Development of novel chemical ligation methods for the synthesis of posttranslationally modified proteins.
Date of Issue2012
School of Biological Sciences
Proteins are the central molecules of life. They play important functional roles in vitually every biological process. Peptide and protein chemical synthesis is an enabling tool for the study of the structure and function of proteins, especially those with posttranslational modifications (PTMs), which are difficult to access by the traditional recombinant techniques. Since the development of solid-phase peptide synthesis (SPPS) and many chemical ligation methods, such as thioester-mediated native chemical ligation, the chemical synthesis of peptides and proteins are revolutionized. Proteins with PTMs remain challenging for biochemists to synthesize utilizing currently available approaches. During my Ph.D study, my work focused on the development of novel chemical methods to overcome these challenges. In this thesis, I am going to present some of my achievements in methodology development for the synthesis of some typical protein PTMs. In chapter 1, a brief introduction to the background of PTMs, the development of modern peptide chemistry and the methods for the preparation of proteins with PTMs were given. In chapter 2, we present the ligation/S-alkylation approach which we successfully applied to the synthesis N-terminal tail acetylated histone H4. Three different H4 variants with combinations of acetylation(s) at lysine 5, 8, 12 and 16 were synthesized using the ligation/S-alkylation approach. In chapter 3 and 4, we focus on the development of methods for the site-specific chemical ubiquitination of peptides and proteins. In chapter 3, we introduce a novel chemical ligation method named dual native chemical ligation at lysine. The dual ligation refers to the ligation at both α- and ε-amine of lysine mediated by the thiol group of 4-thiolysine. This dual ligation approach does not only expand the scope of chemical ligation to lysine residue but also provides an efficient chemical approach for site-specific ubiquitination. In chapter 4, we modify and improve our dual chemical ligation approach and apply it in the synthesis of a K48-linked diubiquitin. This demonstrated the feasibility of our methodology in synthesizing ubiquitinated proteins. In chapter 5, we developed a novel N- to C-terminus sequential chemical ligation approach for protein synthesis. This approach works through the combination of native chemical ligation and peptidyl N,N-bis(2-mercaptoethyl)-amide (BMEA) mediated ligation. We first demonstrated the feasibility of the approach using small model peptides and then applied the approach to the chemical synthesis of ubiquitin. In the concluding chapter, I review and discuss the current achievements in chemical synthesis of acetylated and ubiquitinated proteins since these two PTMs are important but challenging to be accessed by chemical approaches.