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

James R. Banigan, Anindita Gayen, Nathaniel J. Traaseth

Research output: Contribution to journalArticle

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 15N 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 15N 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.

Original languageEnglish (US)
Pages (from-to)391-399
Number of pages9
JournalJournal of Biomolecular NMR
Volume55
Issue number4
DOIs
StatePublished - Apr 2013

Keywords

  • EmrE
  • Magic-angle-spinning
  • Membrane proteins
  • Sequential acquisition assignment methods
  • Small multidrug resistance
  • Solid-state NMR

ASJC Scopus subject areas

  • Biochemistry
  • Spectroscopy

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