title: Neurotoxin II bound to acetylcholine receptors in native membranes studied by dynamic nuclear polarization NMR.
abstract: 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 well-resolved spectra, yet a considerable enhancement (ε = 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.
authors: Leibniz-Institut für Molekulare Pharmakologie (FMP), Robert-Rössle-Str. 10, 13125 Berlin, Germany.
authors: Arne H
authors: AH
authors: Linden
authors: Sascha
authors: S
authors: Lange
authors: W Trent
authors: WT
authors: Franks
authors: Umit
authors: U
authors: Akbey
authors: Edgar
authors: E
authors: Specker
authors: Barth-Jan
authors: BJ
authors: van Rossum
authors: Hartmut
authors: H
authors: Oschkinat
mesh_terms: D000818:Animals; D002462:Cell Membrane / Q000378:metabolism*; D003039:Cobra Neurotoxin Proteins / Q000378:metabolism*; D017815:Elapidae / Q000378:metabolism*; D004557:Electric Organ / Q000166:cytology; D008958:Models, Molecular; D019906:Nuclear Magnetic Resonance, Biomolecular / Q000379:methods*; D011485:Protein Binding; D011950:Receptors, Cholinergic / Q000378:metabolism*; D014101:Torpedo
chemical_list: D003039:Cobra Neurotoxin Proteins; D011950:Receptors, Cholinergic; C076649:neurotoxin II, Naja naja oxiana