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

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.