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


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 Achillesâ?? 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.

• Content Type Journal Article
• Category Article
• Pages 1-8
• DOI 10.1007/s10858-011-9571-8
• Authors
  • Kaustubh R. Mote, Department of Chemistry, University of Minnesota, Minneapolis, MN 55455-0431, USA
  • T. Gopinath, Department of Biochemistry, Molecular Biology and Biophysics, University of Minnesota, 207 Pleasant St. S. E, Minneapolis, MN 55455, USA
  • Nathaniel J. Traaseth, Department of Biochemistry, Molecular Biology and Biophysics, University of Minnesota, 207 Pleasant St. S. E, Minneapolis, MN 55455, USA
  • Jason Kitchen, National High Magnetic Field Laboratory, Tallahassee, FL 32310, USA
  • Peter L. Gorâ??kov, National High Magnetic Field Laboratory, Tallahassee, FL 32310, USA
  • William W. Brey, National High Magnetic Field Laboratory, Tallahassee, FL 32310, USA
  • Gianluigi Veglia, Department of Chemistry, University of Minnesota, Minneapolis, MN 55455-0431, USA

• • Journal Journal of Biomolecular NMR
  • Online ISSN 1573-5001
  • Print ISSN 0925-2738

Source: Journal of Biomolecular NMR