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Facile backbone structure determination of human membrane proteins by NMR spectroscopy

Nature Methods volume 9, pages 834–839 (2012)Cite this article

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Abstract

Although nearly half of today's major pharmaceutical drugs target human integral membrane proteins (hIMPs), only 30 hIMP structures are currently available in the Protein Data Bank, largely owing to inefficiencies in protein production. Here we describe a strategy for the rapid structure determination of hIMPs, using solution NMR spectroscopy with systematically labeled proteins produced via cell-free expression. We report new backbone structures of six hIMPs, solved in only 18 months from 15 initial targets. Application of our protocols to an additional 135 hIMPs with molecular weight <30 kDa yielded 38 hIMPs suitable for structural characterization by solution NMR spectroscopy without additional optimization.

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Figure 1: Workflow for screening and evaluation of 15 selected hIMPs for structural studies.
Figure 2: NMR spectral quality and N-H backbone assignment for six hIMPs.
Figure 3: Solution NMR spectroscopy structures and long-distance constraints used in structure calculations.
Figure 4: Solution NMR spectroscopy structures of six hIMPs.

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Acknowledgements

We thank G. Louie for comments in preparation of the manuscript, A.S. Arseniev for suggestions on the spin-labeling procedure and S. Maslennikov for writing the atomDistancer program. C.K. thanks the Pioneer Foundation for a Pioneer Fund Postdoctoral Scholar Award. This work has been partly supported by US National Institutes of Health (S.C.: GM098630, GM095623; A.S. and U.P.: GM094662, GM094625 FDP, and GM54762), Incheon Free Economic Zone and the World Class University Program (Korea).

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Author notes

  1. Christian Klammt and Innokentiy Maslennikov: These authors contributed equally to this work.

Authors and Affiliations

  1. Structural Biology Laboratory, Salk Institute for Biological Studies, La Jolla, California, USA

    Christian Klammt, Innokentiy Maslennikov, Monika Bayrhuber, Cédric Eichmann, Navratna Vajpai, Ellis Jeremy Chua Chiu, Katherine Y Blain, Luis Esquivies, Witek Kwiatkowski, Roland Riek & Senyon Choe

  2. Joint Center for Biosciences at Gachon University of Medicine and Science, Incheon, Korea

    Christian Klammt, Innokentiy Maslennikov, Ellis Jeremy Chua Chiu, June Hyun Jung Kwon & Senyon Choe

  3. Laboratorium für Physikalische Chemie, ETH Zürich, Zürich, Switzerland.,

    Monika Bayrhuber, Cédric Eichmann, Navratna Vajpai & Roland Riek

  4. Clayton Foundation Laboratories for Peptide Biology, Salk Institute for Biological Studies, La Jolla, California, USA

    Bartosz Balana & Paul A Slesinger

  5. Department of Bioengineering and Therapeutic Sciences, Department of Pharmaceutical Chemistry and California Institute for Quantitative Biosciences, University of California at San Francisco, San Francisco, California, USA

    Ursula Pieper & Andrej Sali

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Contributions

C.K., I.M., W.K., R.R. and S.C. designed experiments, C.K., E.J.C.C., L.E. and J.H.J.K. cloned hIMP targets, performed cell-free expression, evaluated protein expression levels and detergent solubilization; C.K. and E.J.C.C. created single cysteine mutants for PRE experiments, prepared isotopically labeled NMR spectroscopy samples and samples for PRE measurements. C.K. and I.M. recorded NMR spectra and evaluated NMR spectral quality of tested hIMPs; C.K., I.M., M.B., C.E., N.V., E.J.C.C. and K.B. collected and assigned NMR spectra and analyzed data; I.M., M.B. and C.E. calculated the structures. C.K., E.J.C.C., B.B. and P.A.S. analyzed HIGD1A antibody specificity by western blot and by immunostaining; U.P. and A.S. calculated modeling leverage based on hIMP structures; C.K., I.M., W.K. and S.C. wrote the manuscript. All authors discussed the results and commented on the manuscript.

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Correspondence to Senyon Choe.

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The authors declare no competing financial interests.

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Klammt, C., Maslennikov, I., Bayrhuber, M. et al. Facile backbone structure determination of human membrane proteins by NMR spectroscopy. Nat Methods 9, 834–839 (2012). https://doi.org/10.1038/nmeth.2033

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