Journal
JOURNAL OF THE AMERICAN CHEMICAL SOCIETY
Volume 133, Issue 48, Pages 19266-19269Publisher
AMER CHEMICAL SOC
DOI: 10.1021/ja206999c
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Funding
- Deutsche Forschungsgemeinschaft [SFB740, 05106/12-1]
- European Union [FP7/2007-2013, 211800, 201924, 261863]
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Methods enabling structural studies of membrane-integrated receptor systems without the necessity of purification provide an attractive perspective in membrane protein structural and molecular biology. This has become feasible in principle since the advent of dynamic nuclear polarization (DNP) magic-angle-spinning NMR spectroscopy, which delivers the required sensitivity. In this pilot study, we observed well-resolved solid-state NMR spectra of extensively (13)C-labeled neurotoxin II bound to the nicotinic acetylcholine receptor (nAChR) in native membranes. We show that TOTAPOL, a biradical required for DNP, is localized at membrane and protein surfaces. The concentration of active, membrane-attached biradical decreases with time, probably because of reactive components of the membrane preparation. An optimal distribution of active biradical has strong effects on the NMR data. The presence of inactive TOTAPOL in membrane-proximal situations but active biradical in the surrounding water/glycerol glass leads to wellresolved spectra, yet a considerable enhancement (epsilon = 12) is observed. The resulting spectra of a protein ligand bound to its receptor are paving the way for further DNP investigations of proteins embedded in native membrane patches.
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