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

Nature Chemistry volume 6, pages 393–403 (2014)Cite this article

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A Corrigendum to this article was published on 23 January 2014

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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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Figure 1: Optimizing Pyl tRNA(N8)XXXX for incorporating unnatural amino acids in response to quadruplet codons decoded by ribo-Q1.
Figure 2: Evolved Pyl tRNA(N8)XXXX direct the efficient unnatural amino acid incorporation in response to quadruplet codons decoded by ribo-Q1.
Figure 3: Efficient incorporation of multiple distinct unnatural amino acids into a single polypeptide.
Figure 4: Efficient incorporation of a matrix of pairs of unnatural amino acids, including photocrosslinkers and chemical handles (azides, alkenes, alkynes and tetrazines), demonstrates generality.
Figure 5: 5 and 8 do not react with each other in a protein, but can be labelled efficiently with 10 and 9.
Figure 6: Site-specific double-labelling of CaM with a FRET pair to follow changes in protein conformation.

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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.

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  1. Kaihang Wang and Amit Sachdeva: K.W. and A.S. contributed equally to this work

Authors and 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. Department of Biochemistry and Biophysics, Oregon State University, 2011 ALS, Corvallis, 97331, Oregon, 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.

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Correspondence to Jason W. Chin.

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Wang, K., Sachdeva, A., Cox, D. et al. Optimized orthogonal translation of unnatural amino acids enables spontaneous protein double-labelling and FRET. Nature Chem 6, 393–403 (2014). https://doi.org/10.1038/nchem.1919

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