NMR Structure Determinations of Small Proteins Using only One Fractionally 20% 13C- and Uniformly 100% 15N-Labeled Sample

Molecules. 2021 Feb 1;26(3):747. doi: 10.3390/molecules26030747.


Uniformly 13C- and 15N-labeled samples ensure fast and reliable nuclear magnetic resonance (NMR) assignments of proteins and are commonly used for structure elucidation by NMR. However, the preparation of uniformly labeled samples is a labor-intensive and expensive step. Reducing the portion of 13C-labeled glucose by a factor of five using a fractional 20% 13C- and 100% 15N-labeling scheme could lower the total chemical costs, yet retaining sufficient structural information of uniformly [13C, 15N]-labeled sample as a result of the improved sensitivity of NMR instruments. Moreover, fractional 13C-labeling can facilitate reliable resonance assignments of sidechains because of the biosynthetic pathways of each amino-acid. Preparation of only one [20% 13C, 100% 15N]-labeled sample for small proteins (<15 kDa) could also eliminate redundant sample preparations of 100% 15N-labeled and uniformly 100% [13C, 15N]-labeled samples of proteins. We determined the NMR structures of a small alpha-helical protein, the C domain of IgG-binding protein A from Staphylococcus aureus (SpaC), and a small beta-sheet protein, CBM64 module using [20% 13C, 100% 15N]-labeled sample and compared with the crystal structures and the NMR structures derived from the 100% [13C, 15N]-labeled sample. Our results suggest that one [20% 13C, 100% 15N]-labeled sample of small proteins could be routinely used as an alternative to conventional 100% [13C, 15N]-labeling for backbone resonance assignments, NMR structure determination, 15N-relaxation analysis, and ligand-protein interaction.

Keywords: CBM64; NMR; NMR assignment; NMR structure determination; SpaC; biosynthetic pathways; biosynthetically directed fractional 13C-labeling; fractional 13C-labeling; protein structure.

MeSH terms

  • Carbon Isotopes / analysis*
  • Cellulase / chemistry*
  • Nitrogen Isotopes / analysis*
  • Nuclear Magnetic Resonance, Biomolecular / methods*
  • Protein Structure, Secondary
  • Staphylococcal Protein A / chemistry*
  • Tetrahymena thermophila / enzymology


  • Carbon Isotopes
  • Nitrogen Isotopes
  • Staphylococcal Protein A
  • Cellulase