Journal of Biomolecular NMR

, Volume 54, Issue 3, pp 291–305 | Cite as

Solid-state NMR analysis of membrane proteins and protein aggregates by proton detected spectroscopy

  • Donghua H. ZhouEmail author
  • Andrew J. Nieuwkoop
  • Deborah A. Berthold
  • Gemma Comellas
  • Lindsay J. Sperling
  • Ming Tang
  • Gautam J. Shah
  • Elliott J. Brea
  • Luisel R. Lemkau
  • Chad M. RienstraEmail author


Solid-state NMR has emerged as an important tool for structural biology and chemistry, capable of solving atomic-resolution structures for proteins in membrane-bound and aggregated states. Proton detection methods have been recently realized under fast magic-angle spinning conditions, providing large sensitivity enhancements for efficient examination of uniformly labeled proteins. The first and often most challenging step of protein structure determination by NMR is the site-specific resonance assignment. Here we demonstrate resonance assignments based on high-sensitivity proton-detected three-dimensional experiments for samples of different physical states, including a fully-protonated small protein (GB1, 6 kDa), a deuterated microcrystalline protein (DsbA, 21 kDa), a membrane protein (DsbB, 20 kDa) prepared in a lipid environment, and the extended core of a fibrillar protein (α-synuclein, 14 kDa). In our implementation of these experiments, including CONH, CO(CA)NH, CANH, CA(CO)NH, CBCANH, and CBCA(CO)NH, dipolar-based polarization transfer methods have been chosen for optimal efficiency for relatively high protonation levels (full protonation or 100 % amide proton), fast magic-angle spinning conditions (40 kHz) and moderate proton decoupling power levels. Each H–N pair correlates exclusively to either intra- or inter-residue carbons, but not both, to maximize spectral resolution. Experiment time can be reduced by at least a factor of 10 by using proton detection in comparison to carbon detection. These high-sensitivity experiments are especially important for membrane proteins, which often have rather low expression yield. Proton-detection based experiments are expected to play an important role in accelerating protein structure elucidation by solid-state NMR with the improved sensitivity and resolution.


Chemical assignment Solid-state NMR Proton detection Magic-angle spinning 



We thank the National Institutes of Health for financial support (R01 GM-75937 and R01 GM-73770 to C.M.R and R15 GM-097713 to D.H.Z.). We also thank the NMR Facility at the School of Chemical Sciences, University of Illinois at Urbana-Champaign.

Supplementary material

10858_2012_9672_MOESM1_ESM.pdf (3.3 mb)
Supplementary material 1 (PDF 3387 kb)


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Copyright information

© Springer Science+Business Media B.V. 2012

Authors and Affiliations

  • Donghua H. Zhou
    • 1
    Email author
  • Andrew J. Nieuwkoop
    • 2
    • 3
  • Deborah A. Berthold
    • 2
  • Gemma Comellas
    • 4
  • Lindsay J. Sperling
    • 2
    • 5
  • Ming Tang
    • 2
  • Gautam J. Shah
    • 2
  • Elliott J. Brea
    • 2
  • Luisel R. Lemkau
    • 2
  • Chad M. Rienstra
    • 2
    • 4
    Email author
  1. 1.Department of PhysicsOklahoma State UniversityStillwaterUSA
  2. 2.Department of ChemistryUniversity of Illinois at Urbana-ChampaignUrbanaUSA
  3. 3.Leibniz-Institut für Molekulare PharmakologieBerlinGermany
  4. 4.Center for Biophysics and Computational BiologyUniversity of Illinois at Urbana-ChampaignUrbanaUSA
  5. 5.Materials Science DivisionLawrence Berkeley National LaboratoryBerkeleyUSA

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