Multidimensional oriented solid-state NMR experiments enable the sequential assignment of uniformly 15N labeled integral membrane proteins in magnetically aligned lipid bilayers

Kaustubh R. Mote, T. Gopinath, Nathaniel J. Traaseth, Jason Kitchen, Peter L. Gor'Kov, William W. Brey, Gianluigi Veglia

Research output: Contribution to journalArticle

Abstract

Oriented solid-state NMR is the most direct methodology to obtain the orientation of membrane proteins with respect to the lipid bilayer. The method consists of measuring 1H- 15N dipolar couplings (DC) and 15N anisotropic chemical shifts (CSA) for membrane proteins that are uniformly aligned with respect to the membrane bilayer. A significant advantage of this approach is that tilt and azimuthal (rotational) angles of the protein domains can be directly derived from analytical expression of DC and CSA values, or, alternatively, obtained by refining protein structures using these values as harmonic restraints in simulated annealing calculations. The Achilles' heel of this approach is the lack of suitable experiments for sequential assignment of the amide resonances. In this Article, we present a new pulse sequence that integrates proton driven spin diffusion (PDSD) with sensitivity-enhanced PISEMA in a 3D experiment ([ 1H, 15N]-SE-PISEMA-PDSD). The incorporation of 2D 15N/ 15N spin diffusion experiments into this new 3D experiment leads to the complete and unambiguous assignment of the 15N resonances. The feasibility of this approach is demonstrated for the membrane protein sarcolipin reconstituted in magnetically aligned lipid bicelles. Taken with low electric field probe technology, this approach will propel the determination of sequential assignment as well as structure and topology of larger integral membrane proteins in aligned lipid bilayers.

Original languageEnglish (US)
Pages (from-to)339-346
Number of pages8
JournalJournal of Biomolecular NMR
Volume51
Issue number3
DOIs
StatePublished - Nov 2011

Keywords

  • Magnetically aligned bicelles
  • Membrane proteins
  • Oriented solid-state NMR (OSS-NMR)
  • PISEMA
  • Proton driven spin diffusion
  • Sarcolipin
  • Sensitivity-enhancement
  • Sequential assignment

ASJC Scopus subject areas

  • Biochemistry
  • Spectroscopy

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