a Deuterated Background for NMR Studies of High-Molecular-Weight Proteins (Application Note 25)

Selective labeling of a protein is used to simplify crowded spectra in order to aid in spectral interpretation or to provide specific probes for dynamic and structural studies. There are a few procedures available for selective labeling. The most popular method is Amino Acid Type Selective labeling which entails the addition of one or more labeled amino acids (~50->250 mg / L) to unlabeled growth media  prior to protein induction. Ideally, the cells will generally utilize the supplemented amino acids for protein synthesis prior to undergoing the de novo synthesis of the target amino acids. The only labeled residues in the expressed protein will thus be the amino acid types supplemented to the medium. A different method, Selective Unlabeling or reverse labeling utilizes the addition of one or more unlabeled amino acids to a labeled minimal medium.  Therefore, the expressed protein will be fully labeled except for the residue types corresponding to the unlabeled amino acids that were supplemented to the minimal medium. 

Methyl Labeling refers to methods which introduce either 13CH3 or 13CHD2 groups into otherwise a uniform deuterated protein. This can be achieved in both prokaryotic and eukaryotic expression systems by adding the amino acids(s) (in sufficient amounts) directly to the cell culture medium prior to protein induction. For e. coli cultures, selective protonation on methyl groups of ILV can be achieved by adding alpha keto acid precursors to the cell culture medium 1 hr prior to induction. Alpha ketoisobutyrate becomes converted to isoleucine and alpha ketoisovaleric acid becomes converted to Leucine and Valine.

Kevin Millis, PhD

Kevin Millis, PhD

Senior Scientist, Application Development Manager

Kevin Millis, PhD, is the Senior Scientist and Market Development Manager for all NMR and Mass Spectrometry product lines. Kevin is responsible for Technical Services both internally and externally for all CIL customers as well as being responsible for the application and market development for the CIL products.