Abstract
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.
Highlights
nuclear magnetic resonance (NMR) spectroscopy has been routinely used for elucidating three-dimensional structures of proteins in solution [1,2,3]
We demonstrated that one sample using [20% 13 C, 100% 15 N]-labeling is sufficient for various NMR analysis of both small alpha-helical and beta-sheet proteins, including NMR structure determination
We demonstrated that the NMR structures determined by the fractionally 13 C-labeling scheme are comparable to the previously determined crystal structures
Summary
NMR spectroscopy has been routinely used for elucidating three-dimensional structures of proteins in solution [1,2,3]. X-ray crystallography because it does not require any crystallization and can investigate protein structures under various solution conditions, including even in situ [4,5]. One critical bottleneck of NMR analysis compared with other three-dimensional analysis is the requirement of stable isotopic labeling such as 15 N and 13 C-labeling, which is typically desirable even for proteins as small as 5 kDa to speed up reliable NMR analysis. The isotopic labeling procedure for NMR inherently increases the cost and efforts for sample preparations, limiting the broader application of various NMR analysis. NMR analysis of several variants can quickly become time-consuming and costly for various useful NMR analyses, such as investigating protein–
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