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FTIR analysis of GPCR activation using azido probes

Nature Chemical Biology volume 5, pages 397–399 (2009)Cite this article

Abstract

We demonstrate the site-directed incorporation of an IR-active amino acid, p-azido-L-phenylalanine (azidoF, 1), into the G protein–coupled receptor rhodopsin using amber codon suppression technology. The antisymmetric stretch vibration of the azido group absorbs at ∼2,100 cm−1 in a clear spectral window and is sensitive to its electrostatic environment. We used FTIR difference spectroscopy to monitor the azido probe and show that the electrostatic environments of specific interhelical networks change during receptor activation.

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Figure 1: Non-natural amino acid mutagenesis.
Figure 2: FTIR spectroscopy on azidoF rhodopsin mutants.
Figure 3: Structural models of rhodopsin activation.

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References

  1. Pierce, K.L ., Premont, R.T . & Lefkowitz, R.J . Nat. Rev. Mol. Cell Biol. 3, 639–650 (2002).

    Article  CAS  Google Scholar 

  2. Huber, T., Menon, S. & Sakmar, T.P. Biochemistry 47, 11013–11023 (2008).

    Article  CAS  Google Scholar 

  3. Kobilka, B. & Schertler, G.F.X. Trends Pharmacol. Sci. 29, 79–83 (2008).

    Article  CAS  Google Scholar 

  4. Hanson, M.A. & Stevens, R.C. Structure 17, 8–14 (2009).

    Article  CAS  Google Scholar 

  5. Mahalingam, M., Martinez-Mayorga, K., Brown, M.F. & Vogel, R. Proc. Natl. Acad. Sci. USA 105, 17795–17800 (2008).

    Article  CAS  Google Scholar 

  6. Vogel, R. et al. J. Mol. Biol. 380, 648–655 (2008).

    Article  CAS  Google Scholar 

  7. Noren, C.J., Anthonycahill, S.J., Griffith, M.C. & Schultz, P.G. Science 244, 182–188 (1989).

    Article  CAS  Google Scholar 

  8. Wang, L., Brock, A., Herberich, B. & Schultz, P.G. Science 292, 498–500 (2001).

    Article  CAS  Google Scholar 

  9. Sakamoto, K. et al. Nucleic Acids Res. 30, 4692–4699 (2002).

    Article  CAS  Google Scholar 

  10. Schultz, K.C. et al. J. Am. Chem. Soc. 128, 13984–13985 (2006).

    Article  CAS  Google Scholar 

  11. Ohno, S. et al. J. Biochem 141, 335–343 (2007).

    Article  CAS  Google Scholar 

  12. Liu, W.S., Brock, A., Chen, S., Chen, S.B. & Schultz, P.G. Nat. Methods 4, 239–244 (2007).

    Article  CAS  Google Scholar 

  13. Ye, S.X. et al. J. Biol. Chem 283, 1525–1533 (2008).

    Article  CAS  Google Scholar 

  14. Silverman, L.N., Pitzer, M.E., Ankomah, P.O., Boxer, S.G. & Fenlon, E.E. J. Phys. Chem. B 111, 11611–11613 (2007).

    Article  CAS  Google Scholar 

  15. Suydam, I.T., Snow, C.D., Pande, V.S. & Boxer, S.G. Science 313, 200–204 (2006).

    Article  CAS  Google Scholar 

  16. Chin, J.W. et al. Science 301, 964–967 (2003).

    Article  CAS  Google Scholar 

  17. Hubbell, W.L., Altenbach, C., Hubbell, C.M. & Khorana, H.G. Adv. Protein Chem. 63, 243–290 (2003).

    Article  CAS  Google Scholar 

  18. Li, J., Edwards, P.C., Burghammer, M., Villa, C. & Schertler, G.F.X. J. Mol. Biol. 343, 1409–1438 (2004).

    Article  CAS  Google Scholar 

  19. Park, J.H., Scheerer, P., Hofmann, K.P., Choe, H.W. & Ernst, O.P. Nature 454, 183–187 (2008).

    Article  CAS  Google Scholar 

  20. Huber, T., Botelho, A.V., Beyer, K. & Brown, M.F. Biophys. J. 86, 2078–2100 (2004).

    Article  CAS  Google Scholar 

  21. Dunham, T.D. & Farrens, D.L. J. Biol. Chem. 274, 1683–1690 (1999).

    Article  CAS  Google Scholar 

  22. Farrens, D.L., Altenbach, C., Yang, K., Hubbell, W.L. & Khorana, H.G. Science 274, 768–770 (1996).

    Article  CAS  Google Scholar 

  23. Patel, A.B. et al. Proc. Natl. Acad. Sci. USA 101, 10048–10053 (2004).

    Article  CAS  Google Scholar 

  24. Kobilka, B.K. & Deupi, X. Trends Pharmacol. Sci. 28, 397–406 (2007).

    Article  CAS  Google Scholar 

  25. Scheerer, P. et al. Nature 455, 497–502 (2008).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

We thank S. Banerjee, T. Haines, U.L. RajBhandary, C. Köhrer and M.S. Sagredo for invaluable discussions. We are also grateful to the Proteomic Resource Center at The Rockefeller University for providing technical resources. Financial support was provided by the Deutsche Forschungsgemeinschaft (grant Vo 811/4-1 to R.V.) and by a C.H. Li Memorial Scholar Award (to S.Y.).

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Authors and Affiliations

  1. Laboratory of Molecular Biology and Biochemistry, The Rockefeller University, New York, New York, USA

    Shixin Ye, Thomas Huber & Thomas P Sakmar

  2. Biophysics Section, Institute of Molecular Medicine and Cell Research, Albert-Ludwigs-University Freiburg, Freiburg, Germany

    Reiner Vogel

Authors
  1. Shixin Ye
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  2. Thomas Huber
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  3. Reiner Vogel
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  4. Thomas P Sakmar
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Contributions

S.Y., T.H. and R.V. designed and conducted experiments, analyzed data and wrote the manuscript. T.P.S. designed experiments and wrote the manuscript.

Corresponding author

Correspondence to Thomas P Sakmar.

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Supplementary Figures 1 and 2, and Supplementary Methods (PDF 135 kb)

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Ye, S., Huber, T., Vogel, R. et al. FTIR analysis of GPCR activation using azido probes. Nat Chem Biol 5, 397–399 (2009). https://doi.org/10.1038/nchembio.167

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