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Journal of Biomolecular NMR

, Volume 65, Issue 1, pp 7–13 | Cite as

Sparse 13C labelling for solid-state NMR studies of P. pastoris expressed eukaryotic seven-transmembrane proteins

  • Jing Liu
  • Chang Liu
  • Ying Fan
  • Rachel A. Munro
  • Vladimir Ladizhansky
  • Leonid S. Brown
  • Shenlin WangEmail author
Communication

Abstract

We demonstrate a novel sparse 13C labelling approach for methylotrophic yeast P. pastoris expression system, towards solid-state NMR studies of eukaryotic membrane proteins. The labelling scheme was achieved by co-utilizing natural abundance methanol and specifically 13C labelled glycerol as carbon sources in the expression medium. This strategy improves the spectral resolution by 1.5 fold, displays site-specific labelling patterns, and has advantages for collecting long-range distance restraints for structure determination of large eukaryotic membrane proteins by solid-state NMR.

Keywords

Magic-angle-spinning solid-state NMR Sparse 13C isotope labelling Eukaryotic seven-transmembrane proteins P. pastoris expression system 

Notes

Acknowledgments

All NMR experiments were carried out at the Beijing NMR Center or the NMR Facility of the National Center for Protein Sciences at Peking University. This work was supported by the National Natural Science Foundation of China (31470727), by the Beijing National Laboratory for Molecular Sciences, and by the start-up funds from Peking University. S.W. is a recipient of the 1000 plan for young talent program of China. We thank National Center for Protein Sciences at Peking University in Beijing, China, for assistance with MALDI-MS experiments and Dr. Wen Zhou and Dr. Rong Meng for data collection.

Supplementary material

10858_2016_33_MOESM1_ESM.docx (4.8 mb)
Supplementary material 1 (DOCX 4956 kb)

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Copyright information

© Springer Science+Business Media Dordrecht 2016

Authors and Affiliations

  1. 1.Beijing NMR CentrePeking UniversityBeijingChina
  2. 2.College of Chemistry and Molecular EngineeringPeking UniversityBeijingChina
  3. 3.Beijing National Laboratory for Molecular SciencesBeijingChina
  4. 4.The Scripps Research InstituteLa JollaUSA
  5. 5.Department of PhysicsUniversity of GuelphGuelphCanada
  6. 6.Biophysics Interdepartmental GroupUniversity of GuelphGuelphCanada

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