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Journal of Biomolecular NMR

, Volume 49, Issue 2, pp 151–161 | Cite as

Uniform isotope labeling of a eukaryotic seven-transmembrane helical protein in yeast enables high-resolution solid-state NMR studies in the lipid environment

  • Ying Fan
  • Lichi Shi
  • Vladimir Ladizhansky
  • Leonid S. Brown
Article

Abstract

Overexpression of isotope-labeled multi-spanning eukaryotic membrane proteins for structural NMR studies is often challenging. On the one hand, difficulties with achieving proper folding, membrane insertion, and native-like post-translational modifications frequently disqualify bacterial expression systems. On the other hand, eukaryotic cell cultures can be prohibitively expensive. One of the viable alternatives, successfully used for producing proteins for solution NMR studies, is yeast expression systems, particularly Pichia pastoris. We report on successful implementation and optimization of isotope labeling protocols, previously used for soluble secreted proteins, to produce homogeneous samples of a eukaryotic seven-transmembrane helical protein, rhodopsin from Leptosphaeria maculans. Even in shake-flask cultures, yields exceeded 5 mg of purified uniformly 13C,15N-labeled protein per liter of culture. The protein was stable (at least several weeks at 5°C) and functionally active upon reconstitution into lipid membranes at high protein-to-lipid ratio required for solid-state NMR. The samples gave high-resolution 13C and 15N solid-state magic angle spinning NMR spectra, amenable to a detailed structural analysis. We believe that similar protocols can be adopted for challenging mammalian targets, which often resist characterization by other structural methods.

Keywords

Solid-state NMR Magic angle spinning Uniformly 13C, 15N labeled proteins Eukaryotic membrane proteins Pichia pastoris 

Abbreviations

7TM

Seven-transmembrane

ASR

Anabaena sensory rhodopsin

BR

Bacteriorhodopsin

BMD

Buffered minimal dextrose

BMM

Buffered minimal methanol

CHES

N-Cyclohexyl-2-aminoethanesulfonic acid

DMPC

1,2-dimyristoyl-sn-glycero-3-phosphocholine

DMPA

1,2-dimyristoyl-sn-glycero-3-phosphate

DTT

Dithiothreitol

E. coli

Escherichia coli

EDTA

Ethylenediaminetetraacetic acid

FTIR

Fourier-transform infrared

GPCRs

G-protein coupled receptors

LR

Leptosphaeria rhodopsin

MALDI TOF

Matrix-assisted laser desorption/ionization time-of-flight

MAS

Magic angle spinning

NR

Neurospora rhodopsin

P. pastoris

Pichia pastoris

PMSF

Phenylmethylsulfonyl fluoride

PR

Proteorhodopsin

SDS–PAGE

Sodium dodecyl sulfate polyacrylamide gel electrophoresis

SR-II

Sensory rhodopsin II

ssNMR

Solid-state NMR

YPD

Yeast peptone dextrose

Notes

Acknowledgments

This research was supported by the University of Guelph (start-up funds to V.L. and L.S.B.), the Natural Sciences and Engineering Research Council of Canada (discovery grants to L.S.B. and to V.L., and doctoral scholarship to Y.F.), the Canada Foundation for Innovation, and the Ontario Innovation Trust. V.L. holds Canada Research Chair in Biophysics, and is a recipient of an Early Researcher Award from the Ontario Ministry of Research and Innovation. L.S. is a recipient of the MITACS Accelerate scholarship, co-funded by Bruker Ltd. We thank Cambridge Isotope Laboratories for the generous gift of isotopically labeled methanol.

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Copyright information

© Springer Science+Business Media B.V. 2011

Authors and Affiliations

  • Ying Fan
    • 1
    • 2
  • Lichi Shi
    • 1
    • 2
  • Vladimir Ladizhansky
    • 1
    • 2
  • Leonid S. Brown
    • 1
    • 2
  1. 1.Department of PhysicsUniversity of GuelphGuelphCanada
  2. 2.Department of Physics and Biophysics Interdepartmental GroupUniversity of GuelphGuelphCanada

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