Combination of (15)N reverse labeling and afterglow spectroscopy for assigning membrane protein spectra by magic-angle-spinning solid-state NMR: application to the multidrug resistance protein EmrE.
 

Combination of (15)N reverse labeling and afterglow spectroscopy for assigning membrane protein spectra by magic-angle-spinning solid-state NMR: application to the multidrug resistance protein EmrE.

Related Articles Combination of (15)N reverse labeling and afterglow spectroscopy for assigning membrane protein spectra by magic-angle-spinning solid-state NMR: application to the multidrug resistance protein EmrE.

J Biomol NMR. 2013 Mar 29;

Authors: Banigan JR, Gayen A, Traaseth NJ

Abstract
Magic-angle-spinning (MAS) solid-state NMR spectroscopy has emerged as a viable method to characterize membrane protein structure and dynamics. Nevertheless, the spectral resolution for uniformly labeled samples is often compromised by redundancy of the primary sequence and the presence of helical secondary structure that results in substantial resonance overlap. The ability to simplify the spectrum in order to obtain unambiguous site-specific assignments is a major bottleneck for structure determination. To address this problem, we used a combination of (15)N reverse labeling, afterglow spectroscopic techniques, and frequency-selective dephasing experiments that dramatically improved the ability to resolve peaks in crowded spectra. This was demonstrated using the polytopic membrane protein EmrE, an efflux pump involved in multidrug resistance. Residues preceding the (15)N reverse labeled amino acid were imaged using a 3D NCOCX afterglow experiment and those following were recorded using a frequency-selective dephasing experiment. Our approach reduced the spectral congestion and provided a sensitive way to obtain chemical shift assignments for a membrane protein where no high-resolution structure is available. This MAS methodology is widely applicable to the study of other polytopic membrane proteins in functional lipid bilayer environments.


PMID: 23539118 [PubMed - as supplied by publisher]



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