Journal of Biomolecular NMR

, Volume 54, Issue 3, pp 307–316 | Cite as

A spectroscopic assignment technique for membrane proteins reconstituted in magnetically aligned bicelles

  • Wenxing Tang
  • Robert W. Knox
  • Alexander A. Nevzorov


Oriented-sample NMR (OS-NMR) has emerged as a powerful tool for the structure determination of membrane proteins in their physiological environments. However, the traditional spectroscopic assignment method in OS NMR that uses the “shotgun” approach, though effective, is quite labor- and time-consuming as it is based on the preparation of multiple selectively labeled samples. Here we demonstrate that, by using a combination of the spin exchange under mismatched Hartmann-Hahn conditions and a recent sensitivity-enhancement REP-CP sequence, spectroscopic assignment of solid-state NMR spectra of Pf1 coat protein reconstituted in magnetically aligned bicelles can be significantly improved. This method yields a two-dimensional spin-exchanged version of the SAMPI4 spectrum correlating the 15N chemical shift and 15N–1H dipolar couplings, as well as spin-correlations between the (i, i ± 1) amide sites. Combining the spin-exchanged SAMPI4 spectrum with the original SAMPI4 experiment makes it possible to establish sequential assignments, and this technique is generally applicable to other uniaxially aligned membrane proteins. Inclusion of an 15N–15N correlation spectrum into the assignment process helps establish correlations between the peaks in crowded or ambiguous spectral regions of the spin-exchanged SAMPI4 experiment. Notably, unlike the traditional method, only a uniformly labeled protein sample is required for spectroscopic assignment with perhaps only a few selectively labeled “seed” spectra. Simulations for the magnetization transfer between the dilute spins under mismatched Hartmann Hahn conditions for various B 1 fields have also been performed. The results adequately describe the optimal conditions for establishing the cross peaks, thus eliminating the need for lengthy experimental optimizations.


Oriented-sample NMR (OS-NMR) Cross-polarization Membrane proteins Pf1 Spectroscopic assignment Magnetically aligned bicelles Mismatched Hartmann Hahn conditions 









SAMMY pulse sequence with π/4 pulse correction


Polarity index slant angles


Repetitive cross-polarization


Mismatched Hartmann-Hahn


Proton-driven spin diffusion



We would like to thank Prof. Stanley J. Opella and George J. Lu (UCSD) for stimulating discussions. Research supported by a grant from NSF (MCB 0843520).

Supplementary material

10858_2012_9673_MOESM1_ESM.doc (102 kb)
Supplementary material 1 (DOC 102 kb)


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Copyright information

© Springer Science+Business Media B.V. 2012

Authors and Affiliations

  • Wenxing Tang
    • 1
  • Robert W. Knox
    • 1
  • Alexander A. Nevzorov
    • 1
  1. 1.Department of ChemistryNorth Carolina State UniversityRaleighUSA

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