title: Combining Sense and Nonsense Codon Reassignment for Site-Selective Protein Modification with Unnatural Amino Acids.
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 tRNACysCCUis 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.
authors.affiliation: Institute for Molecular Bioscience, The University of Queensland , St Lucia, QLD 4072, Australia.
authors.forename: Zhenling
authors.identifier: 0000-0002-5664-8303
authors.initials: Z
authors.lastname: Cui
authors.affiliation: Institute for Molecular Bioscience, The University of Queensland , St Lucia, QLD 4072, Australia.
authors.forename: Sergey
authors.initials: S
authors.lastname: Mureev
authors.affiliation: StemProtein , 6350 Nancy Ridge Drive, San Diego, California 92121, United States.
authors.forename: Mark E
authors.initials: ME
authors.lastname: Polinkovsky
authors.affiliation: Medical Research Council Laboratory of Molecular Biology , Francis Crick Avenue, Cambridge CB2 0QH, United Kingdom.
authors.forename: Zakir
authors.initials: Z
authors.lastname: Tnimov
authors.affiliation: Institute for Molecular Bioscience, The University of Queensland , St Lucia, QLD 4072, Australia.
authors.forename: Zhong
authors.initials: Z
authors.lastname: Guo
authors.affiliation: Institute for Molecular Bioscience, The University of Queensland , St Lucia, QLD 4072, Australia.
authors.forename: Thomas
authors.initials: T
authors.lastname: Durek
authors.affiliation: Institute for Molecular Bioscience, The University of Queensland , St Lucia, QLD 4072, Australia.
authors.forename: Alun
authors.initials: A
authors.lastname: Jones
authors.affiliation: Institute for Molecular Bioscience, The University of Queensland , St Lucia, QLD 4072, Australia.
authors.forename: Kirill
authors.initials: K
authors.lastname: Alexandrov
mesh_terms: D000596:Amino Acids / Q000235:genetics*; D000604:Amino Acyl-tRNA Synthetases / Q000378:metabolism; D001386:Azides / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism; D002474:Cell-Free System / Q000187:drug effects / Q000378:metabolism; D018389:Codon, Nonsense / Q000235:genetics*; D018388:Codon, Terminator / Q000235:genetics; D004926:Escherichia coli / Q000187:drug effects / Q000235:genetics / Q000378:metabolism; D056890:Eukaryota / Q000187:drug effects / Q000235:genetics / Q000378:metabolism; D010649:Phenylalanine / Q000031:analogs & derivatives / Q000494:pharmacology; D014176:Protein Biosynthesis / Q000187:drug effects / Q000235:genetics*; D011499:Protein Processing, Post-Translational / Q000187:drug effects / Q000235:genetics*; D012343:RNA, Transfer / Q000235:genetics
chemical_list: D000596:Amino Acids; D001386:Azides; D018389:Codon, Nonsense; D018388:Codon, Terminator; C027162:4-azidophenylalanine; D010649:Phenylalanine; D012343:RNA, Transfer; D000604:Amino Acyl-tRNA Synthetases; C019975:cysteinyl-tRNA synthetase; D002118:Calcium
keywords: dual protein labeling; in vitro protein translation; sense codon reassignment; single molecular FRET; site-specific protein modification; synthetic tRNA