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

Uniformly (13)C- and (15)N-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 (13)C-labeled glucose by a factor of five using a fractional 20% (13)C- and 100% (15)N-labeling scheme could lower the total chemical costs, yet retaining sufficient structural information of uniformly [(13)C, (15)N]-labeled sample as a result of the improved sensitivity of NMR instruments. Moreover, fractional (13)C-labeling can facilitate reliable resonance assignments of sidechains because of the biosynthetic pathways of each amino-acid. Preparation of only one [20% (13)C, 100% (15)N]-labeled sample for small proteins (<15 kDa) could also eliminate redundant sample preparations of 100% (15)N-labeled and uniformly 100% [(13)C, (15)N]-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% (13)C, 100% (15)N]-labeled sample and compared with the crystal structures and the NMR structures derived from the 100% [(13)C, (15)N]-labeled sample. Our results suggest that one [20% (13)C, 100% (15)N]-labeled sample of small proteins could be routinely used as an alternative to conventional 100% [(13)C, (15)N]-labeling for backbone resonance assignments, NMR structure determination, (15)N-relaxation analysis, and ligand–protein interaction.