Article | Published:

Optimized orthogonal translation of unnatural amino acids enables spontaneous protein double-labelling and FRET

Nature Chemistry volume 6, pages 393403 (2014) | Download Citation

  • A Corrigendum to this article was published on 23 January 2014

This article has been updated

Abstract

The ability to introduce different biophysical probes into defined positions in target proteins will provide powerful approaches for interrogating protein structure, function and dynamics. However, methods for site-specifically incorporating multiple distinct unnatural amino acids are hampered by their low efficiency. Here we provide a general solution to this challenge by developing an optimized orthogonal translation system that uses amber and evolved quadruplet-decoding transfer RNAs to encode numerous pairs of distinct unnatural amino acids into a single protein expressed in Escherichia coli with a substantial increase in efficiency over previous methods. We also provide a general strategy for labelling pairs of encoded unnatural amino acids with different probes via rapid and spontaneous reactions under physiological conditions. We demonstrate the utility of our approach by genetically directing the labelling of several pairs of sites in calmodulin with fluorophores and probing protein structure and dynamics by Förster resonance energy transfer.

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Change history

  • 07 January 2014

    In the version of this Article originally published, the middle initial of the co-author Nabil M. Wilf was incorrect, and the Acknowledgements section was missing a reference to the European Research Council; the statement should have read: "We thank the Medical Research Council (U105181009, UD99999908) and the European Research Council (MC-A024-PG0A) for financial support. We thank S-P. Chew (MRC-LMB Mass Spectrometry) for obtaining MALDI data." These errors have now been corrected in the online versions of the Article.

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Acknowledgements

We thank the Medical Research Council (U105181009, UD99999908) and the European Research Council (MC-A024-5PG0A) for financial support. We thank S-P. Chew (MRC-LMB Mass Spectrometry) for obtaining MALDI data.

Author information

Author notes

    • Kaihang Wang
    •  & Amit Sachdeva

    K.W. and A.S. contributed equally to this work

Affiliations

  1. Medical Research Council Laboratory of Molecular Biology, Francis Crick Avenue, Cambridge CB2 0QH, UK

    • Kaihang Wang
    • , Amit Sachdeva
    • , Daniel J. Cox
    • , Nabil M. Wilf
    • , Kathrin Lang
    • , Stephen Wallace
    •  & Jason W. Chin
  2. Oregon State University, Department of Biochemistry and Biophysics, 2011 ALS, Corvallis, Oregon 97331, USA

    • Ryan A. Mehl

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Contributions

J.W.C. and K.W. conceived the project. J.W.C., K.W. and A.S. planned and designed experiments and wrote the manuscript, with input from the other authors. R.A.M. provided MjTetPheRS and compound 5 for pilot experiments. All other authors performed experiments or provided reagents. K.W. and A.S. contributed equally to this work.

Competing interests

The authors declare no competing financial interests.

Corresponding author

Correspondence to Jason W. Chin.

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https://doi.org/10.1038/nchem.1919

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