Abstract
Solid-state Nuclear Magnetic Resonance (ssNMR) is an emerging technique to investigate the structures and dynamics of membrane proteins in an artificial or native membrane environment. However, the structural studies of proteins by ssNMR are usually prolonged or impeded by signal assignments, especially the assignments of signals for collection of distance restraints, because of serious overlapping of signals in 2D 13C–13C spectra. Sparse labeling of 13C spins is an effective approach to simplify the 13C spectra and facilitate the extractions of distance restraints. Here, we propose a new reverse labeling combination of six types of amino acid residues (Ile, Leu, Phe, Trp, Tyr and Lys), and show a clean reverse labeling effect on a model membrane protein E. coli aquaporin Z (AqpZ). We further combine this reverse labeling combination and alternate 13C–12C labeling, and demonstrate an enhanced dilution effect in 13C–13C spectra. In addition, the influences of reverse labeling on the labeling of the other types of residues are quantitatively analyzed in the two strategies (1, reverse labeling and 2, reverse labeling combining alternate 13C–12C labeling). The signal intensities of some other types of residues in 2D 13C–13C spectra are observed to be 20–50% weaker because of the unwanted reverse labeling. The extensively sparse 13C labeling proposed in this study is expected to be useful in the collection of distance restraints using 2D 13C–13C spectra of membrane proteins.
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Acknowledgements
This work was supported by grants from the National Key R&D Program of China (Grant Nos. 2016YFA0501200 and 2017YFA0505400) and the National Natural Science Foundation of China (Grant Nos. 31600625, 21425523, 31770798, 21927801, 21991080, 21921004).
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Tong, Q., Tan, H., Li, J. et al. Extensively sparse 13C labeling to simplify solid-state NMR 13C spectra of membrane proteins. J Biomol NMR 75, 245–254 (2021). https://doi.org/10.1007/s10858-021-00372-y
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DOI: https://doi.org/10.1007/s10858-021-00372-y