Off-campus UNL users: To download campus access dissertations, please use the following link to log into our proxy server with your NU ID and password. When you are done browsing please remember to return to this page and log out.

Non-UNL users: Please talk to your librarian about requesting this dissertation through interlibrary loan.

Site-Selective Modification of Proteins through Genetic Encoding Unnatural Amino Acid and Bioorthogonal Reaction Development in Biological Studies

Bin Ma, University of Nebraska - Lincoln

Abstract

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).

Subject Area

Organic chemistry

Recommended Citation

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.
https://digitalcommons.unl.edu/dissertations/AAI29999645

Share

COinS