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Solid-state NMR spectroscopy structure determination of a lipid-embedded heptahelical membrane protein

Nature Methods volume 10, pages 1007–1012 (2013)Cite this article

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Abstract

Determination of structure of integral membrane proteins, especially in their native environment, is a formidable challenge in structural biology. Here we demonstrate that magic angle spinning solid-state NMR spectroscopy can be used to determine structures of membrane proteins reconstituted in synthetic lipids, an environment similar to the natural membrane. We combined a large number of experimentally determined interatomic distances and local torsional restraints to solve the structure of an oligomeric membrane protein of common seven-helical fold, Anabaena sensory rhodopsin (ASR). We determined the atomic resolution detail of the oligomerization interface of the ASR trimer, and the arrangement of helices, side chains and the retinal cofactor in the monomer.

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Figure 1: SSNMR spectra of ASR recorded on sparsely labeled samples.
Figure 2: Schematic representation of iterative long-range cross-peak assignment and convergence of structure calculation.
Figure 3: Overall topology and inter-monomer packing of ASR trimer.
Figure 4: Structural organization of ASR monomer.
Figure 5: A comparison of ASR structures in 3D crystals and in lipids.

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Acknowledgements

This research was funded by Natural Science and Engineering Research Council of Canada, National Research Foundation of Korea (Global Research Network Program), Canada Foundation for Innovation and Ontario Ministry of Economic Development and Innovation. S.W. is a recipient of the Canadian Institutes for Health Research Postdoctoral Fellowship. V.L. is supported by Canada Research Chair in Biophysics (Tier II). We thank B. Bardiaux (Leibniz-Institut für Molekulare Pharmakologie, Berlin) for providing the ARIA 2.3 program before publication, and C.P. Jaroniec and J. Lanyi for carefully reading the manuscript.

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Author notes

  1. Lichi Shi & Izuru Kawamura

    Present address: Present addresses: Department of Medical Genetics and Microbiology, Medical Science Building, University of Toronto, Toronto, Ontario, Canada (L.S.) and Faculty of Engineering, Yokohama National University, Yokohama, Japan (I.K.).,

Authors and Affiliations

  1. Department of Physics, University of Guelph, Guelph, Ontario, Canada

    Shenlin Wang, Rachel A Munro, Lichi Shi, Izuru Kawamura, Leonid S Brown & Vladimir Ladizhansky

  2. Biophysics Interdepartmental Group, University of Guelph, Guelph, Ontario, Canada

    Shenlin Wang, Rachel A Munro, Lichi Shi, Leonid S Brown & Vladimir Ladizhansky

  3. Department of Organic Chemistry for Life Science, Kobe Pharmaceutical University, Kobe, Japan

    Takashi Okitsu & Akimori Wada

  4. Department of Life Science, Sogang University, Seoul, South Korea

    So-Young Kim & Kwang-Hwan Jung

  5. Institute of Biological Interfaces, Sogang University, Seoul, South Korea

    So-Young Kim & Kwang-Hwan Jung

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Contributions

S.W., L.S., I.K. and R.A.M. prepared samples; R.A.M. and L.S.B. performed biochemical and FTIR characterization; V.L. and S.W. collected SSNMR spectroscopy data; S.W. processed and analyzed the data, and calculated the structures; T.O. and A.W. synthesized isotopically labeled retinal; S.-Y.K. and K.-H.J. produced ASR mutants; L.S.B. and V.L. designed the study; S.W., L.S.B. and V.L. wrote the paper. All authors discussed the results of the study.

Corresponding authors

Correspondence to Leonid S Brown or Vladimir Ladizhansky.

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The authors declare no competing financial interests.

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Wang, S., Munro, R., Shi, L. et al. Solid-state NMR spectroscopy structure determination of a lipid-embedded heptahelical membrane protein. Nat Methods 10, 1007–1012 (2013). https://doi.org/10.1038/nmeth.2635

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