Journal of Biomolecular NMR

, Volume 36, Issue 1, pp 55–71 | Cite as

Magic-angle spinning solid-state NMR of a 144 kDa membrane protein complex: E. coli cytochrome bo3 oxidase

  • Heather L. Frericks
  • Donghua H. Zhou
  • Lai Lai Yap
  • Robert B. Gennis
  • Chad M. Rienstra


Recent progress in magic-angle spinning (MAS) solid-state NMR (SSNMR) has enabled multidimensional studies of large, macroscopically unoriented membrane proteins with associated lipids, without the requirement of solubility that limits other structural techniques. Here we present initial sample preparation and SSNMR studies of a 144 kDa integral membrane protein, E. coli cytochrome bo3 oxidase. The optimized protocol for expression and purification yields ∼5 mg of the enzymatically active, uniformly 13C,15N-enriched membrane protein complex from each liter of growth medium. The preparation retains endogenous lipids and yields spectra of high sensitivity and resolution, consistent with a folded, homogenous protein. Line widths of isolated signals are less than 0.5 ppm, with a large number of individual resonances resolved in the 2D and 3D spectra. The 13C chemical shifts, assigned by amino acid type, are consistent with the secondary structure previously observed by diffraction methods. Although the structure is predominantly helical, the percentage of non-helical signals varies among residue types; these percentages agree well between the NMR and diffraction data. Samples show minimal evidence of degradation after several weeks of NMR data acquisition. Use of a triple resonance scroll resonator probe further improves sample stability and enables higher power decoupling, higher duty cycles and more advanced 3D experiments to be performed. These initial results in cytochrome bo3 oxidase demonstrate that multidimensional MAS SSNMR techniques have sufficient sensitivity and resolution to interrogate selected parts of a very large uniformly 13C,15N-labeled membrane protein.


chemical shift correlation spectroscopy membrane protein recoupling sample preparation secondary structure 


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The funding for this work was provided by the University of Illinois (startup funds to C.M.R.), the NIH & NIGMS Roadmap Initiative (GM075937-01), and an Ullyot Fellowship to H.F. The authors would like to thank Dr. Paul Molitor (VOICE NMR Facility of School of Chemical Science, University of Illinois) for technical support and Drs. John Stringer and Charles Mullen (Varian, Inc.) for assistance with installation of the 750 MHz scroll resonator probe.


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Copyright information

© Springer Science+Business Media B.V. 2006

Authors and Affiliations

  • Heather L. Frericks
    • 1
  • Donghua H. Zhou
    • 1
  • Lai Lai Yap
    • 2
  • Robert B. Gennis
    • 1
    • 2
    • 3
  • Chad M. Rienstra
    • 1
    • 2
    • 3
  1. 1.Department of ChemistryUniversity of Illinois at Urbana-ChampaignUrbanaUSA
  2. 2.Department of BiochemistryUniversity of Illinois at Urbana-ChampaignUrbanaUSA
  3. 3.Center for Biophysics and Computational BiologyUniversity of Illinois at Urbana-ChampaignUrbanaUSA

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