Combining Sense and Nonsense Codon Reassignment for Site-Selective Protein Modification with Unnatural Amino Acids

• DOI: 10.1021/acssynbio.6b00245
• Publication Date: 2016-12-14T17:21:37Z
• Authors: Zhenling Cui; Sergey Mureev; Mark E. Polinkovsky; Zakir Tnimov; Zhong Guo; Thomas Durek; Alun Jones; Kirill Alexandrov
• Keywords: Genetic Code; Stop codon

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 multiple uAAs into polypeptides hampered by limited number reassignable nonsense codons. This challenge addressed in current work developing Escherichia coli vitro translation system depleted specific endogenous tRNAs. The translational activity this dependent on addition synthetic tRNAs chosen sense codon. allows uAA pre- or cotranslationally charged with uAA. We demonstrate utility incorporating BODIPY fluorophore unique AGG calmodulin(CaM) open reading frame using precharged BODIPY-tRNACysCCU. deacylated tRNACysCCU poor substrate Cysteinyl-tRNA synthetase, which ensures low background Cys Simultaneously, p-azidophenylalanine mediated amber-codon suppression its post-translational conjugation tetramethylrhodamine dibenzocyclooctyne (TAMRA-DIBO) were performed same polypeptide. simple robust takes advantage compatibility machinery thus requires only one derivatization step introduce two fluorescent labels. Using approach, we obtained CaM nearly homogeneously labeled FRET-forming fluorophores. Single molecule FRET analysis revealed dramatic changes conformation probe upon exposure Ca2+ chelating agent. presented applicable other codons can be directly transferred eukaryotic cell-free systems.