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Site-Selective Modification of Proteins through Genetic Encoding Unnatural Amino Acid and Bioorthogonal Reaction Development in Biological Studies
Post-translational modifications (PTMs) of proteins play key roles in functional pro- teomic by regulating activities, localization, and interaction with other molecules in the cellular environment. The studies of PTMs have become a major concern in chemical biology. Chemical modification of proteins offered a powerful toolbox in protein engineering for PTMs studies. Selectivity and specificity of chemical modi- fication stay as one of the challenges in this area. Genetic code expansion allows site-specific modification of proteins via unnatural amino acid (UAA) mutagenesis. In my research, several UAAs were introduced into a specific site of the target protein, and applications based on bioorthogonal reactions were also demonstrated. In chapter 1, tRNA/aaRS engineering for UAA mutagenesis was introduced with referenced reviews. Typical bioorthogonal reactions for chemical modifica- tion of proteins and recent developments were also discussed. In chapter 2, a proximity-enhanced site-selective protein labeling reaction was introduced. A lysine analog bearing a double bond (K-Alkene) was synthesized and the corre- sponding tRNA/aaRS pair was identified by screening a tRNA/aaRS library. K- Alkene was successfully incorporated into proteins in both E. coli and mammalian cells. Tetrazine-Maleimide linker for proximity-enhanced protein modification was synthesized and applied to the dimerization of Glutathione S-transferase (GST). In chapter 3, a synergistic oxidant- and light- promoted cycloaddition reaction was applied in site-selective protein labeling. we successfully applied cycloaddition of 2-napthoquinone-2-methide (oNQM) in protein labeling via a synergistic action between oxidation (ferricyanide) and light irradiation. In chapter 4, several tyro- sine modification analogs were synthesized. The corresponding tRNA/aaRSs for genetic encoding of p-Carboxymethyl-L-phenylalanine (pCMF) and sulfotyrosine (sTyr) in mammalian cells were identified via directed evolution by our collabora- tors, Dr. Xinyuan He from the Dr. Wei Niu’s lab at the Chemical & Biomolecular Engineering Department in University of Nebraska-Lincoln (UNL).
Ma, Bin, "Site-Selective Modification of Proteins through Genetic Encoding Unnatural Amino Acid and Bioorthogonal Reaction Development in Biological Studies" (2022). ETD collection for University of Nebraska - Lincoln. AAI29999645.