Combining sense and nonsense codon reassignment for site-selective protein modification with unnatural amino acids

Cui, Zhenling, Mureev, Sergey, Polinkovsky, Mark E., Tnimov, Zakir, Guo, Zhong, Durek, Thomas, Jones, Alun and Alexandrov, Kirill (2016) Combining sense and nonsense codon reassignment for site-selective protein modification with unnatural amino acids. ACS Synthetic Biology, 6 3: 535-544. doi:10.1021/acssynbio.6b00245


Author Cui, Zhenling
Mureev, Sergey
Polinkovsky, Mark E.
Tnimov, Zakir
Guo, Zhong
Durek, Thomas
Jones, Alun
Alexandrov, Kirill
Title Combining sense and nonsense codon reassignment for site-selective protein modification with unnatural amino acids
Journal name ACS Synthetic Biology   Check publisher's open access policy
ISSN 2161-5063
Publication date 2016-12-14
Year available 2017
Sub-type Article (original research)
DOI 10.1021/acssynbio.6b00245
Open Access Status Not yet assessed
Volume 6
Issue 3
Start page 535
End page 544
Total pages 10
Place of publication Washington, DC, United States
Publisher American Chemical Society
Language eng
Subject 2204 Biomedical Engineering
1301 Biochemistry, Genetics and Molecular Biology (miscellaneous)
Abstract Incorporation of unnatural amino acids (uAAs) via codon reassignment is a powerful approach for introducing novel chemical and biological properties to synthesized polypeptides. However, the site-selective incorporation of multiple uAAs into polypeptides is hampered by the limited number of reassignable nonsense codons. This challenge is addressed in the current work by developing Escherichia coli in vitro translation system depleted of specific endogenous tRNAs. The translational activity in this system is dependent on the addition of synthetic tRNAs for the chosen sense codon. This allows site-selective uAA incorporation via addition of tRNAs pre- or cotranslationally charged with uAA. We demonstrate the utility of this system by incorporating the BODIPY fluorophore into the unique AGG codon of the calmodulin(CaM) open reading frame using in vitro precharged BODIPY-tRNA . The deacylated tRNA is a poor substrate for Cysteinyl-tRNA synthetase, which ensures low background incorporation of Cys into the chosen codon. Simultaneously, p-azidophenylalanine mediated amber-codon suppression and its post-translational conjugation to tetramethylrhodamine dibenzocyclooctyne (TAMRA-DIBO) were performed on the same polypeptide. This simple and robust approach takes advantage of the compatibility of BODIPY fluorophore with the translational machinery and thus requires only one post-translational derivatization step to introduce two fluorescent labels. Using this approach, we obtained CaM nearly homogeneously labeled with two FRET-forming fluorophores. Single molecule FRET analysis revealed dramatic changes in the conformation of the CaM probe upon its exposure to Ca or a chelating agent. The presented approach is applicable to other sense codons and can be directly transferred to eukaryotic cell-free systems.
Formatted abstract
Incorporation of unnatural amino acids (uAAs) via codon reassignment is a powerful approach for introducing novel chemical and biological properties to synthesized polypeptides. However, the site-selective incorporation of multiple uAAs into polypeptides is hampered by the limited number of reassignable nonsense codons. This challenge is addressed in the current work by developing Escherichia coli in vitro translation system depleted of specific endogenous tRNAs. The translational activity in this system is dependent on the addition of synthetic tRNAs for the chosen sense codon. This allows site-selective uAA incorporation via addition of tRNAs pre- or cotranslationally charged with uAA. We demonstrate the utility of this system by incorporating the BODIPY fluorophore into the unique AGG codon of the calmodulin(CaM) open reading frame using in vitro precharged BODIPY-tRNACysCCU. The deacylated tRNACysCCU is a poor substrate for Cysteinyl-tRNA synthetase, which ensures low background incorporation of Cys into the chosen codon. Simultaneously, p-azidophenylalanine mediated amber-codon suppression and its post-translational conjugation to tetramethylrhodamine dibenzocyclooctyne (TAMRA-DIBO) were performed on the same polypeptide. This simple and robust approach takes advantage of the compatibility of BODIPY fluorophore with the translational machinery and thus requires only one post-translational derivatization step to introduce two fluorescent labels. Using this approach, we obtained CaM nearly homogeneously labeled with two FRET-forming fluorophores. Single molecule FRET analysis revealed dramatic changes in the conformation of the CaM probe upon its exposure to Ca2+ or a chelating agent. The presented approach is applicable to other sense codons and can be directly transferred to eukaryotic cell-free systems.
Keyword Dual protein labeling
In vitro protein translation
Sense codon reassignment
Single molecule FRET
Site-specific protein modification
Synthetic tRNA
Q-Index Code C1
Q-Index Status Provisional Code
Grant ID DP1094080
APP1037320
Institutional Status UQ
Additional Notes http://pubs.acs.org/doi/abs/10.1021/acssynbio.6b00245

Document type: Journal Article
Sub-type: Article (original research)
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Created: Tue, 28 Feb 2017, 17:01:47 EST by Chris Ende on behalf of Aust Institute for Bioengineering & Nanotechnology