Neurotoxin II Bound to Acetylcholine Receptors in Native Membranes Studied by Dynamic Nuclear Polarization NMR

Citations Over TimeTop 10% of 2011 papers

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.

Related Papers

• → Orphan spin operators enable the acquisition of multiple 2D and 3D magic angle spinning solid-state NMR spectra(2013)36 cited
• Effects of acute denervation on ratio change of nicotinic acetylcholine receptor subtypes in the rat gastrocnemius muscle(2008)
• → Characterization of V2O5-AlPO4 catalysts by 51V and 1H magic-angle spinning solid-state nuclear magnetic resonance spectroscopy(1995)7 cited
• → Magic‐Angle Spinning Solid‐State Nuclear Magnetic Resonance: Application to Structural Biology(2009)2 cited
• [Antibody against synthetic peptide corresponding to alpha-subunit residues 128-142 of nicotinic acetylcholine receptor (AChR): the significance in myasthenia gravis (MG)].(1988)