Determination of structural topology of a membrane protein in lipid bilayers using polarization optimized experiments (POE) for static and MAS solid state NMR spectroscopy

Kaustubh R. Mote, T. Gopinath, Gianluigi Veglia

Research output: Contribution to journalArticlepeer-review

30 Scopus citations

Abstract

The low sensitivity inherent to both the static and magic angle spinning techniques of solid-state NMR (ssNMR) spectroscopy has thus far limited the routine application of multidimensional experiments to determine the structure of membrane proteins in lipid bilayers. Here, we demonstrate the advantage of using a recently developed class of experiments, polarization optimized experiments, for both static and MAS spectroscopy to achieve higher sensitivity and substantial time-savings for 2D and 3D experiments. We used sarcolipin, a single pass membrane protein, reconstituted in oriented bicelles (for oriented ssNMR) and multilamellar vesicles (for MAS ssNMR) as a benchmark. The restraints derived by these experiments are then combined into a hybrid energy function to allow simultaneous determination of structure and topology. The resulting structural ensemble converged to a helical conformation with a backbone RMSD ∼0.44 Å , a tilt angle of 24° ± 1°, and an azimuthal angle of 55° ± 6°. This work represents a crucial first step toward obtaining high-resolution structures of large membrane proteins using combined multidimensional oriented solid-stateNMR and magic angle spinning solid-state NMR.

Original languageEnglish (US)
Pages (from-to)91-102
Number of pages12
JournalJournal of biomolecular NMR
Volume57
Issue number2
DOIs
StatePublished - Oct 2013

Bibliographical note

Funding Information:
Acknowledgments We thank Dr. Martin Gustavsson and Dr. Vitaly Vostrikov for helpful discussions. This work is supported by the National Institute of Health (GM64742 to G.V.). The experiments were carried out at the University Of Minnesota NMR Center.

Keywords

  • DUMAS-ssNMR
  • Hybrid method for membrane protein structure determination
  • MEIOSIS
  • Magic angle spinning solid state NMR
  • Magnetically aligned bicelles
  • Membrane proteins
  • Oriented solid-state NMR (OSS-NMR)
  • Sarcolipin

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