The DOI matches its Crossref record; 51 of the 51 references it cites were found in a registry or an index.
Optimized orthogonal translation of unnatural amino acids enables spontaneous protein double-labelling and FRET
Open accessAbstract
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. A series of quadruplet decoding tRNAs has been developed to form an optimized orthogonal translation system. These tRNAs enable efficient, site-specific incorporation of multiple unnatural amino acids into a protein, with a substantial increase in yield over previous methods. The amino acids are then used to site-specifically label a protein with a pair of fluorophores, enabling studies of the protein's dynamics.
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Cites
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Three-dimensional structure of calmodulin
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Cellular Incorporation of Unnatural Amino Acids and Bioorthogonal Labeling of Proteins
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Self-citationCalcium signaling: A tale for all seasons
Carafoli, E. (2002) · Proceedings of the National Academy of Sciences · doi:10.1073/pnas.032427999 · Cited by 844
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Self-citationAddition of a photocrosslinking amino acid to the genetic code of Escherichia coli
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Self-citationHoeflich, K. P., & Ikura, M. (2002) · Cell · doi:10.1016/s0092-8674(02)00682-7 · Cited by 700
Encoding multiple unnatural amino acids via evolution of a quadruplet-decoding ribosome
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Self-citationBioorthogonal Reactions for Labeling Proteins
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Cited by
Scinto, S. L., Bilodeau, D. A., Hincapie, R., Lee, W., Nguyen, S. S., Xu, M., am Ende, C. W., Finn, M. G., Lang, K., Lin, Q., et al. (2021) · Nature Reviews Methods Primers · doi:10.1038/s43586-021-00028-z · Cited by 592
Self-citationContemporary approaches to site-selective protein modification
Hoyt, E. A., Cal, P. M. S. D., Oliveira, B. L., & Bernardes, G. J. L. (2019) · Nature Reviews Chemistry · doi:10.1038/s41570-019-0079-1 · Cited by 537
Histones: At the Crossroads of Peptide and Protein Chemistry
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Labeling and Single-Molecule Methods To Monitor G Protein-Coupled Receptor Dynamics
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Metadata
- Authors
- Wang, K., Sachdeva, A., Cox, D. J., Wilf, N. M., Lang, K., Wallace, S., Mehl, R. A., & Chin, J. W.
- Venue
- Nature Chemistry
- Published
- 2014-04-20
- Volume
- 6
- Issue
- 5
- Pages
- 393-403
- ISSN
- 1755-4330 (ISSN-L)
- 1755-4349
- OpenAlex
- W2059253719
- Cited by
- 269
- References
- 51
- FWCI
- 12.17
- Type
- journal article
- Language
- en
- Open access
- green
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