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The Journal of Membrane Biology

, Volume 247, Issue 9–10, pp 957–964 | Cite as

The Use of Amphipols for NMR Structural Characterization of 7-TM Proteins

  • Shantha Elter
  • Thomas Raschle
  • Sabine Arens
  • Aldino Viegas
  • Vladimir Gelev
  • Manuel Etzkorn
  • Gerhard Wagner
Article

Abstract

While amphipols have been proven useful for refolding of seven transmembrane helical (7-TM) proteins including G-protein-coupled receptors (GPCRs) and it could be shown that an amphipol environment is in principle suitable for NMR structural studies of the embedded protein, high-resolution NMR insights into amphipol refolded and isotopically labeled GPCRs are still very limited. Here we report on the recent progress toward NMR structural studies of the melanocortin-2 and -4 receptors, two class A GPCRs which so far have not been reported to be incorporated into an amphipol environment. Making use of the established 7-TM protein bacteriorhodopsin (BR) we initially tested and optimized amphipol refolding conditions. Most promising conditions were transferred to the refolding of the two melanocortin receptors. Analytical-scale refolding experiments on the melanocortin-2 receptor show very similar behavior to the results obtained on BR. Using cell-free protein expression we could generate sufficient amounts of isotopically labeled bacteriorhodopsin as well as melanocortin-2 and -4 receptors for an initial NMR analysis. Upscaling of the amphipol refolding protocol to protein amounts needed for NMR structural studies was, however, not straightforward and impeded detailed NMR insights for the two GPCRs. While well-resolved and dispersed NMR spectra could only be obtained for bacteriorhodopsin, a comparison of NMR data recorded on the melanocortin-4 receptor in SDS and in an amphipol environment indicates that amphipol refolding induces larger structural modifications in the receptor.

Keywords

Amphipathic polymers Solution-state NMR 7-TM proteins Bacteriorhodopsin Melanocortin receptor 

Notes

Acknowledgments

This work was supported by grants from the German Academic Exchange Service (DAAD) and the DFG (ET 103/2-1) to M. E. as well as grants from the NIH (GM094608, GM075879, EB002026 and S10 RR029236).

Supplementary material

232_2014_9669_MOESM1_ESM.pdf (165 kb)
Supplementary material 1 (PDF 165 kb)

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Copyright information

© Springer Science+Business Media New York 2014

Authors and Affiliations

  • Shantha Elter
    • 1
  • Thomas Raschle
    • 2
    • 4
  • Sabine Arens
    • 1
  • Aldino Viegas
    • 1
  • Vladimir Gelev
    • 3
  • Manuel Etzkorn
    • 1
    • 2
  • Gerhard Wagner
    • 2
  1. 1.Institute of Physical BiologyHeinrich-Heine-UniversityDüsseldorfGermany
  2. 2.Department of Biological Chemistry & Molecular PharmacologyHarvard Medical SchoolBostonUSA
  3. 3.Faculty of Chemistry and PharmacySofia UniversitySofiaBulgaria
  4. 4.BiozentrumUniversity of BaselBaselSwitzerland

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