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Site-Selective Modification of Proteins Through Genetic Code Expansion and Their Application for Biological Study

Xin Shang, University of Nebraska - Lincoln

Abstract

Chemical modification of proteins has proved to be an essential tool in engineering proteins for basic biological research, biotechnological application and pharmaceutical industry. The selectivity of modifications as one of the major challenges intrigues interest from organic chemists and chemical biologists. In order to achieve the selectivity, genetically incorporating unnatural amino acids into recombinant proteins via suppression of amber stop codons with an amino-acyl tRNA synthetase/tRNA pair was exploited in my project. I introduced several novel unnatural amino acids at defined sites of target proteins and demonstrated specific applications.^ In chapter 1, chemical reactions for protein modifications were and the major strategies explored to achieve the site-selectivity were summarized. Moreover, a variety of bioorthogonal reactions to covalently modify proteins was illustrated. Applications of site-selective protein modifications were briefly introduced with referenced reviews.^ In chapter 2, styrene derived unnatural amino acid (KStyr) was site-specifically incorporated into proteins and displayed highly bioorthogonal reactivity with tetrazine and tetrazole. New fluorophores were formed in both reactions, which enabled KStyr to be a versatile fluorogenic chemical reporter for site-selective protein labeling. Protein labelings in vitro and in vivo were successfully demonstrated.^ In chapter 3, vinyl sulfide unnatural amino acid (EVSI) as an oxidative stress-masked electrophilic unnatural amino acid was designed for a proximity-enhanced protein-protein crosslinking. Introducing EVSI at interaction surface of GST proteins allowed for a covalent bond formation with its proximal nucleophilic residue on the interacting GST after oxidation.^ In chapter 4, a photo-enhanced oxidative coupling for bioorthogonal ligation was disclosed by a means of protein-based fluorescent screening. Quinone methide intermediates initiated by ferricyanide displayed high selective reactivity with HOMO-promoted dienophile over cysteine. The reaction rate was dramatically increased by photo-irradiation and enabled site-selective protein labeling in cell lysate within short time.^ In chapter 5, a vinyl protected tyrosine (ViP) was designed and selectively incorporated at Tyr66 of sfGFP and cpsfGFP to turn off their fluorescence. Oxymercuration of vinyl ether was capable of removing protective vinyl group and recovered wild type sfGFP and cpsfGFP. This protection-deprotection based reaction on sfGFP and cpsfGFP variants proved highly efficient in the selective detection of mercury (II).^

Subject Area

Chemistry

Recommended Citation

Shang, Xin, "Site-Selective Modification of Proteins Through Genetic Code Expansion and Their Application for Biological Study" (2017). ETD collection for University of Nebraska - Lincoln. AAI10272437.
http://digitalcommons.unl.edu/dissertations/AAI10272437

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