Abstract
For typical globular proteins, contacts involving aromatic side chains would constitute the largest number of distance constraints that could be used to define the structure of proteins and protein complexes based on NOE contacts. However, the 1H NMR signals of aromatic side chains are often heavily overlapped, which hampers extensive use of aromatic NOE cross peaks. Some of this overlap can be overcome by recording 13C-dispersed NOESY spectra. However, the resolution in the carbon dimension is rather low due to the narrow dispersion of the carbon signals, large one-bond carbon–carbon (C–C) couplings, and line broadening due to chemical shift anisotropy (CSA). Although it has been noted that the CSA of aromatic carbons could be used in TROSY experiments for enhancing resolution, this has not been used much in practice because of complications arising from large aromatic one-bond C–C couplings, and 3D or 4D carbon dispersed NOESY are typically recorded at low resolution hampering straightforward peak assignments. Here we show that the aromatic TROSY effect can optimally be used when employing alternate 13C labeling using 2-13C glycerol, 2-13C pyruvate, or 3-13C pyruvate as the carbon source. With the elimination of the strong one-bond C–C coupling, the TROSY effect can easily be exploited. We show that 1H–13C TROSY spectra of alternately 13C labeled samples can be recorded at high resolution, and we employ 3D NOESY aromatic-TROSY spectra to obtain valuable intramolecular and intermolecular cross peaks on a protein complex.
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Abbreviations
- NMR:
-
Nuclear magnetic resonance
- NOE:
-
Nuclear Overhauser effect
- NOESY:
-
NOE spectroscopy
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Acknowledgments
This research was supported by the National Institutes of Health (Grants GM047467, GM094608 and EB002026).
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Milbradt, A.G., Arthanari, H., Takeuchi, K. et al. Increased resolution of aromatic cross peaks using alternate 13C labeling and TROSY. J Biomol NMR 62, 291–301 (2015). https://doi.org/10.1007/s10858-015-9944-5
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DOI: https://doi.org/10.1007/s10858-015-9944-5