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FTIR analysis of GPCR activation using azido probes

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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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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 Text and Figures

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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